Isabelle J Q2 Schalk
email: schalk@unistra.fr.

Coraline Rigouin

Julien Godet

An overview of siderophore biosynthesis among fluorescent Pseudomonads and new insights into their complex cellular organization Q1

Siderophores are iron-chelating molecules produced by bacteria to access iron, a key nutrient. These compounds have highly diverse chemical structures, with various chelating groups. They are released by bacteria into their environment to scavenge iron and bring it back into the cells. The biosynthesis of siderophores requires complex enzymatic processes and expression of the enzymes involved is very finely regulated by iron availability and diverse transcriptional regulators. Recent data have also highlighted the organization of the enzymes involved in siderophore biosynthesis into siderosomes, multi-enzymatic complexes involved in siderophore synthesis. An understanding of siderophore biosynthesis is of great importance, as these compounds have many potential biotechnological applications because of their metal-chelating properties and their key role in bacterial growth and virulence. This review focuses on the biosynthesis of siderophores produced by fluorescent Pseudomonads, bacteria capable of colonizing a large variety of ecological niches. They are characterized by the production of chromopeptide siderophores, called pyoverdines, which give the typical green colour characteristic of fluorescent pseudomonad cultures. Secondary siderophores are also produced by these strains and can have highly diverse structures (such as pyochelins, pseudomonine, yersiniabactin, corrugatin, achromobactin and quinolobactin).

Introduction

Siderophores are a major family of iron-chelating agents that play a key role in bacterial iron homeostasis. They generally have a molecular weight between 200 and 2000 Da and are characterized by a very strong affinity for ferric iron (Fe 3+ ) [START_REF] Boukhalfa | Chemical aspects od siderophore mediated iron transport[END_REF]. They are produced and secreted by bacteria under ironrestricted conditions to scavenge iron from their environment. In parallel, bacteria express transporters at their cell surface that are able to capture back these chelators once they have chelated ferric iron [START_REF] Schalk | Structure, function and binding selectivity and stereoselectivity of siderophore-iron outer membrane transporters[END_REF].

Fluorescent pseudomonads produce the fluorescent pyoverdines as their major siderophores [START_REF] Cornelis | Diversity of siderophoremediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines[END_REF][START_REF] Meyer | Siderophore typing, a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads[END_REF]. These chelators are produced by the bacteria to access iron and also play an important role in the virulence of Pseudomonad pathogens and, in the case of P. aeruginosa, have been shown to be necessary for the establishment of mature biofilms [START_REF] Meyer | Pyoverdin is essential for virulence of Pseudomonas aeruginosa[END_REF][START_REF] Handfield | In vivo-induced genes in Pseudomonas aeruginosa[END_REF][START_REF] Mirleau | Fitness in soil and rhizosphere of Pseudomonas fluorescens C7R12 compared with a C7R12 mutant affected in pyoverdine synthesis and uptake[END_REF][START_REF] Banin | Iron and Pseudomonas aeruginosa biofilm formation[END_REF][START_REF] Yang | Pyoverdine and PQS mediated subpopulation interactions involved in Pseudomonas aeruginosa biofilm formation[END_REF][START_REF] Taguchi | The siderophore pyoverdine of Pseudomonas syringae pv. tabaci 6605 is an intrinsic virulence factor in host tobacco infection[END_REF]. In addition, diverse secondary siderophores with a lower affinity for Fe 3+ are also produced by Pseudomonads, such as pyochelin (PCH), pseudomonin, corrugatins and ornicorrugatins, yersiniabactin and thioquinolobactin [START_REF] Cornelis | Iron uptake and metabolism in pseudomonads[END_REF]. This review will focus on the biosynthesis of the siderophores produced by fluorescent Pseudomonads, the enzymatic biochemistry involved, the cellular organization of the biosynthetic machinery, and how siderophore synthesis is regulated. Pseudomonas aeruginosa is the archetype among fluorescent Pseudomonads and most of the data found in the literature and presented here will concern this pathogen; however, parallels will be made with siderophore biosynthesis among other fluorescent Pseudomonads, when possible. Pseudomonas aeruginosa strains produce four distinct pyoverdines, called PVDI, PVDII, PVDIII and PVDIV, and PCH as a secondary siderophore [START_REF] Schalk | Pyoverdine biosynthesis and secretion in Pseudomonas aeruginosa: implications for metal homeostasis[END_REF][START_REF] Gasser | Cellular organization of siderophore biosynthesis in Pseudomonas aeruginosa: evidence for siderosomes[END_REF]Ringel and Brüser, 2018;[START_REF] Ronnebaum | Nonribosomal peptides for iron acquisition: pyochelin biosynthesis as a case study[END_REF]. Many detailed reviews have already been published on PVDI and PCH biosynthesis [START_REF] Visca | Pyoverdine siderophores: from biogenesis to biosignificance[END_REF][START_REF] Schalk | Pyoverdine biosynthesis and secretion in Pseudomonas aeruginosa: implications for metal homeostasis[END_REF][START_REF] Gasser | Cellular organization of siderophore biosynthesis in Pseudomonas aeruginosa: evidence for siderosomes[END_REF]Ringel and Brüser, 2018;[START_REF] Ronnebaum | Nonribosomal peptides for iron acquisition: pyochelin biosynthesis as a case study[END_REF]. Thus, we will provide a general description of their biosynthesis and highlight recent and original data concerning the cellular organization of the siderophore biosynthetic machinery. Indeed, high-resolution fluorescence microscopy and approaches such as fluorescence recovery after photobleaching (FRAP) or single-molecule tracking with photoactivated localization microscopy (sptPALM) have provided new insights into the cellular distribution of the biosynthetic enzymes within the bacteria, their dynamics in the bacterial cytoplasm and the possible protein interactions involved [START_REF] Guillon | High cellular organisation of pyoverdine biosynthesis in Pseudomonas aeruginosa: localization of PvdA at the old cell pole[END_REF][START_REF] Guillon | Deciphering protein dynamics of the siderophore pyoverdine pathway in Pseudomonas aeruginosa[END_REF][START_REF] Imperi | Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome[END_REF][START_REF] Gasser | Cellular organization of siderophore biosynthesis in Pseudomonas aeruginosa: evidence for siderosomes[END_REF][START_REF] Gasser | In cellulo FRET-FLIM and single molecule tracking reveal the supra-molecular organization of the pyoverdine bio-synthetic enzymes in Pseudomonas aeruginosa[END_REF]. These recent data provide a new vision of siderophore biosynthesis.

Siderophores produced by Pseudomonas strains

Pyoverdines

All fluorescent Pseudomonads produce pyoverdines and almost 100 distinct pyoverdines, produced by various strains and species of fluorescent Pseudomonas, have been identified to date [START_REF] Demange | Bacterial siderophores: structure and NMR assigment of pyoverdins PaA, siderophores of Pseudomonas aeruginosa ATCC 15692[END_REF][START_REF] Budzikiewicz | Siderophores of fluorescent pseudomonads[END_REF][START_REF] Fuchs | Structural studies of pyoverdins by mass spectrometry[END_REF][START_REF] Budzikiewicz | Siderophores of the Pseudomonadaceae sensu stricto (fluorescent and nonfluorescent Pseudomonas spp.). Fortschritte der Chemie organischer Naturstoffe[END_REF][START_REF] Budzikiewicz | Characterization of the chromophores of pyoverdins and related siderophores by electrospray tandem mass spectrometry[END_REF]. These siderophores all have the same structural organization, consisting of three components (Fig. 1A): (i) a dihydroquinoline-type chromophore, (ii) a strain-specific peptide comprised of six to 14 amino acids, and (iii) a side-chain bound to the nitrogen atom at position C-3 of the chromophore. The chromophore is (1S)-5-amino-2,-3-dihydro-8,9-dihydroxy-1H-pyrimido-[1,2-a]quinolone-1-carboxylic acid and is exactly the same for all pyoverdines, giving specific spectral characteristics to these compounds, consisting of an absorbance at 400 nm (at neutral pH) and an emission of fluorescence at 447 nm when in the apo forms (the ferric form being non-fluorescent) [START_REF] Albrecht-Gary | Bacterial iron transport: coordination properties of pyoverdin PaA, a peptidic siderophore of Pseudomonas aeruginosa[END_REF][START_REF] Folschweiller | The interaction between pyoverdin and its outer membrane receptor in Pseudomonas aeruginosa leads to different conformers: a timeresolved fluorescence study[END_REF][START_REF] Budzikiewicz | Characterization of the chromophores of pyoverdins and related siderophores by electrospray tandem mass spectrometry[END_REF]. The side chain bound to the chromophore is, in most cases, a succinamide, succinate or α-ketoglutaric acid or sometimes also malamide, malic acid or succinic acid.

The sequence and length of the peptide moiety differ between pyoverdines and may contain unusual amino acids, such as D-isomers and amino acids that are not usually found in proteins (such as N 5hydroxycycloornithine or L-2,4-diaminobutyrate) [START_REF] Budzikiewicz | Siderophores of fluorescent pseudomonads[END_REF][START_REF] Fuchs | Structural studies of pyoverdins by mass spectrometry[END_REF][START_REF] Meyer | Siderophore typing, a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads[END_REF]. This peptide moiety can also be cyclic in some pyoverdines. Each pyoverdine is characterized by its sequence, which gives each Pseudomonas strain the ability to access iron using only the pyoverdine produced by itself. This is due to the fact that

Pseudomonas strains produce a specific pyoverdine and express a corresponding specific transporter at the outer membrane that is able to recognize and capture back only the ferric form of the produced pyoverdine or one that is structurally related (with a similar peptide sequence) [START_REF] Greenwald | FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter[END_REF][START_REF] Schalk | Structure, function and binding selectivity and stereoselectivity of siderophore-iron outer membrane transporters[END_REF]. The peptide moiety plays a key role in such transporter recognition, as shown by the x-ray structures of a pyoverdine transporter in complex with several different ferric-pyoverdine complexes [START_REF] Greenwald | FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter[END_REF]. As already mentioned in the Introduction, four distinct pyoverdines are produced by P. aeruginosa strains-PVDI, PVDII, PVDIII and PVDIV. Each is characterized by a different peptide chain (Fig. 1A) [START_REF] Meyer | Use of siderophores to type pseudomonads: the three Pseudomonas aeruginosa pyoverdine systems[END_REF][START_REF] Ruangviriyachai | A new pyoverdin from Pseudomonas aeruginosa R[END_REF] and each has a corresponding outer membrane transporter FpvAI, FpvAII, FpvAIII and FpvAIV. Overall, almost 100 pyoverdines produced by all fluorescent Pseudomonads are divided into four groups or families based on the structural features of the peptide chain [for more details see (Fuchs et al., 2001)].

