Paul Bert

Audrey

Alex Chloé

Lucille Jérémy

Irina Gwen

Noémie, Camille, Victorien, Andréa, Baoloc Manuela Lilly

Kelly

Dinitrogen tetroxide (dimer of NO2) N2OR: Nitrous Oxide Reductase NAD(P)H: Nicotinamide Adenine

Keywords: Pseudomonas fluorescens, adaptation, glycerophospholipid, fatty acids, pollution, nitrogen oxides Pseudomonas fluorescens, adaptation, glycérophospholipide, acides gras, pollution, oxydes d'azote, détoxification La science Iron-Sulfur cluster cyclic Adenosine MonoPhosphate Confocal Laser Scanning Microscopy CO2: Carbon dioxide CO3 -: Carbonate radical anion Ct: Cycle threshold Cti: cis-trans-isomerase Xanthosine-(3', 5 Mass Spectrometry Imaging N2O: Nitrous oxide Nuclear Magnetic Resonance Spectroscopy Nitrogen dioxide Nitric Oxide Reductase Nitric Oxide Synthase NOx: Nitrogen Oxides Optical Density OM: Outer Membrane ONOO Phosphatidic Acid PBAP: Primary Biological Aerosol Particles

Pseudomonas adaptation to stress factors: role of membrane lipids and

Pseudomonas fluorescens response to NO2

The high distribution of Pseudomonas fluorescens group is linked to its ability to adapt to stress factors. This work goaled the response of an airborne P. fluorescens MFAF76a, and its clinical standard P. fluorescens MFN1032 to environmental changes in order to refine the specific adaptation of airborne bacteria. First the HPTLC-MALDI TOF MSI tool defined glycerophospholipid (GP) composition of both strains. In stationary growth phase, an unknown GP, in short UGP, was found and seemed to be involved in temperature adaptation for the clinical strain.

After exposure to 0.1, 5 and 45 ppm concentrations, the bacterial response to NO2 was defined through motility, biofilm formation, antibiotic resistance and expression of several chosen target genes. While no change in parameters was seen in bacteria exposed to 0.1 and 5 ppm of NO2, several alterations were occurred with a bacterial exposure to 45 ppm. NO2 seemed to bias the UGP production, reduced P. fluorescens swim and decreased swarm only for MFN1032 strain. Biofilm formed by NO2-treated MFAF76a showed increased maximum thickness, with no change in c-di-GMP intracellular level. Expression of the hmp-homologue gene involved in NO detoxification was upregulated in response to NO2, suggesting a possible common pathway between NO and NO2 detoxification.

Finally, NO2 was found to increase bacterial resistance to ciprofloxacin and chloramphenicol. Thus the resistance nodulation cell division (RND) MexEF-OprN efflux pump encoding genes were highly upregulated in both strains. Together these findings implement the first model of bacterial response to NO2 toxicity and the role(s) of GP in bacterial adaptation to environmental changes.

Table of content

I. Introduction 1. Properties of Pseudomonas genus

Bacteria of genus Pseudomonas belong to the phylum Proteobacteria, class γ-Proteobacteria, order Pseudomonadales. Pseudomonas are unicellular Gram-negative straight or lightly curved rods, with rounded extremity and average size of 2x0.5 µm [START_REF] Palleroni | Genus I. Pseudomonas migula 1894[END_REF]. These bacteria are motile with one or several polar flagella, able to growth in minimum medium and metabolize several hydrocarbon substrata as energy and carbon source. These aerobic bacteria have a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor.

However, nitrate can be used by several Pseudomonas strains as an alternate electron acceptor, allowing an anaerobic growth. Pseudomonas possess oxidase and catalase activity [START_REF] Brenner | Bergey's Manual® of Systematic Bacteriology[END_REF]. The features of these organisms are the degradation of a large number of organic compounds [START_REF] Barathi | Utilization of petroleum hydrocarbons by Pseudomonas fluorescens isolated from a petroleum-contaminated soil[END_REF][START_REF] Foght | Degradation of polycyclic aromatic hydrocarbons and aromatic heterocycles by a Pseudomonas species[END_REF][START_REF] Leahy | Microbial degradation of hydrocarbons in the environment[END_REF]), the interactions with plants ( [START_REF] Mercado-Blanco | Interactions between plants and beneficial Pseudomonas spp.: exploiting bacterial traits for crop protection[END_REF][START_REF] Nomura | Suppression of host defense in compatible plant-Pseudomonas syringae interactions[END_REF][START_REF] Tao | Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae[END_REF] and the associations in the rhizosphere, which are advantageous for agriculture [START_REF] Lugtenberg | Molecular determinants of rhizosphere colonization by Pseudomonas[END_REF][START_REF] Rainey | Adaptation of Pseudomonas fluorescens to the plant rhizosphere[END_REF]. In addition, Pseudomonas include one of the most dangerous opportunistic pathogens among all bacteria P. aeruginosa [START_REF] Driscoll | The epidemiology, pathogenesis and treatment of Pseudomonas aeruginosa infections[END_REF][START_REF] Srour | Effect of infection with transmissible strains of Pseudomonas aeruginosa on lung transplantation outcomes in patients with cystic fibrosis[END_REF]. Pseudomonas comprise taxa of metabolically versatile organisms capable of living under diverse environmental conditions. Consequently, the members of this group are able to growth in diverse environments [START_REF] Ringen | A study of the incidence of Pseudomonas aeruginosa from various natural sources[END_REF] as illustrated in Figure 1, including water [START_REF] Mena | Risk assessment of Pseudomonas aeruginosa in water[END_REF], soil [START_REF] Couillerot | Pseudomonas fluorescens and closely-related fluorescent pseudomonads as biocontrol agents of soilborne phytopathogens[END_REF][START_REF] Kiely | Exploiting new systems-based strategies to elucidate plant-bacterial interactions in the rhizosphere[END_REF], air [START_REF] Duclairoir Poc | Airborne fluorescent pseudomonads : what potential for virulence?[END_REF][START_REF] Morris | Surprising niche for the plant pathogen Pseudomonas syringae[END_REF], food, and hospital environment [START_REF] Shooter | Food and medicaments as possible sources of hospital strains of Pseudomonas aeruginosa[END_REF][START_REF] Shooter | Isolation of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella from food in hospitals, canteens, and schools[END_REF]. This worldwide distribution seems to be due to a high physiological and genetic adaptability [START_REF] Spiers | The causes of Pseudomonas diversity[END_REF], being the presence of numerous genomic islands in the genus, a key adaptability traits of individual strains to such ubiquitous environments [START_REF] Peix | Historical evolution and current status of the taxonomy of genus Pseudomonas[END_REF]. Thus, the genomes of several species like P. aeruginosa, P. fluorescens, P. syringae, P. putida or P. stutzeri include between 3.7 and 7.1x10 6 base pairs (bps) [START_REF] Spiers | The causes of Pseudomonas diversity[END_REF].

Taxonomic diversity

The name Pseudomonas originates from the work of Migula, 1894 describing this genus as "Cells with polar organs of motility strictly". Defined in this way, the genus Pseudomonas was very heterogeneous and suffers several taxonomic reclassifications. In 1966 Stainer established the Pseudomonas classification based on the capacity of strains to metabolize the different energy sources. This fundamental work described 267 Pseudomonas strains with their biochemical, physiological and phenotypic properties [START_REF] Stanier | The aerobic pseudomonads a taxonomic study[END_REF]. With the emergence of molecular biology, the Pseudomonas genus was reorganized. The study of genomic sequence P a g e 21 | 270 homology by DNA-DNA [START_REF] Palleroni | Deoxyribonucleic acid homologies among some Pseudomonas species[END_REF][START_REF] Palleroni | Phenotypic characterization and deoxyribonucleic acid homologies of Pseudomonas solanacearum[END_REF] or DNA-RNA [START_REF] Palleroni | Nucleic acid homologies in the genus Pseudomonas[END_REF] hybridizations, as well as the phylogenetic analysis based on 16S ribosomal RNA (rRNA) sequence [START_REF] Anzai | Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence[END_REF] have led to the division of Pseudomonas genus on five distinct 'rRNA homology' groups. This division explains an important heterogeneity of the Pseudomonas genus (Vos et al., 1985(Vos et al., , 1989;;Vos & Ley, 1983). The species belonging to the 'group I' conserved the genus name and termed the Pseudomonas sensu stricto; the other species ('group II-V') Pseudomonas sensu lato have been reclassified and generically renamed according to their phylogenetic relationships, include nowadays the related genus, including Ralstonia, Comamonas, Agrobacterium, Brevundimonas, or Burkholderia [START_REF] Anzai | Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence[END_REF]. The genus Pseudomonas sensu stricto was divided on two major lineages (Figure 2) represented by typical species, namely Pseudomonas aeruginosa and Pseudomonas fluorescens [START_REF] Bodilis | Variable copy number, intra-genomic heterogeneities and lateral transfers of the 16S rRNA gene in Pseudomonas[END_REF][START_REF] Mulet | DNA sequence-based analysis of the Pseudomonas species[END_REF]. The P. aeruginosa lineage is the more homogenous and consists of the P. aeruginosa species, but also of more distant species, like P. oleovorans or P. stutzeri. Basing on study of the 16S rRNA gene and targeting one or two hypervariable regions, Bodilis and coworkers divided P. fluorescens lineage on two r-clusters ("r"-clusters, the clusters based on "r"RNA gene data): the "putida" and the "fluorescens" [START_REF] Bodilis | Variable copy number, intra-genomic heterogeneities and lateral transfers of the 16S rRNA gene in Pseudomonas[END_REF]. The putida r-cluster corresponds to the P. putida group; the fluorescens r-cluster contains the P. fluorescens, P. syringae and P. lutea groups, as shown in Figure 2.

The fluorescens r-cluster is the largest in terms of species number (about 60% of the Pseudomonas species described), and the most heterogeneous Pseudomonas group in terms of phenotypes and genotypes [START_REF] Bodilis | Variable copy number, intra-genomic heterogeneities and lateral transfers of the 16S rRNA gene in Pseudomonas[END_REF].

Pseudomonas fluorescens strains

General information

The name Pseudomonas fluorescens was chosen because of the production of complex fluorescent peptidic siderophores called pyoverdines (Figure 3), which are very efficient iron scavengers in bacteria [START_REF] Cornelis | Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines[END_REF]. This species is described as psychrotrophic germ with optimum growth temperature between 25°C and 30°C, able to growth at 4°C, but unable to growth at 41°C, as thermophilic bacteria. Several strains of P. fluorescens are sequenced (58 sequenced genomes, 5 genomes annotated without gap, in April 2015, according to NCBI, http://www.ncbi.nlm.nih.gov/). A whole variety of phenotypes and genotypes allows the classification of P. fluorescens as heterogeneous bacteria, with several biovars and subbiovars. The percentage of DNA/DNA hybridization between strains of the same biovar is frequently less than 50%. Thus, the genotype of this species is difficult to define. The taxonomy of Pseudomonas is currently being renewed [START_REF] Palleroni | Genus I. Pseudomonas migula 1894[END_REF], as evidenced by the reclassification of CHAO or Pf-5 strains to a novel species named P. protegens [START_REF] Ramette | Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin[END_REF]. Here, we defined P. fluorescens as bacteria, belonging to the fluorescens r-cluster and previously published at the name P. fluorescens. (Spiers et al., 2013).

Distribution and adaptive capacity of Pseudomonas fluorescens

The members of P. fluorescens group are highly distributed in all major environments, including water, soil and air as illustrated in Figure 1 [START_REF] Bodilis | Phylogenetic relationships between environmental and clinical isolates of Pseudomonas fluorescens and related species deduced from 16S rRNA gene and OprF protein sequences[END_REF]Duclairoir Poc et al., 2014). These species contribute greatly to the turnover of organic matter and, while present P a g e 24 | 270 in soil, are abundant on the surfaces of plant roots and leave. This applies also the role of P.

fluorescens as rhizosphere biocontrol agents [START_REF] Walsh | Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation[END_REF]. Biocontrol strains have noticeably been observed at the root surface (i.e. the rhizoplane), often forming microcolonies or discontinued biofilms in the grooves between epidermal cells [START_REF] Couillerot | Pseudomonas fluorescens and closely-related fluorescent pseudomonads as biocontrol agents of soilborne phytopathogens[END_REF]. Some of the plant-colonizing strains, such as SBW25, positively affect plant health and nutrition [START_REF] Silby | Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens[END_REF].

The ability to growth at refrigerated temperatures opens to P. fluorescens the possibility to colonize the 'cold environments'. Thus, P. fluorescens was isolated from Antarctic glacier ice melt [START_REF] Attard | Effects of atmospheric conditions on ice nucleation activity of Pseudomonas[END_REF] or from clouds and rain water [START_REF] Ahern | Fluorescent pseudomonads isolated from Hebridean cloud and rain water produce biosurfactants but do not cause ice nucleation[END_REF], where these bacteria can catalyze freezing of supercooled water at a temperature as warm as -2°C [START_REF] Attard | Effects of atmospheric conditions on ice nucleation activity of Pseudomonas[END_REF][START_REF] Joly | Ice nucleation activity of bacteria isolated from cloud water[END_REF]. The low temperature adaptation makes P. fluorescens contamination a particular problem for the food-processing [START_REF] Dogan | Genetic diversity and spoilage potentials among Pseudomonas spp. isolated from fluid milk products and dairy processing plants[END_REF][START_REF] Jonghe | Influence of storage conditions on the growth of Pseudomonas species in refrigerated raw milk[END_REF]. Indeed, at low temperature P. fluorescens is able to product degradation enzymes, including proteases, lipases and gelatinases, modifying organoleptic food quality [START_REF] Russell | Bacterial membranes: the effects of chill storage and food processing. An overview[END_REF]. In addition, P. fluorescens can cause blood infusion-related infections and outbreaks, contaminating the blood products or the equipment associated with intravenous infusions during storage [START_REF] Gibaud | Pseudomonas fluorescens septicemia following transfusion of contaminated blood[END_REF][START_REF] Khabbaz | Pseudomonas fluorescens bacteremia from blood transfusion[END_REF][START_REF] Scales | Microbiology, genomics, and clinical significance of the Pseudomonas fluorescens species complex, an unappreciated colonizer of humans[END_REF].

Being psychrotrophic bacteria, several P. fluorescens strains still grow at increasing temperature (at least 37°C) [START_REF] Picot | Regulation of the cytotoxic effects of Pseudomonas fluorescens by growth temperature[END_REF]. Bodilis and coworkers proposed to split P.

fluorescens into two groups: (1) "true psychrotrophs" able to multiply at temperatures from 4°C

to at least 30°C (but no grow at 37°C); and (2) "thermo-tolerant psychrotrophs" able to grow at temperatures from 4°C to at least 37°C (but no grow at 42°C). Group (1) contains only strains from soil and water. Group ( 2) is composed of clinical isolates and also certain strains with soil origins [START_REF] Bodilis | Phylogenetic relationships between environmental and clinical isolates of Pseudomonas fluorescens and related species deduced from 16S rRNA gene and OprF protein sequences[END_REF]. This thermotolerance associated with virulence factors expression and biofilm formation (Figure 1) allows P. fluorescens to tame eukaryotic hosts. While far less virulent than P. aeruginosa, P. fluorescens can cause acute infections (opportunistic) in humans and was reported in clinical samples from the mouth, stomach and lungs of patients with compromised immune status [START_REF] Chapalain | Comparative study of 7 fluorescent pseudomonad clinical isolates[END_REF][START_REF] Scales | Microbiology, genomics, and clinical significance of the Pseudomonas fluorescens species complex, an unappreciated colonizer of humans[END_REF], as shown in Figure 1. Species of P. fluorescens are associated to agents of pulmonary diseases [START_REF] Bahrani-Mougeot | Molecular analysis of oral and respiratory bacterial species associated with ventilator-associated pneumonia[END_REF][START_REF] Choi | Involvement of epidermal growth factor receptor-linked signaling responses in Pseudomonas fluorescens-infected alveolar epithelial cells[END_REF][START_REF] Dickson | Changes in the lung microbiome following lung transplantation include the emergence of two distinct Pseudomonas species with distinct clinical associations[END_REF], urinary infections [START_REF] Osawa | Evaluation of antibacterial efficacy of drugs for urinary tract infections by genotyping based on pulsed-field gel electrophoresis (PFGE)[END_REF], eye infections [START_REF] Durbán | Bacterial contamination of hydrophilic contact lens solutions marketed in spain[END_REF], and bloodstream infections [START_REF] Benito | Outbreak of Pseudomonas fluorescens bloodstream infection in a coronary care unit[END_REF]. Several strains are able to link to eukaryotic cells, such as intestinal cells Caco2/TC7, to modify their permeability, inducting cytotoxic and pro-inflammatory responses (Madi et al., 2010a, b).

Indeed, P. fluorescens possesses a number of functional traits, which provide to this bacterium the capacity to grow in mammalian hosts. These traits include production of bioactive P a g e 25 | 270 secondary metabolites [START_REF] Gross | Genomics of secondary metabolite production by Pseudomonas spp[END_REF][START_REF] Mavrodi | Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation[END_REF][START_REF] Ramette | Prevalence of fluorescent pseudomonads producing antifungal phloroglucinols and/or hydrogen cyanide in soils naturally suppressive or conducive to tobacco black root rot[END_REF][START_REF] Rossignol | Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens[END_REF][START_REF] Weller | Role of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. in the defense of plant roots[END_REF], synthesis of siderophores [START_REF] Cornelis | Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines[END_REF], type III secretion system [START_REF] Marchi | Genomic analysis of the biocontrol strain Pseudomonas fluorescens Pf29Arp with evidence of T3SS and T6SS gene expression on plant roots[END_REF][START_REF] Mavrodi | Structural and functional analysis of the type III secretion system from Pseudomonas fluorescens Q8r1-96[END_REF][START_REF] Rezzonico | The type III secretion system of biocontrol Pseudomonas fluorescens KD targets the phytopathogenic Chromista Pythium ultimum and promotes cucumber protection[END_REF][START_REF] Sperandio | Virulence of the Pseudomonas fluorescens clinical strain MFN1032 towards Dictyostelium discoideum and macrophages in relation with type III secretion system[END_REF], ability to form biofilms [START_REF] Barahona | Efficient rhizosphere colonization by Pseudomonas fluorescens f113 mutants unable to form biofilms on abiotic surfaces[END_REF][START_REF] Baum | Characterization of structures in biofilms formed by a P a g e 219 | 270 Pseudomonas fluorescens isolated from soil[END_REF], and plasticity of some strains to adapt to grow at higher temperature.

Biofilm formation as P. fluorescens adaptation mode

Bacteria respond to fluctuating environmental signals to coordinate adaptive changes in metabolic pathways and physiological outputs. Integration of environmental factors affords bacteria the ability to make important decisions regarding how to respond to the constantly changing environments. One of the possibilities is the change of the lifestyle. Bacteria can live in a free movement (planktonic) existence or commit to a surface attached lifestyle (biofilm).

There are three major types of bacterial motility (Figure 4), depending on medium viscosity, namely, swimming in aqueous environments, twitching on solid surfaces, and swarming on semisolid, viscous media, such as those containing 0.4 to 0.7% (wt/vol) agar [START_REF] Overhage | Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance[END_REF]. Swimming and swarming motility needs the synthesis of flagella. The swimming is brought about by individual cells independently perceiving chemical signals that trigger adaptive chemotactic responses. The swarming motility is characterized by a multicellular movement of bacteria that migrate above solid substrates in groups of tightly bound cells [START_REF] Calvio | Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon[END_REF].

Swarming motility requires both the functional flagella and the production of surface-wetting agent biosurfactant [START_REF] Caiazza | Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14[END_REF][START_REF] Kearns | A field guide to bacterial swarming motility[END_REF]. Production of the biosurfactants serves P a g e 26 | 270 as an essential aid to swarming motility by acting as a wetting agent to overcome the surface tension of water and facilitates movement across the most surface [START_REF] Caiazza | Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa[END_REF]. The nature of Pseudomonas biosurfactants was described as strain-dependent [START_REF] Duclairoir-Poc | Influence of growth temperature on cyclolipopeptides production and on adhesion behaviour in environmental strains of Pseudomonas fluorescens[END_REF]. Unlike P. aeruginosa, which products biosurfactant rhamnolipid, P. fluorescens secretes cyclic lipopeptides (CLP), including viscosine and viscosinamide [START_REF] Duclairoir-Poc | Influence of growth temperature on cyclolipopeptides production and on adhesion behaviour in environmental strains of Pseudomonas fluorescens[END_REF][START_REF] Nielsen | Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54[END_REF]. Twitching motility is a flagella-independent form of bacterial translocation over moist surfaces. It occurs by the extension, tethering, and then retraction of polar type IV pili [START_REF] Mattick | Type IV pili and twitching motility[END_REF].

When coming in contact with a surface, whether in natural, industrial, or clinical settings, bacteria must evaluate whether the surface and the environment are favorable for attachment and biofilm formation [START_REF] Wei | Biofilm matrix and its regulation in Pseudomonas aeruginosa[END_REF]. [START_REF] Ma | Assembly and development of the Pseudomonas aeruginosa biofilm matrix[END_REF][START_REF] Wei | Biofilm matrix and its regulation in Pseudomonas aeruginosa[END_REF] The biofilm is defined as a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to inert or living surfaces [START_REF] Costerton | Bacterial biofilms: a common cause of persistent infections[END_REF][START_REF] O'toole | c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review[END_REF]. The formation of biofilms occurs as a series of regulated steps displayed in Figure 5 [START_REF] Ma | Assembly and development of the Pseudomonas aeruginosa biofilm matrix[END_REF][START_REF] O'toole | Biofilm formation as microbial development[END_REF]. Bacteria swim using flagellar-mediated motility toward a surface and initiate a reversible attachment (Figure 5.1) [START_REF] O'toole | Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis[END_REF]. A subpopulation of transiently attached bacteria becomes irreversibly attached to the surface to form a monolayer, followed by the formation of small microcolonies (Figure 5.2) [START_REF] Hinsa | Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein[END_REF]. The microcolonies grow into a mature biofilm with a developed architecture (Figure 5 et al., 2003). The biofilm cycle is completed by the biofilm dispersion, when bacterial cells are shed from the biofilm into the medium (Figure 5.5), to found best environmental conditions [START_REF] Chua | Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles[END_REF][START_REF] O'toole | Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis[END_REF].

Figure 6. Scanning electron micrographs of P. fluorescens biofilms

The monocultures of 14-day biofilms form P. fluorescens EvS4-B1 were shown. 1. Flat sheets of material (arrowheads), with some of the sheets wrapped around other structures (arrow). Bar = 1 µM. 2. The inside core of the "wrapped" structures, consisting of bacteria (B) embedded in an extracellular matrix and thin sheet of material (arrow). Bar = 1 µM. 3. A sheet of material (S), consisting of extracellular matrix, covering and attaching to the fiber network (potentially fluid-filled channels) and including associated bacteria (B) and particulate matter (P). Bar = 2µM. Adapted from [START_REF] Baum | Characterization of structures in biofilms formed by a P a g e 219 | 270 Pseudomonas fluorescens isolated from soil[END_REF][START_REF] Scales | Microbiology, genomics, and clinical significance of the Pseudomonas fluorescens species complex, an unappreciated colonizer of humans[END_REF] The biofilm formation involves an array of cellular factors and highly regulated mechanisms, specific for each bacterial species [START_REF] Monds | Phosphate-dependent modulation of c-di-GMP levels regulates Pseudomonas fluorescens Pf0-1 biofilm formation by controlling secretion of the adhesin LapA[END_REF]. Several extracellular components such as exopolysaccharides (EPS), proteins (e.g. adhesins, amyloid familial amyloidotic polyneuropathy (Fap) fibrils), and extracellular DNA (eDNA) are involved in adhesion and biofilm formation by the pseudomonads [START_REF] Dueholm | Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation[END_REF]. However, a unifying theme across bacterial species is the synthesis of the cellular signaling molecule bis- (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), which regulates both biofilm formation and motility [START_REF] D'argenio | Cyclic di-GMP as a bacterial second messenger[END_REF][START_REF] Boyd | LapG, required for modulating biofilm formation by Pseudomonas fluorescens Pf0-1, is a calcium-dependent protease[END_REF][START_REF] Schirmer | Structural and mechanistic determinants of c-di-GMP signalling[END_REF]. The c-di-GMP is a soluble molecule, which functions as a second messenger in bacteria. In general, cdi-GMP stimulates the biosynthesis of adhesins and EPS matrix in biofilms and inhibits various forms of motility. It controls switching between the motile planktonic and sessile biofilmassociated 'lifestyles' in bacteria [START_REF] Hengge | Principles of c-di-GMP signalling in bacteria[END_REF]. This molecule is synthesized by diguanylate cyclases (DGCs) [START_REF] Paul | Cell cycledependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain[END_REF], and is broken down into 5′-phosphoguanylyl-(3'-5')guanosine (pGpG) by specific phosphodiesterases (PDEs) as presented in Figure 7. The active DGC is a dimer of two subunits with GGDEF domains. The PDE harbors EAL or HD-GYR domains [START_REF] Kulesekara | Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence[END_REF][START_REF] Stelitano | C-di-GMP hydrolysis by Pseudomonas aeruginosa HD-GYP phosphodiesterases: analysis of the reaction mechanism and novel roles for pGpG[END_REF]. While the accumulation of c-di-GMP in bacterial cells promotes the biofilm formation, favoring EPS production and suppressing motility, reduction of c-di-GMP level favors planktonic growth and/or dispersion of established biofilm [START_REF] Christen | Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP[END_REF]Newell et al., 2011a). P a g e 28 | 270 Biofilm research has utilized P. fluorescens as a model, because of its high adaptation capacity and typical growth on surface in nature (Newell et al., 2011b). O'Toole and coworkers identified a mechanism of regulation of biofilm formation by phosphate in P. fluorescens Figure 8. This mechanism is based, in majority, on two proteins: a large adhesive protein, LapA, required for the attachment, and a periplasmic cysteine protease LapG that cleaves LapA from the cell surface, as shown in litterature [START_REF] Boyd | Structural features of the Pseudomonas fluorescens biofilm adhesin LapA required for LapGdependent cleavage, biofilm formation, and cell surface localization[END_REF][START_REF] O'toole | c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review[END_REF]. Under high phosphate level, the c-di-GMP accumulates in the cell. LapG activity is regulated by the inner membrane c-di-GMP effector protein LapD [START_REF] O'toole | c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review[END_REF]. LapD binds c-di-GMP and sequesters LapG at the inner membrane, promoting the maintenance of LapA on the cell surface. Under these conditions, LapA remains at the cell surface, forms irreversible attachments to the substratum, promoting biofilm formation (Newell et al., 2011a).

Figure 8. A proposed model for regulation of P. fluorescens biofilm formation

At high concentrations of phosphate, c-di-GMP accumulates in the cell. LapD binds c-di-GMP, sequesters LapG at the inner membrane, promoting the maintenance of LapA on the cell surface. In these conditions, cells attach to the surface and form the biofilm. Conversely, low phosphate concentrations are sensed by PhoR/Pst system complex, the PhoR kinase phosphorylates PhoB, which activates the transcription of rapA. The RapA degrades cdi-GMP to pGpG. This leads to dissociation of c-di-GMP from LapD, and the cleavage of the LapA in the periplasm, promoting the detachment of attached to substratum bacteria, and/or inhibition of future surface attachment of planktonic cells. According Newell et al., 2011a. Systematic analysis of P. fluorescens genomes revealed, that these species harbors over 20 genes, coding for putative DGCs, with 4 confirmed to be involved in the regulation of surface attachment and/or biofilm formation (Newell et al., 2011b). These genes include wspR gene.

WspR is the response regulator (RR) of the Wsp chemosensory system, which is involved in production of c-di-GMP, as its effector. The Wsp system includes the DGC WspR, a predicted membrane-bound methyl-accepting chemotaxis protein (MCP)-like receptor (WspA), two CheW-like scaffolding proteins (WspB and WspD), histidine sensor kinase (WspE), and two methyltransferases WspC and WspF [START_REF] Hickman | A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels[END_REF]. In addition to WspR, three other DGCs were found to promote biofilm formation in P. fluorescens. GcbA mainly controls swimming motility, GcbB preferentially affects localization of the LapA adhesin, and GcbC affects both LapA and motility (Newell et al., 2011b).

Although the clear establishing of positive correlation between biofilm formation and c-di-GMP level, the regulation of c-di-GMP, and as result, the switch between planktonic and biofilm growth, is increasingly complex [START_REF] Petrova | The Pseudomonas aeruginosa diguanylate cyclase GcbA, a homolog of P. fluorescens GcbA, promotes initial attachment to surfaces, but not biofilm formation, via regulation of motility[END_REF]. Several genes (flgZ, sadB, fgtA, flhH or fap) and regulation pathways (AdnA, GacA/GacS, AlgU or FuBA) were described as required for P. fluorescens adhesion and motility [START_REF] Casaz | The Pseudomonas fluorescens transcription activator AdnA is required for adhesion and motility[END_REF][START_REF] Dueholm | Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation[END_REF] P a g e 30 | 270 [START_REF] Martínez-Granero | The Gac-Rsm and SadB signal transduction pathways converge on AlgU to downregulate motility in Pseudomonas fluorescens[END_REF][START_REF] Mastropaolo | Novel genes involved in Pseudomonas fluorescens Pf0-1 motility and biofilm formation[END_REF][START_REF] Nian | Isolation of transposon mutants and characterization of genes involved in biofilm formation by Pseudomonas fluorescens TC222[END_REF][START_REF] Robleto | Genetic analysis of the AdnA regulon in Pseudomonas fluorescens: nonessential role of flagella in adhesion to sand and biofilm formation[END_REF]. These systems do not precise here, and themselves can be a subject of another work.

Biofilm is not only a simple bacterial lifestyle, but also a good adaptation mode to changing environmental conditions. The highly permeable water channels interspersed throughout the biofilm allow bacterial nutrient availability and metabolic cooperativity [START_REF] Davey | Microbial biofilms: from ecology to molecular genetics[END_REF]; the extracellular matrix protects bacteria against environmental stressors [START_REF] O'toole | c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review[END_REF], including pollution [START_REF] Baumgarten | Membrane vesicle formation as a multiplestress response mechanism enhances Pseudomonas putida DOT-T1E cell surface hydrophobicity and biofilm formation[END_REF][START_REF] Baum | Characterization of structures in biofilms formed by a P a g e 219 | 270 Pseudomonas fluorescens isolated from soil[END_REF] or temperature changes [START_REF] White-Ziegler | Low temperature (23 degrees C) increases expression of biofilm-, cold-shock-and RpoS-dependent genes in Escherichia coli K-12[END_REF], allowing bacterial survival in inconvenient environments.

Biofilm formation is one of the strategies to colonize eukaryotic host. As a result, infections caused by bacterial biofilms are persistent and very difficult to eradicate [START_REF] Drenkard | Antimicrobial resistance of Pseudomonas aeruginosa biofilms[END_REF][START_REF] Mah | A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance[END_REF].

Envelope role in adaptation of Pseudomonas fluorescens

Introduction

To be faced with an unpredictable, dilute and often hostile environment, bacteria have evolved a sophisticated and complex cell envelope that protects them playing a role of a first selective barrier between the cell and its environment [START_REF] Russell | Membranes as a target for stress adaptation[END_REF][START_REF] Silhavy | The bacterial cell envelope[END_REF]. The barrier functions of bacterial envelope are known to be depend critically on the physical state of lipid bilayer, including its structure and fluidity [START_REF] Mansilla | Control of membrane lipid fluidity by molecular thermosensors[END_REF]. Microbial envelopes are responsible for a plethora of physiological processes, such as regulating the substances traffic in and out of the cells [START_REF] Uratani | Effect of phospholipid composition on activity of sodiumdependent leucine transport system in Pseudomonas aeruginosa[END_REF]. It stabilizes protein structures for a correct functioning of membrane embedded enzymes and provides a matrix for many biological reactions [START_REF] Bernal | A Pseudomonas putida cardiolipin synthesis mutant exhibits increased sensitivity to drugs related to transport functionality[END_REF][START_REF] Pogozheva | Life at the border: adaptation of proteins to anisotropic membrane environment[END_REF]. The cell wall plays also a role of the first sensor in activating a stress response [START_REF] Los | Membrane fluidity and its roles in the perception of environmental signals[END_REF] and participates in bacteria/host interactions (van der [START_REF] Van Der Meer-Janssen | Lipids in host-pathogen interactions: pathogens exploit the complexity of the host cell lipidome[END_REF]Vromman & Subtil, 2014;[START_REF] Wenk | Lipidomics of host-pathogen interactions[END_REF].

Therefore, the precise regulation of membrane structure and fluidity, known as homeoviscous adaptation, in the face of a constantly changing environment is an important challenge for all bacteria [START_REF] Baysse | The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification[END_REF].

Properties of P. fluorescens membrane

Pseudomonas envelope, as well as envelope of all Gram-negative bacteria, is composed of two membranous structures (Figure 9) [START_REF] Bos | Biogenesis of the Gram-negative bacterial outer membrane[END_REF][START_REF] Glauert | The topography of the bacterial cell wall[END_REF]. The inner one (IM) is composed of a glycerophospholipids (GPs) bilayer [START_REF] Chatterjee | Outer membrane vesicles of bacteria[END_REF].

The outer membrane (OM) is an asymmetrical bilayer, consisting of GPs and lipopolysaccharides (LPS) in the inner and outer leaflet respectively (Freulet-Marrière et al., P a g e 31 | 270 2000). The GPs bilayer of OM immerges proteins, including porins, and receptors [START_REF] Bos | Biogenesis of the Gram-negative bacterial outer membrane[END_REF]. The outer leaflet is composed primarily of LPS projecting outside and the inner leaflet containing GPs.

Figure 9. Pseudomonas cell envelope

Inner membrane (IM) is a typical glycerophospholipid (GP) bilayer, playing a role of permeability barrier of the cell. The outer membrane (OM) is composed of an inner monolayer of GP and an outer monolayer of the Lipid A component of lipopolysaccharide (LPS). Lipid A is a fatty acid substituted phosphorylated disaccharide, connected via the unusual sugar 3-deoxy-D-manno-octulosonic acid (KDO) to a polysaccharide to build up the inner core, outer core and the O-antigen repeat. The prismatic space contains the amino acid-sugar cross-linked peptidoglycan layer, which gives a structural rigidity to the cell envelope. Many proteins and membrane-derived oligosaccharides (MDO) can also be found in periplasm. Adapted from W. Dowhan and M. Bogdanov, 2002. The LPS consists of three different sectors: (i) lipid-A (a fatty acid substituted phosphorylated disaccharide), (ii) the core polysaccharide comprising the inner and the outer cores, and (iii) the O-specific polysaccharide chains, a heterogeneous polymer of branched oligosaccharide units, as shown in Figure 9 [START_REF] Chatterjee | Outer membrane vesicles of bacteria[END_REF][START_REF] Freulet-Marrière | Evidence for association of lipopolysaccharide with Pseudomonas fluorescens strain MF0 porin OprF[END_REF]. The lipid portion of LPS serves as the lipid anchor and is commonly composed of fatty acids (FAs), sugars, and phosphate groups. The P. fluorescens LPS have been described as able to associate to the major outer membrane porin OprF [START_REF] Freulet-Marrière | Evidence for association of lipopolysaccharide with Pseudomonas fluorescens strain MF0 porin OprF[END_REF] and essential for the outer membrane functions, particularly during hostpathogen interactions [START_REF] Picot | Cytotoxic effects of the lipopolysaccharide from Pseudomonas fluorescens on neurons and glial cells[END_REF].