Pyoverdines chelate iron with a 1:1 stoichiometry and an affinity of 10 32 M -1 for ferric iron has been determined for PVDI produced by P. aeruginosa PAO1 [START_REF] Albrecht-Gary | Bacterial iron transport: coordination properties of pyoverdin PaA, a peptidic siderophore of Pseudomonas aeruginosa[END_REF]. The chelating groups always involve the catechol and two bidentate ligands from the peptide moiety (Fig. 1A). As for most siderophores, pyoverdines are able to chelate many metals other than iron (Braud et al., 2009b;Hannauer et al., 2012a) and stability constants have been determined for PVD-Ni 2+ (Ka = 10 10.9 M -1 ), PVD-Cd 2+ (Ka = 10 8.2 M -1 ) and PVD-Cu 2+ (Ka = 10 20.1 M -1 ) [START_REF] Ferret | Effect of pyoverdine supply on cadmium and nickel complexation and phytoavailability in hydroponics[END_REF].

Other siderophores produced by Pseudomonads

Almost all fluorescent Pseudomonads produce another siderophore in addition to pyoverdine, often called the secondary siderophore because of a lower affinity for ferric iron relative to that of pyoverdine [START_REF] Cornelis | Diversity of siderophoremediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines[END_REF][START_REF] Mossialos | Siderophores in fluorescent pseudomonads: new trick from an old dog[END_REF][START_REF] Matthijs | Ornicorrugatin, a new siderophore from Pseudomonas fluorescens AF76[END_REF][START_REF] Matthijs | Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440[END_REF]. Such secondary siderophores can have diverse chemical structures (Fig. 1B). In addition to pyoverdines, P. aeruginosa strains produce PCH. PCH chelates ferric iron with a 2:1 stoichiometry and an affinity of 10 18 M -2 [START_REF] Cox | Pyochelin: novel structure of an iron-chelating growth promoter for Pseudomonas aeruginosa[END_REF][START_REF] Tseng | Bacterial siderophores: the solution stoichiometry and coordination of the Fe(III) complexes of pyochelin and related compounds[END_REF][START_REF] Brandel | Pyochelin, a siderophore of Pseudomonas aeruginosa: physicochemical characterization of the iron(III), copper(II) and zinc(II) complexes[END_REF]. Pseudomonas fluorescens produces enantio-pyochelin (E-PCH)-the optical antipode of PCH- [START_REF] Youard | Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin[END_REF][START_REF] Hoegy | Stereospecificity of the siderophore pyochelin outer membrane transporters in fluorescent Pseudomonads[END_REF], P. syringae DC3000 yersiniabactin (also produced by Yersinia) [START_REF] Jones | Salicylic acid, yersiniabactin, and pyoverdin production by the model phytopathogen Pseudomonas syringae pv. tomato DC3000: synthesis, regulation, and impact on tomato and Arabidopsis host plants[END_REF][START_REF] Petermann | The Yersinia high pathogenicity Island is present in Salmonella enterica subspecies I isolated from turkeys[END_REF] and P. syringae B728a achromobactin (also produced by Erwinia chrysanthemi) [START_REF] Franza | Erwinia chrysanthemi requires a second iron transport route dependent of the siderophore achromobactin for extracellular growth and plant infection[END_REF][START_REF] Berti | Analysis of achromobactin biosynthesis by Pseudomonas syringae pv. syringae B728a[END_REF]. This list shows that secondary siderophores are not always specific to Pseudomonads but are sometimes also produced by other bacterial species. Pyridine-2,6-bis(thiocarboxylate) (PDTC) has been shown to be produced by P. stutzeri [START_REF] Lee | Identification of an extracellular agent [correction of catalyst] of carbon tetrachloride dehalogenation from Pseudomonas stutzeri strain KC as pyridine-2, 6-bis (thiocarboxylate)[END_REF] and P. putida [START_REF] Ockels | An Fe(II) complex of pyridine-2,6-di-(monothiocarboxylic acid) -a novel bacterial metabolic product[END_REF] and is capable of transporting iron [START_REF] Lewis | Physiological and molecular genetic evaluation of the dechlorination agent, pyridine-2,6-bis(monothiocarboxylic acid) (PDTC) as a secondary siderophore of Pseudomonas[END_REF]. The role of secondary siderophores is not clear, but they are often produced in lower iron-restricted conditions than pyoverdines [START_REF] Cunrath | Metallome of Pseudomonas aeruginosa: a role for siderophores[END_REF]. Like pyoverdines, secondary siderophores are able to chelate metals other than iron. PCH has been shown to form complexes with many metals [START_REF] Youard | Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin[END_REF][START_REF] Hoegy | Stereospecificity of the siderophore pyochelin outer membrane transporters in fluorescent Pseudomonads[END_REF], Yersiniabactin and Achromobactin by Pseudomonas syringae [START_REF] Jones | Salicylic acid, yersiniabactin, and pyoverdin production by the model phytopathogen Pseudomonas syringae pv. tomato DC3000: synthesis, regulation, and impact on tomato and Arabidopsis host plants[END_REF][START_REF] Petermann | The Yersinia high pathogenicity Island is present in Salmonella enterica subspecies I isolated from turkeys[END_REF], Pseudomonine by Pseudomonas fluorescens and Pseudomonas entomophila [START_REF] Mercado-Blanco | Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374[END_REF][START_REF] Matthijs | Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440[END_REF], Quinolobactin and Thio-quinolobactin by Pseudomonas fluorescence ATCC17400 [START_REF] Matthijs | The Pseudomonas siderophore quinolobactin is synthesized from xanthurenic acid, an intermediate of the kynurenine pathway[END_REF][START_REF] Matthijs | Thioquinolobactin, a Pseudomonas siderophore with antifungal and anti-Pythium activity[END_REF], PDTC (Pyridine-2,6-bis(thiocarboxylate)) by Pseudomonas stutzeri [START_REF] Lee | Identification of an extracellular agent [correction of catalyst] of carbon tetrachloride dehalogenation from Pseudomonas stutzeri strain KC as pyridine-2, 6-bis (thiocarboxylate)[END_REF] and Pseudomonas putida [START_REF] Ockels | An Fe(II) complex of pyridine-2,6-di-(monothiocarboxylic acid) -a novel bacterial metabolic product[END_REF] and Corrugatin by Pseudomonas acorrugata [START_REF] Matthijs | Ornicorrugatin, a new siderophore from Pseudomonas fluorescens AF76[END_REF] [START_REF] Baysse | Vanadium interferes with siderophore-mediated iron uptake in Pseudomonas aeruginosa[END_REF]Braud et al., 2009a) and the stability constant has been determined for Zn 2+ (Ka = 10 26.0 M -2 ) and Cu 2+ (Ka = 10 25.0 M -2 ) [START_REF] Brandel | Pyochelin, a siderophore of Pseudomonas aeruginosa: physicochemical characterization of the iron(III), copper(II) and zinc(II) complexes[END_REF].

Biosynthesis

The biosynthetic pathways of PVDI and PCH produced by P. aeruginosa PAO1 have been extensively investigated and all steps and enzymes involved have been identified and characterized. An overall description of these two biosynthetic schemes is presented below. As the biosynthesis of other pyoverdines must be very similar to that of PVDI, we also discuss the gene organization of pyoverdine biosynthesis among fluorescent Pseudomonads in general. Much less, and sometimes nothing, is known about the biosynthesis of the other secondary siderophores produced by fluorescent Pseudomonads: hypothetical or incomplete biosynthetic pathways have been proposed for PDTC, quinolobactin, thioquinolobactin and achromobactin (summarized below). The biosynthesis of yersiniabactin has been described in detail in Y. pestis [START_REF] Ahmadi | Total biosynthesis and diverse applications of the nonribosomal peptide-polyketide siderophore yersiniabactin[END_REF] but not in P. syringae, but probably very similar enzymes and biological mechanisms are involved [START_REF] Ahmadi | Total biosynthesis and diverse applications of the nonribosomal peptide-polyketide siderophore yersiniabactin[END_REF].

Pyoverdine biosynthesis

Knowledge of the various steps of PVDI biosynthesis, the pyoverdine produced by P. aeruginosa PAO1, is very complete and precise. PVDI synthesis starts in the bacterial cytoplasm, with the assembly of an 11-amino-acid peptide with an unformed chromophore and a myristic or myristoleic acid chain at its N-terminal end (Hannauer et al., 2012b). This cytoplasmic peptide undergoes maturation in the periplasm to yield PVDI [START_REF] Yeterian | Synthesis of the siderophore pyoverdine in Pseudomonas aeruginosa involves a periplasmic maturation[END_REF]Hannauer et al., 2012b). Its biosynthesis involves the coordinated action of several enzymes, including four non-ribosomal peptide synthesis (NRPS) enzymes, three enzymes that generate atypical amino acids present in the peptide moiety of the siderophore, and several enzymes involved in the maturation of this siderophore in the bacterial periplasm before secretion.

The PVDI peptide backbone contains two unusual amino acids, L-2,4-diaminobutyrate (L-Dab) and L-N 5 -formyl-N 5 -hydroxyornithine (L-fOHOrn). L-Dab is synthesized by the enzyme PvdH, an aminotransferase that catalyses the formation of L-Dab from L-aspartate β-semialdehyde [START_REF] Vandenende | Functional characterization of an aminotransferase required for pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa PAO1[END_REF]) (Fig. 2A). L-fOHOrn is synthesized from L-ornithine by hydroxylation and formylation catalysed by PvdA and PvdF respectively [START_REF] Visca | Cloning and nucleotide sequence of the pvdA gene encoding the pyoverdin biosynthetic enzyme L-ornithine N5-oxygenase in Pseudomonas aeruginosa[END_REF][START_REF] Mcmorran | Involvement of a transformylase enzyme in siderophore synthesis in Pseudomonas aeruginosa[END_REF][START_REF] Ge | Heterologous expression, purification, and characterization of an l-ornithine N(5)hydroxylase involved in pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa[END_REF]. Other unusual amino acids can be found in pyoverdine sequences of Pseudomonas strains, such as β-hydroxy aspartic acid, β-hydroxy histidine, ornithine, cyclo-N 5 -hydroxy ornithine, N 5 -acetyl-N 5 -hydroxy ornithine and N 5hydroxybutyryl-N 5 -hydroxy ornithine (Cezard Q4 and Sonnet, 2014). Some of the enzymes involved in such modifications have been identified. For example, PvdY is the enzyme responsible for the acetylation of hydroxyornithine in the biosynthesis of type II pyoverdine (PVDII) in P. aeruginosa strains [START_REF] Lamont | Characterization of a gene encoding an acetylase required for pyoverdine synthesis in Pseudomonas aeruginosa[END_REF]. PvdY is only present in strains that make PVDII.

The peptide backbone of P. aeruginosa PVDI is composed of 11 amino acids, which are assembled by NRPS enzymes (Fig. 2A), multi-modular enzymes that activate amino acids and assemble them into peptide chains. Each module of one NRPS enzyme activates and modifies a specific amino acid for addition to the growing peptide chain that is then elongated with another amino acid activated and modified by an adjacent module. Consequently, the number and order of the NRPS modules directly dictate the linear sequence of the final peptide chain. A typical module contains three domains: an adenylation (A) domain, a condensation (C) domain and a peptidyl-carrier protein (PCP) domain. The A domain recognizes a specific amino acid and activates the acid by an ATP-dependent adenylation. Then, the acid is transferred to a free thiol of a covalently bound phosphopantetheine cofactor of the adjacent PCP domain by thioesterification to form an acyl-S-PCP domain intermediate. Finally, the C domain catalyses the condensation between upstream and downstream PCP-thioesterified substrates, forming the peptide bond between the two amino acids. A more detailed description of the biochemistry of NRPS enzymes is provided in reviews by [START_REF] Hur | Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology[END_REF], [START_REF] Gulick | Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens[END_REF], and [START_REF] Süssmuth | Nonribosomal peptide synthesis-principles and prospects[END_REF]. The last NRPS of the assembly line usually has a thioesterase domain, adjacent to the terminal PCP domain, which catalyses hydrolysis of the peptide chain from the NPRS, leading to its release [START_REF] Izoré | The many faces and important roles of protein-protein interactions during nonribosomal peptide synthesis[END_REF]. The biosynthesis of the PVDI precursor requires four NPRS enzymes: PvdL, PvdI, PvdJ and PvdD (Fig. 2A). The synthesis starts with PvdL, an NRPS enzyme composed of four modules [START_REF] Mossialos | Identification of new, conserved, non-ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine[END_REF]. The first module of PvdL is unusual and consists of an acyl coenzyme A ligase domain that catalyses the acylation of a myristic acid or a myristoleic acid. It has been suggested that this acylation occurs to maintain the peptide precursor at the membrane and prevent its diffusion during assembly of the peptide (Hannauer et al., 2012b). The second module of PvdL catalyses the activation of L-Glu and its condensation to the myristic acid-coA formed in the first module. The third module binds an L- Tyr amino residue. An epimerization domain embedded in this module isomerizes the L-Tyr residue to the D-Tyr form. Finally, the fourth module adds the L-Dab amino acid to form the acylated tripeptide L-Glu/D-Tyr/L-Tab. The second NRPS involved, PvdI, is composed of four modules and adds the amino acids D-Ser, L-Arg, D-Ser and L-hfOrn to the precursor peptide formed by PvdL. PvdI contains epimerization domains in modules 2 and 4 to generate D-Ser from L-Ser [START_REF] Lehoux | Genomics of the 35-kb pvd locus and analysis of novel pvdIJK genes implicated in pyoverdine biosynthesis in Pseudomonas aeruginosa[END_REF]. The two following NRPS enzymes are PvdJ and PvdD, which are bimodular and add L-lys and L-hfOrn and two L-Thr residues respectively [START_REF] Merriman | Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases[END_REF][START_REF] Ackerley | Substrate specificity of the nonribosomal peptide synthetase PvdD from Pseudomonas aeruginosa[END_REF]. The last module of PvdD has a final thioesterase domain that catalyses the hydrolysis of the peptide chain from the NRPS. Two genes, PA2411 and PA2425 (pvdG), are predicted to encode thioesterase enzymes and are embedded in the PVDI biosynthetic gene cluster, suggesting a role in PVDI biosynthesis. Whether these thioesterases have overlapping functions, act in trans to release the in-forming peptide, or have another function in PVDI biosynthesis is still not clear. Of note, genetic inactivation of PvdG blocks the synthesis of PVDI [START_REF] Lamont | Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa[END_REF]. The last enzyme supposedly involved in PVDI synthesis is MbtH. MbtH-like proteins are small auxiliary proteins that interact with the A domain to enhance its activity [START_REF] Felnagle | MbtH-like proteins as integral components of bacterial nonribosomal peptide synthetases[END_REF][START_REF] Boll | Role of MbtH-like proteins in the adenylation of tyrosine during aminocoumarin and vancomycin biosynthesis[END_REF]. The crystal structure of P. aeruginosa MbtH has been solved [START_REF] Drake | The 1.8 A crystal structure of PA2412, an MbtH-like protein from the pyoverdine cluster of Pseudomonas aeruginosa[END_REF]. Although direct involvement of MbtH in NRPS activity has not been demonstrated, biochemical studies have provided evidence for a role in the production and secretion of PVDI [START_REF] Drake | The 1.8 A crystal structure of PA2412, an MbtH-like protein from the pyoverdine cluster of Pseudomonas aeruginosa[END_REF].

Once assembled, the acetylated precursor peptide is transported to the periplasm by the ATP-binding-cassette (ABC) inner membrane transporter, PvdE. The involvement of PvdE in periplasmic transport was unravelled by the study of a pvdE mutant that led to undetectable PVDI-related fluorescence in cultures of P. aeruginosa cells [START_REF] Yeterian | Synthesis of the siderophore pyoverdine in Pseudomonas aeruginosa involves a periplasmic maturation[END_REF]. Consequently, it was concluded that PvdE exports the non-fluorescent PVDI precursor from the cytosol to the periplasm but is not involved in its extracellular secretion [START_REF] Yeterian | Synthesis of the siderophore pyoverdine in Pseudomonas aeruginosa involves a periplasmic maturation[END_REF]. Once in the periplasm, the PVDI precursor is subjected to modifications that ultimately lead to the final siderophore (Fig. 2B). The first modification is deacetylation of the precursor, performed by the enzyme PvdQ and leading to the removal of the myristic or myristoleic acid moiety from the peptide and formation of a pyoverdine precursor, called ferribactin [START_REF] Drake | Structural characterization and high-throughput screening of inhibitors of PvdQ, an NTN hydrolase involved in pyoverdine synthesis[END_REF]Hannauer et al., 2012b). This precursor then enters an oxidative cyclization cascade that results in chromophore cyclisation from the L-Dab and D-Tyr residues, the second and third residues of the PVDI precursor [START_REF] Dorrestein | Formation of the chromophore of the pyoverdine siderophores by an oxidative cascade[END_REF][START_REF] Dorrestein | Oxidative cascades: a facile biosynthetic strategy for the assembly of complex molecules[END_REF], involving the copper-dependent tyrosinase PvdP [START_REF] Nadal-Jimenez | PvdP is a tyrosinase that drives maturation of the pyoverdine chromophore in Pseudomonas aeruginosa[END_REF][START_REF] Poppe | Pseudomonas aeruginosa pyoverdine maturation enzyme PvdP has a noncanonical domain architecture and affords insight into a new subclass of tyrosinases[END_REF]. This enzyme catalyses the conversion of ferribactin into dihydropyoverdine in three steps: (i) hydroxylation of the D-tyrosine moiety of the tetrahydropyrimidine ring, resulting in a catechol functionality, (ii) formation of a third ring in the chromophore, and (iii) restoration of the catechol functionality [START_REF] Nadal-Jimenez | PvdP is a tyrosinase that drives maturation of the pyoverdine chromophore in Pseudomonas aeruginosa[END_REF][START_REF] Poppe | Pseudomonas aeruginosa pyoverdine maturation enzyme PvdP has a noncanonical domain architecture and affords insight into a new subclass of tyrosinases[END_REF]. The final oxidation of dihydropyoverdine into PVDI has recently been assigned to PvdO [START_REF] Yuan | Crystal structure of PvdO from Pseudomonas aeruginosa[END_REF]Ringel et al., 2018). Ringel et al. (2018) showed that a mutant strain of P. fluorescens A506 lacking PvdO only produces the dihydropyoverdine form of PVDI. The authors raised the possibility that PvdO must be associated with another enzyme or a specific cofactor to be active, as the enzyme was inactive in vitro.