The P. fluorescens LPS is an infamous molecule because they are responsible for the endotoxin shock associated with the septicemia [START_REF] Raetz | Lipopolysaccharide endotoxins[END_REF], apoptosis and a concomitant and limited necrosis [START_REF] Picot | Cytotoxic effects of the lipopolysaccharide from Pseudomonas fluorescens on neurons and glial cells[END_REF].

Peptidoglycan layer is made up of repeating units of the disaccharide N-acetyl glucosamine and N-acetyl muramic acid, which are cross-linked by pentapeptide side chains [START_REF] Heilmann | On the peptidoglycan of the cell walls of Pseudomonas aeruginosa[END_REF] P a g e 32 | 270 [START_REF] Mirelman | Biosynthesis of peptidoglycan in Pseudomonas aeruginosa. The incorporation of peptidoglycan into the cell wall[END_REF]Vollmer et al., 2008). Because of its rigidity, it determines the cell shape. Agents, such as enzymes or antibiotics, that damage the peptidoglycan cause cell lysis owing to the turgor pressure of the cytoplasm [START_REF] Silhavy | The bacterial cell envelope[END_REF].

P. fluorescens possesses a relatively simple GP composition with phosphatidylethanolamine (PE, 60-80%), phosphatidylglycerol (PG; 10-25%), phosphatidylcholine (PC; not defined) and cardiolipin (CL; 8-25%) being the four major GPs found in the cell envelope [START_REF] Cullen | The effects of temperature on the composition and physical properties of the lipids of Pseudomonas fluorescens[END_REF][START_REF] Gill | Effect of growth temperature on the lipids of Pseudomonas fluorescens[END_REF]. Their relative ratio varies according experimental conditions and bacterial strains. However, these data are based on the experiments, performed in the 1970s using the thin-layer chromatography (TLC) [START_REF] Cullen | The effects of temperature on the composition and physical properties of the lipids of Pseudomonas fluorescens[END_REF][START_REF] Gill | Effect of growth temperature on the lipids of Pseudomonas fluorescens[END_REF]. The FAs composition of P. fluorescens has been well described [START_REF] Fouchard | Influence of growth conditions on Pseudomonas fluorescens strains: a link between metabolite production and the PLFA profile[END_REF]. Like all members of Pseudomonas genus, P. fluorescens contains 16-18 carbons in FA chains with or without unsaturation (only one unsaturation is found). In the absence of well systematized review about Pseudomonas GP synthesis and homeostasis we decided to summarize all known today information about this topic in the review format.

Glycerophospholipid synthesis and functions in Pseudomonas

P. fluorescens lipidome adaptation

Environmental changes directly affect structural membrane characteristics [START_REF] Baysse | The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification[END_REF]. As demonstrated in Figure 10, GPs' bilayer forms different phases (i.e. a gel phase or a liquid-crystalline phase) [START_REF] Hazel | The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment[END_REF]. The normal cell function requires membrane GPs bilayers that are largely fluid, in crystalline lamellar phase (planar bilayer) [START_REF] Mansilla | Control of membrane lipid fluidity by molecular thermosensors[END_REF][START_REF] Russell | Membranes as a target for stress adaptation[END_REF].

Figure 10. Adaptation of glycerophospholipid structure and composition in Pseudomonas

In the lamellar liquid-crystalline state, the lipid molecules are melted and disordered. Upon transition to the gel state, the glycerophospholipids (GPs) become ordered and the frequency of rotation and lateral movement is reduced. Bacteria can regulate their membrane fluidity adjusting the GPs head groups and fatty acids (FAs). In order to increase membrane fluidity, the increasing level of cis-unsaturated fatty acids (cis-UFAs) and phosphatidylcholine (PC) is required. In contrast, to decrease membrane fluidity, the levels of saturated (SFAs), trans-unsaturated (trans-UFAs), or cyclopropane FA are increased. Concerning the GP head groups, the increasing of phosphatidylglycerol (PG) and/or cardiolipin (CL) levels negatively regulates membrane fluidity. Adapted from [START_REF] Baysse | The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification[END_REF] Environmental changes can cause the suboptimal membrane fluidity resulting in the subnormal membrane functions. The temperature at the midpoint of this transition is called the transition temperature (Tm), and the change of phase accompanying an increase in temperature is called the lipid phase transition, or most properly, the order-disorder transition. The Tm is a function of the membrane GP composition and mainly depends on the FA composition [START_REF] Mansilla | Control of membrane lipid fluidity by molecular thermosensors[END_REF]. The (overly simplified) rule of thumb is that GPs that contain unsaturated fatty acids (UFAs) have much lower Tm than those lipids made of saturated fatty acids (SFAs). The effect is due to different packing of the two types of GP acyl chains as demonstrated in > Tm (PG) [START_REF] Rühl | The glycerophospholipid inventory of Pseudomonas putida is conserved between strains and enables growth condition-related alterations[END_REF].

Increases resistance to osmotic stress and solvents

The ratio of GP head groups plays a pivotal role in maintain of optimal membrane properties and results in the balance between zwitterionic or neutral GPs (such as PE) and anionic GP (i.e.

PG or CL) [START_REF] Parsons | Bacterial lipids: metabolism and membrane homeostasis[END_REF]. These changes affect the physico-chemical properties of membranes due to the differences in melting temperature between the GP head groups. The increase of the PG level comparing to PE in the membrane, allows the decrease of membrane permeability for lipophilic and polar molecules, and, at the same time, makes membrane more stable. This is because the PG and the CL have a higher transition temperature than PE and are more ordered and less densely packed GPs [START_REF] Fang | Phospholipid compositional changes of five pseudomonad archetypes grown with and without toluene[END_REF][START_REF] Murzyn | Phosphatidylethanolamine phosphatidylglycerol bilayer as a model of the inner bacterial membrane[END_REF]. The PC level plays also an important role in membrane properties. The increase of the PC level, comparing to PE, coincides positively with membrane fluidity. Basing on the size of the GP head group relative to the FA chains, the PC increases the membrane fluidity. Giving that the chemical structure of the choline HG is larger, than that of the PE, the PC has a relatively similar area of head group compared to FA chains. In contrast, the PE decreases the membrane fluidity, because the HG of PE is smaller, than the FA chains, resulting in a larger area of FA chains relative to the HG [START_REF] Fajardo | Influence of phospholipid species on membrane fluidity: a meta-analysis for a novel phospholipid fluidity index[END_REF].

P a g e 51 | 270

From these considerations, it seems to be clear, that bacteria and the most (if not all) poikilothermic organisms must regulate their membrane fluidity in response to environmental modifications. For this, the members of genus Pseudomonas have various pathways, several examples of which will be discussed below.

Control of fatty acid de novo synthesis

The control at the level of FAS is crucial for membrane homeostasis, because the biophysical properties of bacterial membrane are determined in large part by the composition of the FAs that are produced by de novo synthesis [START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. In this way, the long-chain acyl-ACP (see part 3.4.3. Glycerophospholipid synthesis and functions in Pseudomonas) plays a role of a key regulator of the FAS. In Pseudomonas spp., the three most significant enzymes are regulated by acyl-ACP: ACC, FabH and FabI. The ACC activity is inhibited by the longchain acyl-ACP [START_REF] Davis | Inhibition of Escherichia coli acetyl coenzyme A carboxylase by acyl-acyl carrier protein[END_REF] and lower ACC activity decreases the quantity of malonate groups essential for the initiation and the elongation steps of FAS. The acyl-ACPmediated inhibition of catalytic activity of FabH prevents the initiation of new acyl-chains and, as consequence, limits the production of FAs (Heath & Rock, 1996a). The FabI catalytic activity, involved in the FA elongation, is also inhibited by acyl-ACP that could also reduce the total rate of bacterial FAs (Heath & Rock, 1996b). However, it should be noted, that P.

aeruginosa possesses the FabY and FabV enzymes (see part 3.4.3. Glycerophospholipid synthesis and functions in Pseudomonas), and the regulation of de novo FA synthesis in this bacterium could be more complex than in other Pseudomonas spp. species. The intracellular acyl-ACP level is controlled by PslB enzyme, which, in turn, is regulated by guanosine 3',5'bispyrophosphate (ppGpp) [START_REF] Wahl | Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli[END_REF]. The ppGpp is a global regulator of gene expression and RNA synthesis in bacteria [START_REF] Poole | Bacterial stress responses as determinants of antimicrobial resistance[END_REF][START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF], responsible also to peptidoglycan production and antibiotic resistance [START_REF] Nguyen | Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria[END_REF]. It should be nevertheless noted, that only growing Pseudomonas cells can modify their GP and FA composition by de novo synthesis [START_REF] Diefenbach | Synthesis of trans-unsaturated fatty acids in Pseudomonas putida P8 by direct isomerization of the double bond of lipids[END_REF].

Regulation of membrane biophysical properties

As previously demonstrated, the mainly studied membrane regulation pathway in Pseudomonas spp. strains is based on the FAs modifications [START_REF] Heipieper | Bacterial solvent responses and tolerance: cis-trans isomerization[END_REF][START_REF] Pepi | Membrane fatty acids adaptive profile in the simultaneous presence of arsenic and toluene in Bacillus sp. ORAs2 and Pseudomonas sp. ORAs5 strains[END_REF][START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. Several physico-chemical factors (i.e. contamination by organic solvents or increasing temperature) can increase the membrane fluidity. To maintain the optimal membrane functions, this increase is regulated by the production of SFAs. The GPs containing 16:0 SFAs display a transition temperature that is higher than those of the 16:1 cis-UFAs [START_REF] Heipieper | The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism[END_REF][START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. The double bond of a cis-UFA causes an unmovable 30° bend in the acyl chain as presented in Table 1 and Figure 11 (Roach et al.,

P a g e 52 | 270
2004), leading to the increase of the membrane fluidity. The SFA/UFA ratio is commonly quantified by the degree of FA saturation [START_REF] Heipieper | Effect of environmental factors on the trans/cis ratio of unsaturated fatty acids in Pseudomonas putida S12[END_REF]. The pathways, including FabAB and Des enzymes for synthesis of UFA, are discussed in the part 3.4.3.

Glycerophospholipid synthesis and functions in Pseudomonas and will not be included below. Bacteria can introduce a double bond into a saturated fatty acid (SFA) increasing membrane fluidity, via catalytic properties of Des enzymes. The rigid cis-double bond, introduces a pronounced kink of 30° in the acyl chain. GPs that contain cis-unsaturated fatty acids (cis-UFAs) occupy a greater molecular volume (blue shading) and do not pack as densely in the bilayer as GPs that contain SFAs. The cyclopropane FAs (a methyl group introduction across the double bond) formed by Cfa catalysis, have similar biophysical properties to the cis-UFAs, but render the membrane more stable to environmental insults. Pseudomonas cells can replace a cis-double bond with a transdouble bond via a mechanism, catalyzed by Cti isomerase. The trans-UFAs have properties that resemble SFAs, but give rise to the membranes that have higher transition temperatures. Phosphatidylethanolamine modifications are shown as an example. Adapted from [START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF].

The conversion of cis-to trans-UFAs is an another adaptive mechanism documented for several Pseudomonas spp. species, enabling bacteria to decrease their membrane fluidity, [START_REF] Heipieper | The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism[END_REF][START_REF] Heipieper | Bacterial solvent responses and tolerance: cis-trans isomerization[END_REF]. The advantage of the conversion is due to steric differences between cis-and trans-UFAs (Figure 11, Table 1). Contrary to the cis-UFA, the steric structure of the trans-configuration lacks the kink in the FA chain and is able to insert into the membrane as for the SFAs [START_REF] Roach | Comparison of cis-and trans-fatty acid containing phosphatidylcholines on membrane properties[END_REF]. The conversion from the cis-to the trans-unsaturated double bond does not have the same quantitative effect on membrane fluidity as the conversion to SFAs, but still substantially influences the rigidity of the membrane, as illustrated in Figure 12 [START_REF] Heipieper | The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism[END_REF]. Besides FabA and FabZ enzymes discussed previously (see part 3.4.3. Glycerophospholipid synthesis and functions in Pseudomonas), the cis-trans-isomerase (Cti) was described in several Pseudomonas species [START_REF] Holtwick | cis/trans Isomerase of unsaturated fatty acids of Pseudomonas putida P8: evidence for a heme protein of the cytochrome c type[END_REF][START_REF] Pedrotta | Isolation and characterization of the cis-trans-unsaturated fatty acid isomerase of Pseudomonas oleovorans GPo12[END_REF], including P.

fluorescens [START_REF] Heipieper | The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism[END_REF]. It is a constitutively expressed periplasmic enzyme that, to exert its action, necessitates neither adenosine triphosphate (ATP) nor other cofactors, and consistently, is independent of the de novo FAS.

In several conditions, Pseudomonas spp. are able to form the cyclopropane FA (a methyl group across the double bond to form a cyclopropane ring, as presented in Table 1 and Figure 11).

The required methylation reaction is carried out by cyclopropane FA synthase (Cfa), which uses S-adenosylmethionine as the methyl donor to create the cyclopropane group. Studies of acyl chain dynamics by nuclear magnetic resonance spectroscopy (NMR) indicate that the cyclopropane rings restrict the overall mobility and the disorder of the acyl chain between the cis segment and the polar HG, compared to the cis-double bonds [START_REF] Chang | Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli[END_REF][START_REF] Dufourc | A 2H-NMR analysis of dihydrosterculoyl-containing lipids in model membranes: structural effects of a cyclopropane ring[END_REF]. The cyclopropane FAs have similar biophysical properties to the UFAs, but render the membrane more stable to environmental insults, such as acid stress [START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. Recent investigations, used molecular dynamics simulation showed, that the cyclopropane FAs may fulfill a dual function: stabilizing membranes against adverse conditions while simultaneously promoting their fluidity. Marked differences in the effect of cis-and P a g e 54 | 270

trans-cyclopropanated FAs were also observed, suggesting that they may play alternative roles in membranes [START_REF] Poger | A ring to rule them all: the effect of cyclopropane fatty acids on the fluidity of lipid bilayers[END_REF].

Control of membrane glycerophospholipid composition

The GP homeostasis is controlled by the biochemical regulation of enzymes, which are previously discussed in the part 3.4.

Glycerophospholipid synthesis and functions in

Pseudomonas. The importance of maintaining the GP balance is illustrated by strains defective in genes, coding for GP synthesis and results in defects in solute and electron transport, the initiation of DNA replication or cell division [START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. For example, the PS synthase, PssA, is an extrinsic membrane protein that associated with, and activated by, membrane PG and CL [START_REF] Linde | Lipid dependence and activity control of phosphatidylserine synthase from Escherichia coli[END_REF][START_REF] Salamon | Interaction of phosphatidylserine synthase from E. coli with lipid bilayers: coupled plasmon-waveguide resonance spectroscopy studies[END_REF]. Thus, the proportion of PE increases, the membrane association and activity of PssA decreases, slowing the rate of PE formation [START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. The CL production is regulated by induction of cls gene expression in stationary growth phase or in high salt environment, but may also be inhibited by high PA or CL levels, contrary to PE, that inducts the CL synthesis [START_REF] Ragolia | The effects of phosphoglycerides on Escherichia coli cardiolipin synthase[END_REF].

In addition, Pseudomonas spp. strains synthesize the unique lipid, alanyl-phosphatidylglycerol (Ala-PG) [START_REF] Arendt | Identification and characterization of a periplasmic aminoacylphosphatidylglycerol hydrolase responsible for Pseudomonas aeruginosa lipid homeostasis[END_REF][START_REF] Klein | Adaptation of Pseudomonas aeruginosa to various conditions includes tRNA-dependent formation of alanylphosphatidylglycerol[END_REF]. This positively charged GP is synthetized by the aminoacylation of PG by the transmembrane MprF enzyme (PA0919 in P. aeruginosa PAO1). The alanine amino acid residue change the net charge of PG from -1 to 0 [START_REF] Parsons | Bacterial lipids: metabolism and membrane homeostasis[END_REF]. This change is induced by acidic growth and involved in a wide range of resistance, including to several antimicrobial compounds [START_REF] Arendt | Identification and characterization of a periplasmic aminoacylphosphatidylglycerol hydrolase responsible for Pseudomonas aeruginosa lipid homeostasis[END_REF].

Membrane glycerophospholipid turnover

Giving the fact, that the FA synthesis needs a high energy expenditure, the GPs could be recycled to maintain the membrane homeostasis or used, in case of need, as precursors in the synthesis of other structural compounds in the cell. This GP recycling is named GP turnover.

The head group of PG undergoes a rapid turnover by transferring to MDOs, which use the PG head group as a substrate for their synthesis. The residual from PG turnover, DAG, is quickly converted to PA and can be used by DgkA enzyme for re-synthesis of PG [START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. The DgkA is an inner membrane lipid kinase [START_REF] Wahl | Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli[END_REF]. This protein is involved in MDO production and related to the tolerance to osmotic stress [START_REF] Rumley | Mechanisms of regulation of the biosynthesis of membrane-derived oligosaccharides in Escherichia coli[END_REF]. In E.coli, the dgkA in plsB genes are inversely regulated by ppGpp intracellular level [START_REF] Wahl | Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli[END_REF]. The PE is also a subject of GP turnover. The FA at sn-1 position of PE is used for the post-transcriptional acylation of lipoproteins. The resulting 2-acylglycerophosphoethanolamine (2-acyl-GPE) is recycled thanks to the catalytic properties of bifunctional enzyme 2-acyl-GPE acyltransferase/acyl-ACP synthase (Aas) [START_REF] Jackowski | Sequence and function of the aas gene in Escherichia coli[END_REF].

P a g e 55 | 270

It should also be noted, that the lipid synthesis must be coordinated with DNA, RNA and protein synthesis to maintain the correct proportion of GPs to proteins in the membrane [START_REF] Bogdanov | Lipids and topological rules governing membrane protein assembly[END_REF][START_REF] Parsons | Bacterial lipids: metabolism and membrane homeostasis[END_REF]. In conclusion, P. fluorescens envelope is the first barrier between the bacterial cell and its environment. The components of bacterial envelope, including GPs, play a major role in sensoring of environment, and adaptation to environmental modifications. This brief report shows the major pathways for GP adaptation to environmental modifications involved in Pseudomonas spp. strains. In the next section of this study, the GP membrane adaptation to various stressors will be discussed in more detail.

Highlights

 P. fluorescens is a highly heterogeneous group of bacteria, colonizing the large variety of ecological niches and able to adapt to modifications of environment.

 The formation of biofilm is one of the various modes of P. fluorescens adaptation to environmental conditions. The biofilm formation is specific for each P.

fluorescens species and involves an array of cellular factors and highly regulated mechanisms.

 The P. fluorescens envelope plays a role of the first selective barrier between environment and cell, protecting bacteria against hostile environmental modifications.

 The glycerophospholipids are the major constituents of P. fluorescens envelope.

Their synthesis is a complex mechanism, which requires an activation of several enzymes. The glycerophospholipids play a pivotal role in envelope protective functions and in bacterial adaptation to environmental changes.

Chapter 2 Stress factors and Pseudomonas fluorescens lipid adaptation [START_REF] Murínová | The adaptation responses of bacterial cytoplasmic membrane fluidity in the presence of environmental stress factorspolychlorinated biphenyls and 3-chlorobenzoic acid[END_REF]

Stressors and bacterial survival

Bacteria, including Pseudomonas spp., are permanently exposed to a series of various stress factors, such as temperature alterations, availability of nutrients and water, or the presence of toxic molecules, that can make the living conditions far from optimal [START_REF] Ramos | Responses of Gram-negative bacteria to certain environmental stressors[END_REF].

These stressor factors have natural or anthropogenic origins and could be divided in three classes: biological, chemical and physical. The physical stressors, that bacteria cope in the environment, include temperature changes, modifications of osmotic pressure or irradiation.

The chemical stressors comprise chemical pollutants found in natural environments, like organic solvents and metals. Biological factors, in turn, englobe the host defense processes, and include antimicrobials, and stresses caused by competition with other microorganisms. Bacteria have to implement adaptive mechanisms to survive under conditions of stress and cope with the challenges of changing environments. With the development of genomic and proteomic approaches, a myriad of studies based on the changes in gene expression, ensuring bacterial stress response, were realized [START_REF] Boor | Bacterial stress responses: what doesn't kill them can make them stronger[END_REF][START_REF] Kim | Oxidative stress response in Pseudomonas putida[END_REF][START_REF] Reva | Functional genomics of stress response in Pseudomonas putida KT2440[END_REF][START_REF] Ron | Bacterial stress response[END_REF][START_REF] Rowley | Pushing the envelope: extracytoplasmic stress responses in bacterial pathogens[END_REF]. Thus, a combination of transcriptional regulatory pathways, which allow bacteria to sense and convert environmental stimuli, resulting in altered gene expression and enzyme activities (signal transduction) were well established. However, bacterial lipids play also a role of target(s) and adaptive mechanism(s) to environmental changes. Some examples of membrane lipid interactions with environmental stressors will be exposed below.

Impact of physical factors on membrane properties

Temperature modifications

In its natural habitats (soil, water and rhizosphere), Pseudomonas spp. can suffer frequent and long-term changes in temperature, which affect bacterial growth and survival. The effect of temperature on the members of P. fluorescens group and temperature adaptation of these bacteria were finely studied in our laboratory. Thus, it has been established, that growth temperature has an effect on P. fluorescens enzyme activity [START_REF] Gügi | Effect of growth temperature on several exported enzyme activities in the psychrotrophic bacterium Pseudomonas fluorescens[END_REF][START_REF] Merieau | Temperature regulation of lipase secretion by Pseudomonas fluorescens strain MFO1[END_REF]. P. fluorescens, growing at low temperatures, is much more sensitive to β-lactam mezlocillin, that the cells, growing at optimal temperature [START_REF] Orange | Growth temperature regulates the induction of -lactamase in Pseudomonas fluorescens through modulation of the outer membrane permeation of a lactam-inducing antibiotic[END_REF], due, in part, to the permeation of this antibiotic through bacterial membrane and the modifications in outer membrane porin OprF [START_REF] Dé | Growth temperature dependence of channel size of the major outermembrane protein (OprF) in psychrotrophic Pseudomonas fluorescens strains[END_REF][START_REF] Hemery | Growth temperature and OprF porin affect cell surface physicochemical properties and adhesive capacities of Pseudomonas fluorescens MF37[END_REF]. Temperature modifications were found to cause the variations in P. fluorescens cytotoxicity: low temperature increases bacterial adhesion to surface, but optimal temperature enchases the apoptotic effect [START_REF] Picot | Regulation of the cytotoxic effects of Pseudomonas fluorescens by growth temperature[END_REF]. ). The temperature higher than the optimum of growth increases membrane fluidity, allowing a membrane switch into the lamellar liquid phase (Figure 10). In contrast, the temperature, lower than optimum decreases membrane fluidity, switching the membrane into the gel phase. The molecular mechanism of regulation of UFA synthesis to adjust the temperature-mediated changes of membrane fluidity was elucidated more than 30 years ago [START_REF] Marr | Effect of temperature on the composition of fatty acids in Escherichia coli[END_REF][START_REF] Mcgarrity | The effect of temperature and other growth conditions on the fatty acid composition of Escherichia coli[END_REF] and was discussed in a number of studies [START_REF] Mansilla | Control of membrane lipid fluidity by molecular thermosensors[END_REF][START_REF] Russell | Membranes as a target for stress adaptation[END_REF]. The lipidome adaptation to temperature modifications was also reported for members of Pseudomonas genus [START_REF] Bhakoo | Fatty acid and phospholipid composition of five psychrotrophic Pseudomonas spp. grown at different temperatures[END_REF][START_REF] Gill | Effect of growth temperature on the lipids of Pseudomonas fluorescens[END_REF][START_REF] Kropinski | Effect of growth temperature on the lipids, outer membrane proteins, and lipopolysaccharides of Pseudomonas aeruginosa PAO[END_REF]. When the temperature rises, Pseudomonas spp. produce more SFAs, decreasing the membrane fluidity (Figure 10, Table 1). Conversely, at low membrane fluidity, caused by low temperature, bacteria produce more UFAs, increasing membrane fluidity and optimizing the growth [START_REF] Mansilla | Control of membrane lipid fluidity by molecular thermosensors[END_REF][START_REF] Russell | Membranes as a target for stress adaptation[END_REF].

Concerning GP composition, modifications of the growth temperature do not elicit significant changes either in the total quantities of GP synthesized or in the concentration of individual GP components in any of the isolates [START_REF] Bhakoo | Fatty acid and phospholipid composition of five psychrotrophic Pseudomonas spp. grown at different temperatures[END_REF]. However, the tests of the temperature effect on the lipidome of P. fluorescens grown under carbon and nitrogen limited conditions [START_REF] Gill | Effect of growth temperature on the lipids of Pseudomonas fluorescens[END_REF] showed that the low temperature increases the proportion of the PE under carbon-limited conditions, but under nitrogen limitation an increase in the amount of CL is observed. The cis-to trans-UFA isomerization is an another mechanism to be described as response of P. syringae [START_REF] Kiran | Psychrophilic Pseudomonas syringae requires trans-monounsaturated fatty acid for growth at higher temperature[END_REF][START_REF] Kiran | cis-trans Isomerase gene in psychrophilic Pseudomonas syringae is constitutively expressed during growth and under conditions of temperature and solvent stress[END_REF] and Vibrio spp. [START_REF] Okuyama | A trans-unsaturated fatty acid in a psychrophilic bacterium, Vibrio sp. strain ABE-1[END_REF][START_REF] Okuyama | The cis/trans isomerization of the double bond of a fatty acid as a strategy for adaptation to changes in ambient temperature in the psychrophilic bacterium, Vibrio sp. strain ABE-1[END_REF] species in response to increasing temperature (Table 1 and Figure 10). The role of cyclopropane-FAs in the adaptation to temperature changes is not clear so far, but there are studies demonstrated the increasing of cyclopropane-FAs with the rise of temperature [START_REF] Nagamachi | Adaptational changes of fatty acid composition and the physical state of membrane lipids following the change of growth temperature in Yersinia enterocolitica[END_REF].

Osmotic pressure

The osmotic strength of the environment is one of the physical parameters that determines the ability of organisms in given environment. Bacterial cells have to maintain the cell turgor, in other words, the intracellular osmotic pressure greater than in the environment in order to optimize the cell extension, growth and division [START_REF] Sleator | Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence[END_REF]. The mechanisms involved in bacterial osmotic adaptation were well studied. The classical bacterial responses to osmotic stress were reviewed by [START_REF] Csonka | Physiological and genetic responses of bacteria to osmotic stress[END_REF][START_REF] Sleator | Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence[END_REF]Hill, 2002. Briefly, bacteria react to osmotic stress first by taking up potassium ions (K + ) level in cytoplasm, which is followed by the increase of accumulation and/or biosynthesis of osmoprotective compounds. As shown in Figure 13, the latter, named compatible solutes, play a role of effective stabilisators of enzymatic functions, allowing the protection against osmotic stress. While the bacterial P a g e 61 | 270 cytoplasm employs the compatible solutes to protect its compounds against osmotic stress, the bacterial membrane also must undergo a number of adaptive changes. The P. fluorescens adaptation to osmotic stress is characterized by the alterations in porins, lipoproteins, and the flagella synthesis. In agreement with these changes, osmotic stress results in vesicle formation and modifications of bacterial motility and antibiotic susceptibility [START_REF] Guyard-Nicodème | Outer membrane modifications of Pseudomonas fluorescens MF37 in response to hyperosmolarity[END_REF].

Figure 13. Enzyme stabilization at high osmolarity

The compatible solutes (blue circles) help to maintain the enzyme structure and properties at high osmolarity.

Adapted from [START_REF] Sleator | Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence[END_REF] The membrane GPs play an important role in bacterial adaptation to osmotic stress [START_REF] Sleator | Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence[END_REF]. The latter, caused by addition of glycerol, sodium chloride, or sucrose was evaluated in P. putida cells [START_REF] Heipieper | Effect of environmental factors on the trans/cis ratio of unsaturated fatty acids in Pseudomonas putida S12[END_REF]. The cell responses were found to be stressor-dependent: exposure to NaCl and sucrose is correlated with an increase in the ratio of trans/cis UFAs, whereas glycerol does not cause any membrane response, indicating the importance of diffusive properties of these toxic compounds across the membrane. NaCl and sucrose cannot freely diffuse across the membrane and causes the accumulation of K + and synthesis of compatible solutes, while equilibration of a glycerol gradient across biological membranes is fast, which is not correlated with K + and compatible solutes synthesis. The proportion of cyclopropane FAs was also found to be increased in high salinity media [START_REF] Romantsov | Cardiolipin and the osmotic stress responses of bacteria[END_REF].

Another remarkable response to osmotic stress was observed in Rhodococcus erythropolis cells.

The members of Rhodococcus genus are not only able of degrading a wide range of organic compounds [START_REF] De Carvalho | Degradation of hydrocarbons and alcohols at different temperatures and salinities by Rhodococcus erythropolis DCL14[END_REF], but also able to the growth in the presence of up to 7.5 % NaCl (de Carvalho, 2012). In hyperosmotic conditions, caused by NaCl, R.

erythropolis completely changes its net surface charge and FA composition [START_REF] De Carvalho | Rapid adaptation of Rhodococcus erythropolis cells to salt stress by synthesizing polyunsaturated fatty acids[END_REF]. Interestingly, this bacterium formes the polyunsaturated long chain FAs, mainly eicosapentaenoic acid (C20:5ω3), arachidonic acid (C20:4ω6) and docosapentaenoic acid (C22:5ω3). The possible function of these very uncommon for bacteria FAs could be based on P a g e 62 | 270 the decrease in the number of negatively charged groups in ion channels resulting in a repellence of the NaCl.

When bacteria are grown in media of increasing salinity, the proportion of anionic GPs increases, comparing to zwitterionic GPs [START_REF] Romantsov | Cardiolipin and the osmotic stress responses of bacteria[END_REF]. The role of CL seems to be very important in the context of high salinity adaptation. Rhodobacter sphaeroides responds to the hyperosmotic stress by increasing of the amount of CL in its membrane [START_REF] Catucci | Neosynthesis of cardiolipin in Rhodobacter sphaeroides under osmotic stress[END_REF]. The growth of E. coli and Bacillus subtilis cls mutants is impaired in high osmolarity medium [START_REF] López | Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity[END_REF][START_REF] Romantsov | Cardiolipin promotes polar localization of osmosensory transporter ProP in Escherichia coli[END_REF].

It seems appropriate, that the lipid studies of osmotic stress adaptation are mainly performed with halophilic microorganisms that are classified, in majority, into the Archaea domain [START_REF] Oren | Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications[END_REF]. The members of genus Halobacterium are halophile par excellence. In osmotic stress conditions, these bacteria increase the level of the archaeal CL bisphosphatidylglycerol (BPG), and synthesize normally absent in the purple membrane analogs of CL: a glycocardiolipin (GlyC) and a sulfated diglycosyl diphytanylglycerol diether (S-DGD-5-PA). This increase of CL level is correlated with a decrease of the level of sulfo-triglycosyl-diether (S-TGD-1) in the purple membrane [START_REF] Lobasso | Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum[END_REF][START_REF] Lopalco | Osmotic shock stimulates de novo synthesis of two cardiolipins in an extreme halophilic archaeon[END_REF].

It should be however noted, that a wide range of adaptive responses to osmotic stress, based on solute or water efflux mechanisms, osmosensing and osmoregulation were described and reported in a very comprehensive review [START_REF] Sleator | Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence[END_REF].

Impact of chemical factors on membrane properties

Organic solvents

Nowadays, all kinds of motors are widely present in our environment and accidental oil spills are frequent, resulting in soil and water contamination with hydrocarbons. Bacteria are the ability to resist to hydrocarbon contamination and/or degrade these toxic compounds in polluted environments, offering an amazing possibility of bioremediation of contaminated surfaces [START_REF] De Carvalho | Degradation of hydrocarbons and alcohols at different temperatures and salinities by Rhodococcus erythropolis DCL14[END_REF].

The bacterial responses to hydrocarbons were extensively studied during the last decade. A large number of the organic-solvent-tolerant bacteria was reported, including Pseudomonas strains, especially P. putida (Heipieper et al., 1994;[START_REF] Heipieper | Bacterial solvent responses and tolerance: cis-trans isomerization[END_REF]. The primary site of action of organic solvents is the cell membrane.

Although no individual analytical technique is able to determine fully the effects of solvents on a membrane, several mechanisms of solvent toxicity were established (Heipieper & Martínez, P a g e 63 | 270 2010). The accumulation of solvents in bacterial membrane leads to the inhibition of membrane protein functions and synthesis, non-specific membrane permeabilization and its swelling [START_REF] Heipieper | Influence of phenols on growth and membrane permeability of free and immobilized Escherichia coli[END_REF](Heipieper et al., , 1994)). The membrane fluidity is also known to be impacted by organic solvents [START_REF] Heipieper | Toxicity of hydrocarbons to microorganisms[END_REF]. Figure 14 shows a scheme of the toxic effect of these molecules on the cell membrane that could cause the inhibition of bacterial growth and/or lysis of the cells. In order to adapt to the hydrocarbon toxicity, bacteria of genus Pseudomonas use several membrane adaptive pathways, which could be divided to short-and long-term responses. The short-term response is based on a decrease of membrane fluidity by a rapid transformation of the cis-FAs to their trans-conformations (Table 1, Figure 11 and Figure 12) [START_REF] Heipieper | Bacterial solvent responses and tolerance: cis-trans isomerization[END_REF]. The role of cyclopropane-FAs was also demonstrated: the cfa knock out mutant was found to be more sensitive to solvent shock than the wild type strain in stationary phase (Segura et al., 2010). The long-term response, in turn, is based on the changes in FAs and the alterations in the ratio of zwitterionic/anionic GPs. Bacteria increase the degree of FAs saturation, that leads to the decrease of the membrane fluidity (Heipieper & de Bont, 1994). In

this way, the adaptation of the FA composition to the hydrocarbons strongly resembles the wellknown process of thermal adaptation (see part 2.1. Temperature modifications). It has been established, that with addition of solvents the level of CL increases, when the level of PE decreases, leading to the increased membrane rigidity [START_REF] Ramos | Mechanisms for solvent tolerance in bacteria[END_REF]. Recently, the effect of n-butanol on the GP composition of four solvent-tolerant Pseudomonas spp. strains was evaluated [START_REF] Rühl | The glycerophospholipid inventory of Pseudomonas putida is conserved between strains and enables growth condition-related alterations[END_REF], demonstrating strain-dependent GP response to this organic P a g e 64 | 270 molecule. In fact, P. putida DOT-T1E and KT2440 grown with 1% of n-butanol exhibit a shift from PE to PG at slightly decreasing amounts of CL. A different response was observed for P. putida S12 and Pseudomonas sp. VLB120, where the PE and CL levels are enhanced, correlated with the decrease of the relative amounts of PG.