The first residue of the pyoverdine peptide backbones among fluorescent Pseudomonads is always a glutamic acid [START_REF] Hohlneicher | Ferribactins as the biogenetic precursors of Pseudomonas siderophores pyoverdins[END_REF]. This L-Glu residue, bound at position C3 of the chromophore, undergoes modifications that allow its conversion into a range of variants, including succinamide, succinic acid, α-ketoglutaric acid, malamide and malic acid residues [START_REF] Budzikiewicz | Siderophores of the Pseudomonadaceae sensu stricto (fluorescent and nonfluorescent Pseudomonas spp.). Fortschritte der Chemie organischer Naturstoffe[END_REF]. These structural variations do not have a direct impact on pyoverdine function but rather on the adaptation to environmental conditions. The two enzymes involved in these modifications have recently been identified and their function assigned. PvdN is an enzyme that contains a pyridoxal phosphate cofactor as a prosthetic group and requires cytoplasmic cofactor assembly for folding [START_REF] Drake | 1.2 Å resolution crystal structure of the periplasmic aminotransferase PvdN from Pseudomonas aeruginosa[END_REF]. Ringel et al. showed by mass spectrometry that the only pyoverdine produced by a pvdN mutant is the α-ketoglutarate form, indicating that transformation to the succinamide derivatives does not occur and must be carried out by PvdN (Ringel et al., 2018;Ringel and Brüser, 2018). PtaA (PflA506_4424) is also a pyridoxal phosphate-dependent transaminase and requires cytoplasmic cofactor assembly for folding and transport to the periplasm [START_REF] Ringel | The periplasmic transaminase PtaA of Pseudomonas fluorescens converts the glutamic acid residue at the pyoverdine fluorophore to α-ketoglutaric acid[END_REF]. As for PvdN, PtaA is not essential for pyoverdine production or function. However, a P. fluorescens A506 mutant deleted for ptaA was unable to produce the α-ketoglutaric acid variant, suggesting that this enzyme is responsible for the alternative modification of the L- Glu side chain [START_REF] Ringel | The periplasmic transaminase PtaA of Pseudomonas fluorescens converts the glutamic acid residue at the pyoverdine fluorophore to α-ketoglutaric acid[END_REF]. The ΔptaA/ΔpvdN double-deletion strain produced neither the succinamide variant (and further derivatives) nor the α-ketoglutaric-acid variant of PVD A506 . The only detectable products were the chromophore-containing precursor with the original glutamic acid residue and ferribactin. Some Pseudomonas strains carry both ptaA and pvdN in their genome, leading to strains that are able to produce different variants of pyoverdine. On the other hand, some Pseudomonas strains have only pvdN or ptaA and consequently only produce the corresponding variant (α-ketoglutaric acid or succinamide derivative variants) (Ringel and Brüser, 2018). PtaA can act on several different substrates, i.e. ferribactin, dihydropyoverdine and pyoverdine, which raises the question of when these enzymes operate in the periplasm [START_REF] Ringel | The periplasmic transaminase PtaA of Pseudomonas fluorescens converts the glutamic acid residue at the pyoverdine fluorophore to α-ketoglutaric acid[END_REF]. The only enzyme for which the function is unknown is PvdM, which is predicted to be a dipeptidase.

Once PVDI is formed, it is secreted from the periplasm into the bacterial environment by the ATP-dependent efflux pump PvdRT-OpmQ [START_REF] Hannauer | Secretion of newly synthesized pyoverdine by Pseudomonas aeruginosa involves an efflux pump[END_REF]. However, the deletion of this efflux pump does not completely abolish PVDI secretion, highly suggesting that another efflux system is involved in this process. In P. putida KT2440, both PvdRT-OpmQ and MdtABC-OpmB have been proposed to be involved in pyoverdine secretion [START_REF] Henríquez | PvdRT-OpmQ and MdtABC-OpmB efflux systems are involved in pyoverdine secretion in Pseudomonas putida KT2440[END_REF]. However, at least one additional efflux system participates in the export of this siderophore, as double deletion mutants for these efflux pumps still secrete pyoverdine. In P. taiwanensis type VI secretion system also appears to be involved in pyoverdine secretion, suggesting the participation of alternative secretion pathways in the export of this siderophore [START_REF] Chen | Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis[END_REF] Organization and diversity of the pyoverdine genomic region among P. aeruginosa strains and fluorescent Pseudomonads

The pyoverdine region in P. aeruginosa strains corresponds to the region of the genome that contains all the genes involved in PVDI biosynthesis, secretion, iron acquisition and recycling (Fig. 3). This region covers approximately 50 kb and contains more than 30 genes. The genes are present on both DNA strands and are separated by regulator genes. [START_REF] Smith | Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa[END_REF] have analysed the degree and patterns of diversity of the PDVI, PVDII and PVDIII genes in P. aeruginosa. They showed that there are three different types of gene organization, corresponding to the three structural types of pyoverdines. In addition, they found that the pyoverdine region is the most highly divergent region in the P. aeruginosa genome [START_REF] Spencer | Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa[END_REF][START_REF] Smith | Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa[END_REF]. Interestingly, this region has unusual codon and oligonucleotide usage, indicating its acquisition by horizontal gene transfer. Among the NRPS-encoding genes, pvdL is always isolated from the three other NRPS genes, pvdI, pvdJ and pvdD, located in the central part of the region and forming a cluster. Moreover, pvdL is the only NPRS enzyme that is highly conserved, whereas pvdD, pvdI and pvdJ display high divergence among strains, with no sequence similarity between strains. This genetic pattern directly reflects the organization of the peptide backbone of PVDI, which is always composed of two parts. The first consists of the conserved three residues L-Glu, D-Tyr and L-Dab, assembled by PvdL, with D-Tyr and L-Dab giving the chromophore and L-Glu the side chain, structures common to all pyoverdines. The second part is the variable peptide chain assembled by PvdD, PvdI and PvdJ. Two other genes also display high divergence between strains in the pyoverdine region, pvdE [the ABC transporter involved in the transport of the pyoverdine precursor from the cytoplasm into the periplasm [START_REF] Yeterian | Synthesis of the siderophore pyoverdine in Pseudomonas aeruginosa involves a periplasmic maturation[END_REF]] and fpvA [the outer membrane transporter involved in the uptake of ferric loaded pyoverdine [START_REF] Poole | Pyoverdinemediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein[END_REF]]. [START_REF] Smith | Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa[END_REF] demonstrated that fpvA shows evidence of positive selection, suggesting that fpvA drives the diversity of the pyoverdine locus. Indeed, the transporter and pyoverdine peptide must coevolve to maintain mutual specificity and recognition; the evolution of fpvA subsequently led to NRPS gene recombination [START_REF] Ruangviriyachai | A new pyoverdin from Pseudomonas aeruginosa R[END_REF]. Finally, some genes, usually specific for the type of pyoverdine produced, are only present in certain types of P. aeruginosa strains. This is the case for pvdYII, which is only present in type II P. aeruginosa strains [START_REF] Lamont | Characterization of a gene encoding an acetylase required for pyoverdine synthesis in Pseudomonas aeruginosa[END_REF].

The organization and diversity of the pyoverdine genomic region among fluorescent Pseudomonads was studied by [START_REF] Ravel | Genomics of pyoverdinemediated iron uptake in pseudomonads[END_REF] by comparing the pyoverdine regions of P. aeruginosa, P. syringae

Color Figure -Print and Online

Organization of PVDI genes (A) and PCH genes (B) on P. aeruginosa PAO1 genome. In both panels, coloured boxes represent the genes coding for enzymes involved in the biosynthesis of the siderophores. Dashed boxes represent the genes coding for siderophore transporters (export across the inner and outer membranes). Grey boxes represent genes involved in iron import via PVDI or PCH (ferri-siderophore import as well as mechanism of iron release from the siderophore and or siderophore recycling). White boxes are for genes coding for proteins of unknown function. The authors highlighted the similarities in the organization of the pyoverdine region between fluorescent Pseudomonads. Homologous genes involved in pyoverdine pathways are found in every species but the overall organization of the region is different: the pyoverdine region can form a single contiguous cluster [START_REF] Owen | Characterization of pyoverdine and achromobactin in Pseudomonas syringae pv. phaseolicola 1448a[END_REF] or be dispersed in the genome in three or four clusters separated by long stretches of DNA encoding genes for other functions [START_REF] Ravel | Genomics of pyoverdinemediated iron uptake in pseudomonads[END_REF][START_REF] Moon | Genomic, genetic and structural analysis of pyoverdine-mediated iron acquisition in the plant growth-promoting bacterium Pseudomonas fluorescens SBW25[END_REF]. In addition, the genetic context of certain genes is not conserved between Pseudomonads. For example, the ptaA gene in P. aeruginosa is located upstream of the pyoverdine region, which suggests that the PtaA enzyme may have additional functions [START_REF] Ringel | The periplasmic transaminase PtaA of Pseudomonas fluorescens converts the glutamic acid residue at the pyoverdine fluorophore to α-ketoglutaric acid[END_REF].

PCH and Enantio-pyochelin biosynthesis

PCH, which is produced by all P. aeruginosa strains, and E-PCH, which is produced by P. fluorescens strains Pf-5 and CHA0, are both condensation products of salicylate and two molecules of cysteine, with the only difference between PCH and E-PCH being the stereochemical configuration of the two incorporated cysteines. Consequently, E-PCH is the optical antipode or enantiomer of PCH [START_REF] Youard | Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin[END_REF]. PCH biosynthesis involves seven cytoplasmic enzymes (two of them being NRPS), with their corresponding genes organized into two operons, pchDCBA and pchEFGHI (Fig. 3) [START_REF] Serino | Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa[END_REF][START_REF] Reimmann | Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF genes, are induced by extracellular pyochelin in Pseudomonas aeruginosa[END_REF]. PCH biosynthesis (Fig. 4) begins with salicylate synthesis: chorismate is first transformed into isochorismate and subsequently into salicylate by the enzymes PchA (isochorismate synthase) and PchB (isochorismate-pyruvate lyase) respectively [START_REF] Gaille | Isochorismate synthase (PchA), the first and rate-limiting enzyme in salicylate biosynthesis of Pseudomonas aeruginosa[END_REF][START_REF] Meneely | Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited[END_REF]. Salicylate is then activated by PchD and transferred to the NRPS enzyme PchE for coupling to a molecule of cysteine under the control of PchC [START_REF] Serino | Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa[END_REF][START_REF] Reimmann | PchC thioesterase optimizes nonribosomal biosynthesis of the peptide siderophore pyochelin in Pseudomonas aeruginosa[END_REF]. PchC is a thioesterase that removes wrongly charged molecules from the peptidyl carrier protein domains of PchE and PchF (see below) [START_REF] Reimmann | PchC thioesterase optimizes nonribosomal biosynthesis of the peptide siderophore pyochelin in Pseudomonas aeruginosa[END_REF]. PchE also ensures L-Cys epimerization into D-Cys, generating dihydroaeruginoique (Dha) [START_REF] Patel | Epimerization of an L-cysteinyl to a D-cysteinyl residue during thiazoline ring formation in siderophore chain elongation by pyochelin synthetase from Pseudomonas aeruginosa[END_REF]. A second molecule of cysteine is coupled to Dha by another NRPS enzyme, PchF, again under the control of PchC [START_REF] Reimmann | PchC thioesterase optimizes nonribosomal biosynthesis of the peptide siderophore pyochelin in Pseudomonas aeruginosa[END_REF]. This second cysteine undergoes cyclisation by the cycling module of PchF, to form nor-pyochelin, and methylation on the second thiazolidine cycle by the methylation module of PchF [START_REF] Patel | Epimerization of an L-cysteinyl to a D-cysteinyl residue during thiazoline ring formation in siderophore chain elongation by pyochelin synthetase from Pseudomonas aeruginosa[END_REF][START_REF] Ronnebaum | Stuffed Methyltransferase catalyzes the penultimate step of pyochelin biosynthesis[END_REF]. The synthesized PCH is then released by the reductase PchG [START_REF] Patel | In vitro reconstitution of the Pseudomonas aeruginosa nonribosomal peptide synthesis of pyochelin: characterization of backbone tailoring thiazoline reductase and N-methyltransferase activities[END_REF][START_REF] Reimmann | Essential PchGdependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa[END_REF]. PCH biosynthesis occurs in the cytoplasm and nothing is currently known about PCH secretion or the proteins and mechanisms involved. More details concerning PCH biosynthesis can be found in an excellent recent review by [START_REF] Ronnebaum | Nonribosomal peptides for iron acquisition: pyochelin biosynthesis as a case study[END_REF].

The biosynthesis of E-PCH in P. fluorescens has not yet been biochemically investigated but is probably quite similar to the PCH pathway in P. aeruginosa. A closely related gene cluster is present in the chromosome of the P. fluorescens strains Pf-5 and CHA0, although the arrangement of the individual genes is different from that in P. aeruginosa and there is no gene with obvious sequence homology to pchG [START_REF] Paulsen | Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5[END_REF][START_REF] Youard | Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin[END_REF][START_REF] Youard | Iron acquisition with the natural siderophore enantiomers pyochelin and enantio-pyochelin in Pseudomonas species[END_REF].

Quinolobactin and thioquinolobactin biosynthesis

Quinolobactin, an 8-hydroxy-4-methoxy-2-quinolone carboxylic acid, is produced by P. fluorescens ATCC17400 from xanthurenic acid. The biosynthetic pathway is still quite speculative (Fig. 4), involves at least four enzymes, and starts from xanthurenic acid [START_REF] Matthijs | The Pseudomonas siderophore quinolobactin is synthesized from xanthurenic acid, an intermediate of the kynurenine pathway[END_REF]. The first step requires the AMPligase QbsL, which activates the carboxylic group of xanthurenic acid via its N-terminal domain and methylates the hydroxyl group in the fourth position via its Nterminal methylase domain. Then, QbsCDE enzymes transfer sulphur from an unknown sulphur donor molecule to form 8-hydroxy-4-methoxy-2-quinoline thiocarboxylic acid (thioquinolobactin). The formation of this compound probably also involves the putative oxidoreductase QbsK. Quinolobactin is probably then formed by spontaneous hydrolysis of thioquinolobactin.

PDTC biosynthesis

PDTC is produced by P. stutzeri [START_REF] Lee | Identification of an extracellular agent [correction of catalyst] of carbon tetrachloride dehalogenation from Pseudomonas stutzeri strain KC as pyridine-2, 6-bis (thiocarboxylate)[END_REF] and P. putida [START_REF] Ockels | An Fe(II) complex of pyridine-2,6-di-(monothiocarboxylic acid) -a novel bacterial metabolic product[END_REF] and its currently proposed biosynthetic pathway involves three major steps and five enzymes (Fig. 4) [START_REF] Sepúlveda-Torre | Analysis of regulatory elements and genes required for carbon tetrachloride degradation in Pseudomonas stutzeri strain KC[END_REF]. It starts with the reduction of 2,3-dihydro-dipicolinic acid by the reductase OrfI into diplicolinic acid, which is then activated by OrfJ. The activated compound undergoes sulfation, probably involving the three enzymes OrfFGH, to give PDTC. The exact role of each of the three enzymes OrfFGH is still unknown.

Achromobactin biosynthesis

Achromobactin is produced by P. syringae and results from the condensation of one citrate molecule and one each of ethanolamine, 2,4-diaminobutyrate and α-ketoglutarate (Fig. 4). The currently proposed scheme for the biosynthesis of achromobactin involves four enzymes and starts from citrate (Berti and

Cellular organization of siderophore (PVDI) biosynthesis

Many biosynthetic pathways are based on networks of enzymes that are able to form multi-enzyme complexes [START_REF] Schmitt | Spatial organization of metabolic enzyme complexes in cells[END_REF], with their spatial organization depending on their protein-protein interactions. Moreover, the amount and activity of each enzyme in these biosynthetic pathways have evolved to be carefully regulated to minimize their production cost to the cells and maximize their efficiency. In P. aeruginosa PAO1, the enzymes involved in the cytoplasmic biosynthesis of PVDI and PCH have been proposed to assemble into siderosomes, i.e. siderophore-specific assemblies of enzymes involved in the synthesis of specific siderophores [START_REF] Guillon | High cellular organisation of pyoverdine biosynthesis in Pseudomonas aeruginosa: localization of PvdA at the old cell pole[END_REF][START_REF] Imperi | Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome[END_REF][START_REF] Cunrath | A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa[END_REF][START_REF] Gasser | Cellular organization of siderophore biosynthesis in Pseudomonas aeruginosa: evidence for siderosomes[END_REF]. Siderosomes were first hypothesized for the cytoplasmic PVDI enzymes on the basis of pull-down assays using a recombinant 6His-PvdA protein as bait to capture low amounts of the NRPS enzymes PvdJ and PvdL [START_REF] Imperi | Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome[END_REF]. The small fraction of PvdJ and PvdL trapped by PvdA suggested transient and dynamic interactions between these proteins. PvdA was also shown to interact with the isolated M2 module of PvdJ in yeast two-hybrid experiments [START_REF] Imperi | Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome[END_REF]. By fluorescence microscopy, PVDI-related proteins appear to be spatially organized in live cells, with clusters of PvdA colocalizing with PvdD, PvdL and PvdJ [START_REF] Guillon | High cellular organisation of pyoverdine biosynthesis in Pseudomonas aeruginosa: localization of PvdA at the old cell pole[END_REF][START_REF] Imperi | Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome[END_REF]. These clusters appear as fluorescent spots located at the cell poles and are linked to iron-restriction and high levels of PVDI production [START_REF] Guillon | High cellular organisation of pyoverdine biosynthesis in Pseudomonas aeruginosa: localization of PvdA at the old cell pole[END_REF][START_REF] Imperi | Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome[END_REF]. In the context of the PVDI cytoplasmic precursor, for which its biosynthesis occurs through the sequential addition of amino acids by NRPS enzymes, the interactions and spatial organization of these enzymes are thought to optimize the transfer of siderophore precursors between them and avoid their diffusion throughout the cytoplasm to prevent deleterious intra-cell metal chelation. Similar spatial patterns were observed for the PCH pathway, with the NRPS enzyme PchE colocalizing with PchA [START_REF] Cunrath | A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa[END_REF]. PchE clustering at the bacterial poles was found to be dependent on PchA expression, whereas PchA clustering and association with the membrane was PchE-independent. This suggests a complex interplay between the various partners that form siderosomes [START_REF] Cunrath | A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa[END_REF]. Classical fluorescence microscopy is constrained by the diffraction of light, which limits its spatial resolution to approximately 250-300 nm. Given the size of Pseudomonas bacteria (rod shaped with long and short axes of approximately 1.5-2 and 0.6-0.8 μm respectively), the ability of fluorescence microscopy to provide precise and accurate information on the localization of proteins is limited. At this diffraction-limited level of resolution, proteins can localize to the same subcellular region without interacting, rendering colocalization experiments difficult to interpret.