Metal pollutants

Several metals, including Ca, Cu, or Zn, are essential for bacterial functioning, as cofactors of various enzymes, or regulators of osmotic pressure. On the other hand, many metals, such as Hg, Pb, or Au, have no biological functions and are toxic to bacterial cells. The bacterial envelope protects cell compounds against environmental pollutants, including toxic metals [START_REF] Beveridge | Bacterial cell wall structure and implications for interactions with metal ions and minerals[END_REF]. As a consequence, the metal resistance pathways are often based on membrane functions, including metal exclusion by permeability barrier, cellular sequestration, and efflux of the metal ions from the cell [START_REF] Markowicz | Response of bacteria to heavy metals measured as changes in FAME profiles[END_REF]. In addition, the enzymatic transformation and reduction of metal to less toxic forms play also a pivotal role in bacterial metal decontamination. Bacterial cell surface carries a negative charge under optimal pH conditions, and can interact with the electropositive metal ions. The evidence of these interactions was observed by Breveridge and coworkers using electron microscopy and illustrated in Figure 15 [START_REF] Beveridge | Binding of metals to cell envelopes of Escherichia coli K-12[END_REF]. While the metals penetrate in bacterial cells through the channel for metal ion acquisition [START_REF] Hohle | Bacterial outer membrane channel for divalent metal ion acquisition[END_REF], bacterial GPs play a significant protective role against metal pollution. The anionic GPs, including PG and CL or phosphoryl groups, are able to react with metal cations in order to sequester these toxic compounds within the membrane. An alternate mechanism P a g e 65 | 270 involved in response to metal contamination is based on the FAs response to the metal binding [START_REF] French | The dynamic nature of bacterial surfaces: implications for metal-membrane interaction[END_REF]. It ws reported that an increase in the level of UFAs results in a two-fold decrease in Ca 2+ binding to the lipid membranes [START_REF] Huster | Strength of Ca 2+ binding to retinal lipid membranes: consequences for lipid organization[END_REF]. In fact, the increased distances between adjacent HGs in cis-UFAs or cyclopropane-FAs comparing to the SFAs (Figure 11), may reduce lipid-metal binding [START_REF] French | The dynamic nature of bacterial surfaces: implications for metal-membrane interaction[END_REF]. The work of Markowicz and coworkers demonstrated the alterations in the cyclopropane-FAs level and the degree of FA saturation in bacteria, grown with several metals. Unfortunately, the observed effects are strainand metal-dependent, making it difficult to establish the mechanism of FA adaptation to metal contamination. It was also shown, that P. putida adapts its membrane FAs to the heavy metals pollution by the transformation of the cis-UFAs to their trans steric conformations [START_REF] Heipieper | Effect of environmental factors on the trans/cis ratio of unsaturated fatty acids in Pseudomonas putida S12[END_REF].

Since the last years, the notion of nanoparticles (particles of size between 1 and 100 nm) became an important subject of pharmaceuticals and industrial science researches [START_REF] Arvizo | Intrinsic therapeutic applications of noble metal nanoparticles: past, present and future[END_REF][START_REF] Whitesides | The 'right' size in nanobiotechnology[END_REF]. The membrane adaptive response of P. putida to silver nanoparticles was studied [START_REF] Hachicho | Effect of silver nanoparticles and silver ions on growth and adaptive response mechanisms of Pseudomonas putida mt-2[END_REF]. The silver nanoparticles were found to cause a strong increase in the trans/cis ratio of UFAs, as a mechanism of control of the increasing membrane fluidity.

Bacteria involved in bioremediation processes must cope the multicontaminated environment, and very often la contamination is accompanied by organic pollutants [START_REF] Cébron | Rapid impact of phenanthrene and arsenic on bacterial community structure and activities in sand batches[END_REF].

The bacterial response to mixed organic and metal pollution were evaluated [START_REF] Pepi | Membrane fatty acids adaptive profile in the simultaneous presence of arsenic and toluene in Bacillus sp. ORAs2 and Pseudomonas sp. ORAs5 strains[END_REF].

Concerning membrane lipids, Bacillus sp. ORAs2 is able to increase the degree of FA saturation at high arsenic concentrations that can probably counteract the increase of arsenic uptake.

Pseudomonas sp. ORAs5 also increases the degree of FAs saturation, but also forms the trans-UFAs. However, its response to heavy metals is a much lower than that to toluene.

Impact of biological factors on membrane properties

Antimicrobial agents

As previously reported in the part 3.4.

Glycerophospholipid synthesis and functions in

Pseudomonas, bacterial lipidome becomes a target for antibiotic development. The chemical nature of antimicrobial agents is quite diverse. However, many of these toxic compounds are positively charged. In addition to the net negative charge, bacterial membrane has a negatively charged GP domains (i.e. domains enriched by CL). There is also evidence, that bacteria are able to rearrange their membrane GPs, forming, for example, anionic GP domains to adapt to stress impact [START_REF] Vanounou | Coexistence of domains with distinct order and polarity in fluid bacterial membranes[END_REF]. Antimicrobial agents are able to interact with the negatively charges bacteria GPs, as reported in Figure 16. This may protect bacteria against P a g e 66 | 270 positively charged antibacterial agents, sequestering antimicrobial agents outside the cell, but also lead to the lower membrane permeability and alterations in bacterial membrane functioning [START_REF] Epand | Lipid domains in bacterial membranes and the action of antimicrobial agents[END_REF].

Figure 16. Glycerophospholipid rearrangement that can occur on binding cationic antimicrobial agents

Clustering of anionic GPs (purple head groups) into a domain occurs as a consequence of binding to the antimicrobial agent. This causes a rearrangement of GPs around the domain, and membrane defects.

Adapted from [START_REF] Epand | Lipid domains in bacterial membranes and the action of antimicrobial agents[END_REF].

The antimicrobial host defense peptides provide a good example of interactions with bacterial membrane. These molecules are small in size and have a cationic structure. The helical peptide NK-2 binds to the anionic PG, causing a rigidification of the membrane and an increase in the phase transition temperature without destroying the membrane structure. In addition, a negligible interaction was observed with pure zwitterionic PC model membranes. Whereas NK-2 has no effect on PC liposomes, it enhances the fluidity of the PE acyl chains and lowers the phase transition enthalpy of the gel to liquid crystalline transition. The most dramatic effect, however, was observed for the lamellar hexagonal transition of the PE, which is reduced by more than 10 °C. Negative charges will increase the probability of the presence of the peptide at the membrane surface [START_REF] Willumeit | Structural rearrangement of model membranes by the peptide antibiotic NK-2[END_REF]. In a similar way, the high concentrations of the tryptophan-rich antimicrobial peptide indolicidin lower the ratio of the gel-phase fluid domains in model membranes [START_REF] Shaw | Mechanisms of antimicrobial peptide action: studies of indolicidin assembly at model membrane interfaces by in situ atomic force microscopy[END_REF]. Alternatively, it was reported that the decrease in the membrane level of PC promotes the resistance of P. aeruginosa to benzalkonium chloride [START_REF] Loughlin | Pseudomonas aeruginosa cells adapted to benzalkonium chloride show resistance to other membrane-active agents but not to clinically relevant antibiotics[END_REF].

The antibiotic tetracycline was reported to alter the membrane fluidity in B. cereus and P. putida membranes. Whereas it has a positive effect on the cytoplasmic membrane fluidity of B. cereus, the opposite was observed in P. putida, reflecting the difference in stress induced behavior between different bacteria species [START_REF] Vincent | Sepsis in European intensive care units: results of the SOAP study[END_REF] The role of the recently identified Ala-PG in P. aeruginosa antibiotic resistance should be mentioned. Ala-PG conferrs a growth advantage when the bacteria are cultured in the presence of a diverse group of substances, including the cationic antimicrobial peptides, such as protamine, the heavy metal chromium (III), the osmolyte sodium lactate and the β-lactam cefsulodin [START_REF] Klein | Adaptation of Pseudomonas aeruginosa to various conditions includes tRNA-dependent formation of alanylphosphatidylglycerol[END_REF]. The biological significance of Ala-PG is not clear, but the P a g e 67 | 270 addition of Ala could neutralize the negative charge of PG imparting a net positive membrane charge and decreasing the interactions of cationic antimicrobials with negatively charged PG.

In addition, it was proposed that the Ala modification might be involved in changing of membrane biological properties, such as permeability and fluidity [START_REF] Roy | Adaptation of the bacterial membrane to changing environments using aminoacylated phospholipids[END_REF].

In conclusion, various stress factors impact bacterial functioning. The bacterial membrane plays a role of the first barrier between the toxic compounds and the cell and is often altered with modifications of environmental conditions. As consequence, bacteria, including the members of Pseudomonas genus, can adapt to these modifications. The membrane GP adaptive responses to several stress factors discussed in this chapter are summarized in Table 2.

Adaptation of polar head groups

High temperature

No modification

Low temperature

No modification

Osmotic stress

Increase of the level of anionic GPs, particularly CL.

Solvents (shortterm response) Solvents (longterm response)

Strain-dependent response. Modifications of ration zwitterionic/anionic GPs

Metals

Sequestration of metal cations into anionic GP domains

Nanoparticles ND Antimicrobial agents ND ND ND ND ND

Sequestration of metal cations into anionic GP domains; increase of Ala-PG level and modification of net membrane charge

The mechanisms of bacterial adaptation to various stress factors based on the glycerophospholipid polar head groups and/or fatty acids (FAs) modifications are summarized in this table. The modifications in FAs include the formation of saturated FAs (SFA), cis-, trans-monounsaturated FAs (cis-UFA, trans-UFA), cyclopropane-FAs (cyclo-FA) and polyunsaturated FAs (poly-UFA). The green square shows the increasing level of displayed parameter, the red square shows the decreasing level of displayed parameter. ND: no determined.

Briefly, the increase of the membrane fluidity, caused by several stress factors, is controlled by the increasing levels of SFAs and trans-UFAs in bacterial membrane. The cyclopropane-FAs play an important role in response to osmotic stress and solvent toxicity. The GP polar head groups are also involved in response to stress. Particularly, the anionic GPs, including PG and CL, are involved in the response to osmotic stress and in the adaptation to cationic toxic compounds, such as antimicrobial agents and metal cations. This overproduction and/or rearrangements of GP bilayer cause both the sequestration of toxic compounds outside of the bacterial cells, but also the alterations in the membrane functions.

P a g e 68 | 270

This chapter discussed only several and the most studied stress factors in terms of the membrane GP adaptation. However, the topic of bacterial adaptation is much vaster that described above.

The stressors, like light [START_REF] Elmnasser | Pulsed-light system as a novel food decontamination technology: a review[END_REF][START_REF] Gómez-López | Pulsed light for food decontamination: a review[END_REF], nutrient limitation [START_REF] Poole | Bacterial stress responses as determinants of antimicrobial resistance[END_REF] and pH [START_REF] Baatout | Benefits of human presence in space -historical, scientific, medical, cultural and political aspects[END_REF][START_REF] Padan | Alkaline pH homeostasis in bacteria: new insights[END_REF] should also be mentioned.

Oxidative stress

Several previously discussed stressors, like metal ions [START_REF] Lemire | Antimicrobial activity of metals: mechanisms, molecular targets and applications[END_REF] and antibiotics [START_REF] Marrakchi | Oxidative stress and antibiotic resistance in bacterial pathogens: state of the art, methodologies, and future trends[END_REF][START_REF] Poole | Bacterial stress responses as determinants of antimicrobial resistance[END_REF] can cause the oxidative stress in bacterial cells. It has been reported, that free radicals can attack directly the polyunsaturated FAs in membranes and initiate the lipid peroxidation. The latter is primary reflected in a decrease of the membrane fluidity, altering the membrane properties. This effect acts as an amplifier, more radicals are formed, more polyunsaturated FAs are oxidized [START_REF] Cabiscol | Oxidative stress in bacteria and protein damage by reactive oxygen species[END_REF]. However, these data should be interpreted with caution. A biochemical mechanism of lipid peroxidation was defined for membranes of eukaryotic organisms, which contain the high levels of polyunsaturated FAs.

By contrast, most bacterial membranes, including Pseudomonas species, are composed of monounsaturated FAs, which are described as unreactive in lipid peroxide chain reactions in vitro [START_REF] Imlay | Pathways of oxidative damage[END_REF]. However, others discussed the repair of E. coli lipid peroxidation (Bonamore et al., 2003a, b). Therefore, it is still unclear whether the lipid peroxidation occurs in bacterial membranes [START_REF] Lemire | Antimicrobial activity of metals: mechanisms, molecular targets and applications[END_REF]. In the case of doubt, in this study, the bacterial lipid peroxidation is not taken into account. The oxidative stress is closed to the nitrosative stress, caused by nitrogen oxides [START_REF] Van Der Heijden | Direct measurement of oxidative and nitrosative stress dynamics in Salmonella inside macrophages[END_REF]. The sources of nitrogen oxides, their chemistry and bacterial adaptation to these toxic compounds will be discussed in the next chapter of this work.

Highlights

 Several environmental stressors can alter bacterial homeostasis. Giving the fact, that the membrane plays an important role of the first barrier between bacterial cell and its environment, several (if not all) environmental stress factors alter membrane functions, that often results in modifications of the membrane fluidity.

 Bacteria, including members of Pseudomonas genus, involve a membrane adaptive response to control the membrane fluidity and optimize growth, which is based, in part, on the modifications in membrane GPs.

 The high temperatures, solvents and metals are known to increase the membrane fluidity, which is controlled by the increasing of the level of SFAs and the rapid transformation of cis-UFAs to their trans-conformations.

 The cyclopropane-FA synthesis is required for the response to the solvent toxicity and osmotic stress. The latter is found to induce the synthesis of unusual for bacteria poly-UFAs.

 The modifications in GP polar head groups are also involved in bacterial response to stress factors. The modifications in ratio of the membrane zwitterionic/anionic GPs allows bacteria to control the membrane fluidity. The anionic GP, including PG and CL, are involved in the response to osmotic stress and in the adaptation to cationic toxic compounds, such as antimicrobial agents and metal cations. The rearrangements of GP bilayer allow the formation of the anionic GP domains, which can capture toxic molecules, but also alter the membrane properties.

Chapter 3 Nitrogen oxides, their chemistry, biological roles and atmospheric pollution

1. Nitrogen oxides (NO x )

1.1.

NOx basic notions

Nitrogen is a basic element for life because it is a component of the two preeminent biological macromolecules: proteins and nucleic acids. Nitrogen exists in several oxidation states, from N(-III) to N(+V). Interconversions of the nitrogen species constitute the global biogeochemical nitrogen cycle, in which bacteria play a predominant role [START_REF] Moreno-Vivián | Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases[END_REF]. Several types of nitrogen oxides (NOx) exist in the environment: N2O, NO, NO2, N2O3, N2O4, NO3, and N2O5. However, the abbreviation NOx mainly relates to nitrogen monoxide (NO) and nitrogen dioxide (NO2) [START_REF] Skalska | Trends in NOx abatement: A review[END_REF]. The structure, reactivity and functions of these molecules were extensively studied. NO and NO2 are more soluble in polar solvents than in water [START_REF] Lide | CRC Handbook of Chemistry and Physics[END_REF][START_REF] Schwartz | Membrane composition and modifications in response to aromatic hydrocarbons in Gram-negative bacteria[END_REF]. From a Lewis structure presented in Figure 17, it becomes evident, that  NO and  NO2 are the free radicals with one unpaired electron. In order to homogenize the text below, these two radicals are named as NO and NO2.

Nitrogen monoxide (NO)

NO is a simple inorganic molecule, possessing unique and fascinating properties. On one hand,

NO is found to be one of the major air pollutants (see part 6.1. NOx as a major air pollutant).

On the other hand, NO is known to be synthetized in several (if not all) living organisms, including mammals, plants and naturally bacteria (see part 2. NO formation in living organisms). These NO functions have reinvigorated research into the fundamental chemistry, and led Science magazine to designate NO as Molecule of the Year in 1992 [START_REF] Koshland | The molecule of the year[END_REF].

However, in presence of O2 and light, NO is unstable and is quickly oxidized to form NO2. The latter reacts with NO in aqueous solution to give N2O3, as shown in Figure 18. When the reaction occurs in air, NO2 dimerizes to give N2O4. Given that NO is a lipophilic molecule, the reaction of NO with oxygen will be accelerated in the cell membrane [START_REF] Hughes | Chapter one -Chemistry of nitric oxide and related species[END_REF], with various and, in majority, negative consequences for the cell.

1.3.Nitrogen dioxide (NO2)

NO2 is described as a toxic air pollutant (see part 6.1. NOx as a major air pollutant), which, however, can be synthesized (at low concentrations) by several organisms (i.e. during P a g e 74 | 270 denitrification process, see part NO in bacteria). In atmosphere, this molecule exists in form of its dimer N2O4 (at 27 °C, 80 % N2O4 for 20 % NO2) [START_REF] Fukuto | The chemical properties of nitric oxide and related nitrogen oxides[END_REF]. Moreover, N2O4

is about 100 times more soluble in water than its monomer NO2 that results in generation of nitrite (NO2 -) and nitrate (NO3 -). In cells, NO2 and its dimer are, in majority, concentrated in the hydrophobic areas (i.e. cell membrane) [START_REF] Hughes | Chapter one -Chemistry of nitric oxide and related species[END_REF]. As previously reported and shown

in Figure 18, in aqueous solution, NO2 reacts with NO to form dinitrogen trioxide (N2O3), which hydrolyzes to form NO2 -. The N2O3 and N2O4 are also known for their oxidative properties and the possible reactions with proteins (see part Proteins) [START_REF] Espey | Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide[END_REF].

Because the NOx are able to react in the cellular environment to give a wide range of products with different biological effects, the relationship between the biological activity of NOx and their chemistry is particularly complex [START_REF] Fukuto | The chemical properties of nitric oxide and related nitrogen oxides[END_REF]. Some of these reactions are represented in Figure 18 and give rise to the generation of the reactive nitrogen species (RNS) [START_REF] Wink | Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide[END_REF]. According the strict chemical definition, the RNS include a wide range of compounds, with contrasting and distinct properties, and their only unifying trait is related to the common origins from NO [START_REF] Patel | Biological aspects of reactive nitrogen species[END_REF]. However, more and more of studies take into account the biological effect of RNS, considering as the RNS only toxic for living organism molecules. The chemistry and biological functions of RNS will be discussed below.

1.4.

Nitrogen oxides and reactive nitrogen species: chemistry and biological role A wide range of RNS with various chemical structures and different (and often opposite) biological roles may be cited. First of all, the RNS play a role of a source of nitrogen. In this way, the physiological level of RNS helps to maintain the normal cell homeostasis and produce macromolecules, and/or energy for growth [START_REF] Fang | Antimicrobial reactive oxygen and nitrogen species: concepts and controversies[END_REF]. However, the pathological level of RNS can cause the nitrosative stress, which negatively impacts the cell functioning. Due to the complex RNS chemistry, the excess of these compounds may induce the generation of other and probably toxic species. Nitrosative stress may lead to nitrosylation reactions that alter the structure of proteins and inhibit their normal functions. There is a difficulty with classifying the RNS according their role in the cells. However, the peroxynitrite (ONOO -), among others, is recognized as a very strong oxidizing agent, causing the DNA damages and lipid oxidation [START_REF] Valko | Free radicals and antioxidants in normal physiological functions and human disease[END_REF]. In this part of the manuscript, we tried to summarize the currently known information about several RNS, their chemistry and function in biology and areas of application.

Peroxynitrite (ONOO -)

Notable example of highly reactive RNS species is peroxynitrite (ONOO -) that is formed through the reaction of NO with the superoxide radical O2 as shown in Figure 18. The chemical properties of peroxynitrite make it a strong, and highly reactive species, which gives rise to both oxidative and nitrosative stresses [START_REF] Ferrer-Sueta | Chemical biology of peroxynitrite: kinetics, diffusion, and radicals[END_REF]. Thus, this molecule plays a role of a central pathogenic mediator in a variety of diseases including cardiovascular, neurodegenerative, and inflammatory disorders. The half-life of this species is thought to be as little as 10 ms in biological systems; however, this time is sufficient for damaging bacterial cells [START_REF] Mclean | Peroxynitrite toxicity in Escherichia coli K12 elicits expression of oxidative stress responses and protein nitration and nitrosylation[END_REF]. Once penetrated in bacterial cytoplasm, peroxynitrite is able to react with a variety of targets (see part NO targets in cells) causing protein modifications, lipid oxidations, and/or DNA damages. The formation rate of peroxynitrite has been estimated at up to 100 M/min in selected cellular compartments [START_REF] Mclean | Peroxynitrite toxicity in Escherichia coli K12 elicits expression of oxidative stress responses and protein nitration and nitrosylation[END_REF]. However, the toxicity of this species seems to be highly dependent on ratio between the cells number and the stressor.

In addition, the toxicity of peroxynitrite seems to be largely influenced by the presence of scavering compounds in culture medium. As demonstrated in Figure 18, ONOO -possesses nucleophilic character and is able to react with electrophiles, such as carbon dioxide (CO2) [START_REF] Fukuto | The chemical properties of nitric oxide and related nitrogen oxides[END_REF]. The obtained nitrosoperoxycarbonate anion (ONOOCO2 -) is unstable and gives two highly reactive radicals: NO2 and carbonate radical anion CO3 -. The nitrosoperoxycarbonate ion, in turn, can be isomerized in unstable nitrocarbonate ion (O2NOCO2 -), which in aqueous solution decomposes to nitrate and bicarbonate [START_REF] Hughes | Chapter one -Chemistry of nitric oxide and related species[END_REF].

Nitroxyl HNO

The nitroxyl (HNO) is a RNS that has emerged as a promising therapy for treating the cardiovascular diseases [START_REF] Ge | Nitroxyl, redox switches, cardiac myofilaments, and heart failure a prequel to novel therapeutics?[END_REF]. Its pharmaceutical utilization is due to the chemistry of this species. HNO is a weak acid (acidity constant (pKa) 11.5), that means, that this RNS is a predominant species at physiological pH. As previously reported for peroxynitrite, HNO is able to react with proteins, which are located in key components of the cardiac electromechanical machinery, ruling myocardial function. This RNS is used to increase the cardiac force production and modulate the cardiac contractility [START_REF] Tocchetti | Playing with cardiac 'redox switches': the 'HNO way' to modulate cardiac function[END_REF].

Nerveless, Fukudo et al. reported the metastable nature of HNO. This properties is due, in the major part, to the self-reactivity of this species: one HNO molecule can react with another to give hyponitrous acid (HONNOH) (Figure 18), which, in turn, is unstable and forms nitrous oxide and water [START_REF] Fukuto | The chemical properties of nitric oxide and related nitrogen oxides[END_REF].

Nitrous oxide (N2O)

It has been established that N2O is one of the greenhouse gases [START_REF] Skalska | Trends in NOx abatement: A review[END_REF]. It has a ~300-fold greater warming potential than CO2, and is involved in destruction of the atmospheric ozone layer. Unlike NO or NO2, N2O has a long half-life around 100 to 150 years, that makes the increasing atmospheric level of this species a serious global problem. Atmospheric N2O concentrations have increased by 19% since pre-industrial times [START_REF] Butterbach-Bahl | Nitrous oxide emissions from soils: how well do we understand the processes and their controls?[END_REF].

The major source of N2O is soil ecosystems, which emit approximately 65% of the total N2O into the atmosphere. Here, the major N2O producer are denitrifying microorganisms (see part

Nitrogen cycle and denitrifying bacteria).

In recent years, the excessive use of nitrogen-based fertilizers in agriculture has also greatly contributed to the conspicuous elevation in atmospheric N2O concentration [START_REF] Hu | Microbial regulation of terrestrial nitrous oxide formation: understanding the biological pathways for prediction of emission rates[END_REF].

Ammonia (NH3)

Ammonia is a basic gas and one of the most abundant nitrogen-containing compounds in the atmosphere. The role of microorganisms in ammonium transformation in sols is impressive.

The soil nitrification, the oxidation of ammonia to nitrate (Figure 18) are performed by several bacterial species, named nitrifying bacteria (see part Nitrogen cycle and denitrifying bacteria). This results in enormous commercial losses of ammonium-based fertilizers, with associated atmospheric and groundwater pollution by nitrous oxide/acid and nitrate [START_REF] Anderson | Airborne reduced nitrogen: ammonia emissions from agriculture and other sources[END_REF].

Nitrous acid (HONO) and nitrite (NO2 -)

Nitrous acid and nitrite are short-lived intermediates of RNS. The nitrous acid (HONO) is a RNS of high importance in urban atmosphere chemistry. In particular, HONO plays an P a g e 78 | 270 important role in the initiation of daytime photochemistry by its rapid photolysis to NO and OH radicals [START_REF] Arens | Heterogeneous reaction of NO2 on diesel soot particles[END_REF]. The photolysis of NONO can provide a significant increasing of the HO-radical rate and generate the products of photochemical smog [START_REF] Czader | Modeling nitrous acid and its impact on ozone and hydroxyl radical during the Texas Air Quality Study[END_REF]. As shown in Figure 18, the nitrite, in turn, can be oxidized to the nitrate (NO3 -). The microorganisms, including denitrifying bacteria, can perform this reaction (see part Nitrogen cycle and denitrifying bacteria). The nitrate can be removed from atmosphere by plants (see part NO in plants) or through wet and dry deposition, leading to the acidification of ecosystems [START_REF] Lammel | Nitrous acid and nitrite in the atmosphere[END_REF].

In conclusion, it should be noted, that NOx, and the derived RNS, have a complex chemistry and, as consequence, various biological, pharmaceutical, and ecological functions. All these species are in interaction with living organisms, including bacteria. However, thanks to its unique properties, the effect of NO on bacterial cells is well studied. Thus, the synthesis and roles of this molecule in living cells, including bacteria will be discussed below.

NO formation in living organisms

NO is known to be synthetized in mammalian [START_REF] Fang | Nitric oxide production by human macrophages: there's NO doubt about it[END_REF], plant [START_REF] Planchet | Nitric oxide production in plants[END_REF] and bacterial (Plate & Marletta, 2013a) cells as a biological effector and/or mediator [START_REF] Choudhari | Nitric oxide and cancer: a review[END_REF]. When engulfed by macrophages, microorganisms are exposed to the NO defense mechanisms [START_REF] El-Gayar | Translational control of inducible nitric oxide synthase by IL-13 and arginine availability in inflammatory macrophages[END_REF]. In the same way, the plants have a NO defense against pathogens [START_REF] Delledonne | Nitric oxide functions as a signal in plant disease resistance[END_REF]. NO functions as a messenger in defense responses and as a general key factor associated with basal resistance in various plant-pathogen systems (Arasimowicz-Jelonek & Floryszak-Wieczorek, 2014). Thus, NO assimilation and synthesis in bacterial hosts seems to be inescapable step in the understanding of the bacterial NOx response.

Mammalian NO

NO is an important enzymatically synthesized signaling molecule responsible for a variety of physiological functions ranking from neurotransmission, relaxation of vascular endothelium, to toxicity to tumor and pathogen cells [START_REF] Fang | Nitric oxide production by human macrophages: there's NO doubt about it[END_REF][START_REF] Ignarro | Biosynthesis and metabolism of endothelium-derived nitric oxide[END_REF][START_REF] Marletta | Nitric oxide: biosynthesis and biological significance[END_REF]. As illustrated in Figure 19, the majority of mammalian NO is synthesized enzymatically from nitric oxide synthase (NOS), through the conversion of L-arginine to L-citrulline in an O2 and NADPH-dependent reaction [START_REF] Thomas | The chemical biology of nitric oxide. Implications in cellular signaling[END_REF]. NO could be formed by three NOS homodimers depending on cellular localization, functions and biochemical characteristics. Two of the NOS proteins, the neuronal NOS (nNOS; NOS-1) and the endothelial NOS (eNOS; NOS-3), are dependent on elevated intracellular Ca 2+ , besides the inducible macrophage NOS (iNOS;

NOS-2) is independent on Ca 2+ level [START_REF] Bruckdorfer | The basics about nitric oxide[END_REF][START_REF] Moncada | XIV. International Union of Pharmacology Nomenclature in nitric oxide research[END_REF].

P a g e 79 | 270 The nNOS was isolated from mammalian brain and named neuronal NOS playing several important roles in regulation of synaptic signaling and plasticity. High levels of nNOS protein are present in skeletal muscle [START_REF] Brenman | Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy[END_REF], where this molecule controls muscle contractility and local blood flow [START_REF] Mungrue | nNOS at a glance: implications for brain and brawn[END_REF]. As the name implies, the eNOS is originally from vascular endothelium, and NO generated by eNOS regulates blood pressure, platelet aggregation, leukocyte adherence, and mitogenesis of vascular smooth muscle cells [START_REF] Shaul | Regulation of endothelial nitric oxide synthase: location, location, location[END_REF]. Deficient eNOS production was implicated in the pathogenesis of systemic and pulmonary hypertension [START_REF] Huang | Hypertension in mice lacking the gene for endothelial nitric oxide synthase[END_REF] and in other vascular disorders including atherosclerosis [START_REF] Oemar | Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis[END_REF] The role of macrophage iNOS is the most interesting in terms of host-bacteria interactions. NO synthesized by iNOS is an important proinflammatory cytotoxic mediator, which defends the host toward various pathogens by inactivating and destroying infectious agents [START_REF] Macmicking | Nitric oxide and macrophage function[END_REF]. Thus, NO assures the resistance to microbial growth and antitumor immunity [START_REF] Fang | Antimicrobial reactive oxygen and nitrogen species: concepts and controversies[END_REF].

NO in plants

As previously reported, NO and the derived RNS play an important role in soil (see part 1.4.

Nitrogen oxides and reactive nitrogen species: chemistry and biological role).

As consequence, the role of NO in plants appears even more multi-faceted than in animals. Briefly, NO is a signaling molecule, controlling the plant growth and development, as well as the plant responses to biotic and abiotic stresses [START_REF] Planchet | Nitric oxide production in plants[END_REF]. In plants, NO is involved in mitochondrial [START_REF] Zottini | Nitric oxide affects plant mitochondrial functionality in vivo[END_REF] and chloroplast functionality [START_REF] Jasid | Chloroplasts as a nitric oxide cellular source. Effect of reactive nitrogen species on chloroplastic lipids and proteins[END_REF], root growth and cell wall lignification (Franciele Mara Lucca Zanardo Böhm, 2010), stomatal closure [START_REF] Garcia-Mata | Nitric oxide regulates K + and Clchannels in guard cells through a subset of abscisic acid-evoked signaling pathways[END_REF], senescence [START_REF] Hung | Nitric oxide counteracts the senescence of rice leaves induced by abscisic acid[END_REF], cell death [START_REF] Pedroso | A nitric oxide burst precedes apoptosis in angiosperm and gymnosperm callus cells and foliar tissues[END_REF] P a g e 80 | 270 and plant responses toward a variety of abiotic stresses, such as wounding, salinity, drought, and hypoxia [START_REF] Neill | Nitric oxide signalling in plants[END_REF].

Figure 20. Schematic model of NOx synthesis and assimilation in plants

Nitrate (NO3 -) is taken up by nitrate transporters (NRT) in roots and reduced in leaves to nitrite (NO2 -) by nitrate reductase (NR). In chloroplasts, nitrite is reduced to ammonium (NH4 + ) and incorporated into amino acids (AA).

In cytosol, nitrite can also be reduced to nitric oxide (NO). NO, in turn, can be generated from other sources, such as L-arginine. NO reacts with reduced glutathione (GSH) producing S-nitrosoglutathione (GSNO), the major plant reservoir of NO. The levels of NO and GSNO are controlled by the enzyme GSNO reductase (GSNOR). Adapted from [START_REF] Frungillo | S-nitrosothiols regulate nitric oxide production and storage in plants through the nitrogen assimilation pathway[END_REF] As demonstrated in Figure 20, the NO production in plants is based on both the synthesis of NO by NOS (e.g. arginine based NO synthesis, like in mammalian cells) and reductive pathways [START_REF] Gupta | On the origins of nitric oxide[END_REF]. Although the mechanism of arginine-dependent NO production in plants is similar to that of mammalian cells, up to now, no mammalian NOS homologs were identified in higher plants. It was described, that the polyamines and the hydroxylamines could be a potential source for the NO synthesis in higher plants [START_REF] Fröhlich | The hunt for plant nitric oxide synthase (NOS): is one really needed?[END_REF]. In addition, S-nitrosoglutathione (GSNO), which is formed by spontaneous reaction of NO with glutathione, is a major NO reservoir and NO donor in plant cells. GSNO level is controlled by the GSNO reductase, which is known to be the main enzyme responsible for the modulation of S-nitrosothiol pools (Figure 20) [START_REF] Frungillo | S-nitrosothiols regulate nitric oxide production and storage in plants through the nitrogen assimilation pathway[END_REF].

Assimilation of nitrate involves the sophisticated transport systems, which relies mainly on membrane-bound nitrate transporters (NRTs) [START_REF] Tsay | Nitrate transporters and peptide transporters[END_REF][START_REF] Wang | Uptake, allocation and signaling of nitrate[END_REF]. Once taken up by roots, nitrite is transported to shoots for assimilation and reduced to nitrite (NO2 -) thanks to activity of the nicotinamide adenine dinucleotide (NAD(P)H)-dependent cytosolic nitrate reductase (NR). NO2 -, in turn, is promptly reduced by nitrite reductase into ammonium (NH4 + )

for further assimilation into organic compounds [START_REF] Crawford | Nitrate: nutrient and signal for plant growth[END_REF][START_REF] Zheng | Crystal structure of a nitrate/nitrite exchanger[END_REF]. In addition to NH4 + production, the nitrite anion can be reduced to NO via non-enzymatic and various enzymatic pathways [START_REF] Salgado | Nitric oxide signaling and homeostasis in plants: a focus on nitrate reductase and S-nitrosoglutathione reductase in stress-related responses[END_REF]. Non-enzymatic nitrite reduction occurs in P a g e 81 | 270 the apoplasts [START_REF] Bethke | Apoplastic synthesis of nitric oxide by plant tissues[END_REF]. The NO2 -reduction by enzymatic mechanism includes the reaction catalyzed by NO2 -:NO reductase (Ni:NOR) in the plasma membrane [START_REF] Stöhr | A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite[END_REF] and in the plant mitochondria [START_REF] Frungillo | S-nitrosothiols regulate nitric oxide production and storage in plants through the nitrogen assimilation pathway[END_REF][START_REF] Salgado | Nitric oxide signaling and homeostasis in plants: a focus on nitrate reductase and S-nitrosoglutathione reductase in stress-related responses[END_REF] and the reaction catalyzed by NR in the cytosol [START_REF] Rockel | Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro[END_REF].

NO in bacteria

Bacteria, like animals and plant, tame NO in order to use this free radical as second messenger in their functioning and signaling. Here, once more, the high bacterial evolutionary potential and the ability to adapt to environmental changes are reflected. Thus, two major pathways of NO metabolism were described in bacteria. The first one includes the primary NOS-mediated NO synthesis [START_REF] Sudhamsu | Bacterial nitric oxide synthases: what are they good for?[END_REF]; and the second one is based on the NO reductase activity, similar to that previously described in plants [START_REF] Arai | Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa[END_REF].