Protein interactions in living cells can be indirectly inferred from diffusion properties, as large complexes diffuse more slowly than smaller ones or free unbound proteins. FRAP, a technique that measures the repopulation of fluorescently labelled proteins in a photobleached area, is able to quantify the two-dimensional lateral diffusion of proteins in situ [START_REF] Axelrod | Mobility measurement by analysis of fluorescence photobleaching recovery kinetics[END_REF] and provides information about possible interactions between proteins. FRAP has been used to characterize the diffusion properties of proteins of the PVDI siderophore pathway [START_REF] Guillon | High cellular organisation of pyoverdine biosynthesis in Pseudomonas aeruginosa: localization of PvdA at the old cell pole[END_REF]. PvdA was found to diffuse homogeneously in the cytoplasm, with an average diffusion rate that was slightly lower than that predicted from its molecular weight and a free diffusion model. The accumulation of PvdA at the cell pole was found to be reversible, as the fluorescence of a bleached out-of-spot area in the cytoplasm completely recovered due to diffusion of fluorescent PvdA coming from the fluorescent spots [START_REF] Guillon | High cellular organisation of pyoverdine biosynthesis in Pseudomonas aeruginosa: localization of PvdA at the old cell pole[END_REF].

More recently, the diffusion of PvdA has been investigated using sptPALM [START_REF] Gasser | In cellulo FRET-FLIM and single molecule tracking reveal the supra-molecular organization of the pyoverdine bio-synthetic enzymes in Pseudomonas aeruginosa[END_REF]. sptPALM enables the characterization of the diffusion trajectories of single proteins with nanometric precision and high temporal resolution [START_REF] Manley | High-density mapping of single-molecule trajectories with photoactivated localization microscopy[END_REF]. The statistical description of thousands of single PvdA traces in live cells showed that PvdA diffuses throughout the cytoplasm, without any evident spatial constraints or structural organization at 40-nm resolution (Fig. 5), with the exception of preferential accumulation at one pole in some cells. Heterogeneous velocities of PvdA displacements corresponded to two diffusing populations, assigned to a trapped (or restrained) fraction of PvdA (approximately 15%) and diffusing PvdA (Fig. 5). Consistent with the transient nature of siderosomes, which associate and dissociate in vivo, these two populations were found to be exchangeable, and transition from diffusing to restrained or restrained to diffusing was observed in single traces within the time-scale of observation. Finally, the diffusion rate of the diffusing PvdA was in very good agreement with that characterized by FRAP, leading to the hypothesis that PvdA is mostly bound to complexes that can slowly diffuse throughout the cytoplasm of the cells.

Finally, the interactions of PvdA with the NRPS enzymes of the PVDI pathway were explored using Förster resonance energy transfer measured by fluorescence lifetime imaging (FLIM-FRET enables the monitoring of protein-protein interactions and the mapping of their spatial organization in a living cell with diffraction-limited spatial resolution [START_REF] Duncan | Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells[END_REF]. However, two labelled proteins that undergo FRET have to physically interact because of the strong inter-dye distance dependence required for FRET to occur and the relatively large size of fused fluorescent proteins. FRET-FLIM was used to characterize the interactions of PvdA with the four different NRPS enzymes of the PVD pathway [START_REF] Gasser | In cellulo FRET-FLIM and single molecule tracking reveal the supra-molecular organization of the pyoverdine bio-synthetic enzymes in Pseudomonas aeruginosa[END_REF]. Surprisingly, FRET-FLIM clearly showed that PvdA physically interacts with all four NRPS enzymes of the PVDI pathway in the cellular context and not only with PvdJ and/or PvdI, the two NRPS enzymes that use fOHOrn, the molecule produced by PvdA, as a building block. Even more interestingly, the stoichiometry of the interacting complex was not the same depending on the NRPS enzyme bound by PvdA. Several PvdA molecules interacted with PvdI, whereas PvdA formed one-to-one (or close to one-toone) complexes with PvdJ. The M2 module of PvdJ, previously identified by two-hybrid studies to be an interacting partner of PvdA [START_REF] Imperi | Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome[END_REF] and responsible for fOHOrn insertion, is a good candidate to harbour the binding site for PvdA. In contrast to PvdJ, multiple binding of PvdA to PvdI likely fulfils the necessity for an excess of locally available substrate to optimize the activity of PvdI. These observations also suggest that, in addition to physical coordination between active sites of tailored enzymes and NRPS modules, the colocalization of such enzymes may be sufficient to promote metabolic efficiency, making siderosomes even more relevant for efficient siderophore production.

PvdA has also been shown to have a hydrophobic, inner-membrane-anchoring domain at the N terminus [START_REF] Meneely | Kinetic mechanism of ornithine hydroxylase (PvdA) from Pseudomonas aeruginosa: substrate triggering of O 2 addition but not flavin reduction[END_REF][START_REF] Imperi | Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome[END_REF]. This association of PvdA with the inner membrane and the presence of a myristic acid chain attached to the first amino acid of the PVDI backbone (Hannauer et al., 2012b) also led to the suggestion that PVDI is synthesized on the cytoplasmic face of the inner membrane, with siderosomes associated with the inner membrane leaflet. Imperi et al. analysed isolated inner membranes of P. aeruginosa by matrix-assisted laser desorption/ionization time-of-flight and reported that a fraction of each of the four NRPS enzymes (PvdL, PvdI, PvdJ and PvdD) involved in PVDI biosynthesis was associated with the inner membrane [START_REF] Imperi | Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome[END_REF], this was also confirmed by cell fractionation assays using fluorescent labelled NRPS of the PVDI and PCH pathways [START_REF] Imperi | Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome[END_REF][START_REF] Cunrath | A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa[END_REF][START_REF] Gasser | Cellular organization of siderophore biosynthesis in Pseudomonas aeruginosa: evidence for siderosomes[END_REF]. These observations led to the hypothesis that siderosomes can exist in the bacterial cytoplasm, but they may also associate with the inner leaflet of cytoplasmic membranes (Fig. 6). Many questions about siderosomes remain unanswered, including how they interact with the inner membrane, the role of the myristic chain present in the PVDI precursor, whether and how NRPS enzymes interact with each other in siderosomes, how the organization of siderosomes affect the activity of the enzymes, whether they are always active in producing PVDI molecules and whether the enzymes involved in the biosynthetic pathways of any siderophores are organized in siderosomes.

Regulation

Siderophore production is generally highly regulated at the transcriptional level through regulation of the expression of the genes encoding the enzymes involved in their biosynthesis. Expression of these genes is repressed by the presence of iron and activated under iron-restricted conditions via molecular mechanisms that require transcriptional regulators [START_REF] Cornelis | Iron uptake regulation in Pseudomonas aeruginosa[END_REF].

Negative regulation for the biosynthesis of all the siderophores described above involves the transcriptional regulator Fur (Ferric Uptake Regulator). For a review dedicated to Fur see [START_REF] Fillat | The FUR (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators[END_REF]. Fur senses the cytoplasmic concentration of Fe 2+ . Once the concentration of Fe 2+ in the bacterial cytoplasm reaches a certain concentration, it binds to Fur and the Fur-Fe 2+ complexes repress the transcription of any genes involved in iron acquisition and consequently those encoding siderophore biosynthetic enzymes (Fig. 7). Such repression involves the interaction of Fur-Fe 2+ with a conserved sequence, called the Fur-box, in the promoter regions of all ironregulated genes [START_REF] Escolar | Opening the iron box: transcriptional metalloregulation by the fur protein[END_REF]. When iron becomes limiting, fewer Fur-Fe 2+ complexes form in the bacterial cytoplasm and the Fur-Fe 2+ complexes dissociate from the Fur-boxes, allowing a basal level of gene expression [START_REF] Escolar | Opening the iron box: transcriptional metalloregulation by the fur protein[END_REF]. Fur-dependent repression has been shown for the pchDCBA, pchEFGHI genes of PCH biosynthesis under iron-rich conditions and for all genes of the PVDI pathways [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].

Conversely, under iron-restricted conditions, Fur no longer acts as a repressor. However, basal expression of the genes encoding the biosynthetic enzymes is low and positive activating loops come into play to achieve high production of pyoverdine and PCH siderophores. The positive regulation of PCH biosynthesis in P. aeruginosa involves the AraC transcriptional regulator PchR, which activates transcription of the pchDCBA and pchEFGHI genes, encoding enzymes involved in PCH biosynthesis (Fig. 7) [START_REF] Heinrichs | Cloning and sequence analysis of a gene (pchR) encoding an AraC family activator of pyochelin and ferripyochelin receptor synthesis in Pseudomonas aeruginosa[END_REF][START_REF] Heinrichs | PchR, a regulator of ferripyochelin receptor gene (fptA) expression in Pseudomonas aeruginosa, functions both as an activator and as a repressor[END_REF][START_REF] Reimmann | Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF genes, are induced by extracellular pyochelin in Pseudomonas aeruginosa[END_REF]. PCH-Fe 3+ complexes and their uptake into the bacterial cytoplasm are required for this activation process. They act as effectors of PchR, by which PchR-PCH-Fe 3+ complexes bind to the conserved PchR-box sequence in the promoter regions of the biosynthetic genes [START_REF] Michel | PchR-box recognition by the AraCtype regulator PchR of Pseudomonas aeruginosa requires the siderophore pyochelin as an effector[END_REF][START_REF] Michel | Ferripyochelin uptake genes are involved in pyochelinmediated signalling in Pseudomonas aeruginosa[END_REF]. Such activation allows the production of approximately 40 μM of PCH for a culture of P. aeruginosa PAO1 cells of optical density at 600 nm of 1, grown under iron restriction conditions [START_REF] Cunrath | Metallome of Pseudomonas aeruginosa: a role for siderophores[END_REF]. pchR transcription is itself negatively regulated by Fur and PchR itself, as the PchR box of pchD is located downstream of the pchR transcription start site [START_REF] Michel | PchR-box recognition by the AraCtype regulator PchR of Pseudomonas aeruginosa requires the siderophore pyochelin as an effector[END_REF]. A similar regulatory mechanism involving a PchR regulator is involved in E-PCH production in P. fluorescens [START_REF] Lin | In vitrobinding of the natural siderophore enantiomers pyochelin and enantiopyochelin to their AraC-type regulators PchR in Pseudomonas[END_REF].