NO synthesis by NO synthase

Several strains of mostly Gram-positive bacteria express homologs of the mammalian NOS (bNOS from bacterial NOS). Recent genome sequencing has revealed that NOS-like protein exists in many bacteria, including Streptomyces, Deinococcus, Staphylococcus, and Bacillus species [START_REF] Crane | The enzymology of nitric oxide in bacterial pathogenesis and resistance[END_REF]. However, to the best of our knowledge, bacteria of genus Pseudomonas do not possess the NOS coding genes.

Nitrogen cycle and denitrifying bacteria

As previously reported in Figure 18, the interconversion of the nitrogen species (oxidation states from -III to +V) constitutes the global biogeochemical nitrogen cycle [START_REF] Watmough | Nitric oxide in bacteria: synthesis and consumption[END_REF]. As represented in Figure 21, the inorganic nitrogen is converted to a biologically useful form (N+V) by N2 fixation or nitrate assimilation. Nitrogen is removed from the environment by both nitrification -the oxidative conversion of ammonia (NH4 + ) to nitrate (NO3 -) (Figure 21, green color) -and denitrification -a respiratory process whereby nitrate is reduced by several enzymatic steps to nitrogen gas (Figure 21 Ammonia is not an intermediate of the denitrification pathway. The overall respiratory reduction of nitrate to nitrogen gas is coupled to ATP generation, allowing the organism to grow in the absence of oxygen [START_REF] Sias | The assimilatory and dissimilatory nitrate reductases of Pseudomonas aeruginosa are encoded by different genes[END_REF][START_REF] Takaya | Aerobic denitrifying bacteria that produce low levels of nitrous oxide[END_REF]. Many bacterial species, especially the members of Pseudomonas genus (in majority P. aeruginosa), can perform the denitrification under anaerobic conditions [START_REF] Almeida | Nitrite inhibition of denitrification by Pseudomonas fluorescens[END_REF][START_REF] Kumita | NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa: characterization of reaction intermediates that appear in the single turnover cycle[END_REF].

Nitrate reductases

In the first step of the denitrification pathway, bacteria have to reduce the nitrate -N(+V)-to the nitrite -N(+III)-thanks to the catalytic properties of nitrate reductases (Figure 22) [START_REF] Baysse | The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification[END_REF][START_REF] Carlson | Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa[END_REF]. Two different nitrate reductases were found in denitrifying P. aeruginosa [START_REF] Baysse | The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification[END_REF]. One is known as a respiratory membrane-bound nitrate reductase (NAR) [START_REF] Carlson | Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa[END_REF], which is directly associated with denitrification and P a g e 83 | 270 anaerobic nitrate respiration. The second is a periplasmic nitrate reductase (NAP) [START_REF] Van Alst | Compensatory periplasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa PAO1 in the absence of membrane nitrate reductase[END_REF]. Why have two enzymes, catalyzing the same reaction remains to be understood.

The membrane-bound nitrate reductase complex consists of three polypeptides α, β, and γ, which are encoded by the narK1K2GHJI operon (Figure 23A). The α-and β-subunits NarGH extend into the cytoplasm and are associated with a molybdopterin cofactor. The enzyme is attached to the cytoplasmic membrane by the γ subunit NarI, which accepts electrons from the quinol pool and transfers them via β-heme to NarG. The NarJ is a chaperone involved in NarGHI assembly, and NarK1K2 are possibly involved in nitrate and nitrite transport [START_REF] Van Alst | Compensatory periplasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa PAO1 in the absence of membrane nitrate reductase[END_REF].

The periplasmic nitrate reductase, encoded by the napEFDABC operon (Figure 23B), consists of two subunits, NapA and NapB. The cytochrome c-type protein NapC is involved in the electron transfer to NapAB. NapA is the large subunit with the molybdenum cofactor and a [4Fe-4S] cluster. NapB is a c-type cytochrome [START_REF] Carlson | Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa[END_REF]. The narK1K2GHJI operon was described as involving in the nitrate respiration, whereas the physiological role of the NAP periplasmic nitrate reduction may vary in different organisms or P a g e 84 | 270 even in the same bacterium under different metabolic conditions [START_REF] Moreno-Vivián | Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases[END_REF].

The physiological role of NAP in P. aeruginosa is not certain in present. Bedzyk and colleges supposed, that the latter is used for the denitrification during aerobic to anaerobic transition, as well as to maintain the redox balance during the reduction of NO3 -to NO2 - [START_REF] Baysse | The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification[END_REF]. Thus, the 'aerobic' denitrification was found to be performed by the coupling of the NAP to the NIR and NOR enzymes [START_REF] Bell | Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha. The periplasmic enzyme catalyzes the first step in aerobic denitrification[END_REF][START_REF] Berks | Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions[END_REF]. Aerobic denitrification can be an advantage for bacteria growing under microaerobic conditions (i.e. biofilm) or in environments, rapidly changing between aerobic and anaerobic conditions [START_REF] Moreno-Vivián | Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases[END_REF].

Nitrite reductases

As previously reported (see part 1.4. Nitrogen oxides and reactive nitrogen species: chemistry and biological role), the nitrite is a toxic molecule, which must be quickly reduced in nitric oxide using the nitrite reductases (Figure 22). Three different types of NIR were found in bacteria. The cytochrome c NIR reduces nitrite to ammonia while the other two classes of NIR (a cytochrome cd1 NIR and a trimeric copper containing NIR) located in the periplasm and catalyze the one electron reduction of NO2 -to NO [START_REF] Watmough | Nitric oxide in bacteria: synthesis and consumption[END_REF]. The cytochrome cd1 was isolated from a number of denitrifying bacteria [START_REF] Van Wonderen | Activation of the cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. Reaction of oxidized enzyme with substrate drives a ligand switch at heme c[END_REF][START_REF] Zajicek | Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase[END_REF], including members of Pseudomonas genus [START_REF] Cutruzzolà | NO production by Pseudomonas aeruginosa cd1 nitrite reductase[END_REF][START_REF] Rinaldo | The catalytic mechanism of Pseudomonas aeruginosa cd1 nitrite reductase[END_REF]. This enzyme functions as a homodimer [START_REF] Nurizzo | Conformational changes occurring upon reduction and NO binding in nitrite reductase from Pseudomonas aeruginosa[END_REF], when each subunit contains a covalently attached heme c and a non-covalently bound heme d1 [START_REF] Nurizzo | Nterminal arm exchange is observed in the 2.15 A crystal structure of oxidized nitrite reductase from Pseudomonas aeruginosa[END_REF]. In P. aeruginosa, the NIR is encoded by the nirSMCFDLGHJEN gene cluster (Figure 23C), where nirS is the structural gene for the enzyme [START_REF] Silvestrini | Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein[END_REF]. The nirM and nirC encode two cytochromes c, and both these cytochromes mediate electron transfer from the cytochrome bc1 complex to the nitrite reductase [START_REF] Arai | Cloning and sequencing of the gene encoding cytochrome c-551 from Pseudomonas aeruginosa[END_REF][START_REF] Hasegawa | Two c-type cytochromes, NirM and NirC, encoded in the nir gene cluster of Pseudomonas aeruginosa act as electron donors for nitrite reductase[END_REF]. The nirFDLGHJE genes are necessary for the biosynthesis of the heme d1 [START_REF] Arai | Regulation and function of versatile aerobic and anaerobic respiratory metabolism in Pseudomonas aeruginosa[END_REF].

The nirN encodes a c-type cytochrome, which is similar to nirS, but its function is not certain [START_REF] Hasegawa | Two c-type cytochromes, NirM and NirC, encoded in the nir gene cluster of Pseudomonas aeruginosa act as electron donors for nitrite reductase[END_REF].

Nitric oxide reductases

Three types of bacterial NORs have been identified: cNOR, qNOR and qCuANOR [START_REF] Shiro | Structural basis for nitrous oxide generation by bacterial nitric oxide reductases[END_REF]. Cytochrome c-dependent NOR (cNOR) was characterized in P. aeruginosa and P. stutzeri [START_REF] Field | The respiratory nitric oxide reductase (NorBC) from Paracoccus denitrificans[END_REF][START_REF] Kastrau | Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome bc complex. Phospholipid requirement, electron paramagnetic resonance and redox properties[END_REF]. It is a heterodimeric enzyme consisting of a small (NorC) and a large (NorB) subunits. NorC is a membrane-bound subunit with a heme c, which accepts electrons from an external protein electron donor. NorB contains three iron centers (two b-type hemes and one non-heme iron, FeB). The b-type heme mediates electron P a g e 85 | 270 transfer from the heme c in the NorC subunit to the binuclear catalytic center, which consists of another b-type heme and FeB [START_REF] Hino | Structural basis of biological N2O generation by bacterial nitric oxide reductase[END_REF][START_REF] Shiro | Structure and function of bacterial nitric oxide reductases: Nitric oxide reductase, anaerobic enzymes[END_REF]. In P. aeruginosa, the NOR is encoded by the norCBD operon. The norC and norB encode cytochrome c and b subunits, respectively, and norD encodes a soluble protein involved in the enzyme activation [START_REF] Arai | Regulation and function of versatile aerobic and anaerobic respiratory metabolism in Pseudomonas aeruginosa[END_REF]Arai et al., 1995a;[START_REF] Zumft | Nitric oxide reductases of prokaryotes with emphasis on the respiratory, hemecopper oxidase type[END_REF].

Nitrous oxide reductase

As shown in Figure 22, the last step of the denitrification, including the reduction of N2O to N2, is catalyzed by the multicopper enzyme N2O reductase (N2OR) (Zumft, 1997;Zumft & Kroneck, 2007). In P. aeruginosa, the N2OR is encoded in the nosRZDFYL operon (Figure 23D), where nosZ is a structural gene [START_REF] Arai | Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa[END_REF]. The nosDFY genes products are involved in the copper insertion into the enzyme, the nosL gene product is an outer membrane disulfide isomerase, and the nosR gene codes for a membrane protein with unknown functions.

Transcriptional regulation of denitrification genes in P. aeruginosa

Two transcriptional regulators, ANR (ANaerobic Regulator of arginine deiminase and nitrate reductase) and DNR (Dissimilatory Nitrate respiration Regulator), which both belong to the FNR (Fumarate and Nitrate reductase Regulator)/cAMP (cyclic adenosine monophosphate) receptor protein family, hierarchically control the expression of the denitrification genes in P. aeruginosa (Figure 24) [START_REF] Kuroki | Fine-tuned regulation of the dissimilatory nitrite reductase gene by oxygen and nitric oxide in Pseudomonas aeruginosa[END_REF]. ANR senses intracellular oxygen levels by the [4Fe-4S] 2+ cluster and is activated upon the oxygen depletion [START_REF] Sawers | Identification and molecular characterization of a transcriptional regulator from Pseudomonas aeruginosa PAO1 exhibiting structural and functional similarity to the FNR protein of Escherichia coli[END_REF]. DNR is a NOsensing regulator, which senses NO molecules through heme [START_REF] Castiglione | The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli[END_REF][START_REF] Giardina | NO sensing in Pseudomonas aeruginosa: structure of the transcriptional regulator DNR[END_REF]. Expression of the dnr gene is under control of ANR and is induced under low oxygen or anaerobic conditions [START_REF] Arai | Cascade regulation of the two CRP/FNR-related transcriptional regulators (ANR and DNR) and the denitrification enzymes in Pseudomonas aeruginosa[END_REF]. The transcription of NAR complex is regulated by NarXL two-component nitrate sensor-Response Regulator (RR) proteins (Figure 24) [START_REF] Van Alst | Compensatory periplasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa PAO1 in the absence of membrane nitrate reductase[END_REF][START_REF] Arat | A network biology approach to denitrification in Pseudomonas aeruginosa[END_REF]. NarXL is a classic two-component bacterial regulatory system, in which a histidyl-aspartyl phosphorelay controls the gene expression in response to specific environmental stimuli [START_REF] Härtig | Nitrate and nitrite control of respiratory nitrate reduction in denitrifying Pseudomonas stutzeri by a two-component regulatory system homologous to NarXL of Escherichia coli[END_REF]. The periplasmic domain of the sensor protein NarX, contains a highly conserved nitrate recognition region. The nitrate recognition results in the autophosphorylation of a conserved histidyl residue in the NarX cytoplasmic transmitter domain. Transfer of phosphate to the conserved aspartyl residue in the receiver domain of the RR, NarL results in the transcriptional regulation of the target operons.

NarL, in conjunction with Anr and Dnr, induces the narK1K2GHJI operon expression, and potentially represses the expression of napEFDABC operon in P. aeruginosa [START_REF] Van Alst | Compensatory periplasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa PAO1 in the absence of membrane nitrate reductase[END_REF][START_REF] Schreiber | The anaerobic regulatory network required for Pseudomonas aeruginosa nitrate respiration[END_REF]. NirQ is an ATP-binding protein, which is required for the activation of nitrite reduction (Figure 24). The nirQ expression is dependent on dnr and narL P a g e 86 | 270 expression and strongly induced under anaerobic conditions [START_REF] Arai | Structure and ANR-dependent transcription of the nir genes for denitrification from Pseudomonas aeruginosa[END_REF][START_REF] Schreiber | The anaerobic regulatory network required for Pseudomonas aeruginosa nitrate respiration[END_REF]. The transcription of norC is also dependent on Anr and Dnr and the presence of nitrite (Arai et al., 1995b). However, it should be noted, that not all Pseudomonas spp. strains possess nitrate respiration coding genes. Among currently sequenced strains, the genomes of most P. aeruginosa, several P. stutzeri, P. brassicacearum subsp. brassicacearum NFM421 and just P. fluorescens F113 are found to contain such genes coding for Nar, Nap, and Nir enzymes (www. pseudomonas.com).

NO targets in cells

NO and RNS were found to damage bacterial cells. The complexity of RNS chemistry and their high reactivity (especially in presence of O2 and reactive oxygen species (ROS)) make difficult to assign the specific antimicrobial actions to individual molecular species (see part 1.4.

Nitrogen oxides and reactive nitrogen species: chemistry and biological role). The dependence of antimicrobial action of RNS on the redox environment in and outside the bacterial cell is an additional complication. However, the RNS were found to interact with numerous targets in a microbial cell, including thiols, metal centers, tyrosines, nucleotide bases and lipids [START_REF] Fang | Antimicrobial reactive oxygen and nitrogen species: concepts and controversies[END_REF][START_REF] Nathan | Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens[END_REF]. Many of these interactions are reversible, which reflects once again, the high bacterial adaptability to counteract the nitrosative stress.

Figure 25. Bacterial targets of RNS

A scheme of simplified bacterial cell is shown. Reactive nitrogen species (RNS) mainly interact with thiols, metal centers and DNA. RNS inhibit bacterial respiration and alter DNA replication through the inactivation of zinc metalloproteins. RNS can mobilize iron from iron-sulphur [Fe-S] proteins. The reactive oxygen species (ROS) provoke mainly a direct DNA damage and, like RNS, interact with bacterial proteins. Adapted from [START_REF] Fang | Antimicrobial reactive oxygen and nitrogen species: concepts and controversies[END_REF].

Membrane glycerophospholipids

As previously reported in Chapter 2 Stress factors and Pseudomonas fluorescens lipid adaptation, bacterial membrane plays a role of the first barrier between the environment and the cell. However, the GP membrane itself is not inert towards several toxic compounds and could be chemically modified. The prototypical effect of RNS on membrane is reflected in lipid oxidation and/or nitrosation [START_REF] Möller | Chapter 2 The interaction of reactive oxygen and nitrogen species with membranes[END_REF][START_REF] Pryor | Reaction of nitrogen dioxide with alkenes and polyunsaturated fatty acids: addition and hydrogenabstraction mechanisms[END_REF]. It was shown, that several RNS affect mainly polyunsaturated FAs, leading to form lipid hydroperoxides, lipid alcohols, and aldehydes. The NO2 effect on GPs is well studied [START_REF] Augusto | Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology[END_REF][START_REF] Pryor | Reaction of nitrogen dioxide with alkenes and polyunsaturated fatty acids: addition and hydrogenabstraction mechanisms[END_REF][START_REF] Pryor | Mechanisms of nitrogen dioxide reactions: initiation of lipid peroxidation and the production of nitrous Acid[END_REF]. It was proposed that the reaction is dependent on the O2 level. At high O2 concentrations, NO2 will predominantly mediate lipid oxidation (see part 1.1. Oxidative stress). Conversely, at low O2 level, nitration reactions may preferentially occur (Figure 26) [START_REF] O'donnell | Nitration of unsaturated fatty acids by nitric oxidederived reactive nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide, and nitronium ion[END_REF]. The GP nitration reaction proceeds via addition of NO2 to the double bond of FA chain, forming an instable nitro alkyl radical, which will be efficiently transformed in the presence of O2 or additional NO2 to peroxynitroalkyl and dinitroalkyl respectively (Figure 26.1 and 2) [START_REF] Pryor | Reaction of nitrogen dioxide with alkenes and polyunsaturated fatty acids: addition and hydrogenabstraction mechanisms[END_REF][START_REF] Pryor | Mechanisms of nitrogen dioxide reactions: initiation of lipid peroxidation and the production of nitrous Acid[END_REF]. In the absence of adequate concentrations of O2 and NO2, nitrous acid is dissociated from alkyl radical, leading to the double bond formation (Figure 26.3) [START_REF] Augusto | Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology[END_REF]. This reaction drives to cis/trans isomerization [START_REF] Jiang | Nitrogen dioxide induces cis-trans-isomerization of arachidonic acid within cellular phospholipids. Detection of transarachidonic acids in vivo[END_REF]. Conversely, the hydrogen abstraction reaction can support the formation of alkyl radical, which, in turn, can be nitrated or oxidized as described previously As the NO oxidation by O2 is accelerated in hydrophobic conditions of lipid membrane [START_REF] Liu | Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes[END_REF], the damage of GPs is not the major role of NO radical. As consequence, NO alone (without O2 or ONOO -) does not modify bacterial FAs [START_REF] Rubbo | Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives[END_REF]. et al., 1999). Concerning another RNS species, peroxynitrite can both oxidize and nitrate polyunsaturated FAs via NO2 formation (Figure 26) [START_REF] O'donnell | Nitration of unsaturated fatty acids by nitric oxidederived reactive nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide, and nitronium ion[END_REF].

(
It should be noted, that all previously discussed mechanisms of NOxlipids interactions were established in eukaryotic cells, containing polyunsaturated FAs. The question "Can NOx modify bacterial monounsaturated FAs or GPs composition?" is still an enigma.

Proteins

Among the RNS targets in bacterial cell, the proteins play one of the most important roles.

Nitrosative stress can cause several types of protein damage. Binding to the protein metal centers, the RNS impact several vital functions in bacteria, such as biofilm formation, or DNA repair. The reaction of RNS with phenols provokes a tyrosine nitration, and the ones with thiol groups of cysteines cause the S-nitrosylation of several proteins [START_REF] Hill | What part of NO don't you understand? Some answers to the cardinal questions in nitric oxide biology[END_REF][START_REF] Mclean | Peroxynitrite toxicity in Escherichia coli K12 elicits expression of oxidative stress responses and protein nitration and nitrosylation[END_REF]. All these mechanisms are of high importance in bacterial functioning, reflecting once more the complex RNS chemistry and the high reactivity of these species, but also a high bacterial adaptability to the RNS toxicity. The brief description of RNS reactions with proteins are sum up below.

Reaction of RNS with thiols

Thiols is organosulfur compounds that contain a carbon-bonded sulfhydryl group (-C-SH or R-SH, where R represents alkane, alkene, or other carbon-containing chemical group). The chemistry of thiols is intimately associated with the chemistry of RNS, which lead to the reversible nitrosation and/or oxidation of thiols [START_REF] Fukuto | The chemical properties of nitric oxide and related nitrogen oxides[END_REF]. The involvement of NO2, N2O4 or N2O3 in these reactions was particularly studied. The thiol nitrosation results in the formation of S-nitrosothiols (RS-NO) [START_REF] Hughes | Chapter one -Chemistry of nitric oxide and related species[END_REF] or S-nitrothiol species (RS-NO2) [START_REF] Antelmann | Thiol-based redox switches and gene regulation[END_REF]. The different modifications appear to be dependent on the nature of RNS, the protein itself, and its subcellular localization. The protein S-nitrosylation was found to be an obligate concomitant of anaerobic respiration on nitrate in E. coli, controlled by OxyR transcription factor (see part 4.6. Secondary NO sensors) [START_REF] Seth | Endogenous protein S-nitrosylation in E. coli: regulation by OxyR[END_REF]. Overall, the thiol group belongs to cysteine, an indispensable α-amino acid for all forms of life. It serves as a precursor for protein synthesis. The unique chemistry of its free thiol group forms the active moiety of several essential metabolites, such as CoA, glutathione and its derivatives [START_REF] Dominy | Identification and characterization of bacterial cysteine dioxygenases: a new route of cysteine degradation for Eubacteria[END_REF]. Nitrosation may cause toxic effects on bacterial cells, altering protein 'normal'

functions [START_REF] Sigfrid | Cytokines and nitric oxide inhibit the enzyme activity of catalase but not its protein or mRNA expression in insulin-producing cells[END_REF]. However, the impact of RNS on bacterial proteins is not always negative. For instance, the S-nitrosylation could play a role of a conserved mechanism for control of protein function. This non common control pathway appears to activate several P a g e 90 | 270 proteins, essential in response to nitrosative stress (see part 5.1. [Fe -S] cluster repair). The mechanism of this activation is based on the reaction of RNS with protein metal centers, which will be described below.

RNS complexes with metal centers

NO is able to reversibly bind to ferrous (Fe 2+ ) and ferric (Fe 3+ ) irons of several prokaryotic and eukaryotic proteins. This binding could both inhibit enzyme activity (i.e. catalase inhibition, [START_REF] Brown | Reversible binding and inhibition of catalase by nitric oxide[END_REF]) and activates several enzymes, such as guanylate cyclase (GCyc) [START_REF] Ding | Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator[END_REF]. The notable example of the NO signaling in bacterial cells is the NO binding to iron-sulfur protein clusters [Fe-S] [START_REF] Cooper | Nitric oxide and iron proteins[END_REF]. The [Fe-S] clusters are common, almost ubiquitous, prosthetic groups that are essential for the function of proteins, involved in a wide range of biological processes, including electron transfer chains, redox and non-redox catalysis of reactions involved in metabolic pathways, and gene regulation [START_REF] Justino | Escherichia coli di-iron YtfE protein is necessary for the repair of stress-damaged iron-sulfur clusters[END_REF]. These prosthetic groups are inorganic moieties, whose iron atoms are coordinated to inorganic sulfides and usually linked to protein by cysteine thiolates [START_REF] Drapier | Interplay between NO and [Fe-S] clusters: relevance to biological systems[END_REF]. clusters that activates the protein [START_REF] Ding | The redox state of the [2Fe-2S] clusters in SoxR protein regulates its activity as a transcription factor[END_REF][START_REF] Gaudu | SoxR, a [2Fe-2S] transcription factor, is active only in its oxidized form[END_REF]. The RNS, as well, activate SoxR in E. coli [START_REF] Nunoshiba | Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages[END_REF] generating, in turn, dinitrosyl-iron-dithiol clusters (Figure 28B) [START_REF] Ding | Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator[END_REF]. The RNS react with phenols, occurring in the protein tyrosine residues nitration to form the 3nitrotyrosine (Figure 29). This post-translational protein modification draws attention of biochemical researches, because it may alter the protein functions. RNS do not able to directly perform the nitration, because they have to react with previously formed tyrosine radical. These latter are formed via tyrosine oxidation by CO3 -and OH radicals, or oxo-metal complexes. An alternative radical mechanism for tyrosine nitration involves the reaction of tyrosyl radicals with NO, giving 3-nitrosotyrosine, which, after sequential two-electron oxidation, forms tyrosine iminoxyl radical and 3-nitrotyrosine [START_REF] Radi | Nitric oxide, oxidants, and protein tyrosine nitration[END_REF]. Although stabilization of tyrosyl radicals, they could form a dimer, 3,3'-dityrosine. Several studies found that RNS, particularly peroxynitrite, cause the massive tyrosine nitration [START_REF] Alvarez | Peroxynitrite reactivity with amino acids and proteins[END_REF][START_REF] Lindemann | Redox proteomics uncovers peroxynitrite-sensitive proteins that help Escherichia coli to overcome nitrosative stress[END_REF][START_REF] Mclean | Peroxynitrite toxicity in Escherichia coli K12 elicits expression of oxidative stress responses and protein nitration and nitrosylation[END_REF]. Unlike in the case of cysteines, the tyrosine nitration is fairly unspecific, affecting a large number of E. coli proteins. These modifications are thought to be irreversible and probably damages proteins irreparably [START_REF] Lindemann | Redox proteomics uncovers peroxynitrite-sensitive proteins that help Escherichia coli to overcome nitrosative stress[END_REF].

In conclusion, the reactivity of RNS with proteins results in various noxious consequences for bacterial cells. The reactions with heme iron centers are known to be involved in respiratory arrest, limiting the ability of cells to maintain the redox balance and generate the energy for P a g e 92 | 270 growth [START_REF] Richardson | A nitric oxide-inducible lactate dehydrogenase enables Staphylococcus aureus to resist innate immunity[END_REF][START_REF] Stevanin | Flavohemoglobin Hmp affords inducible protection for Escherichia coli respiration, catalyzed by cytochromes bo′ or bd, from nitric oxide[END_REF]. Thus, nitrosative stress helds to severely impair oxidative phosphorylation (OP), a major process generating ATP in an O2-dependent manner in bacteria. This ATP-generating system relies on the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) [START_REF] Appanna | Fumarate metabolism and ATP production in Pseudomonas fluorescens exposed to nitrosative stress[END_REF]. NO directly reacts with metal prosthetic groups of cytochromes in the ETC and [Fe-S] clusters of dehydratases (i.e. aconitase) [START_REF] Husain | Nitric oxide evokes an adaptive response to oxidative stress by arresting respiration[END_REF]. This underscores the tendency of RNS to inhibit aerobic respiration, necessitating the expression of anaerobic metabolic genes. The sensitivity of aconitase [Fe-S] cluster to NO affects the TCA cycle [START_REF] Gardner | Nitric oxide sensitivity of the aconitases[END_REF][START_REF] Richardson | A nitric oxide-inducible lactate dehydrogenase enables Staphylococcus aureus to resist innate immunity[END_REF]. Bacterial survival under conditions of nitrosative stress indicates that diverse bacterial species respond to nitrosative stress by altering the expression of metabolic pathways (see part 4.5. Metabolic reprogramming).

DNA damage

As previously reported, RNS react with [Fe-S] clusters to inactivate proteins that contribute to the DNA stability, such as ribonucleotide reductase (necessary for normal DNA replication)

and several enzymes involved in DNA repair (e.g. Fpg DNA glycosylase, DNA repair methyltransferase and DNA ligase) [START_REF] Spek | Recombinational repair is critical for survival of Escherichia coli exposed to nitric oxide[END_REF]. In addition to the potentially deleterious effects of loss of function of these important DNA-metabolizing proteins, RNS may induce DNA modifications such as cross-links and base damage (reviewed in [START_REF] Burney | The chemistry of DNA damage from nitric oxide and peroxynitrite[END_REF].

NO does not directly react with DNA, but can become a potent DNA damaging agent once it has been transformed to N2O3, or peroxynitrite. These RNS react with DNA via multiple pathways [START_REF] Burney | The chemistry of DNA damage from nitric oxide and peroxynitrite[END_REF][START_REF] Tamir | DNA damage by nitric oxide[END_REF], which are briefly mentioned here and

. Repair of DNA damage). As reported in

Figure 30, in addition to deamination, the N2O3 can also cause the formation of single-and double-strand breaks in DNA (SSB and DSB respectively) [START_REF] Burney | The chemistry of DNA damage from nitric oxide and peroxynitrite[END_REF].

Figure 30. DNA damage pathways resulting from RNS exposure

Adapted from [START_REF] Burney | The chemistry of DNA damage from nitric oxide and peroxynitrite[END_REF].

In contrast to N2O3, which is primarily involved in the deamination of DNA, most of the DNA damages caused by peroxynitrite is oxidative (Figure 30). DNA treatment with peroxynitrite generally leads to the deoxyribose oxidation and oxidation of bases, most notably guanine [START_REF] Hughes | Repair of nitric oxide-damaged DNA in β-cells requires JNK-dependent GADD45α expression[END_REF]. This reaction results in predominantly 8-oxoguanine (8-oxoG) and 8nitroguanine (8-nitro-dG) formation. Its biological consequences include both cytotoxicity and mutagenicity. In fact, 8-nitro-dG is unstable and readily depurinates to form abasic sites. The formed 8-oxoG can actually react again with peroxynitrite to form secondary oxidation products, whose biological consequences are currently under investigation. In addition to base lesions, peroxynitrite, like N2O3, can also lead to SSBs [START_REF] Spek | Recombinational repair is critical for survival of Escherichia coli exposed to nitric oxide[END_REF].

Bacterial strategies of response to RNS

It becomes increasingly evident, that NO and derived RNS damage several (if not all) compounds of bacterial cells, but bacteria have developed a wide range of adaptive responses to counteract the RNS toxicity. These responses, including NO sensing, metabolism modifications, NO detoxification pathway(s) and GP barrier, will be discussed in this part of the work.

Membrane glycerophospholipid barrier

As described before, bacterial membrane offers a significant resistance to the passage of most toxic species. It is therefore relevant to know whether GP membranes are permeable to RNS, to define if these molecules could penetrate into the cell and react with another targets within bacteria. As shown in Figure 31, the lipid membranes do not present a barrier against the diffusion of NO and O2 [START_REF] Signorelli | Nitrogen dioxide solubility and permeation in lipid membranes[END_REF], but significantly decrease the diffusion of HNO3 and H2O2, and prevent the diffusion of ONOO -, O2 and carbonate radical (CO3 -) [START_REF] Möller | Chapter 2 The interaction of reactive oxygen and nitrogen species with membranes[END_REF]. The NO2 membrane permeability was investigated in several studies [START_REF] Khairutdinov | Permeation of phospholipid membranes by peroxynitrite[END_REF][START_REF] Möller | Chapter 2 The interaction of reactive oxygen and nitrogen species with membranes[END_REF][START_REF] Signorelli | Nitrogen dioxide solubility and permeation in lipid membranes[END_REF]. Khairutdinov et al.

estimated the NO2 permeability coefficient is between 4  10 -4 and 10 cm s -1 [START_REF] Khairutdinov | Permeation of phospholipid membranes by peroxynitrite[END_REF], a range too wide to make appropriate predictions, ranging from minimal to significant membrane resistance to NO2 permeation. Signorelli and colleagues estimated the NO2 permeability coefficient at 5 cm s -1 , suggesting that the GP membrane is permeable to this free radical, but 10 times slower than to NO [START_REF] Signorelli | Nitrogen dioxide solubility and permeation in lipid membranes[END_REF]. So, the order of permeability across lipid membrane is: NO, O2 then ONOOH, NO2, HOO, H2O, which are quicker than O2 •-, ONOO -, CO3 •-as illustrated in Figure 31.

Figure 31. Permeability of reactive species across lipid membrane

Pm/Pw is the ratio between membrane permeability and the permeability through an equivalent layer of water. Lipid membranes are not barrier to NO, but a moderate barrier to the permeation of ONOOH or NO2. The permeability of charged molecules, like ONOO -is not significant. Adapted from [START_REF] Möller | Chapter 2 The interaction of reactive oxygen and nitrogen species with membranes[END_REF] While the charged molecules do not able to freely cross GP bilayer, they are able to penetrate bacterial membrane. The peroxynitrite, for example, can enter the cell either by passive diffusion (as peroxynitrous acid, ONOOH) or by anion channels (as peroxynitrite anion, ONOO -) [START_REF] Ferrer-Sueta | Chemical biology of peroxynitrite: kinetics, diffusion, and radicals[END_REF]. However, it should be noted, that these studies were P a g e 95 | 270 performed in PC membrane models to establish the response of eukaryotic membrane to free radical toxicity. Bacterial membrane composition is quite different of that of eukaryotic cell.

We assume, that the permeability of NOx through bacterial membrane could (or could not) be different of previously studied.

Repair of DNA damage

All living organisms have evolved several mechanisms to repair the DNA damages, preventing the genome instability and limiting the accumulation of deleterious mutations. As previously demonstrated, The RNS can damage bacterial genomes that result in aberrant base pairing, DNA crosslinks, abasic sites, SSBs or DSBs (see part DNA damage). These modifications lead to the reduction in viability by impairing the transcription of essential genes and the stalling of DNA replication; furthermore, the incorporation of incorrect bases can itself be harmful and affects the viability of progeny. The DNA repair was extensively studied in both prokaryotes and eukaryotes [START_REF] Boiteux | Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae[END_REF][START_REF] Eisen | A Phylogenomic study of DNA repair genes, proteins, and processes[END_REF][START_REF] Van Der Veen | The BER necessities: the repair of DNA damage in humanadapted bacterial pathogens[END_REF]. Given the diverse nature of DNA damages caused by RNS, several repair pathways have evolved to rectify them and to restore lost genetic information, including the base excision repair (BER), nucleotide excision repair (NER) or mismatch repair (MMR) pathways. The BER pathway assures the cells protection from the deamination, alkylation and hydrolysis of bases [START_REF] Huffman | DNA base damage recognition and removal: new twists and grooves[END_REF]. Briefly, in this pathway, damaged bases are recognized and removed by DNA glycosylases. The DNA backbone is then cut and the single-strand gap is replaced with nucleotide(s) (van der Veen & Tang, 2015). The NER pathway recognizes bulky adducts that distort the DNA helix [START_REF] Kisker | Prokaryotic nucleotide excision repair[END_REF]. There is another NER pathway, which is coupled to the transcription machinery, when the NER system recognizes RNA polymerases that are stopped at an abnormal base during transcription [START_REF] Deaconescu | Structural basis for bacterial transcriptioncoupled DNA repair[END_REF]. The MMR pathway is involved in repair of mismatched nucleotides and insertions/deletions, which occur during DNA replication or direct DNA damage [START_REF] Jiricny | Postreplicative mismatch repair[END_REF]. Repair of DSBs is mediated by the homologous recombination (HR) or non-homologous end-joining (NHEJ) systems [START_REF] Ayora | Double-strand break repair in bacteria: a view from Bacillus subtilis[END_REF]. All these systems are well described in E. coli cells. For more information about the DNA repair we encourage readers to refer to a recent and a very comprehensive review of van der Veen and Tang, 2015.

Detoxification

In order to copy the toxic NOx effects, bacteria have evolved a number of detoxification mechanisms based on NOx transformation, such as reduction, or oxygenation. In Pseudomonas spp., two major NO detoxification pathways were demonstrated. The first is the reduction of NO by nitric oxide reductase [START_REF] Arai | Role of nitric oxidedetoxifying enzymes in the virulence of Pseudomonas aeruginosa against the silkworm, Bombyx mori[END_REF][START_REF] Shiro | Structure and function of bacterial nitric oxide reductases: Nitric oxide reductase, anaerobic enzymes[END_REF]. NO2 reduction can be P a g e 96 | 270 performed with two purposes: nitrite/nitrate respiration (see part Nitrogen cycle and denitrifying bacteria), and the utilization of nitrite as a nitrogen source for growth (assimilation) (Figure 21). Since the NO2 and NO reductases involved in respiration were discussed above, here, we focus on nitrite reductase involved in the nitrite assimilation. In this assimilatory pathway, conducted by Nas enzymes (for nitrate assimilation), nitrate is reduced to nitrite, which is then reduced by assimilatory nitrite reductase (NirBD in Pseudomonas spp. species) to ammonia, which serves as a source of nitrogen for biosynthesis (Figure 21).

In nitrate-assimilating Pseudomonas strains, ammonium is generated in the cytoplasm by a NADH-dependent assimilatory nitrite reductase (NirBD) to detoxify the nitrite that accumulates in anaerobic nitrate-respiring cells and to regenerate NAD + [START_REF] Romeo | Transcriptional regulation of nitrate assimilation in Pseudomonas aeruginosa occurs via transcriptional antitermination within the nirBD-PA1779-cobA operon[END_REF].

NirBD contains a single siroheme, a [4Fe-4S] center, NADH-and flavin adenine dinucleotide (FAD)-binding domain [START_REF] Campbell | Functional domains of assimilatory nitrate reductases and nitrite reductases[END_REF][START_REF] Guerrero | The assimilatory nitrate-reducing system and its regulation[END_REF].

Figure 32. Model of NalA-and NasT/Sdependent expression of nitrate assimilation operon in P. aeruginosa PAO1

In absence of nitrate, the ratio of glutamate/2ketoglutarate decreases, which leads to the activation of NtrBC system and σ 54 dependent nalA promoter. Thus, NalA is synthesized and NasT is sequestrated by NasS. In the presence of nitrate, the NasS-mediated NasT sequestration is decreased, resulting in antitermination within the nalA leader and transcription of operon. Adapted from [START_REF] Romeo | Transcriptional regulation of nitrate assimilation in Pseudomonas aeruginosa occurs via transcriptional antitermination within the nirBD-PA1779-cobA operon[END_REF] The nirBD genes were found to encode the two subunits of nitrite reductase, large and small respectively and, as shown in Figure 32, are located in operon with nitrate reductase coding gene [START_REF] Romeo | Transcriptional regulation of nitrate assimilation in Pseudomonas aeruginosa occurs via transcriptional antitermination within the nirBD-PA1779-cobA operon[END_REF]. The expression of these genes is subject to dual regulation, mediated by the antiterminator protein NasT, activity of which is negatively controlled by nitrate sensitive regulator NasS (Figure 32) [START_REF] Lin | Nitrate assimilation by bacteria[END_REF][START_REF] Romeo | Transcriptional regulation of nitrate assimilation in Pseudomonas aeruginosa occurs via transcriptional antitermination within the nirBD-PA1779-cobA operon[END_REF]. In the absence of nitrate, transcription terminates in the operon leader nalA as NasS inactivates NasT. In contrast, binding of nitrate to NasS leads to antitermination within nalA, and consequently to nirBD expression.

Transcription of this operon is carried out by a σ 54 -dependent promoter and mediated by P a g e 97 | 270 activation of the two-component system NtrBC (Figure 32). The Nas pathway is not regulated by oxygen level, and could be expressed under anaerobic and aerobic conditions. In addition, it seems to be largely distributed among Pseudomonas spp. species, and was found in majority of sequenced strains, including several P. aeruginosa, P. fluorescens, P. putida, P. stutzeri and P. syringae strains (www. pseudomonas.com).

However, the most fully studied and understood NO detoxification pathway is based on the flavohemoglobin (FlavoHb) (Cruz- [START_REF] Cruz-Ramos | NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp[END_REF]. FlavoHb is a NO detoxifying protein distributed among a wide variety of bacteria [START_REF] Poole | Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12[END_REF]Poole & Hughes, 2000), including P. aeruginosa Fhp [START_REF] Arai | Transcriptional regulation of the flavohemoglobin gene for aerobic nitric oxide detoxification by the second nitric oxide-responsive regulator of Pseudomonas aeruginosa[END_REF]. Functional properties of this enzyme is dependent on the O2 level. Aerobically, FlavoHb detoxifies NO by acting as an NO dioxygenase (Gardner et al., 1998a) and affords inducible protection of aconitase activity (Gardner et al., 1998b) and respiration [START_REF] Stevanin | Flavohemoglobin Hmp affords inducible protection for Escherichia coli respiration, catalyzed by cytochromes bo′ or bd, from nitric oxide[END_REF]. Anaerobically, FlavoHb serves as a NO reductase, generating nitrous oxide [START_REF] Hausladen | Nitrosative stress: metabolic pathway involving the flavohemoglobin[END_REF]. The E. coli flavohaemoglobin (Hmp) was the first described microbial globin [START_REF] Wu | Microbial globins[END_REF]. This is a monomer with Cterminal domain that closely resembles to ferredoxin-nicotinamide adenine dinucleotide phosphate (NADP + ) reductase with FAD and NAD(P)H binding sites. In addition to its role in NO detoxification, FlavoHb is involved in bacterial protection against reactive oxygen species (Koskenkorva-Frank & Kallio, 2009). Furthermore, FlavoHb is involved in the repair of the polyunsaturated FAs in the cell membranes, which are damaged during oxidative/nitrosative stress (see part Membrane glycerophospholipids) (Bonamore et al., 2003b). In P. aeruginosa, transcription of the fhp gene is activated by FhpR, a NorR-type σ54-dependent transcriptional activator, located upstream of fhp. The studies of fhp promoter region demonstrated that the expression of fhp is independent on DNR or ANR. Thus, DNR activates the genes for denitrification, which utilizes NO as an electron acceptor under anaerobic conditions. FhpR activates the fhp transcription, which detoxifies NO under aerobic conditions [START_REF] Arai | Transcriptional regulation of the flavohemoglobin gene for aerobic nitric oxide detoxification by the second nitric oxide-responsive regulator of Pseudomonas aeruginosa[END_REF]. It addition, two another genes, asrA, and PA3697 coding for aminoglycoside-induced stress response ATP-dependent protease and hypothetical protein, were described as regulators of fhp transcription in P. aeruginosa. Interestingly, AsrA protein is involved in short-term protection of P. aeruginosa against high concentrations of tobramycin [START_REF] Kindrachuk | Involvement of an ATPdependent protease, PA0779/AsrA, in inducing heat shock in response to tobramycin in Pseudomonas aeruginosa[END_REF], suggesting that NO detoxification could modify P. aeruginosa antibiotic sensitivity.

NO release

Bacteria are able not only to transform NO through detoxification pathways, but also to release this toxic molecules from the cells. This release seems to be entrust to RND (Resistancenodulation-cell division) family efflux pumps, and particularly MexEF-OprN [START_REF] Poole | Stress responses as determinants of antimicrobial resistance in Pseudomonas aeruginosa: multidrug efflux and more[END_REF].

Like other tripartite RND family pumps, the MexEF-OprN efflux system consists of an inner P a g e 98 | 270 membrane drug-proton antiporter (MexF), an outer membrane channel-forming component (OprN), and a prismatic membrane fusion protein (MexE) (Figure 33) [START_REF] Alvarez-Ortega | RND multidrug efflux pumps: what are they good for?[END_REF]. In P. aeruginosa the mexEF-oprN is positively controlled by MexT, which, in turn, is probably negatively regulated by MexS [START_REF] Uwate | Two routes of MexS-MexTmediated regulation of MexEF-OprN and MexAB-OprM efflux pump expression in Pseudomonas aeruginosa[END_REF]. Moreover, that the mexEF-oprN multidrug efflux locus is induced by nitrosative stress [START_REF] Fetar | mexEF-oprN Multidrug efflux operon of Pseudomonas aeruginosa: regulation by the MexT activator in response to nitrosative stress and chloramphenicol[END_REF]. This induction is dependent on both the MexT transcriptional activator and the MexS repressor (Poole, 2005a), which together with mexEF-oprN are involved in response to NO detoxification.

Figure 33. Schematic illustration of the MexEF-OprN RND efflux pump

The MexEF-OprN system is formed by the inner membrane MexF protein, the outer membrane protein OprN and the membrane fusion protein MexE. The activity of the MexE RND protein is coupled to the proton gradient. This efflux pump can extrude different compounds, including antibiotics and NOx from the bacterial cytoplasm and the periplasm. Adapted from [START_REF] Alvarez-Ortega | RND multidrug efflux pumps: what are they good for?[END_REF] The primarily role of RDN pumps is an antibiotic resistance. Activation of MexEF-OprN gives rise to the bacterial resistance to, among others, chloramphenicol, fluoroquinolones, trimethoprim and triclosan [START_REF] Köhler | Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa[END_REF]. The activity of MexEF-OprN pump also influences the quorum sensing, and reduces virulence factors (biofilm formation, pyocyanin production) and swarming motility [START_REF] Lamarche | MexEF-OprN efflux pump exports the Pseudomonas quinolone signal (PQS) precursor HHQ (4-hydroxy-2-heptylquinoline)[END_REF]. As the overproduction of MexEF-OprN pump is involved in response to NO, it seems likely that NO could modify bacterial sensitivity to antimicrobial agents, targeted by these pump.

Metabolic reprogramming

As reported in the part Proteins, nitrosative stress causes the alterations in the cell respiration, altering the ATP production via oxidative phosphorylation (OP) [START_REF] Husain | Nitric oxide evokes an adaptive response to oxidative stress by arresting respiration[END_REF]. Recent investigations found, that to adapt to these aspects of RNS toxicity, bacteria of P. fluorescens group reprogram their metabolism [START_REF] Auger | The metabolic reprogramming evoked by nitrosative stress triggers the anaerobic utilization of citrate in Pseudomonas fluorescens[END_REF][START_REF] Auger | A novel ATPgenerating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation[END_REF]. To counteract the ineffectivity of aconitase and TCA cycle caused by NO donor, P. fluorescens was found to P a g e 99 | 270 use the citrate lyase (CiLy), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) to convert citrate into pyruvate and ATP (Figure 34).

Figure 34. Reprogramming of P. fluorescens metabolism in order to adapt to nitrosative stress

The RNS reactions with [Fe-S] clusters and metal centers of tricarboxylic acid (TCA) cycle and ETC alter the ATP production via oxidative phosphorylation pathway (red). A supercomplex involving citrate lyase (CiLy), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) (green) is assembled by the organism to effectively produce ATP, the phosphotransfer networks, including adenylate kinase (AK) and nucleoside diphophate kinase (NDPK), permit the storage of this crucial moiety. This supercomplex can be disassociated by dephosphorylation (gray). Adapted from [START_REF] Auger | A novel ATPgenerating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation[END_REF] Interestingly, these enzymes appear to be transiently assembled as a supercomplex to form a metabolon, which completly reprogramms the metabolic networks assuring a bacterial surviving in presence of RNS. In this way, the CiLy degrades citrate into oxaloacetate and acetate. The dicarboxylic acid delivers the two compounds, pyruvate and ATP, allowing the survival of P. fluorescens under nitrosative stress conditions. This machinestry is coupled to the increased activities of phosphotransfer enzymes like adenylate kinase (AK) and nucleoside diphophate kinase (NDPK) (Figure 34), which ensure the efficacy of this ATP-making machine [START_REF] Appanna | Fumarate metabolism and ATP production in Pseudomonas fluorescens exposed to nitrosative stress[END_REF][START_REF] Auger | A novel ATPgenerating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation[END_REF]. This elegant strategy allows bacteria to form ATP. Thus, metabolic modifications present the underlying force at the center of bacterial adaptation in order to generate and store ATP anaerobically when the electron transport chain is defective [START_REF] Auger | A novel ATPgenerating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation[END_REF].

Secondary NO sensors

The NO sensing in bacterial cell is a complex mechanism, involving a wide range of 'secondary' sensors. The discussed below systems, as the name of chapter indicates, have as principal function to sense another signal, but NO can modulate their activity and so, potentially influences processes mediated by these sensors. One of these mechanisms is based on the cellular redox signaling/regulation and mediated through proteins that have redox-sensitive cysteines [START_REF] Antelmann | Thiol-based redox switches and gene regulation[END_REF]. In fact, redox-regulated proteins typically contain one or several redox-sensing cysteines, which are in the free thiol state in the reducing environment of the cytosol. When the cell encounters RNS, the redox-sensitive cysteine(s) become(s) nytrosated (see part Reaction of RNS with thiols), which leads to a change in protein structure, often activating protein. Once the cell has overcome the redox stress, cellular systems, such as glutaredoxin or thioredoxin, reduce the redox-sensing cysteines and return the proteins activity back to 'normal' [START_REF] Brandes | Nitrosative stress treatment of E. coli targets distinct set of thiol-containing proteins[END_REF][START_REF] Lindemann | Redox proteomics uncovers peroxynitrite-sensitive proteins that help Escherichia coli to overcome nitrosative stress[END_REF].

The SoxRS provides one of the prototypical examples of this mechanism. In E. coli the SoxRS regulon protects bacteria against NO [START_REF] Ding | Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator[END_REF]. As previously described in part RNS complexes with metal centers, the sensor of this regulon is SoxR protein, activated by NO (Figure 28). Its target soxS, in turn, increases the transcription of several genes, including superoxide dismutase, sodA, glucose-6-phosphate dehydrogenase, zwf, two flavodoxins fdlA and fdlB and fur regulator, involved in iron metabolism. The sodA gene allows the removing O2 •-that may react with NO to form NO2 and ONOO - [START_REF] Feld | Bactericidal antibiotics do not appear to cause oxidative stress in Listeria monocytogenes[END_REF]. The zwf is involved in the repair of [Fe-S] clusters [START_REF] Giró | Glucose-6-phosphate dehydrogenase and ferredoxin-NADP(H) reductase contribute to damage repair during the soxRS response of Escherichia coli[END_REF]. SoxRS activation protects E. coli from macrophage killing [START_REF] Nunoshiba | Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages[END_REF]. However, SoxRS is still a minor player in the physiological response to the RNS toxicity. SoxR orthologues are present in many bacteria, including P. aeruginosa. However, its target, SoxS, was not found in P. aeruginosa [START_REF] Kobayashi | Activation of SoxR-dependent transcription in Pseudomonas aeruginosa[END_REF]. Thus, in this bacterium, SoxR is a functional redox-responsive activator, which is probably directly linked to O2 •-or NO. Its activity is required for P. aeruginosa virulence and antibiotic resistance [START_REF] Palma | Pseudomonas aeruginosa SoxR does not conform to the archetypal paradigm for SoxRdependent regulation of the bacterial oxidative stress adaptive response[END_REF].

The transcription factor OxyR is a major H2O2 transcriptional activator. H2O2 is a toxic for bacteria oxidative species, produced by plants [START_REF] Ohwada | Susceptibility to hydrogen peroxide and catalase activity of root nodule bacteria[END_REF] and macrophages [START_REF] Loegering | Macrophage hydrogen peroxide production and phagocytic function are decreased following phagocytosis mediated by Fc receptors but not complement receptors[END_REF], during hostpathogen interactions. Like SoxR, OxyR is activated by oxidation under aerobic conditions, and also by S-nitrosylation under anaerobic conditions. Activated by H2O2, OxyR induces the gene expression in a distinct regulon through alternative post-translational modifications. However, the S-nitrosylated OxyR induces the gene expression in other regulon, whose the components protect bacteria against nitrosative stress at least by limiting the S-nitrosylation [START_REF] Seth | Endogenous protein S-nitrosylation in E. coli: regulation by OxyR[END_REF].

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Catalase is an enzyme involved in bacterial response to oxidative stress, and able to catalyze the decomposition of H2O2 to O2 and H2O [START_REF] Chelikani | Diversity of structures and properties among catalases[END_REF]. P. aeruginosa defense against this oxidizing biocide is mediated by three catalases: KatA, KatB and KatC (Elkins et al., 1999). The transcription of katA is regulated, in part, by the intercellular signaling system called quorum sensing [START_REF] Hassett | Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide[END_REF], OxyR [START_REF] Heo | The major catalase gene (katA) of Pseudomonas aeruginosa PA14 is under both positive and negative control of the global transactivator OxyR in response to hydrogen peroxide[END_REF], IscR (an [Fe-S] cluster assembly regulator) [START_REF] Kim | IscR modulates catalase A (KatA) activity, peroxide resistance and full virulence of Pseudomonas aeruginosa PA14[END_REF] and ANR [START_REF] Trunk | Anaerobic adaptation in Pseudomonas aeruginosa: definition of the Anr and Dnr regulons[END_REF]. The high level of katA expression was detected in anaerobic P. aeruginosa cultures, indicating that KatA possesses functions beyond the removal of H2O2. KatA may directly interact with NO to protect bacterial cells against NO toxicity by buffering of free NO when potentially toxic concentrations of NO are approached [START_REF] Su | Catalase (KatA) plays a role in protection against anaerobic nitric oxide in Pseudomonas aeruginosa[END_REF] (Figure 35). Ferric uptake protein (Fur) is a global regulator, conserved in Gram-negative bacteria. It acts as a transcriptional repressor when it binds ferrous ion [START_REF] Cornelis | Iron uptake regulation in Pseudomonas aeruginosa[END_REF]. This dimeric protein is activated by the Fe 2+ ion. The active form is able to bind to specific DNA sequences, located in the promoter region of genes involved in iron acquisition [START_REF] Ochsner | Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters[END_REF]. This binding leads to the repression of the downstream genes. In contrast, when iron concentration is not sufficient, the active Fur releases the Fe 2+ irons. In this case, Fur is not able to bind specific DNA sequences and iron acquisition is then stimulated [START_REF] D'autréaux | Spectroscopic description of the two nitrosyl-iron complexes responsible for fur inhibition by nitric oxide[END_REF]. The iron centers of Fur can react with NO molecules to form iron-nitrosyl complex, unable to bind DNA, causing a derepression of Fur-regulated promoters [START_REF] D'autréaux | Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron[END_REF]. It is particularly interesting, because the functions of Fur are not only limited to iron homeostasis. A wide variety of genes involved in various mechanisms, such as manganese superoxide dismutase or catalase, are under Fur control [START_REF] Hassett | Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophoremediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities[END_REF]. The Fur-mediated bacterial response to NO seems to be dependent on experimental conditions (i.e. growing medium) [START_REF] Flatley | Transcriptional responses of Escherichia coli to S-nitrosoglutathione under defined chemostat conditions reveal major changes in methionine biosynthesis[END_REF]. Fur was found to be insensitive to NO in vivo in minimal medium, and, in contrast, was inactivated in rich medium. The suggested explanation for this paradox is therefore that the rich medium is P a g e 102 | 270 sufficiently poor in iron to reveal Fur-mediated, NO-sensitive repression of genes involved in iron acquisition [START_REF] Flatley | Transcriptional responses of Escherichia coli to S-nitrosoglutathione under defined chemostat conditions reveal major changes in methionine biosynthesis[END_REF][START_REF] Spiro | Regulators of bacterial responses to nitric oxide[END_REF].

In response to NO, bacteria product the enzymes, which reduce or oxidize this molecule to decrease its toxic effect, and the regulatory proteins that respond to NO [START_REF] Spiro | Regulators of bacterial responses to nitric oxide[END_REF]. As NO is formed in bacterial hosts, the pathogens require mechanisms to detect and detoxify this molecule [START_REF] Stevanin | Nitric oxide detoxification systems enhance survival of Neisseria meningitidis in human macrophages and in nasopharyngeal mucosa[END_REF]. Metabolism of NO is important for virulence of P. aeruginosa [START_REF] Arai | Role of nitric oxidedetoxifying enzymes in the virulence of Pseudomonas aeruginosa against the silkworm, Bombyx mori[END_REF]. Anaerobic biofilms of P. aeruginosa actively denitrify, and NO reduction is required for biofilm viability [START_REF] Yoon | Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis[END_REF]. Thus, it is not surprising that bacteria possess numerous and diverse NO-mediated regulatory networks. So, in bacteria, NO is a signal molecule, which regulates several important processes. This NO-mediated regulation is discussed in the next part of this work.

NO-mediated regulation in bacteria

[Fe -S] cluster repair

As previously discussed in this study, NO is involved in the redox regulation within bacterial cells (see part 4.6. Secondary NO sensors). Since the protein dinitrosyl iron complexes are stable in vivo, they are not degraded, but efficiently repaired in bacterial cells [START_REF] Ding | Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator[END_REF][START_REF] Rogers | Cysteine-mediated destabilization of dinitrosyl iron complexes in proteins[END_REF]. Ferredoxin dinitrosyl iron complex may be converted to the ferredoxin [2Fe-2S] cluster thanks to ISC (iron-sulfur cluster) system [START_REF] Yang | Repair of nitric oxide-modified ferredoxin [2Fe-2S] cluster by cysteine desulfurase (IscS)[END_REF]. The latter consists of cysteine desulfurase IscS, which provides the atomic sulfur during cluster assembly. The IscU protein provides scaffold for IscS-mediated assembly. IscA is an assembly protein, involved in the delivery of irons to the sites of assembly. IscR plays a role of their regulator, that senses the demand for [Fe-S] cluster formation/repair [START_REF] Rogers | Cysteine-mediated destabilization of dinitrosyl iron complexes in proteins[END_REF][START_REF] Schwartz | IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins[END_REF]. These proteins, in conjunction with chaperons hscAB and ferredoxin fdx, are involved in both repair of [Fe -S] cluster and its de novo assembly [START_REF] Djaman | Repair of oxidized iron-sulfur clusters in Escherichia coli[END_REF].

In fact, IscS is able to directly convert the ferredoxin dinitrosyl iron complex to the [2Fe-2S] cluster in the presence of L-cysteine. All these proteins are encoded by iscSUA-hscBA-fdx operon in E. coli [START_REF] Rogers | Cysteine-mediated destabilization of dinitrosyl iron complexes in proteins[END_REF]. Furthermore, only IscS protein was described as mediated the repair process [START_REF] Djaman | Repair of oxidized iron-sulfur clusters in Escherichia coli[END_REF]. ISC system is well distributed among bacteria [START_REF] Jones-Carson | Inactivation of [Fe-S] metalloproteins mediates nitric oxide-dependent killing of Burkholderia mallei[END_REF], including Pseudomonas spp. strains [START_REF] Choi | Identification of Pseudomonas aeruginosa genes crucial for hydrogen peroxide resistance[END_REF]. In P. aeruginosa ISC system is encoded by yfhJ-fdx2-hscAB-iscAUSR operon (www.pseudomonas.com). In addition, the ISC system is involved in many physiological pathways, such as iron homeostasis or resistance to oxidative stress [START_REF] Romsang | The iron-sulphur cluster biosynthesis regulator IscR contributes to iron homeostasis and resistance to oxidants in Pseudomonas aeruginosa[END_REF].

P a g e 103 | 270

Other proteins are implicated in [Fe-S] cluster repair. The YtfE of E. coli, ScdA of S. aureus and NorA of Ralstonia eutropha are homologous to Dnr of P. aeruginosa. These proteins are important for the repair process of [Fe-S] clusters of aconitase and furmatase, damaged by nitrosative stress [START_REF] Justino | Escherichia coli di-iron YtfE protein is necessary for the repair of stress-damaged iron-sulfur clusters[END_REF][START_REF] Overton | Widespread distribution in pathogenic bacteria of di-iron proteins that repair oxidative and nitrosative damage to iron-sulfur centers[END_REF][START_REF] Strube | Formation of a dinitrosyl iron complex by NorA, a nitric oxide-binding di-iron protein from Ralstonia eutropha H16[END_REF]. A ytfE deletion mutant grows poorly under anaerobic conditions, has increased sensibility to iron starvation, and decreased activity of [Fe-S] cluster containing enzymes [START_REF] Justino | Escherichia coli di-iron YtfE protein is necessary for the repair of stress-damaged iron-sulfur clusters[END_REF].

Biofilm and motility

Bacteria employ NO in non-lethal concentrations as signaling agent to control biofilm formation/dispersion and motility. NO-mediated biofilm dispersion was observed in a wide range of mono-or multispecies bacterial biofilms. Addition of NO donors was shown to induce the biofilm dispersion in E. coli, Vibrio cholera, Bacillus licheniformis, and Serratia marcescens (Barraud et al., 2009a), Neisseria gonorrhoeae [START_REF] Potter | Thioredoxin reductase is essential for protection of Neisseria gonorrhoeae against killing by nitric oxide and for bacterial growth during interaction with cervical epithelial cells[END_REF], Staphylococcus aureus [START_REF] Davidson | NO means 'yes' in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association[END_REF]. In B. subtilis, inhibition of endogenous NO production leads to the decrease of biofilm biomass [START_REF] Schreiber | The role of nitric-oxide-synthasederived nitric oxide in multicellular traits of Bacillus subtilis 3610: biofilm formation, swarming, and dispersal[END_REF]. Addition of NO donors induces biofilm dispersion in drinking water and recycled-water systems (Barraud et al., 2009a). In Pseudomonas spp. species, NO also causes biofilm dispersion. In fact, addition of low NO doses (25 to 500 nM) promotes dispersion of P. aeruginosa biofilm. P. aeruginosa mutant, lacking NO reductase exhibits greatly enhanced biofilm dispersion [START_REF] Barraud | Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa[END_REF]. In P. putida, heterologous expression of NOS genes is involved in increase of motility and biofilm dispersion (Liu et al., 2012b). In Pseudomonas spp., NO stimulates PDE activity, which decreases the c-di-GMP level interlocking the biofilm dispersion (Barraud et al., 2009b).

Several P. aeruginosa PDE are involved in NO-dependent biofilm dispersion, including DipA, RbdA and NdbA [START_REF] Li | NO-induced biofilm dispersion in Pseudomonas aeruginosa is mediated by an MHYT domain-coupled phosphodiesterase[END_REF][START_REF] Roy | The phosphodiesterase DipA (PA5017) is essential for Pseudomonas aeruginosa biofilm dispersion[END_REF]. In addition, the chemotaxis transducer BdlA is essential for NO-mediated biofilm dispersion (Morgan et al., 2006). However, none of these proteins are specific to NO. In fact, without NO addition, nbdA is only weakly expressed and does not affect the c-di-GMP level (Figure 36).

Upon NO exposure, transcription of nbdA is significantly induced, leading to enhanced PDE activity and biofilm dispersion. The MHYT domain of NbdA serves as a copper-binding gas sensor is responsible to the NO-mediated biofilm regulation [START_REF] Hay | MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa[END_REF][START_REF] Li | NO-induced biofilm dispersion in Pseudomonas aeruginosa is mediated by an MHYT domain-coupled phosphodiesterase[END_REF].

The RbdA and DipA are required to biofilm dispersion in response to NO. These PDEs directly interact with BdlA, regulating the c-di-GMP level in order to enable the process of dispersion (Petrova & Sauer, 2012a). Recent evidence also suggests a feedback regulation between c-di-GMP and BdlA. While BdlA indirectly regulates c-di-GMP level via PDE activity, activation of BdlA is, in turn, dependent on c-di-GMP abundance (Petrova & Sauer, 2012b). In addition, the c-di-GMP-synthesizing DGC GcbA is required for biofilm dispersion by regulating the posttranscriptional processing of BdlA [START_REF] Petrova | The Pseudomonas aeruginosa diguanylate cyclase GcbA, a homolog of P. fluorescens GcbA, promotes initial attachment to surfaces, but not biofilm formation, via regulation of motility[END_REF]. In fact, GcbA contributes to the cleavage of BdlA in P. aeruginosa, allowing an initial cell attachment to the surface. So, GcbA indirectly plays a surprising for DGC role in biofilm dispersion (Figure 37). The both GcbA and BdlA are dependent on the c-di-GMP level and are inversely regulated during the sessilemotile and motile-sessile transitions. In eukaryotes, the major receptor for NO is the GCyc, which has both haem and nonhaem binding site for NO [START_REF] Cary | Nitric oxide signaling: no longer simply on or off[END_REF]. Similar domains are encoded in bacteria and revealed a NO receptor family, named Heme-Nitric Oxide/Oxygen binding (H-NOX) proteins [START_REF] Iyer | Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins[END_REF]. The H-NOX genes are found in operons with diverse bacterial signaling genes (Plate & Marletta, 2013a), the majority of which can be divided in two classes: (i) containing histidine kinase (HK) and (ii) DGCs and PDE.

As shown in Figure 38B, H-NOX proteins control the HK activity in Shewanella oneidensis and Vibrio cholerae [START_REF] Arora | Nitric oxide regulated two-component signaling in Pseudoalteromonas atlantica[END_REF][START_REF] Henares | Discovery of a nitric oxide responsive quorum sensing circuit in Vibrio harveyi[END_REF][START_REF] Price | Shewanella oneidensis MR-1 H-NOX regulation of a histidine kinase by nitric oxide[END_REF]. The NObound HnoX state inhibits associated HK (HnoK) autophosphorylation. HnoK, in turn, targets three different RR: HnoB, HnoC and HnoD. HnoB contains a PDE effector domain and its phosphorylation by HnoK stimulates hydrolysis of c-di-GMP [START_REF] Plate | Nitric oxide modulates bacterial biofilm formation through a multicomponent cyclic-di-GMP signaling network[END_REF]. HnoD functions as an inhibitor of HnoB. HnoC mediates transcriptional control for all of the signaling compounds of this network to regulate the dynamics of the NO response (Plate & Marletta, 2013b). All together, these proteins increase c-di-GMP levels in response to NO that leads to increased biofilm formation (Figure 38B). As shown in Figure 38A, in Shewanella woodyi H-NOX protein plays an opposite role. This protein contains both PDE and DGC domains, capable of the synthesis and degradation of cdi-GMP [START_REF] Liu | Characterization of a diguanylate cyclase from Shewanella woodyi with cyclase and phosphodiesterase activities[END_REF]. Without NO, H-NOX activates DGC, leading to 'normal' biofilm formation. NO-bound H-NOX increases PDE activity, allowing the decrease of intracellular cdi-GMP level and decrease of biofilm formation (Liu et al., 2012a). Sequences with 15-40% identity to the GCyc H-NOX were identified in many bacteria, nonetheless, certain bacterial genus, such as Bacillus or Pseudomonas, lack H-NOX containing genes [START_REF] Iyer | Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins[END_REF][START_REF] Karow | Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis[END_REF][START_REF] Karow | Characterization of functional heme domains from soluble guanylate cyclase[END_REF].

Overall, NO-mediated regulation of biofilm formation and motility has emerged as a common theme, regulated by c-di-GMP level [START_REF] D'argenio | Cyclic di-GMP as a bacterial second messenger[END_REF]. This process can be carried out through either direct control of DGC and PDE activity or through more sophisticated multicomponent systems, based on H-NOX NO-mediated activation. The simpler direct system seems to cause dispersion of biofilm in response to NO, while the more sophisticated systems seem to react in opposite way, increasing the biofilm formation.

It should be noted, that NO-mediated effect on bacterial biofilm is described as to be concentration-dependent. At low NO concentrations, in the micromolar and nanomolar ranges, a decrease in biofilm biomass is observed. In contrast, at high (millimolar) concentrations, NO causes an increase in biofilm biomass and a decrease in planktonic biomass, related to a rapid P a g e 107 | 270 conversion of NO to NO3 -and enhances anaerobic metabolism [START_REF] Barnes | Optimal dosing regimen of nitric oxide donor compounds for the reduction of Pseudomonas aeruginosa biofilm and isolates from wastewater membranes[END_REF][START_REF] Barraud | Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa[END_REF].

Unfortunately, to the best of our knowledge, the studies of effect of RNS on bacterial biofilm and/or motility are practically non-existent, only nitrate sensing and metabolism were a subject of study [START_REF] Van Alst | Nitrate sensing and metabolism modulate motility, biofilm formation, and virulence in Pseudomonas aeruginosa[END_REF]. Analyze of narXL and NAR genes mutants shows altered swimming and swarming motility. No modification is noted in biofilm biomass, but in biofilm architecture compared to the wild-type P. aeruginosa. As authors proposed, these biofilm alterations are due to the inefficient motility and biosurfactant production. However, a NAR mutant, as well as a NAR/NAP double mutant had no defect in swimming, but in swarming motility and in biofilm formation. The deletion of P. aeruginosa nirS gene demonstrates the decrease of swarming, but not swimming motility. The norC mutant does not show any motility modification [START_REF] Van Alst | Nitrate sensing and metabolism modulate motility, biofilm formation, and virulence in Pseudomonas aeruginosa[END_REF]. This study suggests a possible link between nitrate sensing, metabolism and biofilm formation/motility in P. aeruginosa. However, future investigation is necessary to rule out the mechanism of these interactions.

Antibiotic resistance

Several studies of NO-mediated resistance to antibiotics were performed [START_REF] Gusarov | Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics[END_REF][START_REF] Mccollister | Nitric oxide protects bacteria from aminoglycosides by blocking the energydependent phases of drug uptake[END_REF][START_REF] Van Sorge | Methicillin-resistant Staphylococcus aureus bacterial nitric oxide synthase affects antibiotic sensitivity and skin abscess development[END_REF]. Gusarov and coworkers [START_REF] Gusarov | Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics[END_REF] investigated the role of NOS genes (bNOS) of B. subtilis and S. aureus in resistance to antimicrobial agents. Thus, the effect of a wide range of antibiotics on wild type and nos mutated strains was compared. The bNOS protects Gram-positive bacteria against antimicrobial agents, through a direct reaction of NO with antimicrobials, making the latter less toxic, and against oxidative stress, caused by these antimicrobials. It is remarkable, that the pretreatment of Δnos strains with exogenous NO also temporarily protects cell against antibiotic toxicity [START_REF] Gusarov | Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics[END_REF]. McCollister et al. investigated the mechanism, by which NO induces resistance of Gram-positive and Gram-negative bacteria to aminoglycosides [START_REF] Mccollister | Nitric oxide protects bacteria from aminoglycosides by blocking the energydependent phases of drug uptake[END_REF]. Aminoglycosides are often used in the treatment of infections caused by Gram-positive [START_REF] Bayer | Initial low-dose aminoglycosides in Staphylococcus aureus bacteremia: good science, urban legend, or just plain toxic?[END_REF] or Gram-negative [START_REF] Poole | Aminoglycoside resistance in Pseudomonas aeruginosa[END_REF] bacteria. These antibiotics are a vital component of antipseudomonal chemotherapy [START_REF] Cheer | Inhaled tobramycin (TOBI): a review of its use in the management of Pseudomonas aeruginosa infections in patients with cystic fibrosis[END_REF]. Aminoglycosides cause bacterial cell death targeting the 30S subunit of ribosomes, and the protein synthesis (Morita et al., 2014). Given the intracellular localization of ribosomal targets, aminoglycosides must penetrate bacterial membrane thanks to electrochemical gradient and electron flow of the respiratory chain [START_REF] Taber | Bacterial uptake of aminoglycoside antibiotics[END_REF]. As NO represses bacterial respiration (see part Proteins), the authors proposed, that this repression of respiration prevents drug uptake, protecting bacteria from the aminoglycoside activity [START_REF] Mccollister | Nitric oxide protects bacteria from aminoglycosides by blocking the energydependent phases of drug uptake[END_REF]. Surprisingly, van Sorge and colleagues (van Sorge et al., 2013) demonstrated, that, bNOS-deficient S. aureus gains P a g e 108 | 270 resistance to aminoglycosides. These results are in opposition to previous studies, suggesting that the role of NO in bacterial aminoglycoside resistance still a mysterious and complex question. All together these data indicate, that both endogenous and exogenous NO seem to affect bacterial antibiotic sensitivity, but the mechanism of NO-dependent modifications of antibiotic resistance remains to be defined.