The positive autoregulation loop of PVDI biosynthesis involves a completely different mechanism than that of PCH biosynthesis, with transcriptional regulators of another family (Fig. 7): two cytoplasmic sigma factors (PvdS and FpvI) and the inner membrane anti-sigma factor (FpvR) [START_REF] Visca | In Iron Regulation and Siderophore Signalling in Virulence by Pseudomonas aeruginosa. Pseudomonas[END_REF][START_REF] Llamas | Cell-surface signaling in Pseudomonas: stress responses, iron transport, and pathogenicity[END_REF]. PvdS activates the transcription of PVDI biosynthetic genes, as well as some that encode virulence factors [START_REF] Prince | Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene[END_REF][START_REF] Cunliffe | Cloning and characterization of pvdS, a gene required for pyoverdine synthesis in Pseudomonas aeruginosa: PvdS is probably an alternative sigma factor[END_REF][START_REF] Ochsner | Exotoxin A production in Pseudomonas aeruginosa requires the iron-regulated pvdS gene encoding an alternative sigma factor[END_REF][START_REF] Vasil | The fur-regulated gene encoding the alternative sigma factor PvdS is required for iron-dependent expression of the LysR-type regulator ptxR in Pseudomonas aeruginosa[END_REF][START_REF] Wilson | Characterization of an ECF sigma factor protein from Pseudomonas aeruginosa[END_REF][START_REF] Wilderman | Characterization of an endoprotease (PrpL) encoded by a PvdSregulated gene in Pseudomonas aeruginosa[END_REF][START_REF] Visca | In Iron Regulation and Siderophore Signalling in Virulence by Pseudomonas aeruginosa. Pseudomonas[END_REF][START_REF] Gaines | Regulation of the Pseudomonas aeruginosa toxA, regA and ptxR genes by the iron-starvation sigma factor PvdS under reduced levels of oxygen[END_REF] activates transcription of the fpvA gene, encoding the outer membrane transporter that imports PVDI-Fe 3+ complexes from the environment [START_REF] Redly | Pyoverdine-mediated regulation of FpvA synthesis in Pseudomonas aeruginosa: involvement of a probable extracytoplasmic-function sigma factor, FpvI[END_REF][START_REF] Redly | FpvIR control of fpvA ferric pyoverdine receptor gene expression in Pseudomonas aeruginosa: demonstration of an interaction between FpvI and FpvR and identification of mutations in each compromising this interaction[END_REF]. Broadly [for more details see the reviews [START_REF] Llamas | Cell-surface signaling in Pseudomonas: stress responses, iron transport, and pathogenicity[END_REF]], this regulatory mechanism first requires the binding of PVDI-Fe to the outer membrane transporter FpvA, which leads to the interaction of the periplasmic domain of FpvA with the inner membrane anti-sigma factor FpvR [START_REF] Brillet | A β-strand lockexchange for signal transduction in TonB-dependent transducers on the basis of a common structural motif[END_REF], resulting in the release of the sigma factors PvdS and FpvI into the cytoplasm. They then activate transcription of the genes that they regulate, such as those that encode PVDI biosynthetic enzymes, resulting in an increase in PVDI production [START_REF] Wilson | Analysis of promoters recognized by PvdS, an extracytoplasmic-function sigma factor protein from Pseudomonas aeruginosa[END_REF]Redly andPoole, 2003, 2005;[START_REF] Spencer | Role of cell surface signaling in proteolysis of an alternative sigma factor in Pseudomonas aeruginosa[END_REF][START_REF] Draper | Differential proteolysis of sigma regulators controls cell-surface signalling in Pseudomonas aeruginosa[END_REF][START_REF] Bastiaansen | Processing of cell-surface signalling anti-sigma factors prior to signal recognition is a conserved autoproteolytic mechanism that produces two functional domains[END_REF]. If PVDI is unable to chelate Fe 3+ in the bacterial environment, FpvR sequesters most of the FpvI and PvdS present in the bacterial cells, blocking activation of the transcription of the regulated genes [START_REF] Redly | FpvIR control of fpvA ferric pyoverdine receptor gene expression in Pseudomonas aeruginosa: demonstration of an interaction between FpvI and FpvR and identification of mutations in each compromising this interaction[END_REF][START_REF] Minandri | Role of iron uptake systems in Pseudomonas aeruginosa virulence and airway infection[END_REF]. However, as less FpvR is expressed than sigma factors in P. aeruginosa cells, basal levels of PvdS and FpvI are still present in the cytoplasm, resulting in a low level of PVDI production that can prime activation of the regulatory loop [START_REF] Edgar | Integrated activities of two alternative sigma factors coordinate iron acquisition and uptake by Pseudomonas aeruginosa[END_REF]. Such regulation of pyoverdine production involving sigma and anti-sigma factors is also used by other fluorescent Pseudomonads, such as P. putida and P. protegens [START_REF] Llamas | Cell-surface signaling in Pseudomonas: stress responses, iron transport, and pathogenicity[END_REF]. Signals other than the iron concentration, such as the level of bis-(3 0 -5 0 )-cyclic dimeric guanosine monophosphate, phosphate starvation, sulphur availability, biofilm formation, and alginate production and other transcriptional regulators have been shown to regulate PVDI production in P. aeruginosa PAO1 cells. However, the molecular mechanisms have not yet been clearly elucidated [START_REF] Delic-Attree | Cloning, sequence and mutagenesis of the structural gene of Pseudomonas aeruginosa CysB, which can activate algD transcription[END_REF][START_REF] Zaborin | Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1[END_REF][START_REF] Imperi | Transcriptional control of the pvdS iron starvation sigma factor gene by the master regulator of sulfur metabolism CysB in Pseudomonas aeruginosa[END_REF][START_REF] Balasubramanian | Deep sequencing analyses expands the Pseudomonas aeruginosa AmpR regulon to include small RNA-mediated regulation of iron acquisition, heat shock and oxidative stress response[END_REF][START_REF] Chen | Multiple diguanylate cyclasecoordinated regulation of pyoverdine synthesis in Pseudomonas aeruginosa[END_REF]. Under strong iron-restricted conditions, PVDI production can reach concentrations of approximately 80 μM for a culture of P. aeruginosa cells of an optical density at 600 nm of (iron restriction growth conditions) [START_REF] Cunrath | Metallome of Pseudomonas aeruginosa: a role for siderophores[END_REF].

Moreover, several studies have shown that the presence of metals other than iron in the bacterial environment can also modulate the bacterial production of siderophores [START_REF] Huyer | Zn increases Siderophore production in Azotobacter vinelandii[END_REF][START_REF] Hofte | Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2[END_REF][START_REF] Hu | Siderophore-mediated aluminum uptake by Bacillus megaterium ATCC 19213[END_REF]. In P. aeruginosa, no metals are able to significantly activate PVDI or PCH production above that induced by iron restriction [START_REF] Lopez | Non-specific interference of cobalt with siderophore-dependent iron uptake pathways[END_REF]. However, PCH synthesis in P. aeruginosa is repressed by Co 2+ and Ni 2+ , with the same efficiency as that by Fe 3+ for Co 2+ (Carballido Lopez et al., 2019). As described above, the transcriptional repressor Fur becomes loaded with ferrous iron in the presence of increasing Fe 3+ concentrations and represses the expression of all pch genes. Fur is not involved in the decrease of PCH production in the presence of increasing Co 2+ or Ni 2+ concentrations, but rather the transcriptional activator PchR [START_REF] Lopez | Non-specific interference of cobalt with siderophore-dependent iron uptake pathways[END_REF]. This regulator becomes loaded with PCH-Co and PCH-Ni, as both complexes can enter bacterial cells. Consequently, PchR is no longer able to activate transcription of the pch genes due to a decrease in the intracellular concentration of PchR-PCH-Fe complexes. The repression of PCH production in the presence of Co 2+ , and probably that of Ni 2+ , is due to a non-specific interaction of PCH-Co with PchR, which is no longer able to activate PCH production.

At last, once all the enzymes involved in the biosynthesis of a siderophore are expressed, the regulation of their enzymatic activity may also be regulated. However, nothing is known yet about such a possible regulation. The organization in siderosomes could play such a regulating role. Moreover, bacteria live in communities with some producing siderophores and some acting as cheater (do not produce siderophores but use those produced by other bacteria); such social interactions in communities also affect the regulation of siderophore production [START_REF] Butaitė | Siderophore cheating and cheating resistance shape competition for iron in soil and freshwater Pseudomonas communities[END_REF][START_REF] Granato | The path to reevolve cooperation is constrained in Pseudomonas aeruginosa[END_REF][START_REF] Butaitė | Environmental determinants of pyoverdine production, exploitation and competition in natural Pseudomonas communities[END_REF][START_REF] Özkaya | Cheating on cheaters stabilizes cooperation in Pseudomonas aeruginosa[END_REF][START_REF] Stilwell | The effect of cheats on siderophore diversity in Pseudomonas aeruginosa[END_REF]. This question needs to be investigated further in the future at the level of diverse communities involving different Pseudomonads but also other bacterial species since P. aeruginosa is able to use many different siderophores produced by other bacteria (exosiderophores) [START_REF] Cornelis | Diversity of siderophoremediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines[END_REF][START_REF] Cornelis | Pseudomonas aeruginosa adapts its iron uptake strategies in function of the type of infections[END_REF]. It has been shown that the presence of exosiderophores clearly impacts the expression levels of the proteins of the different iron uptake pathways of P. aeruginosa [START_REF] Llamas | The heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas aeruginosa[END_REF][START_REF] Llamas | Characterization of five novel Pseudomonas aeruginosa cellsurface signalling systems[END_REF][START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF].