Abiotic RNS and air pollution

NOx as a major air pollutant

The air pollution is one of the major problems especially in urban areas, because of the urban population growth, combined with development of industrial areas, where the airborne pollutant are concentrated [START_REF] Chaloulakou | Compliance with the annual NO2 air quality standard in Athens. Required NOx levels and expected health implications[END_REF][START_REF] Mayer | Air pollution in cities[END_REF]. The notion of 'air pollutant' includes all substances, which can damage human health, animals, vegetation and also materials [START_REF] Skalska | Trends in NOx abatement: A review[END_REF]. Air pollution exists as a complex mixture, whose the effects in majority are attributed to NOx, sulfur dioxide and particulate matter (PM). As reported by World Health Organization [START_REF] Harrison | Sources of air pollution[END_REF] and shown in Figure 39, the major atmospheric source of NOx is anthropogenic, including principally the combustion processes in stationary or mobile sources, such as emissions from diesel vehicles and fossil fuels [START_REF] Brunekreef | Air pollution and health[END_REF][START_REF] Mayer | Air pollution in cities[END_REF].

Figure 39. NOx sources in atmosphere

According [START_REF] Harrison | Sources of air pollution[END_REF] However, the atmospheric NOx can be naturally produced by lightning [START_REF] Schumann | The global lightning-induced nitrogen oxides source[END_REF], and volcano eruption (B. [START_REF] Huebert | Volcano fixes nitrogen into plant-available forms[END_REF], considered as a natural sources for NOx production [START_REF] Boichu | On bromine, nitrogen oxides and ozone depletion in the tropospheric plume of Erebus volcano (Antarctica)[END_REF].

The chemical, biological [START_REF] Ghenu | Dispersion of pollutants and sstimation of emissions in a street canyon in Rouen, France[END_REF][START_REF] Ramaroson | Field measurements of airport emissions and air pollutants including aerosols at Paris CDG airports. The AIRPUR project[END_REF] and toxicological [START_REF] Fall | Réponse toxicologique d'une culture organotypique de tissu pulmonaire exposée à des gazes d'échappement issus d'un moteur à combustion[END_REF] studies of these air pollutants were made in order to estimate their effect on human P a g e 109 | 270 health and environment and develop the new technologies of air decontamination. As described in part 1. Nitrogen oxides (NOx), these nitrogen derivatives cause several environmental problems, including photochemical smog, acid rain, ozone layer depletion, and, as consequence, global warming. According WHO [START_REF] Sivertsen | Global ambient air pollution concentrations and trends[END_REF], this pollution effect could be explained by: (i) the capacity of NOx to absorb visible solar radiation and their contribution to impaired atmospheric visibility; (ii) the NOx ability to absorb visible radiation and a direct role in global climate change; (iii) their regulation of the oxidizing capacity of the atmosphere by controlling the radical species level, including hydroxyl radicals; and (iv) the role of NOx in determining of ozone concentrations. However, NOx, especially NO2, are known for their toxicity to human health, as reported below.

NOx effect on human health

As humans, we are always in contact with air all around us. Due to the low NOx solubility in aqueous solutions, the major pathway of NOx action in humans is pulmonary (inhalation). The effects of exposure to urban air pollution on health are now well established. Several studies performed during the 1990s, demonstrated, that the traffic related NOx emission enhances the risk of respiratory symptoms and allergic diseases especially in children [START_REF] Ciccone | Road traffic and adverse respiratory effects in children. SIDRIA Collaborative Group[END_REF][START_REF] Duhme | The association between self-reported symptoms of asthma and allergic rhinitis and self-reported traffic density on street of residence in adolescents[END_REF][START_REF] Duhme | Asthma and allergies among children in West and East Germany: a comparison between Munster and Greifswald using the ISAAC phase I protocol. International Study of Asthma and Allergies in Childhood[END_REF][START_REF] Van Vliet | Motor vehicle exhaust and chronic respiratory symptoms in Vromman[END_REF]. For example, traffic-related air pollution is correlated with symptoms of cough during the first 2 years of life [START_REF] Gehring | Trafficrelated air pollution and respiratory health during the first 2 yrs of life[END_REF].

Exposure to combustion products, containing NOx, is related to the development of wheezing bronchitis in children [START_REF] Pershagen | Air pollution involving VII. References P a g e 233 | 270 nitrogen dioxide exposure and wheezing bronchitis in children[END_REF]. In animal experiment, NO2 alters lung barrier function, suggesting the possible NO2 damage of alveolar epithelium [START_REF] Mcelroy | Biochemical detection of type I cell damage after nitrogen dioxideinduced lung injury in rats[END_REF].

Unfortunately, these data were confirmed by recent researches, which highlight the effect of air pollutants on cardiovascular disease [START_REF] Brook | Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association[END_REF][START_REF] Declercq | Impact sanitaire de la pollution atmosphérique dans neuf villes françaises[END_REF]. Others identified a possible link between the air pollution and life expectancy [START_REF] Pope | Fine-particulate air pollution and life expectancy in the United States[END_REF]. The low air pollution increases the life expectancy in European cities, and save about €7.8 billion per year [START_REF] Declercq | Impact sanitaire de la pollution atmosphérique dans neuf villes françaises[END_REF]. According a similar calculation mode, the French citizens could gain about 6 month of life expectancy thanks to the 'good' air quality. However, this simple comparison is not very convincing, because of a myriad of factors, except air quality, that must impact the life expectancy. Nonetheless, these data aim to highlight the impact of air pollution on the human health and the importance of studying this topic.

The effects of air pollutants on human health are dependent on pollutant concentration and on time of exposure, demonstrating short-and long-term effects. Thus, individuals exposed for 2 hours to 1 ppm of NO show a significant decrease of specific airway conductance, without any clinical sign [START_REF] Kagawa | Respiratory effects of 2-hr exposure to 1.0 ppm nitric oxide in normal subjects[END_REF]. The short-term exposure at low NO2 concentrations was found to slightly affects human bronchial responsiveness in subjects with mild asthma (Bylin et al., P a g e 110 | 270 1988). With rise of concentration, NO2 induces a neutrophilic inflammation in the airways that was detectable in bronchial washing at 6 h after NO2 exposure. The increase in neutrophils could be related to the enhanced IL-8 secretion observed at 1.5 h after exposure [START_REF] Blomberg | The inflammatory effects of 2 ppm NO2 on the airways of healthy subjects[END_REF]. The French Environment and Energy Management Agency (ADEME) reports, that the long-term exposure to air pollutants may increase the risk of respiratory allergy, inflammatory response of blood vessels, affect the cardiac function and cause the cardiovascular diseases especially in children and elderly persons. The short-term exposures cause the discomfort, eye stinging effects, irritation to eyes, nose and throat. However, the more important effects, like nausea, cough, respiratory disorders or worsening of asthma symptom, are also observed (ADEME, 2015). critic point of this decreasing. As previously reported (see part 1. Nitrogen oxides (NOx)), in atmosphere, NO is quickly transformed to NO2. Consequently, this reaction is regarded as the most important route for atmospheric NO2 production. Given that NO2 is much more toxic than NO, the ratio of NO2/NOx in air must to be taken into consideration. However, the main feature of recently obtained results is the elevation of the NO2/NOx ratio, indicating the drastic increase of NO2 concentration. The increase of NO2 concentration in road tunnel was found to be based on the post-processing techniques of the exhaust gas from road vehicles [START_REF] Bernagaud | Observations du ratio NO2/NOx en tunnel routier[END_REF]. The study of diesel emission toxicological impacts shows that increased NO2 levels correlates with the production of a systemic oxidant stress in rats lung tissue (Morin et al., 2013a).

NOx level in atmosphere

According WHO, the rural annual concentrations of NO2 in industrialized countries were measured at around 15-30 μg/m 3 . In urban areas, the annual NO2 concentrations exceed 40 μg/m 3 [START_REF] Sivertsen | Global ambient air pollution concentrations and trends[END_REF]. However, the NO2 concentrations may vary considerably within cities and from time to time during the day and night. In 2002, the annual average NO2 concentrations in France was reported to 44 μg/m 3 , although the annual NO2 level in Paris was found to be higher, and reported to 57 μg/m 3 . Hourly average values of NO2 near very busy roads can exceed 940 μg/m 3 (0.5 ppm). The personal exposure in vehicles in tunnels can be very high, a range of 179-688 μg/m 3 of NO2 inside a car in a road tunnel during rush hours was established [START_REF] Svartengren | Short-term exposure to air pollution in a road tunnel enhances the asthmatic response to allergen[END_REF].

During last decades, the rising restrictions regarding NOx emission were imposed. The ratification of the Geneva Convention, Kyoto and Gothenburg Protocols require the 'Parties' to control the emission of NOx, recognizing the harmful effect of these pollutants [START_REF] Reilly | Multi-gas assessment of the Kyoto Protocol[END_REF][START_REF] Unece | Protocol concerning the Control of Emissions of Nitrogen Oxides[END_REF]. According the National competence center for Industrial Safety and Environmental Protection (INERIS), NOx are classed as dangerous chemicals and their exposure thresholds were established (INERIS, 2011a, b) and reported in Table 3. The 'lethal effect threshold' corresponds to the NO/NO2 concentrations, above which mortality can be observed in the exposed population. The 'irreversible and reversible effect thresholds' correspond to the NO/NO2 concentrations, above which the irreversible and reversible effects P a g e 112 | 270 can be observed in the exposed population. On this basis, European Environmental commission and French government developed the NOx standards ranking from hourly NOx limits to industrial NOx standards ('WHO | Ambient (outdoor) air quality and health', 2015).

NOx standards

The annual and 1-hour means of NO2 were set to 40 μg/m 3 and 200 μg/m 3 respectively (Table 4). Annual NO2 threshold is calculated as average value of daily NO2 concentrations. According 'WHO | Ambient (outdoor) air quality and health', 2015

In order to limit pollution caused by road vehicles, European Union introduced the stricter limits on NO2 for emissions from motor vehicles and their specific replacement parts (Euro 5 and Euro 6 standards) ('Reduction of pollutant emissions from light vehicles', 2015). The standards for pollutant emissions from road vehicles are set separately for light-duty (cars and light vans)

and for heavy-duty (trucks and buses) vehicles. For light-duty vehicles, the maximal NOx emissions from diesel is set to 180 mg/km; the NOx emissions from petrol vehicles or those running on natural gas is 60 mg/km.

In conclusion, it should be mentioned, that NOx, especially NO2, present a toxic atmospheric pollutant, which has a negative effect on human health, but also on environment. The latter, in turn, includes, among others, the microorganisms.

Microorganisms in atmosphere

The air is a biotic environment, which contains the myriads of primary biological aerosol particles (PBAP). The latter are defined as solid airborne particles derived from biological organisms, including microorganisms and fragments of biological materials, such as plant debris and animal dander [START_REF] Després | Primary biological aerosol particles in the atmosphere: a review[END_REF]. To gain a better understanding of their composition, concentration, seasonal fluctuation and regional diversity, several methods were developed and/or adapted [START_REF] Georgakopoulos | Microbiology and atmospheric processes: biological, physical and chemical characterization of aerosol particles[END_REF] ranking from cell culture, immunological detection, microscopy, flow cytometry, to methods of molecular biology. In this way, bacteria, archaea and fungi are known as the major compounds of PBAP. With regard to airborne fungi, Fröhlich-Nowoisky team detected Basidiomycota (club fungi, 64%) as major phylum in a semi-urban environment in central Europe [START_REF] Fröhlich-Nowoisky | High diversity of fungi in air particulate matter[END_REF].

However, the preponderance of Ascomycota (sac fungi, 80-92%) was noted at a mountain site P a g e 113 | 270 in North America [START_REF] Bowers | Characterization of airborne microbial communities at a high-elevation site and their potential to act as atmospheric ice nuclei[END_REF], indicating the regional differences and possible seasonal variations in airborne fungal distribution [START_REF] Fröhlich-Nowoisky | Biogeography in the air: fungal diversity over land and oceans[END_REF]. The sequencing of 16S rRNA and amoA genes in samples of air particulate matter allows to identify the airborne archaea as members of phylums Thaumarchaeota and Euryarchaeota [START_REF] Fröhlich-Nowoisky | Diversity and seasonal dynamics of airborne archaea[END_REF]. Bacteria are one of the major compounds of PBAP (Burrows et al., 2009a;[START_REF] Després | Primary biological aerosol particles in the atmosphere: a review[END_REF]. Mean bacterial concentrations in ambient air can be greater than 1  10 4 cells m -3 [START_REF] Bauer | The contribution of bacteria and fungal spores to the organic carbon content of cloud water, precipitation and aerosols[END_REF][START_REF] Burrows | Bacteria in the global atmosphere -Part 2: Modeling of emissions and transport between different ecosystems[END_REF]Morin et al., 2013b). Due to their size, bacteria have a long atmospheric residence time and can be transported by wind over long distances [START_REF] Womack | Biodiversity and biogeography of the atmosphere[END_REF]. Although a wide range of research on bacteria in the atmosphere, it remains difficult to establish a clear picture of the actual composition of bacteria in the air. The bacterial composition in the atmosphere is strongly dependent on many factors, such as seasonality, climate and meteorological factors, or anthropogenic influence [START_REF] Després | Primary biological aerosol particles in the atmosphere: a review[END_REF]. Thus, bacterial diversity in rural areas is generally higher than at urban sites [START_REF] Després | Characterization of primary biogenic aerosol particles in urban, rural, and high-alpine air by DNA sequence and restriction fragment analysis of ribosomal RNA genes[END_REF]. The concentrations of bacteria in urban seem to be higher than in rural environments [START_REF] Bovallius | Long-range air transmission of bacteria[END_REF][START_REF] Fang | Culturable airborne bacteria in outdoor environments in Beijing, China[END_REF][START_REF] Di Giorgio | Atmospheric pollution by airborne microorganisms in the city of Marseilles[END_REF]. There is evidence that the bacterial communities in urban environment are influenced by human activities [START_REF] Bovallius | Long-range air transmission of bacteria[END_REF][START_REF] Fang | Culturable airborne bacteria in outdoor environments in Beijing, China[END_REF]. Although instability of bacterial composition in air, the literature often reports members of Pseudomonas genus to be found among airborne species [START_REF] Després | Primary biological aerosol particles in the atmosphere: a review[END_REF][START_REF] Dybwad | Characterization of airborne bacteria at an underground subway station[END_REF][START_REF] Fang | Culturable airborne bacteria in outdoor environments in Beijing, China[END_REF][START_REF] Pearce | Biodiversity of air-borne microorganisms at Halley Station, Antarctica[END_REF][START_REF] Šantl-Temkiv | Characterization of airborne ice-nucleationactive bacteria and bacterial fragments[END_REF]. However, to the best of our knowledge, the effect of air pollution on bacterial functioning and/or mechanisms of adaptation of airborne bacteria to air pollution were poorly studied.

Highlights

 NOx are free radicals, which include NO and NO2. Thanks to an unpaired electron, NOx can react with other species, containing unpaired electron, forming reactive nitrogen species (RNS). The chemistry of NOx and derived RNS is complex and dependent on a myriad of factors. However, the oxygen level plays a major role in RNS reactions.

 NO occupies a central position in RNS chemistry and may be reduced and/or oxidized to form a large number of reactive and frequently toxic species.

 Bacterial membrane is permeable to NO and NO2, but prevents the diffusion of charged RNS, like ONOO -. The membrane glycerophospholipids are not inert towards NOx. The polyunsaturated fatty acid chains may be oxidized and/or nitrated by NOx depending on the oxygen level.

 NO and its derivatives can react with proteins, causing the protein nitration, which may alter their functions and sometimes activate proteins. The related protein nitration and/or P a g e 114 | 270 nitrosylation are efficiently repaired thanks to several very elegant mechanisms, including ISC (iron-sulfur cluster) system.

 The RNS are able to damage the DNA directly (DNA deamination), or indirectly (alteration of DNA replication). These damages are also quickly repaired.

 The major NOx sources are usually into two classes: abiotic and biotic. Abiotic NOx principally come from combustion processes. Biotic NOx production is based primarily on the NO synthesis by mammalian cells, plants and bacteria.

 In bacterial cells, NO is synthesized by NO synthases and nitrate/nitrite reductases.

Several members of Pseudomonas genus are denitrifying bacteria, able to transform nitrate to nitrogen by reduction pathway. NO synthase coding genes are not found in sequenced genomes of Pseudomonas spp. species.

 Bacteria can detoxify NO using nitrate/nitrite reductase and flavohemoglobin catalytic properties.

 NO is involved in a wide range of processes in bacterial cells, including regulation of bacterial biofilm formation and antibiotic resistance.

 NOx are one of major air pollutant, which appear to have a harmful effect on human health, but also on environment.

 European Union with World Health Organization and French government established the NOx emission standards in order to control the air quality.

 The air contains a wide variety of primary biological aerosol particles (PBAPs), including bacteria, fungi or archaea. Bacterial composition of atmosphere is diverse and depends on various factors. However, the members of Pseudomonas genus are often detected among airborne microbiota.

II. Goals

With growth of the urban population, air pollution, caused in majority by urban activities, including motor traffic and industry, attracts global attention. Airborne bacteria undergo the air pollution effect and have to adapt to both fast natural and anthropogenic modifications in the environment. The high diversity and ubiquitous distribution of P. fluorescens suggest a remarkable degree of physiological and genetic adaptability. Physiologically, bacterial adaptability to various ecological niches and environmental modifications depends on the structure and organization of the envelope, composed of glycerophospholipids, which plays a major role in bacterial functioning.

In this context the aims of this work are: The enzymes and kits for qRT-PCR assays (RNase, TurboDnase I, cDNA RT Kit, SYBR Green DNA Poymerase, dNTPs and dUTPs), as well as the PCR primers were obtained from Life Technology (Life Technology, Brumath, France).



Bacterial strains

Two P. fluorescens strains MFN1032 and MFAF76a were used in this study. P. fluorescens MFN1032 is a clinical strain isolated from the sputum of a pneumonia patient [START_REF] Rossignol | Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens[END_REF][START_REF] Sperandio | Cell-associated hemolysis activity in the clinical strain of Pseudomonas fluorescens MFN1032[END_REF]. The airborne strain P. fluorescens MFAF76a was collected in the dust cloud of cereal docks in harbor installations of Rouen (France) (Morin et al., 2013b). The both strains are psychrotrophic bacteria with optimum of growth at 28°C. However, they are able to growth at 37°C [START_REF] Chapalain | Comparative study of 7 fluorescent pseudomonad clinical isolates[END_REF][START_REF] Duclairoir Poc | Airborne fluorescent pseudomonads : what potential for virulence?[END_REF].

Methods

Methods of microbiology

Bacterial culture

The strains are kept on glass beads at -80°C in 15% glycerol solution, from a bacterial culture at the end of exponential growth phase in Luria-Bertani (LB) medium. Precultures were realized

III. Materials and methods

P a g e 120 | 270 from one bead added in 10 mL of LB. After overnight incubation at 28°C, precultures were used to prepare bacterial culture. The added volume of preculture was calculated to ensure the optical density (OD) of culture A580=0.08 (Spectrometer Termo Spectronic, Cambridge, UK).

Bacterial cultures were grown at 28°C or at 37°C under limited agitation (180 rpm) in LB or DMB (Davis Medium Broth) minimal medium with glucose as carbon source (2.16 g per liter).

The compositions of growth media are presented in Table 5.

Growth monitoring

The bacterial growth was monitored by turbidimetry at A580 using Bioscreen C MBR (Oy Growth Curves Ab Ltd., Helsinki, Finland). Bacteria were resuspended in DMB minimal medium and added to Bioscreen Honeycomb plates (Oy Growth Curves Ab Ltd., Helsinki, Finland) in a total volume of 200 µL of DMB (A580=0.08). Growth was measured every 15 min for 24 h. The generation time was calculated following formula: TEGP / ((log10 Nt -log10 N0) / log102). Where TEGP: time of exponential growth phase; Nt: CFU at the end of exponential growth phase; N0: CFU at the beginning of exponential growth phase. The bacterial growth was assayed in at least three independent experiments with two replicates for each experimental condition.

Minimum inhibitory concentration (MIC) assays

Bacterial sensitivity to ciprofloxacin, chloramphenicol and two aminoglycosides (tobramycin and kanamycin) was tested. The MICs of antibiotics were determined by broth microdilution method in DMB minimal medium. Bacterial solution were diluted in the appropriate broth to targeted bacteria was incubated at 28°C for 24 h. MIC was defined to be the lowest concentration of antibiotic that inhibited bacteria growth as determined by turbidimetry at A580.

Motility assays

Swimming and swarming motility assays were performed on agar plates using DMB medium containing 0.2% (wt/vol) and 0.5 % (wt/vol) agar, respectively as previously described [START_REF] Déziel | Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities[END_REF]. The resultant diameters of swim and swarm zones were measured after 24 h of incubation at 28 °C. All motility types were assayed in at least three independent experiments with three replicates for each experimental condition.

Study of biofilm monitoring by confocal laser scanning microscopy (CLSM)

Samples for CLSM were prepared in 175 µm glass-bottom dishes (SensoPlate™, VWR, Fontenay-sous-Bois, France) by inoculation of 300 µL of bacterial suspension in sterile saline solution (A580 =1). After 2 h of incubation at 28°C, planktonic bacteria were removed and the adhesion of P. fluorescens was observed using confocal laser scanning microscope (LSM 710, ZEISS) with an immersion objective 63x. After addition of DMB medium, bacterial biofilms were incubated at 28°C for 24 h. DMB medium was removed, biofilms were rinsed with 1 mL of saline solution and observed using CLSM. All biofilm assays were analyzed using the fluorescence of Cyan Fluorescent Protein (CFP, excitation by the 405 nm laser line and detection at 485 nm) through at least three independent experiments with two replicates for each experimental condition. The biofilm thickness and related biovolumes (bacterial volume (µm 3 ) by µm 2 ) were estimated from 6 fields on 3 independent experiments using COMSTAT software [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program comstat[END_REF].

Physico-chemical methods

Bacterial contact angle measurements by sessile drop

The contact angle formed by water droplet, w, on microbial lawns was measured by sessile drop method using a goniometer DSA 100 (Krüss GmbH, Germany). Bacterial lawns were prepared in suspension of physiological saline in order to obtain at least 2 mL with A580 of at least 5. To obtain homogeneous, continuous bacterial lawns, the suspension was slowly deposited on the filter by applying negative pressure, rinsed with 10 mL of physiological saline and dried at room temperature for 2 h under standardized conditions. To capture the contact angle of water w on the bacterial lawn, four water droplets were observed on each biological triplicate. To determine this contact angle, the drop contour was mathematically described by the Young -Laplace equation using DSA 100, and the contact angle was evaluated as the slope

Evaluation of bacterial surface hydrophobicity

The hydrophobicity of bacterial surface was evaluated by the microbial adhesion to solvent (MATS) test [START_REF] Bellon-Fontaine | Microbial adhesion to solvents: a novel method to determine the electron-donor/electron-acceptor or Lewis acid-base properties of microbial cells[END_REF]. It consisted in assessing the affinity of the cells to two solvents duos consisting of a monopolar solvent and an apolar solvent. In each set, both solvents have similar surface tension, but the monopolar one is acidic (electron accepting, e.g. chloroform) or basic (electron donating, e.g. ethyl acetate). The apolar solvents are respectively hexadecane and decane. For the experiments, bacterial cells were resuspended in saline solution to A400=0.8 (Abs1). This bacterial suspension was mixed with each solvent at 1/6 (v/v) by vigorous agitation for 1 min in order to form an emulsion. This mixture was then left for at least 15 min until the separation of the two phases. The aqueous phase absorbance (Abs2) was measured and the percentage of adhesion was expressed as: %affinity = (1-Abs2/Abs1) x100.

Exposition to NO2

Bacterial culture at the end of exponential growth phase (about 3x10 7 bacteria per filter) was transferred on cellulose nitrate membrane filter (pore size 0.2 µm, diameter 47 mm, Sartorius Biolab Products, Gottingen, Germany) and grown in DMB agar plates at 28°C for 4 h to obtain the single layer's bacterial population. After 4 h of incubation, the cellulose membranes were placed on agar 1 well dishes (size 127.8 x 85.5 mm, Thermo Scientific Nunc, Roshester, USA) that were directly transferred in gas delivery device.

In order to restore the environmental conditions, bacterial NO2 exposure was done in gas phase for 2 h, according to Ghaffari et al., 2005. The delivery device consisted of two sterile cylindrical Plexiglas exposure chambers (one for the NO2 exposure, the second one for the control -exposure to the synthetic air), deposed in drying oven at 28°C creating a thermally isolated environment. The NO2, N2, and O2 obtained to Air Liquide GMP Europe (Mitry-Mory, France) were mixed together using digital mass flowregulators (Alicat Scientific, Inc., Tucson, USA) to obtain pre-calculated concentration of NO2 and maintain the O2/N2 ratio at 2/8, (v/v).

The obtained gas mixture and synthetic air (Air Liquide GMP Europe, Mitry-Mory, France)

were routed independently to each of the exposure chambers at a constant flow rate of 2 L/min.

The parallel NO2/synthetic air circuits allow utilization of the same bacterial culture for exposed to NO2 and control samples. After passing through exposure chamber, the NO2 concentrations were monitored by AC32M nitrogen oxides analyzer (Environnement S.A, Poissy, France) and safely vented to a chemical hood. Chamber temperature and relative humidity data were Lipids were extracted according to the method of Bligh and Dyer [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF]. About 1 g of lyophilized bacteria was resuspended in 3 mL of CHCl3/CH3OH (1/2, v/v). The obtained mixture was completed by 1 mL of CHCl3, and 1.8 mL of H2O successively. Samples were centrifuged 15 min at 3,000 g. The chloroform phase containing lipids was collected and stored at -20°C under N2 atmosphere.

Extraction of fatty acid methyl esters (FAME)

The FAME were prepared from lipid extract samples by incubation for 15 min at 95°C in a boron trifluoride/ethanol mixture (140 g BF3 per liter of CH3OH) and extracted with hexane as described by [START_REF] Morrison | Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol[END_REF]. Briefly, the FAME were added to 0.3 mL of H2O and 0.5 mL of hexane. After shaking the hexane phase was transferred in GC-autosampler flasks.

The hexane was removed by N2-gasstream. To unify volumes, 0.5 mL of hexane was added in each simple before GC analysis.

Glycerophospholipid analysis

Separation of GPs by high performance thin-layer chromatography (HPTLC)

The GPs were separated by high performance thin-layer chromatography (HPTLC) using method adapted from Fuchs et al., 2007a. Beforehand, HPTLC plates were washed with the separation solvent system: CHCl3/ CH3-CH2OH/ H2O/ N(CH2CH3)3 (35/35/7/35, v/v/v/v), and activated at 110°C under vacuum for 2 hours. Bacterial GP extracts were spotted in triplicates on HPTLC plates and separated at room temperature.

After development, GPs spots were labeled with spray of 0.05% primuline dye in acetone/water, (8/2, v/v) and visualized by UV fluorescence (365 nm). The primuline binds non-covalently to the apolar fatty acyl residues of lipids and does not affect a subsequent MS analysis [START_REF] Richter | The reaction between phosphatidylethanolamines and HOCl investigated by TLC: fading of the dye primuline is induced by dichloramines[END_REF]. Moreover, the staining with ninhydrin and azure A were performed in order to identify the lipid classes having a free amino groups and sulfatides and/or sulfoglycolipids respectively [START_REF] Kates | Techniques of lipidology: isolation, analysis and identification of lipids[END_REF][START_REF] Kean | Rapid, sensitive spectrophotometric method for quantitative determination of sulfatides[END_REF]. The 3% solution of ninhydrin prepared in absolute

Glycerophospholipid identification by MALDI TOF MS

GLs were identified directly on HPTLC plates by matrix-assisted laser desorption ionization mass spectrometrytime of flight (MALDI-TOF) using an Autoflex III mass spectrometer equipped with a laser Optibeam TM Nd/YAG (355 nm, 200-Hz tripled-frequency) (Bruker Daltonics, Bremen, Germany). DHB was chosen as MALDI matrix since it generates positive ion spectra of lipids and does not interfere with the silica of HPTLC plates. HPTLC plate containing GP spots was coated by DHB matrix solution (200 g/L in C2H3N /0.1% TFA, 90/10, v/v) (Fuchs et al., 2007a) and immediately dried under dust-free atmosphere. Matrix coating was made twice to reach a mean of DHB matrix of 5 mg/cm 2 . Matrix coated HPTLC plate were fixed on a TLC-MALDI target developed by Bruker Daltonics (Bremen, Germany).

The Autoflex III mass spectrometer was run in the reflector positive ion mode using the TLC-MALDI software (v. 1.1.7.0,Bruker Daltonics,Bremen,Germany). The excitation voltage was 19.5 kV, the reflector voltage was 21.0 kV. The laser strength was kept about 30% above threshold to have a good signal-to-noise ratio. A major strength is needed to desorb lipids from silica [START_REF] Lobasso | Coupled TLC and MALDI-TOF/MS analyses of the lipid extract of the hyperthermophilic archaeon Pyrococcus furiosus[END_REF]. 200 single laser shoots were averaged for each point. The distance between two points was determined as 1 mm. Obtained 2D mass spectra enable the rapid MS analysis of lipid spots. Post Source Decay (PSD) spectra were acquired on a Bruker Autoflex III mass spectrometer (Bruker Daltonics), as previously described [START_REF] Fuchs | MALDI-TOF MS of phosphatidylethanolamines: different adducts cause different post source decay (PSD) fragment ion spectra[END_REF]. The precursor ions were isolated using a time ion selector. The fragment ions were refocused onto the detector by stepping the voltage applied to the reflectron in appropriate increments. This was done automatically by using the "FAST" ("fragment analysis and structural TOF") subroutine of the FlexAnalysis software.

Imaging HPTLC-MALDI TOF MS assays

Mass spectrometry imaging (MSI) of GP spots was performed using the FlexImaging software (v. 2.1.,Bruker Daltonics,Bremen,Germany). A polygon measurement region was defined according the GP spots of single sample with a spatial resolution of 200 µM. The GP spots were imaged with 500 laser shots per pixel with multiple additions of single position acquisition run (every 40 shots). Each MS spectrum was baselined and smoothed in FlexAnalysis software.

Following previous MS and PSD identification (see part Glycerophospholipid identification by MALDI TOF MS), individual GP spots were labeled by a specific color code according to their m/z.

Fatty acid analysis

Separation and identification of fatty acids (FAs)

The FAs were separated and analyzed by gas chromatography (GC) coupled to flame ionization detection using an Agilent Technology, 6890 Network GC System, 7683 Series Injector equipped with a split/splitless injector. The apparatus was equipped with a CP-Sil 88 capillary column (length, 50m; inner diameter, 0.25 mm; 0.25 mm film; Chrompack, Middelburg, the Netherlands). FAs were identified by coinjection as internal standards of reference compounds obtained from Supelco (Bellefonte, Pennsylvania, USA).

Quantification of fatty acids

The FAs were quantified on the basis of their peak areas in total ion chromatograms. All experiments were performed in triplicate. The degree of FA saturation was determined as the ratio between the saturated FAs and the unsaturated FAs [START_REF] Heipieper | Effect of environmental factors on the trans/cis ratio of unsaturated fatty acids in Pseudomonas putida S12[END_REF].

Methods of molecular biology

. C-di-GMP extraction

Extraction and quantification of intracellular c-di-GMP was performed as previously described [START_REF] Spangler | A liquid chromatography-coupled tandem mass spectrometry method for quantitation of cyclic di-guanosine monophosphate[END_REF][START_REF] Strehmel | The sensor kinase PA4398 modulates swarming motility and biofilm formation in Pseudomonas aeruginosa PA14[END_REF]. Aliquots of bacterial culture (CFU=3.3x10 as an internal standard. After 15 min of incubation on ice, bacterial cells were lysed at 95°C for 10 min, followed by centrifugation at 20,000 g, 4°C for 5 min. Supernatants were collected and stored on ice. Remaining pellets were resuspended in 400 µL of extraction buffer, incubated on ice for 15 min, and centrifuged at 15,000 g, 4°C for 5 min. The extraction was repeated thrice, the supernatants were pooled and evaporated using a DNA SpeedVac ® DNA 120 (Thermo Savant, Holbrook, USA) at 43°C. The remaining residues were resuspended in 500 µL of H2O.

Extraction of total RNA

The total RNA were extracted as previously described by [START_REF] Gicquel | The extracytoplasmic function sigma factor SigX modulates biofilm and virulence-related properties in Pseudomonas aeruginosa[END_REF] One volume of absolute ethanol was added to collected bacterial cells (about 10 8 of bacteria). The pellet bacteria were harvested by centrifugation at 13,000 g, 10 min and lysed in 300 µL of buffer I (0.02 M sodium acetate; 0.5% sodium dodecyl sulfate (SDS); 1 mM ethylenediaminetetraacetic acid (EDTA)). After homogenization, the total RNA were extracted twice in 600 µL of hot acid phenol (pH 4.3; 60°C). After 3 min incubation at 60°C, the aqueous phase containing the RNA was separated from organic phase, containing the genomics DNA and proteins by centrifugation at 13,000 g for 4 min. 500 µL of chloroform/isoamyl alcohol (24/1, v/v) were added to aqueous phase. The centrifugation at 13,000 g for 2 min was performed to separate two phases. Aqueous phase, containing the total RNA, was collected. The total RNA was precipitated in cold 100% ethanol with 100 mM sodium acetate, harvested by centrifugation at 13,000 g 30 min at 4°C, rinsed with 1 mL of 75% ethanol and dissolved in 50 µL of H2O treated by diethylpyrocarbonate (DEPC), inactivating RNase enzyme.

The contamination by genomic DNA was eliminated with addition of 10U TurboDnase I (Life Technology, Brumath, France) in reaction mixture containing 6.25 mM MgCl2. The reaction was performed at 37°C for 2 h. The absence of genomic DNA was controlled by PCR with the pair of primers FmexX/RmexX (

Reverse transcription reaction

Reverse transcription (RT) of total RNA into complementary DNA (cDNA) was performed using "High capacity cDNA RT Kit" (Applied Biosystems ® , Life Technology, Brumath, France) according to provider recommendations. The cDNA synthesis was performed in a final volume of 50 µL from 2.5 µL of RNA during 2 h at 37°C. The reaction was stopped by samples heating at 85°C during 5 min. The control samples (negative RT) were made at the same conditions without addition of reverse transcriptase in reaction mixture. The control of cDNA synthesis was performed by 2% agarose gel electrophoresis.

Design and optimization of primers

The sequences of target genes were identified using Blast+ v2.2.30 software [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. The primers of target genes were designated using Primer Express ® software v3.0.1 (Applied biosystem ® , Life Technology, Fontenay-sous-Bois, France) and are compiled in Table 6. Temperature of melting of qRT-PCR primers was 60°C. quantity spectrum, the slope was obtained using a linear equation to determine the PCR efficiency (E): E=10 (1/slope) . The reaction specificity was tested using melting curves of amplification products, amplification of negative RT and H2O replacing the cDNA.

Real-time quantitative amplification

Real time quantitative amplification was realized using 7500 Fast Real Time PCR system (Applied biosystem ® , Life Technology, Fontenay-sous-Bois, France). The reaction was realized according to provider recommendations. The reaction was made in 13 µL with 6.5 µL of SYBR Green DNA Poymerase, dNTPs and dUTPs. The simples contained 300 nM of each primer and 7.5 of cDNA. For amplification the following program was used: one step at 95°C

-20 min, 40 cycles at 95°C -10 s, 60°C -30 s and 72°C -6 s. The relative RNA quantification was realized by comparison of cycle threshold (Ct) (2 -ΔΔCt ) using 16S RNA as control.

Transformation of Pseudomonas by electroporation

Plasmid preparation

The Cyan Fluorescent Protein (CFP) transformed P. fluorescens MFN1032 and MFAF76a were used in this study. The fluorescent gene cfpopt was introduced in pPSV35 vector [START_REF] Rietsch | ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa[END_REF][START_REF] Vallet-Gely | Local and global regulators linking anaerobiosis to cupA fimbrial gene expression in Pseudomonas aeruginosa[END_REF]. The CFP cassette was obtained from pTetONCFPopt plasmid containing 729-bp cfpopt gene [START_REF] Sastalla | Codonoptimized fluorescent proteins designed for expression in low-GC Gram-positive bacteria[END_REF] by double digesting with PstI and Xmal. This gene was inserted into the lacZ site of the polylinker of pPSV35 by ligation into the pPSV35 vector digested with the same enzymes.

Preparation of P. fluorescens competent cells

The competent cells of P. fluorescens were prepared as follows [START_REF] Enderle | Electroporation of freshly plated Escherichia coli and Pseudomonas aeruginosa cells[END_REF] bacteria were collected on the agar plate and washed twice in 500 µL of distilled water at 4°C.

The pellet cells were collected by two centrifugations at 10,000 g, 4°C for 1 min and resuspended in 40 µL of cold distillated water.

Transformation by electroporation

The transformation by electroporation helps to make the bacterial cells permeable to DNA through electric shock. About 150 ng of plasmid were added to 100 µL of competent cells. The mixture was transferred into electroporation chamber with electrical voltage 1,800 V during 5 ms (Gene transformer GTF100, Savant). The reaction volume was transferred in 1 mL of LB medium and incubated at 28°C during 1 h to obtain the first phenotypic expression. After chosen the clones by antibiotic selection (gentamycin 15µg/mL), the transformation was confirmed by confocal laser scanning microscope (LSM 710, ZEISS). The c-di-GMP levels were quantified by HPLC MS/MS as described previously [START_REF] Strehmel | The sensor kinase PA4398 modulates swarming motility and biofilm formation in Pseudomonas aeruginosa PA14[END_REF]. The chromatographic separation was performed on a 1100 Series HPLC system (Agilent, Waldbronn, Germany) using a Multospher AQ RP 18.5 µm, 250 x 4.0 mm HPLC column (CS Chromatography Service GmbH, Landenwehe, Germany) in a gradient mode using 10 mM ammonium acetate with 0.1% acetic acid as eluent A and methanol a eluent B. The injection volume of each sample was set to 40 µL and the flow rate was 0.4 mL/min. The gradient program was as follows: from 0-4 min 100% A, followed by a linear gradient from 100% to 80% A in 1 min, held for 2 min at 80% A, followed by a linear gradient from 80% to 60% A in 1 min and held for additional 9 min at 60%. Finally, re-equilibration of the column was obtained by constantly running 100% A for 16 min. All experiments were performed in at least three independent experiments with three replicates for each experimental condition.

Quantification of c-di-GMP by Electrospray Ionization (ESI) MS

ESI MS was performed on an API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Toronto, Canada) using a turbo ion spray interface in positive mode. The ionization potential was of 5,000 V and a temperature of 400°C using nitrogen a curtain, nebulizer and collision gas. The parameter settings were optimized by infusion experiments. Data were acquired in multiple reaction monitoring (MRM) mode using the Analyst software version 1.6.

(Applied Biosystems, Toronto, Canada). Identification and quantification of c-di-GMP was performed by using three specific mass transitions from molecule ion m/z 691 to the product ions: m/z 152, m/z 135 and m/z 540. The external calibration was carried out at c-di-GMP concentrations ranging from 10 ng to 200 ng in 500 µL H2O using the internal standard cXMP (50 ng). Obtained concentrations of c-di-GMP were normalized against total protein contents of respective cultures, which was determined by the bicinchoninic acid assay [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF]. All experiments were performed in at least three replicates for each experimental condition.

III. Materials and methods

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Methods of bioinformatics

Gene sequences identification

The nucleotide sequences identified in this study were obtained using the non-annotated

Phylogenetic analyses

The nucleic sequences of 12 Pseudomonas type strains used in this study were obtained from a previous study [START_REF] Bodilis | A long-branch attraction artifact reveals an adaptive radiation in Pseudomonas[END_REF]. Some nucleic sequences from 8 sequenced genomes were also included in this analysis, from the PAO1 [START_REF] Stover | Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen[END_REF], PA14 [START_REF] Lee | Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial[END_REF], SBW25 [START_REF] Rainey | Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome[END_REF], Pf0-1 [START_REF] Silby | Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens[END_REF], CHAO (Jousset et al., 2014, p. 0), PF-5 [START_REF] Silby | Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens[END_REF], MFN1032 (KP119846 for 16S RNA [START_REF] Mazurier | Type III secretion system and virulence markers highlight similarities and differences between human-and plantassociated pseudomonads related to Pseudomonas fluorescens and P. putida[END_REF];

unpublished data: KT350505 for gyrB, KT350506 for rpoB, KT350507 for rpoD), and MFAF76a (this study: KT350501 for 16S RNA, KT350502 for gyrB, KT350503 for rpoB, KT350504 for rpoD ).

From the 20 Pseudomonas strains, nucleic alignments of four partial housekeeping genes (gyrB gene, 798 bp; rpoB gene, 642 bp; rpoD gene, 564bp; and 16S rRNA gene, 1182 bp) were optimized individually then concatenated, leading to 3,186 nucleotide positions. Protein sequences were aligned with ClustalW2 with default parameters [START_REF] Bodilis | A long-branch attraction artifact reveals an adaptive radiation in Pseudomonas[END_REF] implemented in Seaview 4.2.4 [START_REF] Gouy | SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF]. Conserved blocks of the multiple alignment were selected by using Gblocks 0.91b with default parameters [START_REF] Talavera | Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments[END_REF] and then, nucleic alignment was deduced from this corrected protein alignment. For the 16S rRNA gene alignment, a preliminary step was conducted before selecting the conserved blocks by using Gblocks 0.91b: the regions in which the secondary structures differed were eliminated, as described previously by [START_REF] Anzai | Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence[END_REF] Phylogenetic analysis was conducted by using BioNJ implemented in Seaview 4.2.4, with JC correction. Bootstrap analysis was conducted from 1000 replicates. The maximum likelihood (ML) tree was inferred using RAxML version 7.2.6 [START_REF] Stamatakis | RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models[END_REF] with partitioned analysis for the third codon position. A third partition corresponding to the 16S rRNA gene was defined.

The generalised time-reversible (GTR) model parameters, branch lengths, proportion of invariant sites and alpha parameter of the gamma distribution had been estimated independently for each partition. Support values were evaluated based on 100 bootstrap replicates.

The pcs nucleic sequences of 6 P. fluorescens strains Pf-5, CHAO, Pf0-1, SBW25, MFN1032

(GenBank accession number KT338639) and MFAF76a (GenBank accession number KT338638) and P. aeruginosa PAO1 were obtained as described before. The pcs nucleic sequence form A506 sequenced genome [START_REF] Loper | Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions[END_REF] was also included in this analysis.

The sequences were analyzed using the neighbor-joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF].

Analyses were conducted using CLC Sequence Viewer 7 (CLC bio, a QIAGEN Company, Denmark). Bootstrap analysis was performed from 1000 replicates.

III. Materials and methods

P a g e 132 | 270

Highlight

 A new HPTLC-MALDI TOF MSI tools may be successively used in study of bacterial lipidome. This method has many advantages in bacterial lipidome studies, including promptness of analysis and low detection limit. However, the quantification of glycerophospholipids with this technic appears to be difficult.

 The fatty acid composition of P. fluorescens reveals 16 and 18 C-atoms in alkyl chains.

 The phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol are the major compounds of P. fluorescens lipidome.

 The phosphatidylcholine is identified in P. fluorescens lipidome for the first time and this glycerophospholipid could be involved in a wide range of bacterial functions, including interactions with eukaryotic hosts. This is all the more important in view of the fact that 37°C is an optimal temperature of human body, and several P. fluorescens strains were identified as the indigenous microbiota of various body sites. As previously mentioned, MFAF76a was characterized as able to grow at 37°C [START_REF] Duclairoir Poc | Airborne fluorescent pseudomonads : what potential for virulence?[END_REF]. These adaptive properties could be used by this bacterium to adapt to climate warming and/or to tame the 'hot environments', like human body, and would involve the changes in its membrane properties. In this way, we decided to add to this study the clinical P. fluorescens strain MFN1032, isolated from the sputum of a pneumonia patient. This bacterium was characterized as growing at 37°C and showing increased virulence against human cells [START_REF] Rossignol | Phenotypic variation in the Pseudomonas fluorescens clinical strain MFN1032[END_REF][START_REF] Sperandio | Virulence of the Pseudomonas fluorescens clinical strain MFN1032 towards Dictyostelium discoideum and macrophages in relation with type III secretion system[END_REF]. It shows intense secretiondependent hemolytic activity [START_REF] Rossignol | Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens[END_REF][START_REF] Sperandio | Cell-associated hemolysis activity in the clinical strain of Pseudomonas fluorescens MFN1032[END_REF], involving the production of phospholipases C and biosurfactants, and has the common characteristics of opportunistic pathogen, similar to that seen for P. aeruginosa. After establishment of P.

fluorescens response to increasing temperature, the membrane response to NO2 was studied.

For this, bacteria were exposed to three NO2 concentrations, corresponding to the 1-hour NO2 standard, the threshold, causing reversible effects on human health, and the high NO2 concentration, provoking irreversible effects, according World Health Organization.

P a g e 182 | 270

Highlight

 The members of P. fluorescens group are highly adaptable and ubiquitously distributed bacteria. Physiologically, these properties could be related with membrane lipid changes, as the first barrier between bacterial cell and its environment.

 The lipidome of clinical P. fluorescens strain MFN1032 includes the phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol, as major glycerophospholipid compounds produced in exponential and stationary growth phases.

 In stationary growth phase, a new glycerophospholipid (UGP) is found in both P. fluorescens strains. Its identification is not completed yet, but it is clear, that this molecule has a PSD fragmentation mechanism characteristic for a glycerophospholipid. Interestingly, in airborne MFAF76a, this molecule is found only in stationary growth phase, contrary to clinical strain MFN1032, which seems to product the UGP in exponential growth phase at 37°C, but not at 28°C, indicating the potential role of this membrane compound in increasing temperature adaptation of clinical strain.

 The supra-optimal temperature modifies the membrane fluidity that is compensated by increasing of degree of fatty acid saturation in both tested P. fluorescens strains.

 Both P. fluorescens strains do not increase the degree of fatty acid saturation after exposure to NO2. Thus, NO2 does not alter membrane fluidity, can freely cross bacterial membrane and penetrates in bacterial cytoplasm.

 No modification in membrane properties of P. fluorescens exposed to hourly NO2 standard, or industrial limit is found.

 In both studied bacteria exposed to high NO2 concentration, the new UGP is not found, indicating the sensitivity of this glycerophospholipid to NO2.

 The exposure to high NO2 concentration does not change the surface charge of airborne MFAF76a, but decreases its electron-accepting character. The latter modifications could be caused by the decreasing (if not complete loss) of UGP, but also by the increasing level of phosphatidylcholine comparing to the phosphatidylethanolamine.

Highlight

 The human exposure to well-known air pollutant, NO2, results in several toxic effects, but bacterial response to NO2 is not established yet. The NO2 effects on bacteria were evaluated in terms of motility, biofilm formation, antibiotic resistance, as well as expression of several chosen target genes.

 No modification in tested phenotypes and genes expression was found in bacteria exposed to 0.1 and 5 ppm of NO2. However, several alterations were observed when the bacteria were exposed to 45 ppm of NO2.

 P. fluorescens strains exposed to NO2 show reduced swimming motility, and decreased swarming in case of the strain MFN1032.

 Biofilm formed by NO2-treated airborne P. fluorescens MFAF76a shows increased maximum thickness, while NO2 had no apparent effect on the clinical MFN1032 biofilm structure. However, the c-d-GMP, which is involved in Pseudomonas spp. biofilm regulation, seems not to be affected by NO2 treatment.

 The both NO2-treated P. fluorescens were found to be more resistant to ciprofloxacin and chloramphenicol. Accordingly, the resistance nodulation cell division (RND) MexEF-OprN efflux pump encoding genes were highly upregulated in the two P. fluorescens strains.

 Expression of the hmp-homologue gene in both tested bacteria, which encodes a NO dioxygenase that is involved in NO detoxification into nitrites, was upregulated in response to NO2, suggesting a possible common pathway between NO and NO2 detoxification.

V. General discussion

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The members of P. fluorescens group are defined as ubiquitously distributed and highly adaptive bacteria, found in majority environmental niches [START_REF] Scales | Microbiology, genomics, and clinical significance of the Pseudomonas fluorescens species complex, an unappreciated colonizer of humans[END_REF]. This high adaptation capacity is linked with the ability of these bacteria to respond to various stress factors. In this study, the response of P. fluorescens to important air pollutant, NO2 was studied.

For this purpose, the airborne P. fluorescens MFAF76a strain was exposed to gaseous NO2 to mimic as far as possible the natural atmospheric conditions. Three NO2 concentrations were chosen in correlation with NO2 standards and toxic thresholds. Thus, bacteria were exposed to hourly NO2 standard 0.1 ppm, recommended by WHO ('WHO | Ambient (outdoor) air quality and health', 2015). In addition, 5 ppm and 45 ppm of NO2 were studied as concentrations, caused the reversible and irreversible effects on human health respectively. In order to establish the specific to airborne bacteria response to NO2, the P. fluorescens strain MFN1032 with clinical origins was used. In our study for the first time, the bacterial response to the NO2 contamination was evaluated in terms of membrane response, phenotypic modification, such as motility, biofilm formation, antibiotic resistance, as well as expression of several chosen target genes. After exposure to 0.1 ppm of NO2 any detectable modification in tested parameters was not found. While the establishment of NO2 standards on the basis of human health effects, our results could enlarge the NO2 guideline values to the air microbiota. The similar response was found for bacteria treated with 5 ppm of NO2. The short-term human exposure to NO2 in concentrations, close to 5 ppm has been examined in several studies, demonstrating various responses. In healthy volunteers, a two-hour exposure to NO2 in concentrations from 2.5 ppm to 5 ppm shows a slight, but significant increase of bronchial hyper responsiveness [START_REF] Beil | Effect of NO2 in workroom concentrations on respiratory mechanics and bronchial susceptibility to acetylcholine in normal persons[END_REF]. By contrast, the same clinical symptom is not observed after an 75 min exposure to 4 ppm of NO2 [START_REF] Linn | Effects of exposure to 4 ppm nitrogen VII. References P a g e 229 | 270 dioxide in healthy and asthmatic volunteers[END_REF]. Although the absence of detectable modification in bacteria exposed to 0.1 and 5 ppm of NO2, this free radical could however alter other non-tested targets (i.e. DNA and/or protein damages).

Several alterations were observed when the bacteria were exposed to 45 ppm of NO2. Contrarily to NO, which is involved in biofilm dispersion [START_REF] Barraud | Nitric oxide: a key mediator of biofilm dispersal with applications in infectious diseases[END_REF][START_REF] Cutruzzola | Origin and impact of nitric oxide in Pseudomonas aeruginosa biofilms[END_REF], we did not observe the NO2-mediated modifications of bacterial biomass. However, biofilm formed by NO2-treated airborne strain MFAF76a showed increased maximum thickness, while NO2 had no apparent effect on the clinical MFN1032 biofilm structure. Given that this phenotype was observed only in airborne P. fluorescens, its apparent inability to produce biosurfactant may not counteract the biofilm expansion, contrary to MFN1032, producer of cyclolipopeptides CLP [START_REF] Rossignol | Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens[END_REF] or to P. aeruginosa, rhamnolipid producer [START_REF] Caiazza | Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa[END_REF][START_REF] Ochsner | Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa[END_REF]. In addition, NO2 completely inhibits bacterial swarming motility and provokes a slight decreasing of swimming. It is P a g e 208 | 270 increasingly evident that biofilm and motility are inversely regulated. The well-known second messenger c-di-GMP plays a central role in this regulation [START_REF] Hengge | Principles of c-di-GMP signalling in bacteria[END_REF]. The c-di-GMP level, as well as the expression of genes, coding for several PDEs was however not affected in response to NO2 treatment, suggesting that the observed modifications in biofilm architecture and motility seem not to be linked with c-di-GMP pathway, and could be correlated, among other factors, with alterations in flagellar motion, type IV pili and surfactant production.

In addition, our study showed the NO2-mediated upregulation of the hmp-homologue gene in P. fluorescens strains MFAF76a and MFN1032, suggesting a possible common pathway between NO and NO2 detoxification. While the mechanism of NO2 detoxification was proposed to carry out through the NO2 reduction pathway [START_REF] Poole | Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12[END_REF], the expression of the nirBhomolog gene, coding for the assimilatory nitrite reductase, which reduces nitrite to ammonium, was not found to be impacted by NO2. We incline to think, that in presence of ammonium in DMB medium (see III. Materials and methods), the production of supplementary ammonium through the nitrite reduction is not appropriate.

The analyses of NO2-mediated gene expression demonstrated, that NO2, like its homolog NO, induces the expression of mexEF-oprN genes, coding for the RND efflux pump. Its overexpression could be involved in the observed increase of P. fluorescens resistance to ciprofloxacin and chloramphenicol. Thus, we could suppose, that NO2 may stimulate bacterial antibiotic resistance, probably creating antibiotic resistant bacteria, diminishing the effectiveness of antibiotic therapies in polluted area. Although a potential role of MexEF-OprN efflux pump in NO2-mediated increase of antibiotic resistance, several other mechanisms could also be highlighted. As described in part 3.2. Proteins, NO is able to impair the oxidative phosphorylation, altering the TCA cycle and ATP production [START_REF] Appanna | Fumarate metabolism and ATP production in Pseudomonas fluorescens exposed to nitrosative stress[END_REF]. The antibiotic uptake is an energy-dependent process, which requires both an electrochemical gradient across the membrane and an electron flow through the respiratory chain [START_REF] Fernandes | Ciprofloxacin interactions with bacterial protein OmpF: Modelling of FRET from a multi-tryptophan protein trimer[END_REF][START_REF] Mccollister | Nitric oxide protects bacteria from aminoglycosides by blocking the energydependent phases of drug uptake[END_REF][START_REF] Pagès | The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria[END_REF]. In this context, the recently reported NO-mediated metabolism reprogramming and utilization of CiLy-PEPC-PPDK complex for ATP production [START_REF] Auger | A novel ATPgenerating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation[END_REF] could also be correlated with the NO-mediated modification of bacterial antibiotic resistance. Taken together, these findings let us suspect that, in the similar to NO manner, NO2 could alter P. fluorescens respiration, decreasing the drug uptake, and making bacteria more resistant to antibiotics. In addition to antibiotic resistance, the overexpression of mexEF-oprN genes could also impact bacterial motility, as previously described (see part 4.4. NO release) and reported by [START_REF] Lamarche | MexEF-OprN efflux pump exports the Pseudomonas quinolone signal (PQS) precursor HHQ (4-hydroxy-2-heptylquinoline)[END_REF].

P a g e 209 | 270

Therefore, the question of NO2 impact on bacterial virulence remains to be studied in more

details. An arsenal of virulence factors was reported and appears to impact on the pathogenicity of P. fluorescens. Included among these are cell-associated factors such as lipopolysaccharide [START_REF] Picot | Cytotoxic effects of the lipopolysaccharide from Pseudomonas fluorescens on neurons and glial cells[END_REF], the exopolysaccharide alginate [START_REF] Bakkevig | Role of the Pseudomonas fluorescens alginate lyase (AlgL) in clearing the periplasm of alginates not exported to the extracellular environment[END_REF], and secreted factors including toxins, proteases and lipases [START_REF] Russell | Bacterial membranes: the effects of chill storage and food processing. An overview[END_REF], phospholipases [START_REF] Rossignol | Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens[END_REF][START_REF] Sperandio | Cell-associated hemolysis activity in the clinical strain of Pseudomonas fluorescens MFN1032[END_REF] and small molecules such as phenazines [START_REF] Laursen | Phenazine natural products: biosynthesis, synthetic analogues, and biological activity[END_REF][START_REF] Weller | Role of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. in the defense of plant roots[END_REF], cyclolipopeptides [START_REF] Duclairoir-Poc | Influence of growth temperature on cyclolipopeptides production and on adhesion behaviour in environmental strains of Pseudomonas fluorescens[END_REF], hydrogen cyanide [START_REF] Ramette | Prevalence of fluorescent pseudomonads producing antifungal phloroglucinols and/or hydrogen cyanide in soils naturally suppressive or conducive to tobacco black root rot[END_REF]), 2,4-diacetylphloroglucinol (Moynihan et al., 2009), and rhizoxin [START_REF] Gross | Genomics of secondary metabolite production by Pseudomonas spp[END_REF]. Quorum-sensing, type 3 and type 6 secretion [START_REF] Decoin | A type VI secretion system is involved in Pseudomonas fluorescens bacterial competition[END_REF][START_REF] Marchi | Genomic analysis of the biocontrol strain Pseudomonas fluorescens Pf29Arp with evidence of T3SS and T6SS gene expression on plant roots[END_REF][START_REF] Mavrodi | Structural and functional analysis of the type III secretion system from Pseudomonas fluorescens Q8r1-96[END_REF] and many other regulatory systems are important for P. fluorescens virulence [START_REF] Scales | Microbiology, genomics, and clinical significance of the Pseudomonas fluorescens species complex, an unappreciated colonizer of humans[END_REF]. To refine the role of NO2 in the regulation of bacterial virulence, the mechanism of bacterial response to its toxicity could be clarified with a better knowledge of the effect of such exposition on the expression of those virulence factors.

Furthermore, in study of bacterial response to stressor factors, the membrane response must to be kept in mind. First of all, the lipidome of two tested P. fluorescens strains was characterized using a HPTLC-MALDI-TOF MSI tool. The PE, PG and PC are found as major compounds of lipidome of these bacteria. In addition, the presence of another, new and not identified yet GP spot (UGP) was detected. This GP is found predominantly in stationary growth phase, indicating its role at this stage of bacterial development. Interestingly, in exposed to 45 ppm of NO2 bacteria, the UGP was not detected, that means at least a decrease, if not a complete loss of this lipid with a possible modification of its production. Opposite results are demonstrated in study of P. fluorescens adaptation to the increasing temperature. In clinical MFN1032 grown at 37°C, the UGP is detected even in exponential growth phase indicating its potential role in temperature adaptation. All together, these results let us suppose, that the UGP may play a role in membrane adaptation to several stress factors. As reported in Chapter 2 Stress factors and Pseudomonas fluorescens lipid adaptation and Table 1, CL often plays an important role in bacterial adaptation to stress [START_REF] Romantsov | Cardiolipin and the osmotic stress responses of bacteria[END_REF]. Like our UGP, CL has a "high" molar mass for bacterial GP and is synthesized, in majority, in stationary growth phase (Kondakova et al., 2015;[START_REF] Mileykovskaya | Cardiolipin membrane domains in prokaryotes and eukaryotes[END_REF]. In correlation with the similarities of PSD spectra of UGP with that ones of PG, these observations let us imagine a possible analogy between CL and UGP, especially since the various analogs of CL were found in Archaea [START_REF] Corcelli | The cardiolipin analogues of Archaea[END_REF]. Naturally, without a complete identification of its chemical structure and metabolic pathway(s) for its synthesis, the role(s) of UGP is still unknown.

Many studies reported the involvement of FAs in membrane adaptive response to stress factors [START_REF] Hachicho | Effect of silver nanoparticles and silver ions on growth and adaptive response mechanisms of Pseudomonas putida mt-2[END_REF][START_REF] Heipieper | Bacterial solvent responses and tolerance: cis-trans isomerization[END_REF]. Our results demonstrate that NO2 does not V. General discussion P a g e 210 | 270 modify the degree of FA saturation, indicating that bacterial FAs are not involved in response to NO2. In fact, in atmosphere, NO2 and its dimer, N2O4 are simple, not charged and lipophilic molecules (see part 1. Nitrogen oxides (NOx)), which freely penetrate bacterial membrane [START_REF] Signorelli | Nitrogen dioxide solubility and permeation in lipid membranes[END_REF] without any modification of FA composition. However, the study of bacterial surface after NO2 exposure demonstrates a significant decrease in membrane electronaccepting properties. Thus, the membrane response to NO2 may be based, among others, on the modifications in GP head groups [START_REF] Möller | Chapter 2 The interaction of reactive oxygen and nitrogen species with membranes[END_REF][START_REF] Pryor | Reaction of nitrogen dioxide with alkenes and polyunsaturated fatty acids: addition and hydrogenabstraction mechanisms[END_REF]. The MS and PSD analyses, performed in our study, did not show any modification (nitration and/or oxidation) of GPs, suggesting the absence of NO2 reactions with bacterial monounsaturated FA chains.

However, the GP composition, in other words the ratio of anionic to zwitterionic GPs, could be modified in order to adapt to nitrosative stress. Thus, the increase of PC production, as well as a possible loss of UGP may explain this membrane modification. In addition, the membrane electron-accepting properties are directly linked with respiratory chain, which was found to be damaged by RNS [START_REF] Auger | The metabolic reprogramming evoked by nitrosative stress triggers the anaerobic utilization of citrate in Pseudomonas fluorescens[END_REF][START_REF] Husain | Nitric oxide evokes an adaptive response to oxidative stress by arresting respiration[END_REF]. In accord with the NO-mediated inhibition of P. aeruginosa respiration [START_REF] Mccollister | Nitric oxide protects bacteria from aminoglycosides by blocking the energydependent phases of drug uptake[END_REF], NO2 could also cause the nitrosylation of [Fe-S] clusters of respiratory chain proteins (for more details see part Proteins), decreasing the electron-accepting membrane properties. In the context of recently demonstrated involvement of bacterial lipids in biofilm regulation [START_REF] Blanka | Constitutive production of c-di-GMP is associated with mutations in a variant of Pseudomonas aeruginosa with altered membrane composition[END_REF][START_REF] Davies | A fatty acid messenger is responsible for inducing dispersion in microbial biofilms[END_REF], the modifications in GP composition could be related with NO2-mediated biofilm modifications. The NO2-mediated alterations of membrane GPs could also impact membrane permeability, facilitating the penetration of several antibiotics, including aminoglycosides, into bacterial cells. Altogether, these data indicate a possible link between the NO2-mediated decrease of aminoglycoside resistance observed in our study and the observed alterations in membrane GP composition.

VI. Conclusions and perspectives

P a g e 213 | 270

For the first time, the response of airborne P. fluorescens to stress factors, such as a major air pollutant NO2, was studied. The tests of the effect of NO2 contamination in terms of membrane response, motility, biofilm formation, antibiotic resistance, as well as expression of several chosen target genes did not show any detectable modification in bacteria, treated with 0.1 and 5 ppm of NO2. While this absence of modification, NO2 could cause bacterial functioning alterations, which are unrevealed through used approaches. In order to better understand the NO2 effect on airborne bacteria, the protein modifications (i.e. S-nitrosylation, reactions of NO2 with metal centers, or tyrosine nitration) and NO2-mediated DNA damages, as well as the possible NO2 sensors and repair mechanisms may be investigated. In this context, it will be interesting to add for this study other NO2 concentrations, such as the mean annual NO2 concentration in France (44 µg/m 3 -0.02 ppm, see part 6.3. NOx level in atmosphere) and to test various exposure times and/or cycles of exposure. In addition, NO in low concentrations between 0.001 and 0.02 ppm (52 and 500 nM respectively) has an effect on bacterial metabolism and homeostasis, altering, bacterial biofilm formation and damaging DNA [START_REF] Barraud | Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa[END_REF][START_REF] Woodmansee | A mechanism by which nitric oxide accelerates the rate of oxidative DNA damage in Escherichia coli[END_REF]. Thus, the assays of such NO2 concentrations might clarify the correlation between the NO and NO2 stress, and establish the response of airborne bacteria to atmospheric NO2 level. Finally, to better mimic environmental conditions, bacteria could be directly exposed to the exhaust gas stream.

Several alterations were observed when the bacteria were treated with 45 ppm of NO2.

Contrarily to the well-studied NO induction of biofilm dispersion, we did not observe the NO2mediated modifications of bacterial biomass. However, the alteration in the biofilm architecture of NO2-treated airborne P. fluorescens MFAF76a is monitores. NO2-exposed P. fluorescens strains show reduced swimming motility, and decreased swarming in case of the strain MFN1032. These NO2-mediated modifications seem to not be linked to c-di-GMP regulatory network, and could be correlated, among other factors, with alterations in flagellar motion, type IV pili and surfactant production. In this way, the supplementary studies concerning NO2medited surfactant production, and flagellar synthesis will complete this study, allowing an enhanced understanding of the NO2 effect on bacterial cells.

The analyses of NO2-mediated gene expression demonstrated, that NO2, like its homolog NO, upregulated the hmp-homologue gene in P. fluorescens strains MFAF76a and MFN1032 expression, suggesting a possible common pathway between NO and NO2 detoxification. While the Hmp-meidiated NO2 detoxification was proposed to carry out through the NO2 reduction pathway [START_REF] Poole | Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12[END_REF], the expression of nirB-homolog genes, coding for assimilatory nitrite reductase NirBD was not found to be impacted by NO2 treatment in the present work.

However, in order to better understand the mechanism of NO2 detoxification, the Hmp-, Nir-P a g e 214 | 270

and Nas-mediated mechanisms should be investigated in more details. Thus, the supplementary proteomic studies in combination with the site-directed mutagenesis could be performed. In addition, NO2 appears to induce the expression of mexEF-oprN genes, coding for the RND efflux pump. Its overexpression could be involved in the observed increase of P. fluorescens resistance to ciprofloxacin and chloramphenicol, while the resistance to aminoglycosides, including tobramycin and kanamycin is decreased. This decrease seems to be independent on the expression of mexXY genes, and could be linked, among other factors, with observed in our study NO2-mediated alterations of membrane GPs.

Concerning the lipidome studies, for the first time, in the lipidome of P. fluorescens, a new and currently unknown glycerophospholipid (UGP) is observed. Remarkably, this GP is undetected in bacteria exposed to high NO2 concentration. Interestingly, in airborne P. fluorescens grown without stress addition, this UGP is only detected in stationary growth phase. Conversely, in clinical P. fluorescens strain growing at human body temperature, the UGP is supposed to be produced even in exponential phase of growth. All together, these data indicate, that UGP may be involved in P. fluorescens adaptation to stress conditions. In this way, the complete chemical characterization of this GP, with the establishment of pathway(s) for its synthesis is an attractive research opportunity. No NO2 effect on fatty acid composition was observed, indicating that NO2 does not modify the membrane fluidity. While the absence of NO2-mediated modification of the net membrane charge, the membrane electron-accepting properties were deceased after NO2 exposure. Given that several factors could be involved in these NO2-mediated surface modifications, the supplementary quantitative studies of NO2 effect on membrane GPs and proteins, as well as the bacterial respiration upon NO2-mediated nitrosative stress conditions could be evaluated.

Finally, the bacterial response to NO2 contamination, established here, is just the beginning of the long road ahead to achieve the goal(s) of a wide area of researches about the effect of atmospheric pollution on air microbiota. The ultimate goals of this topic would be to establish the NO2 effect on bacterial functioning, as well as toxicity and pathogenicity against eukaryotic hosts. In this context, the virulence factors expression, in combination with genomic studies and cytotoxicity tests should be used. S2. Primer sequences of the indicated genes used for quantitative RT-PCR assay. The high distribution of Pseudomonas fluorescens group is linked to its ability to adapt to stress factors. This work goaled the response of an airborne P. fluorescens MFAF76a, and its clinical standard P. fluorescens MFN1032 to environmental changes in order to refine the specific adaptation of airborne bacteria. First the HPTLC-MALDI TOF MSI tool defined glycerophospholipid (GP) composition of both strains. In stationary growth phase, an unknown GP, in short UGP, was found and seemed to be involved in temperature adaptation for the clinical strain.

VII. References

Supplementary Table

After exposure to 0.1, 5 and 45 ppm concentrations, the bacterial response to NO2 was defined through motility, biofilm formation, antibiotic resistance and expression of several chosen target genes. While no change in parameters was seen in bacteria exposed to 0.1 and 5 ppm of NO2, several alterations were occurred with a bacterial exposure to 45 ppm. NO2 seemed to bias the UGP production, reduced P. fluorescens swim and decreased swarm only for MFN1032 strain. Biofilm formed by NO2-treated MFAF76a showed increased maximum thickness, with no change in c-di-GMP intracellular level. Expression of the hmp-homologue gene involved in NO detoxification was upregulated in response to NO2, suggesting a possible common pathway between NO and NO2 detoxification. Finally, NO2 was found to increase bacterial resistance to ciprofloxacin and chloramphenicol. Thus the resistance nodulation cell division (RND) MexEF-OprN efflux pump encoding genes were highly upregulated in both strains. Together these findings implement the first model of bacterial response to NO2 toxicity and the role(s) of GP in bacterial adaptation to environmental changes.

FiguresFigure 1 .

 1 Figures Figure 1.Functional range and environmental niches of the Pseudomonas genus, highlighting the broad distribution of the P. fluorescens species ..............................................................................................................

  Figure 1. Functional range and environmental niches of the Pseudomonas genus, highlighting the broad distribution of the P. fluorescens species ..............................................................................................................

Figure 2 .

 2 Figure 2. Phylogenetic relationships of Pseudomonas genus bacteria among 16S ribosomal RNA genes ...........

Figure 3 .

 3 Figure 3. Microscopic and macroscopic aspect of P. fluorescens .........................................................................

Figure 4 .

 4 Figure 4. Swimming, swarming, and twitching motilities of P. aeruginosa PAO1 ..............................................

Figure 5 .

 5 Figure 5. Pathway of biofilm development in P. aeruginosa ................................................................................

Figure 6 .

 6 Figure 6. Scanning electron micrographs of P. fluorescens biofilms ....................................................................

Figure 7 .

 7 Figure 7. Structure and physiological functions of c-di-GMP ..............................................................................

Figure 8 .

 8 Figure 8. A proposed model for regulation of P. fluorescens biofilm formation ..................................................

Figure 9 .

 9 Figure 9. Pseudomonas cell envelope ...................................................................................................................

Figure 10 .

 10 Figure 10. Adaptation of glycerophospholipid structure and composition in Pseudomonas ................................

Figure 11 .

 11 Figure 11. Modifications of fatty acids structure in Pseudomonas spp. ................................................................

Figure 12 .

 12 Figure 12. Schematic model of regulation of the Cti enzymatic mechanism by membrane fluidity.....................

Figure 13 .

 13 Figure 13. Enzyme stabilization at high osmolarity ..............................................................................................