Conclusions

Siderophore biosynthetic pathways can be highly complex, involving highly diverse enzymes. We now have Transcriptional regulation of genes coding for proteins involved in the PVDI and PCH iron-uptake pathways. Under iron-restricted conditions, transcription of the genes of the PCH pathway is activated via the transcriptional regulator PchR. This protein, in a complex with PCH-Fe 3+ , activates the transcription of all the genes of the PCH pathway, except pchR, by interacting with the PchR box. In the PVDI pathway, two sigma factors, PvdS and FpvI, are involved in activation of the transcription of the genes of the pvd locus. FpvI activates the transcription of only fpvA, the outer membrane importer of ferri-PVDI. PvdS activates the transcription of all other genes, except pvdS, fpvI and fpvR (FpvR being the antisigma factor of PvdS and FpvI). In the presence of iron, gene transcription in both pathways is repressed by the transcriptional regulator Fur in a complex with Fe 2+ . The Fur-Fe 2+ complex binds to the Fur box in the promoter region of the various genes and operons encoding the enzymes involved in PVD and PCH biosynthesis and also represses transcription of the transcriptional regulators PvdS, FpvI and PchR. For more details see the text.

© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology precise knowledge of the various enzymatic steps involved in the PVDI and PCH pathways. However, many questions remain for most of the biosynthesis pathways of the other siderophores produced by fluorescent Pseudomonads and a major effort is also necessary to unravel all the existing subtleties and variations in the biosynthesis of the divers pyoverdines produced by these bacteria. The cellular organization of these enzymes, their distribution in the bacterial cells, the existence of siderosomes and the diverse protein interactions involved in these potential enzymatic complexes also still raise many questions and concerns. Having precise knowledge of the biosynthetic pathways of siderophores can be a true asset in biotechnology. Indeed, because of the strong metal chelating properties of these compounds and their importance in bacterial iron homeostasis, siderophores have many applications in either biomedical [START_REF] Bedford | Iron chelation in the treatment of cancer: a new role for deferasirox?[END_REF][START_REF] Mislin | Siderophoredependent iron uptake systems as gates for antibiotic Trojan horse strategies against Pseudomonas aeruginosa[END_REF][START_REF] Schalk | Bacterial iron uptake pathways: gates for the import of bactericide compounds[END_REF][START_REF] Schalk | Siderophore-antibiotic conjugates: exploiting iron uptake to deliver drugs into bacteria[END_REF] or bioremediation [START_REF] Cornu | Contrasting effects of pyoverdine on the phytoextraction of Cu and Cd in a calcareous soil[END_REF][START_REF] Ferret | Effect of pyoverdine supply on cadmium and nickel complexation and phytoavailability in hydroponics[END_REF][START_REF] Hazotte | Effect of Pseudomonas fluorescens and pyoverdine on the phytoextraction of cesium by red clover in soil pots and hydroponics[END_REF]

Fig. 1 .

 1 Fig. 1. Color Figure -Print and Online A. General structure of pyoverdines and the structure of PVDI, the pyoverdine produced by P. aeruginosa PAO1. The chromophore is in green, the side chain in black and the peptide moiety in blue. The sequences of the peptide moiety of the four pyoverdines produced by P. aeruginosa strains are also shown. B. Structures of the secondary siderophores produced by fluorescent Pseudomonads. Pyochelin (PCH) is produced by Pseudomonas aeruginosa, Enantio-pyochelin (E-PCH) by Pseudomonas fluorescens[START_REF] Youard | Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin[END_REF][START_REF] Hoegy | Stereospecificity of the siderophore pyochelin outer membrane transporters in fluorescent Pseudomonads[END_REF], Yersiniabactin and Achromobactin by Pseudomonas syringae[START_REF] Jones | Salicylic acid, yersiniabactin, and pyoverdin production by the model phytopathogen Pseudomonas syringae pv. tomato DC3000: synthesis, regulation, and impact on tomato and Arabidopsis host plants[END_REF][START_REF] Petermann | The Yersinia high pathogenicity Island is present in Salmonella enterica subspecies I isolated from turkeys[END_REF], Pseudomonine by Pseudomonas fluorescens and Pseudomonas entomophila[START_REF] Mercado-Blanco | Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374[END_REF][START_REF] Matthijs | Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440[END_REF], Quinolobactin and Thio-quinolobactin by Pseudomonas fluorescence ATCC17400[START_REF] Matthijs | The Pseudomonas siderophore quinolobactin is synthesized from xanthurenic acid, an intermediate of the kynurenine pathway[END_REF][START_REF] Matthijs | Thioquinolobactin, a Pseudomonas siderophore with antifungal and anti-Pythium activity[END_REF], PDTC (Pyridine-2,6-bis(thiocarboxylate)) by Pseudomonas stutzeri[START_REF] Lee | Identification of an extracellular agent [correction of catalyst] of carbon tetrachloride dehalogenation from Pseudomonas stutzeri strain KC as pyridine-2, 6-bis (thiocarboxylate)[END_REF] and Pseudomonas putida[START_REF] Ockels | An Fe(II) complex of pyridine-2,6-di-(monothiocarboxylic acid) -a novel bacterial metabolic product[END_REF] and Corrugatin by Pseudomonas acorrugata[START_REF] Matthijs | Ornicorrugatin, a new siderophore from Pseudomonas fluorescens AF76[END_REF].

Fig. 2 .

 2 Fig. 2. Color Figure -Print and Online Biosynthesis pathways of PVDI in P. aeruginosa PAO1. A. Synthesis of the pyoverdine precursor in the bacterial cytoplasm. Each of the four NRPS (PvdL, PvdI, PvdJ and PvdD) involved in the synthesis is divided into modules (M). Each module is composed of a condensation domain (C), an acetylation domain (A) and a peptidyl carrier protein (PCP) domain. PvdL and PvdI contain epimerization domains (E) and PvdD a thioesterase domain (Te). The amino acids incorporated in the chain are indicated below each module, depicted by the corresponding colour and represented by coloured circles in the final precursor. B. Maturation of pyoverdine precursor into PVDI isoforms in the bacterial periplasm. After translocation of pyoverdine precursor from the cytoplasm into the periplasm via PvdE, pyoverdine precursor undergoes a maturation process involving five enzymes (PvdQ, PvdP, PvdO, PvdN and PtaA). The green and red circles on pyoverdine isoforms represent succinic acid and α-ketoglutaric acid respectively. For more details on PVDI biosynthesis see the text.

Fig

  Fig. 3.

  2009). The first step consists of the conversion of citrate to O-citryl-serine by the synthetase AcsD, followed by decarboxylation by AcsE to obtain O-citryl-ethanolamine[START_REF] Schmelz | AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosynthesis[END_REF]. Then, AcsC synthetase transforms diaminobutyryl-citryl-ethanolamine into O-citryl-ethanolamine. The last step consists of the addition of two molecules

Fig. 4 .

 4 Fig. 4. Biosynthesis pathways of pyochelin, quinolobactin, thioquinolobactin, PDTC and achromobactin. See the text for details of each biosynthesis pathway.

Fig. 5 .

 5 Fig. 5. Color Figure -Print and Online Single-molecule tracking of PvdA-PAmCherry. A. Raw fluorescence signal (upper) and focus on an ROI (lower) of a single PvdA-PAmCherry molecule in a live cell (yellow) observed over time at 62.5 Hz. Five frames from the temporal stack of images separated by approximately 50 ms are represented. The contour of the bacterial cell (red overlay) was determined from the corresponding phase-contrast microscopy image. The localization of single PvdA-PAmCherry molecules is highlighted by the blue or purple overlaid circles. The size of the circle approximately corresponds to the diffraction limit (~250 nm). The precision, which determines the uncertainty of the estimated position of the PvdA-PAmCherry, is approximately 40 nm. The time-trace linking the localization at different time points for a given PvdA-PAmCherry molecule is represented by the cyan segments. Scale bar = 1 μm. B. PvdA-PAmCherry diffusion map in a single representative cell. Approximately 2500 localizations, generating approximately 400 fluorescence traces, are represented. Each displacement step is colour coded according to the displacement jump distance since the previous frame (in micrometre). C. Amplitude of the constrained and diffusing species of PvdA-PAmCherry as a function of their diffusion coefficients averaged per cell (n = 23 cells, N = 3 independent experiments) (data from Gasser et al., 2020).

Fig. 6 .

 6 Fig. 6. Color Figure -Print and Online Scheme of the siderosome involved in PVDI biosynthesis. The seven enzymes involved in the cytoplasmic biosynthesis of the PVDI precursor are represented. The four NRPS (PvdL, PvdI, PvdJ and PvdD) are responsible for the synthesis of the 11 amino acid peptides, and PvdH, PvdA and PvdF are the accessory proteins involved in the synthesis of L-Dab and L-fOH Orn. Enzyme diameters are proportional to their molecular weight (MW). On the right of the figure a schematic view of the enzymatic complex called siderosome (for more details see the text).

Fig. 7 .

 7 Fig. 7. Color Figure -Print and Online

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Cézard, C., Farvacques, N., and Sonnet, P. (2015) Chemistry and biology of pyoverdines, Pseudomonas primary siderophores. Curr Med Chem 22: 165-186. © 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology J. Schalk, C. Rigouin and J. Godet

I. J. Schalk, C. Rigouin and J. Godet

I. J. Schalk, C. Rigouin and J. Godet