Figure 14 .

 14 Figure 14. Scheme of the toxicity of hydrocarbons in the cell membranes ..........................................................

Figure 15 .

 15 Figure 15. Interactions of cell envelope with metals .............................................................................................

Figure 16 .

 16 Figure 16. Glycerophospholipid rearrangement that can occur on binding cationic antimicrobial agents............

Figure 17 .

 17 Figure 17. Lewis structure of (A) NO and (B) NO2 ..............................................................................................

Figure 18 .

 18 Figure 18. Redox relationships of NOx with other RNS species ...........................................................................

Figure 19 .

 19 Figure 19. Scheme of biosynthesis of nitric oxide from L-arginine in mammalian cells ......................................

Figure 20 .

 20 Figure 20. Schematic model of NOx synthesis and assimilation in plants ............................................................

Figure 21 .

 21 Figure 21. Bacterial nitrogen cycle .......................................................................................................................

Figure 22 .

 22 Figure 22. Denitrification pathway in Pseudomonas spp. species and enzymes, catalyzed the NOx reduction ....

Figure 23 .

 23 Figure 23. Schematic representation of the denitrification genes locus in Pseudomonas aeruginosa PAO1 .......

Figure 24 .

 24 Figure 24. Schematic model of the regulatory network controlling the denitrification genes in P. aeruginosa ...

Figure 25 .

 25 Figure 25. Bacterial targets of RNS ......................................................................................................................

Figure 26 .

 26 Figure 26. Schematic representation of NO2 reaction with polyunsaturated lipids ...............................................

Figure 27 .

 27 Figure 27. Reaction of NO and lipid peroxyl radicals ..........................................................................................

Figure 28 .

 28 Figure 28. Reaction of RNS with [Fe-S] cluster ...................................................................................................

Figure 29 .

 29 Figure 29. Free radical pathways to tyrosine nitration ..........................................................................................

Figure 30 .

 30 Figure 30. DNA damage pathways resulting from RNS exposure........................................................................

Figure 31 .

 31 Figure 31. Permeability of reactive species across lipid membrane ......................................................................

Figure 32 .

 32 Figure 32. Model of NalA-and NasT/S-dependent expression of nitrate assimilation operon in P. aeruginosa PAO1 ....................................................................................................................................................................

Figure 33 .

 33 Figure 33. Schematic illustration of the MexEF-OprN RND efflux pump ...........................................................

Figure 34 .

 34 Figure 34. Reprogramming of P. fluorescens metabolism in order to adapt to nitrosative stress .........................

Figure 35 .

 35 Figure 35. The proposal model of the KatA-mediated NO sensing ....................................................................

Figure 36 .

 36 Figure 36. Model for the transcriptional regulation of nbdA through NO...........................................................

Figure 37 .

 37 Figure 37. GcbA-mediated modulation of the BdlA-dependent biofilm dispersion ...........................................

Figure 38 .

 38 Figure 38. H-NOX-dependent control of biofilm formation through the regulation of c-di-GMP level ............

Figure 39 .

 39 Figure 39. NOx sources in atmosphere ................................................................................................................

Figure 40 .

 40 Figure 40. Evolution of NOx emission in metropolitan France from 1990 to 2013 ............................................

Figure 1 .

 1 Figure 1. Functional range and environmental niches of the Pseudomonas genus, highlighting the broad distribution of the P. fluorescens species Adapted from Scales et al., 2014.

Figure 2 .

 2 Figure 2. Phylogenetic relationships of Pseudomonas genus bacteria among 16S ribosomal RNA genesAdapted form[START_REF] Bodilis | Variable copy number, intra-genomic heterogeneities and lateral transfers of the 16S rRNA gene in Pseudomonas[END_REF]

Figure 3 .

 3 Figure 3. Microscopic and macroscopic aspect of P. fluorescens A. Scanning electron micrograph of P. fluorescens (www.scienceclarified.com). B. Macroscopic aspect of P. fluorescens SBW25, pyoverdine production (Spiers et al., 2013).

  Figure 4. Swimming, swarming, and twitching motilities of P. aeruginosa PAO1A. Macroscopic motility assays. Swimming motility on a semi-liquid agar plate (Left); Swarming motility on a semisolid agar plate (Middle); Macroscopic assay of twitching motility (Right). B. Microscopic aspect of Pseudomonas motility. Modified according M. Harunur and A. Kornberg, 2000.

Figure

  Figure 5. Pathway of biofilm development in P. aeruginosa

. 3 and 4 )

 34 . The mature biofilm is typically characterized by macrocolonies, located in extracellular matrix and separated by fluid-filled channels as shown in Figure 6. These channels transport nutrients and oxygen to the bacteria and aid in waste removal (Davey & O'Toole, 2000; Hinsa P a g e 27 | 270

Figure 7 .

 7 Figure 7. Structure and physiological functions of c-di-GMP In bacteria, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is controlled by diguanylate cyclases (DGC) with GGDEF domains (red) and specific phosphodiesterases that carry EAL or HD-GYP domains (blue).The high c-di-GMP level reduces motility and stimulates various biofilm-associated functions, such as the formation of fimbriae and other adhesins and various matrix exopolysaccharides. pGpG, 5'-phosphoguanylyl-(3'-5')-guanosine. pGpG is subsequently split into two GMP molecules. Adapted from[START_REF] Hengge | Principles of c-di-GMP signalling in bacteria[END_REF]

  Tatiana Kondakova; François D'Heygère; Marc J. Feuilloley; Nicole Orange; Hermann J. Heipieper; Cécile Duclairoir Poc Chemistry and Physics of Lipids, 190 (2015), 27-42; doi:10.1016/j.chemphyslip.2015.06.006

Figure 11 .

 11 Figure 11. Modifications of fatty acids structure in Pseudomonas spp.

Figure 12 .

 12 Figure 12. Schematic model of regulation of the Cti enzymatic mechanism by membrane fluidity

  The bacterial response on temperature fluctuations is one of the most studied topics of bacterial lipid adaptation. The temperature can change bacterial membrane fluidity(Mansilla et al.,

Figure 14 .

 14 Figure 14. Scheme of the toxicity of hydrocarbons in the cell membranes According Heipieper and Martínez, 2010.

Figure 15 .

 15 Figure 15. Interactions of cell envelope with metals Thin section of E. coli K12 envelope with A. Cu and B. Mg. The arrows point to the membrane of a single vesicle. No stain other than the initial binding metal was used for contrast. Adapted from Beveridge & Koval, 1981.

Figure 17 .

 17 Figure 17. Lewis structure of (A) NO and (B) NO2

Figure 19 .

 19 Figure 19. Scheme of biosynthesis of nitric oxide from L-arginine in mammalian cellsAdapted from[START_REF] Moncada | XIV. International Union of Pharmacology Nomenclature in nitric oxide research[END_REF][START_REF] Bruckdorfer | The basics about nitric oxide[END_REF]

  Since NO could be formed during the denitrification pathway in Pseudomonas species, this mechanism will be discussed below. As demonstrated in Figure22, the denitrification consists of four sequential steps catalyzed by the denitrification enzymes, such as nitrate reductase (NAR/NAP), nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (N2OR).

Figure 22 .

 22 Figure 22. Denitrification pathway in Pseudomonas spp. species and enzymes, catalyzed the NOx reduction

Figure 23 .

 23 Figure 23. Schematic representation of the denitrification genes locus in Pseudomonas aeruginosa PAO1 A. The nar locus. The narXL and narK1K2GHJI share a divergently oriented promoter region. B. The nap locus. C. The nir locus. D. The nos locus. The scale is made for each locus. bp: base pair. According: www.pseudomonas.com.

Figure 24 .

 24 Figure 24. Schematic model of the regulatory network controlling the denitrification genes in P. aeruginosaUnder anaerobic or low O2 conditions, ANR (red color) activates the expression of DNR (blue color). The latter activates the expression of all denitrification genes in response to NOx. A two-component nitrate sensor regulator, NarXL (green color) positively regulates expression of the nar genes, and represses the expression of nap genes. The NirQ (orange color) is involved in the fine tuning of the activities of nir and nor genes. Adapted from[START_REF] Arai | Regulation and function of versatile aerobic and anaerobic respiratory metabolism in Pseudomonas aeruginosa[END_REF]

  P a g e 87 | 270 Some of the most important antibacterial actions of RNS are shown in Figure 25 and discussed below.

  Figure 26.4 and 5).

Figure 26 .

 26 Figure 26. Schematic representation of NO2 reaction with polyunsaturated lipids Adapted from Augusto et al., 2002.

Figure 27 .

 27 Figure 27. Reaction of NO and lipid peroxyl radicals Adapted from O'Donnell et al., 1999.Therefore, NO potentially interacts with oxidized lipid at several levels. Rubbo proposed that, depending of the rate of ROS production, NO both stimulates and inhibits lipid peroxidation and yields to the nitrogen-containing derivatives of oxidized lipids. Its protective role includes (i) redirection of O2 --and ONOO --mediated cytotoxic reactions to other oxidative pathways, and (ii) termination of lipid peroxidation propagation reaction (Figure27.1)[START_REF] Rubbo | Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives[END_REF]. Thus, NO reacts with a lipid peroxyl radical (LOO -) to give the lipid peroxynitrite (LOONO -) intermediate, which is rapidly hydrolyzed to secondary radical species (Figure 27.2). Two free radicals, NO2 and lipid alkoxyl radical (LO) may be formed (Figure 27.3) and could react with additional NO (Figure 26.4), or undergo a termination of lipid peroxidation

Figure

  Figure 28. Reaction of RNS with [Fe-S] cluster The RNS reaction with [Fe-S] cluster is demonstrated in example of SoxR activation. Green ovals: DNA binding domains; colorless ovals: ironbinding domains. SoxR is shown as a homodimer. (A) Redoxregulation. When the [2Fe-2S] centers of SoxR are in the reduced state, the protein transcriptionally inactive. Oneelectron oxidation converts the protein to a potent transcription activator. (B) Model for activation of SoxR by nitrosylation. The intact [2Fe-2S] cluster of SoxR is directly modified by NO to generate dinitrosyl-iron-dithiol clusters. Adapted from Ding and Demple, 2000. One of attractive examples of this reaction is presented in Figure 28 by SoxR transcriptional regulator that controls an oxidative stress response in E. coli (Ding & Demple, 2000). When the [2Fe-2S] centers of SoxR are in the reduced state [2Fe-2S] 1+ , the protein is transcriptionally inactive. As shown in Figure 28A, the ROS cause one-electron oxidation to yield [2Fe-2S] 2+

Figure 29 .

 29 Figure 29. Free radical pathways to tyrosine nitrationAdapted form[START_REF] Radi | Nitric oxide, oxidants, and protein tyrosine nitration[END_REF]

reported in Figure 30 .

 30 The N2O3 leads particularly to either direct or indirect nitrosative DNA damage. Direct damage results in the nitrosation of primary amines on DNA bases leading to the deamination. Any DNA base containing an exocyclic amino group can undergo deamination, whose the end result is the net replacement of an amino group by a hydroxyl group. Therefore, adenine, cytosine, 5-methylcytosine, and guanine can all be deaminated to form hypoxanthine, uracil, thymine and xanthine respectively, all of which are potentially mutagenic if unrepaired. The DNA repair pathways present a very interesting area of current researches and resumed in this work (see part 4.2

Figure 35 .

 35 Figure 35. The proposal model of the KatA-mediated NO sensing Under anaerobic or low O2 conditions, ANR activates katA expression, resulting in KatA production. Using an unidentified mechanism, KatA may sense a toxic concentration of NO and buffers NO to protect bacteria against nitrosative stress.

Figure

  Figure 36. Model for the transcriptional regulation of nbdA through NO (A) Without NO addition nbdA expression is low, resulting in only a few NbdA proteins that do not significantly contribute to the cellular c-di-GMP concentration. (B) NO induces expression of nbdA, resulting in elevated level of NbdA proteins, reduced cellular c-di-GMP levels and increased biofilm dispersion.Adapted from[START_REF] Li | NO-induced biofilm dispersion in Pseudomonas aeruginosa is mediated by an MHYT domain-coupled phosphodiesterase[END_REF]

Figure 37 .

 37 Figure 37. GcbA-mediated modulation of the BdlA-dependent biofilm dispersion In planktonic and initially attached cells, GcbA is produced and synthesizes the c-di-GMP (blue line). This promotes the transition from reversible to irreversible surface attachment. Via c-di-GMP level, GcbA contributes to the cleavage of BdlA in initially attached cells (black line). Other DGC(s) (green line) potentially act in concert with GcbA to regulate the c-di-GMP level and, as consequence, BdlA. Conditions of reduced cellular motility and NO production lead to a reduction in GcbA. BdlA directly interacts with the PDEs RbdA and DipA, modulating their activity to reduce c-di-GMP levels. During the dispersion process, DipA and RbdA reduce c-di-GMP level to facilitate the sessile-motile transition and to induce GcbA production, activating a mechanism, which will allow the detached cells to attach to a new surface. The relative sizes of symbols indicating proteins and c-di-GMP correlate with their relative abundances in the cell during the indicated growth mode. Adapted from Petrova et al., 2015.

Figure 38 .

 38 Figure 38. H-NOX-dependent control of biofilm formation through the regulation of c-di-GMP level (A) H-NOX signaling in S. woodyi. The H-NOX protein HnoX interacts with a dual diguanylate cyclase/phosphodiesterase (DGC/PDE) enzyme. The NO-bound H-NOX state inhibits DGC, and increases PDE activity, leading to increased c-di-GMP hydrolysis and lower biofilm formation in response to NO. (B) H-NOX signaling promotes biofilm formation in response to NO in S. oneidensis and V. cholerae. The NO-bound H-NOX state inhibits autophosphorylation of the associated histidine kinase HnoK. HnoK possesses three phosphotransfer targets: HnoB, HnoC, and HnoD. Adapted from Plate & Marletta, 2013a.

  According the Interprofessional Technical Centre for Studies on Air Pollution (CITEPA), the NOx emission trends observed during the period from 1990 to 2013 are on the decrease (CITERA, 2014). These observations are globally encouraging and especially reflect the reduction actions implemented by European Union and French government (see par 6.4. NOx standards).The Figure 40 below summarizes total emissions over the period 1990 -2013 for NOx in France.

Figure 40 .

 40 Figure 40. Evolution of NOx emission in metropolitan France from 1990 to 2013 The data are presented as total NOx emission according Interprofessional Technical Centre for Studies on Air Pollution. According CITEPA, 2014. These data are all the more encouraging, because the NOx emission value in 2013 (990 kiloton) is close to the objective for 2010, fixed in Gothenburg Protocol as 860 kiloton of NOx per year (Review of the Gothenburg Protocol. Report of the Task Force on Integrated Assessment Modelling and the Centre for Integrated Assessment Modelling, 2007). However, there is a



  Characterization of the lipidome of airborne P. fluorescens strain using a new mass spectrometric method HPTLC-MALDI TOF MSI.  Establishment of the membrane response of airborne P. fluorescens to environmental modifications, like increasing temperature in the context of climate change, and NO2 as a marker of air pollution. For this, the membrane response of airborne P. fluorescens was compared to that of clinical P. fluorescens strain. This analogy of P. fluorescens strains from different ecological niches could give supplementary information about the specific for airborne bacteria mechanism(s) of adaptation to environmental modifications. Evaluation of the response of airborne P. fluorescens to air pollution. This investigation includes the first screen of potential mechanism(s) of adaptation of airborne bacteria to NO2 air contamination, including NO2 detoxification pathways, modification of phenotypes and genes expression caused by NO2.GPs standards, MALDI matrix (2,5-dihydroxybenzoic acid; DHB), solvents (chloroform, methanol, ethanol, trimethylamine, acetonitrile, hexane, ethyl acetate, hexadecane and decane) and GP dyes (ninhydrin, azure A and primuline) were obtained from Sigma-Aldrich (Saint-Quentin-Fallavier, France). The 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-nonadecanoyl [19:0-PE (18:1/18:1)+Na] + was provided by Avanti Polar Lipids, Inc.(Alabaster, Alabama, USA). HPTLC silica gel 60 plates F254 (75 x 50 mm in size, on aluminum backs) were obtained from Merck (Darmstadt, Germany).Sodium acetate, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), acid phenol, chloroform/isoamyl alcohol, diethylpyrocarbonate, tris-acetate-ethylenediamine tetraacetic acid (TAE), used in RNA manipulations, as well as xanthosine-(3',5')-cyclic monophosphate (cXMP) used in c-di-GMP quantification, were provided by Sigma-Aldrich (Saint-Quentin-Fallavier, France). All chemicals were of the highest commercially available purity and used without any further purification.

  A580=0.08 and added to a 96-well test plate (Nunc TM , Roskilde, Denmark) containing different concentrations of antibiotics in triplicate. The test plate containing antibiotic dilutions and III. Materials and methods P a g e 121 | 270

  line at the three -phase contact point. The w acquisition was made in the first 10 s once the drop stabilized.

  to control reliable steady-state environmental conditions inside the exposure chambers. Three concentrations of NO2 were used in this study: 0.1 ppm; 5 ppm and 45 ppm. After exposure bacteria were resuspended to A580=2 in 20 mL of sterile saline solution.

  Calibration was performed in two steps. The external calibration of the apparatus is performed daily in FlexControl software. This calibration was made using the DHB matrix and the Peptide Calibration Standard II (Bruker Daltonics, Bremen, Germany) covering a mean mass range between 200 and 3500 Da. Supplementary internal calibration was made using sphingomyelin (1 μg/mL) mixed with the matrix solution before dipping. Peaks generated by these standards were used to calibrate TLC MALDI software. This double calibration improved the measurement accuracy of bellow 0.1 Da for lipid identification. Lipids were identified manually III. Materials and methods P a g e 125 | 270 using the LIPID MAPS database. All experiments were performed in at least three independent experiments with three replicates for each experimental condition.

  9 ) were harvested by centrifugation for 2 min, 8,000 g, 4°C. Bacterial pellets were resuspended in 600 µL ice-cold extraction buffer, composing to C2H3N/ CH3OH/ H2O (2/2/1, v/v/v) with and without of 5 µL of a 10 µg/mL solution of xanthosine-(3',5')-cyclic monophosphate (cXMP)

  fluorescens IV. Results. Chapter 4 Study of P. fluorescens lipidome P a g e 139 | 270 A new study of the bacterial lipidome: HPTLC-MALDI-TOF imaging enlightening the presence of phosphatidylcholine in airborne Pseudomonas fluorescens MFAF76a Tatiana Kondakova, Nadine Merlet-Machour, Manuel Chapelle, David Preterre, Frédéric Dionnet, Marc Feuilloley, Nicole Orange, Cécile Duclairoir Poc Research in Microbiology 166 (2015) 1-8; doi:10.1016/j.resmic.2014.11.003 Framework In previous studies, several P. fluorescens strains have been isolated from harbor terminal in Rouen (France), including MFAF76a strain, characterized as able to growth at 37°C and express noticeable virulence, particularly against human epithelial pulmonary cells. Although a myriad of proteomics and genomics studies of P. fluorescens adaptability to ecological niches and environmental conditions, the lipidome of P. fluorescens and its role(s) in bacterial adaptation were poorly studied. The P. fluorescens glycerophospholipid studies undergo the excessive development of genomic and proteomic methods and were realized only using thin-layer chromatography (TLC) methods, currently used in the 1970s. In this study, we were interested to characterize the membrane glycerophospholipid composition of this airborne P. fluorescens MFAF76a in order to (i) establish a complete picture of P. fluorescens lipidome; and (ii) use obtained data in future studies of response of P. fluorescens lipidome to environment modifications.

Chapter 5

 5 Effect of stress factors on membrane of P. fluorescens IV. Results. Chapter 5 Effect of stress factors on membrane of P. fluorescens P a g e 151 | 270FrameworkAs humans, airborne bacteria undergo the effect(s) of atmospheric changes, including urban air pollution. After characterization of lipidome of airborne P. fluorescens MFAF76a, we decided to initiate the study of membrane response of this bacterial strain to well-known air pollutant NO2. To the best of our knowledge, the effect of NO2 on membrane of airborne bacteria was not established yet. In this study the lipidome of airborne P. fluorescens was investigated in exponential and stationary growth phase using the new HPTLC-MALDI TOF MSI tool presented in Chapter 4 Study of lipidome of airborne P. fluorescens. First, the effect of increasing temperature (37°C) on P. fluorescens membrane was studied in order to establish the pathway(s) employed by P. fluorescens membrane to adapt to environmental modifications.

Chapter 6

 6 Pseudomonas fluorescens response to NO 2 toxicity IV. Results. Chapter 6 Pseudomonas fluorescens response to NO2 toxicity P a g e 185 | 270 P. fluorescens airborne strain MFAF76a and clinical strain MFN1032 response to NO 2 stress Tatiana Kondakova, Chloé Catovic, Magalie Barreau, Michael Nusser, Gerald Brenner-Weiss, Sylvie Chevalier, Frédéric Dionnet, Nicole Orange, Cécile Duclairoir Poc Submitted in Frontiers in Microbiology Research Topic Atmospheric Microbiology Framework After the establishment of P. fluorescens membrane response to NO2 contamination, we were interested by the question "what happens when NO2 penetrate in bacterial cytoplasm?" Given the high adaptability of P. fluorescens to various environmental conditions, these bacteria have to develop the pathway(s) for NO2 detoxification. In addition, NO2 may change genetic and/or phenotypic expression, including surface adhesion or motility. Given the lack of information about bacteria -NO2 interactions, this study was based on the hypothesis of the similarity of bacterial response to NO2 and, its homolog, NO. Thus, as previously described, two P. fluorescens strains, airborne MFAF76a and clinical MFN1032 were exposed to three NO2 concentrations, in order to (i) establish concentration-dependent response of both P. fluorescens to NO2 and (ii) to test the bacterial adaptation mode to NO2 thresholds. As results, this study establishes the pathways, employed by both P. fluorescens to detoxify and release NO2 from cytoplasm, as well as the modifications in bacterial biofilm formation and antibiotic resistance, caused by NO2 exposure. IV. Results. Chapter 6 Pseudomonas fluorescens response to NO2 toxicity P a g e 203 | 270

  Des particules au NO2 comme effecteur toxique principal des émissions de moteurs diesel, un problème lié à l'évolution des réglementations Euro successives. Pollution-Atmospherique. text, . . text, . Morita, Y., Tomida, J. & Kawamura, Y. (2014). Responses of Pseudomonas aeruginosa to antimicrobials. Front Microbiol 4.

THESE

  Pseudomonas adaptation to stress factors: role of membrane lipids andPseudomonas fluorescens response to NO2

Keywords:

  Pseudomonas fluorescens, adaptation, glycerophospholipid, fatty acids, pollution, nitrogen oxides Adaptation de Pseudomonas aux facteurs de stress : rôle des lipides membranaires et réponse de Pseudomonas fluorescens au NO2La large distribution des Pseudomonas fluorescens est liée à leur grande adaptabilité aux facteurs de stress tels que les variations environnementales. Ce travail avait pour objet la réponse spécifique au milieu aéroporté de P. fluorescens, comme l'aéroportée MFAF76a et la clinique MFN1032, comme standard. La technique récente HPTLC-MALDI-TOF MSI a permis de caractériser les divers glycérophospholipides (GP) des deux souches. En phase stationnaire de croissance, un GP inconnu (UGP -unknown GP) a été isolé et semble intervenir dans l'adaptation à la température de la souche clinique MFN1032. Quant au stress NO2 gaz, les deux souches ont été exposées aux concentrations: 0.1, 5 et 45 ppm. Leurs phénotypes ont été confrontés à l'expression de quelques gènes ciblés. Pour les valeurs standard 0,5 and 5 ppm en NO2, aucun paramètre n'est modifié. Par contre, une réponse bactérienne est constatée suite à l'exposition à 45 ppm de NO2. Cette exposition semble impacter la production d'UGP. De plus hmp et mexEF-oprN, codant respectivement pour la flavohémoglobine et pour la pompe à efflux RND, se trouvent surexprimés, corroborant l'évolution de la résistance bactérienne aux antibiotiques. Contrairement au NO, aucune altération de la biomasse de biofilm n'est observée pour le NO2, qui favorise, cependant, l'augmentation de son épaisseur chez MFAF76a, mais aussi l'inhibition du swarming et la diminution du swimming, avec un taux de c-di-GMP stable. Ce faisceau de résultats offre, pour la première fois, la réponse bactérienne et le rôle des GP lors de stress comme NO2 ou à la température, autre modification environnementale.

Table

Table 1 . Chemical structures of membrane fatty acids

 1

	Fatty acid	Structure	Effect on membrane fluidity
	C16:0		Decreases membrane fluidity
	cis-C16:1		Increases membrane fluidity
			Increases resistance to
	trans-C16:1		solvents and
			temperature
	cyclopropane-
	C17:0

Table 2 . Summary table of pathways, involved in bacterial lipid adaptation to various stress factors Stress factor Lipid adaptation mechanism SFA cis- UFA trans- UFA cyclo- FA Poly- UFA

 2

Bacterial nitrogen cycle

	Nitrate assimilation (red color)
	leads to the conversion of
	inorganic nitrogen N(+V) to a
	biologically useful form N(-III).
	The nitrification (green color) is
	the oxidative conversion of
	ammonia N(-III) to nitrate
	N(+V). Denitrification (blue
	color) is a respiratory process
	when	nitrate	N(+V)	is
	successively reduced to nitrite
	N(+III), nitric oxide N(+II),
	nitrous oxide N(+I), and
	dinitrogen N(0). The oxidation
	state of nitrogen is shown in
	bracket.
	Adapted from Watmough et al.,
	1999.

, blue color). P a g e 82 | 270

Figure 21.

Table 3 . Threshold values for NOx

 3

				Time (min)
	NOx threshold	10		20		30		60
		NO	NO2	NO	NO2	NO	NO2	NO NO2
	Significant lethal effect (ppm)	NF	118	NF	98	NF	88	NF	73
	Irreversible effects (ppm)	150	60	120	55	100	50	80	40
	Reversible effects (ppm)	NF	5	NF	5	NF	5	NF	5
	According INERIS, 2011a, b. NF: not found

Table 4 . NO2 air quality standards

 4

	NO2 standard	µg/m 3	Concentration	ppb
	Hourly NO2 standard	200		106
	Annual NO2 standard	40		21.2

Table 5 . Composition of growth media

 5

	Medium	Composition (per liter)
		Tryptone	10 g
	Luria Bertani (LB) (pH 7)	Yeast extract NaCl Agar (solid medium)	5 g 5 g 15 g
		H2O qsp
		K2HPO4	10.450 g
		KH2PO4	3.810 g
		(NH4)SO4	2.010 g
	Davis Medium Broth (DMB)	MgSO4	0.049 g
		Glucose	4.320 g
		Agar	15.000 g
		H20 qsp

  on HPTLC plate. The plate was heated at 110°C for 15 min to visualize the phospholipid spots. The 2% azure A in 1 mM sulfuric acid was applied to HPTLC plate.

	III. Materials and methods
	ethanol was sprayed Retention factors (Rf) are calculated using the Sweday JustTLC software (v. 4.0.3, Lund,
	Sweden).
	P a g e 124 | 270

Table 6 )

 6 . If necessary the samples were retreated with

	TubroDnase for 1 h. The TurboDnase was inactivated by adding of 1 µL of 0.5M EDTA at
	75°C for 10 min. The quality of total RNA was controlled by 2% agarose gel electrophoresis
	with Tris-Acetate-Ethylenediamine tetraacetic acid (TAE) 1X. The RNA concentration was
	measured by spectrophotometry at A260. The absence of contamination by proteins and organic
	solvents was controlled by measurement of ratios A260/A280 and A260/A230.

Table 6 . Primer sequences used for quantitative RT-PCR assay

 6

	Gene	Forward primer (5'-3')	Reverse primer (5'-3')
	Pseudomonas fluorescens MFAF76a
	hmp	AAACCGCGATCTATGACCAG	GCTTCATGCCGATGTACTGA
	Pfl76a_nirB	AATTGAAAGTCACCGGCATC	GAATCTGCCGGAAATACCAA
	bdlA	ACATTCTTACCGCCAACCAG	ACTGTCCGTGGAAGAACTCG
	ndbA	AGATGAAAGAGCCGATCGAA	TGAAGAAGTGCAGACCGTTG
	mexE	ACTCAAACATTTGCGCTTCC	AATTCGTCCCACTCGTTGAC
	mexF	GAGTGGACCGACCTGACCTA	GCAAGTCCCACCAGTACGAT
	oprN	ACCTCAACAACCAGCAGGAG	AGGTCGACGGTCAGTTTGTC
	mexX	CGCAGCGAGTTTCCCAAT	TGGATGGTTGCCTGCTCAA
	mexY	GGCTGGGTCGGCTATGC	TCAGCAGGACCACGTAGATCAT
	Pseudomonas fluorescens MFN1032
	hmp	GATAAACCGCTGGTGCTGAT	ATCCTCGGCGTAGCAGTAGA
	Pfl1032_nirB	CGCCCTATGTGCTGGTGTT	ATCTGCCGGGCCATCTC
	dipA	ACGAAGACATCACCCAGACC	TTGATGCGCTTGAAGTTGTC
	mucR	CCTTGTCGTGATTTCCCTGT	GCCAACATCCCGATAAAGTG
	mexE	CCGACAAGGTTTACGCCTAC	GTGTATTCGCCCTTGCTGTT
	mexF	CTGACCCTGACCATCACCTT	ATTGAGGATCGCGTAGTTGG
	oprN	GTTGCTGGCATTGGAAGAGT	GTCGAGCAACACCAGGAAGT
	mexX	CCGTTGCCGGGTAAATTG	GAACTCGCTGCGCAGGAT
	mexY	CAGTGGTTTTGAGTTCCGTTTG	GCAGCGTCCAGCAATTCAGT
	All pairs of primers were tested with serial dilutions of cDNA mixture obtained previously.
	Each dilution of cDNA quantity spectrum was analyzed in duplicate. Based on results of cDNA

Table 7 .

 7 genome drafts of P. fluorescens MFN1032 and MFAF76a. First of all, a research of homologous sequences in P. fluorescens annotated genomes was performed using The genome alignment and the studies of genomic neighborhood were performed in multiple genome alignment software Mauve v. 2.3.1 (Darling lab, Sydney, Australia), according to[START_REF] Darling | Mauve: multiple alignment of conserved genomic sequence with rearrangements[END_REF]

	Pseudomonas genome database (http://pseudomonas.com/). The conserved nucleotide
	sequences were identified in P. fluorescens MFN1032 and MFAF76a using Blast+ (Stand-
	alone) software (v. 2.2.30, NCBI, USA) according to Altschul et al., 1997 and compiled in

Table 7 . Genes identified with the Blast 2.2.30+ software

 7 Study of lipidome of airborne P.

				GenBank accession			References
			Gene name	number	Protein name	Protein description
				MFAF76a MFN1032			Strain	Bibliographies
	NO2	detoxification	hmp Pfl1032_nirB Pfl76a_nirB	KR818822 KT186428	KR818823 KT070320	flavohemoglobin subunit NirB putative nitrite reductase large	nitric oxide dioxygenase assimilatory nitrite reductase P. aeruginosa E. coli P. aeruginosa	Arai et al., 2005; Poole et al., 1996; Gardner et al., 1998 Romeo et al., 2012
			bdlA	KT186436		putative protein BdlA	biofilm dispersion protein	P. aeruginosa	Petrova and Sauer,
							BdlA		2012b
			dipA		KT186437	putative phosphodiesterase	protein, possessing EAL	P. aeruginosa	Roy et al., 2012
	Biofilm and motility		mucR ndbA	KT186444	KT186445	DipA putative phosphodiesterase MucR putative phosphodiesterase	domain and involved in biofilm dispersion protein possessing both GGDEF and EAL domains and involved in NO-mediated biofilm dispersion protein possessing both	P. aeruginosa P. aeruginosa	Li et al., 2013 Li et al., 2013
						NdbA	GGDEF and EAL domains and
							involved in NO-mediated
							biofilm dispersion
			mexE	KT070324	KT070323	putative membrane fusion	RND family efflux	P. aeruginosa	Maseda et al.,
						protein MexE	transporter		2000; Fetar et al.,
	Antibiotic	resistance	mexF oprN	KT070321 KT070325	KT070322 KT186432	putative cation efflux protein MexF putative multidrug efflux RND transporter, outer membrane	multidrug efflux transporter outer membrane component of multidrug efflux system		2011; Poole K., 2005b
						factor lipoprotein OprN

Mots clefs: Pseudomonas fluorescens, adaptation, glycérophospholipide, acides gras, pollution, oxydes d'azote, détoxification

Remerciements

This work was realized in Laboratory of Microbiology Signals and Microenvironment EA4312, of University of Rouen. I am grateful to the Haute Normandie region for the financial support for this research work.

I would like to thank Pr. Angela CORCELLI from University of Bari Aldo Moro, and Dr.

Janine FRÖHLICH from Max Planck Institute for Chemistry my sincere gratitude. Special thanks to Dr. Hermann HEIPIEPER from Helmholtz Centre for Environmental Research of Leipzig for agreeing to

The analyses were performed for three independent bacterial lipid extracts. Analysis of fatty acids in hexane was performed using a quadruple GC System (HP5890, Hewlett &Packard, Palo Alto, USA). The fatty acids were identified by GC and co-injection of authentic reference compounds obtained from Supelco (Bellefonte, USA).

The peak areas of the fatty acids were used to determine their relative amounts.

VIII. Annexes

P a g e 244 | 270

Chapter 5 Effect of stress factors on membrane of P. fluorescens The fatty acids were identified and quantified by the GC in at least three replicates. The peak areas of fatty acids were used to determine their relative amounts. The results are presented as the percentage of average value relative to the sum ± SEM. b EGP: exponential growth phase; c SGP: stationary growth phase.

Effect of air pollution on membrane of airborne bacteria: NO2 modifies both Pseudomonas fluorescens glycerophospholipids and membrane electron-accepting character

Figure S1. Schematic representation of NO2 gas delivery system

Bacterial NO2 exposure was done in gas phase for 2 h, according (Ghaffari et al., 2005). Two exposure chambers (one for the NO2 exposure, the second one for the control simple -exposure to the synthetic air) were used. The gases NO2, N2 and O2 were mixed together to obtain pre-calculated concentration of NO2 and maintain the O2/N2 ratio at 2/8, (v/v). All experimental parameters (NO2 concentration, temperature, relative humidity) were controlled.

P a g e 245 | 270 a The fatty acids were identified and quantified by GC in at least three replicates. The peak areas of the fatty acids were used to determine their relative amounts b . The results are presented as the percentage of average value relative to the sum ± SEM.

Effect of temperature and NO2 on membrane glycerophospholipids of clinical Pseudomonas fluorescens strain MFN1032

Figure S1. Schematic representation of NO2 gas delivery system

Bacterial NO2 exposure was done in gas phase for 2 h, according (Ghaffari et al., 2005). Two exposure chambers (one for the NO2 exposure, the second one for the control simple -exposure to the synthetic air) were used. The gases NO2. N2 and O2 were mixed together to obtain pre-calculated concentration of NO2 and maintain the O2/N2 ratio at 2/8. (v/v). All experimental parameters (NO2 concentration, temperature, relative humidity) were controlled.

P a g e 246 | 270