Mexjk / Oprm

Tet Ery

/ Mexjk

Opmh

Tri

Mexvw / Oprm

CHL, EB, ERY, FQ, QL, TC MuxABC- Acr

Charlotte Richardot

Paulo Juarez

Katy Jeannot

Isabelle Patry

Patrick Plésiat
email: patrick.plesiat@univ-fcomte

Catherine Llanes
email: cllanesb@univ-fcomte.fr.

-Me-PCA

Keywords: Pseudomonas isolation agar RNAseq: high-throughput RNA sequencing RND: resistance nodulation cell division reactive oxygen species rpm: rotations per minute rRNA: ribosomal RNA mexR, nalC, nalD, armR, rocS1/2-A2, brlR, mexT, cpxR, czcRS, copRS Cd 2+ , Zn , acriflavine, AG, aminoglycosides, ATM, aztreonam, AZI, azithromycin, BL, β-lactams (except carbapenems), BPR, ceftobiprole, CHIR, LpxC inhibitor

.

. In fact, P. aeruginosa has been recently classified by the World Health Organization as a critical priority urgently needing new antibiotics, as the emergence of multi-resistant strains in the clinical setting is constantly increasing (Tacconelly and Magrini, 2017). Such resistance is due to intrinsic mechanisms including the low permeability of the outer membrane, production of enzymes capable to degrade or modify antibiotics (AmpC, OXA-50 and APH(3')-IIb) and the action of two efflux systems (MexAB-OprM and MexXY/OprM) able to export multiple molecules outside the cell (Poole, 2004).

Nevertheless, the spectrum of this resistance can be expanded or intensified by acquired mechanisms, usually selected in presence of antibiotics, resulting from the transfer of mobile genetic elements or from genetic mutations altering expression of resistance-associated genes.

.

Genomic analysis had shown that most of P. aeruginosa strains harbor operons coding for 12 RND-type transporters. The substrate profiles of these transporters have been partially Table 1: Substrate specificities and regulatory genes of characterized RND efflux pumps of Pseudomonas aeruginosa Efflux pump Regulatory gene Substrates

and suggestions will be very valuable for me and for our team.

To Dr. Thilo Köhler, "father" of MexEF-OprN, for accepting to participate to the jury and for all the recommendations and suggestions that you gave to me during the annual thesis committee.

A special greeting to Dr. Christophe Bordi. More than thanking you for your participation to the jury and to my thesis committee, I would especially like to thank you for always being available when I needed some advice or an external opinion about unusual regulation mechanisms. I really appreciated all your good will and your enthusiasm when talking about science.

During this work I could collaborate with Pr. Isabelle Broutin (Université Paris Descartes), Pr.

Mustapha Si-Tahar and Dr. Eric Morello (Université de Tours). I would like to thank them for all the suggestions and ideas that they gave to the "CmrA project"; working with you was very pleasant. Thanks to Sylvie Elsen (CEA, Grenoble) for all the discussions that we had concerning the AraC-like regulators. Your passion for science and your charism were always inspiring.

To my PhD advisors, Pr. Patrick Plésiat and Dr. Catherine Llanes; words would not be enough to thank all that you have done for me. Thank you for all the advices, the time that you spent discussing with me and your good will.

To my team co-workers; Isabelle Patry, Anaïs Potron, Katy Jeannot, Damien Fournier, Arnaud Bolard, Hélène Puja, Eleni Liapis, Loïs Andrey. To the mycology team members; Steffi, Audrey, Adéline, Coralie, Alice, Jenny. To the hygiene team members; Didier, Benoît, Marie.

Thank you for helping me and giving me advice when I needed; for all the sharing and for keeping the good mood at the lab. I told you that I would be brief, but I hope you know how much I appreciated to work with you.

To the former and temp members of the team; Charlotte Richardot, Aurélie Noguès, Mélanie Grosjean, Corentin Daguinot, Luana Silva, Camille Robin, Xiyu Tian, Camil Hadjar, Alexandre Tetard. Thank you all for all your laughter, your advice, your technical support and so other things. For those who are starting this adventure I wish you all the best.

A very special thanks to my co-workers at Smaltis SAS from whom I got a lot of support during the last phases of this work. Cédric and Sophie, I would like to thank you for trusting me and for giving me the opportunity to work with you, I have no doubt that I will learn a lot from both of you; your work and your innovation are very inspiring. I would also like to thank Marjorie and Elise for giving me the support and the words that I needed when finishing this work. I am quite sure that working with you will be awesome. *** Quisiera hacer un agradecimiento especial a mi familia y amigos. Frieda y Roberto. Ustedes me abrieron las puertas de su casa cuando esta aventura comenzaba solamente y durante los días que pasé con ustedes aprendí muchísimas cosas que de no haber estado con ustedes puede ser que no hubiera logrado quedarme. Les estaré siempre agradecido. A mis padres, Sandra y Héctor y mis hermanos; José y Paula. Siempre les estaré agradecidos por todo el esfuerzo que han hecho durante toda la vida. Ustedes cuatro son un ejemplo de trabajo, prosperidad, honestidad, humildad y responsabilidad. Cualidades y valores morales que todo científico necesita.

A mis amigos, Car, Seba, Ate, Lili, Juanpa, Gil, Alex, Aleja, Eli. Gracias por ser parte de esta aventura y por ser tan comprensivos cuando moría de estrés.

List of Figures

List of Tables

I. Introduction

established while the contribution of four pumps to antibiotic resistance has been confirmed

(namely, MexAB-OprM, MexXY/OprM, MexCD-OprJ and MexEF-OprN) (Li et al., 2015).

The MexEF-OprN efflux pump encoded by operon mexEF-oprN, is produced at very-low levels in wild-type strains grown under standard laboratory conditions; therefore it does not significantly contribute to the natural resistance of the species to antimicrobials (Köhler et al., 1997;Li et al., 2000). When upregulated in nfxC mutants, MexEF-OprN tends to decrease intracellular accumulation of a rather limited spectrum of antibiotics (fluoroquinolones, chloramphenicol, trimethoprim and tetracycline) (Köhler et al., 1997), of biocides [START_REF] Chuanchuen | Cross-Resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ[END_REF]) and of quorum sensing precursors [START_REF] Lamarche | MexEF-OprN efflux pump exports the Pseudomonas quinolone signal (PQS) precursor HHQ (4-hydroxy-2-heptylquinoline)[END_REF]. Of note, nfxC mutants are resistant to carbapenems and more susceptible to β-lactams and aminoglycosides when compared to wild-type strains, due to concomitant repression of antibiotic-resistance genes such as oprD, mexAB-oprM and mexXY, respectively (Köhler et al., 1997).

The mode of regulation of operon mexEF-oprN is quite different from that of other efflux operons. Indeed, while these latter are regulated by local repressors, mexEF-oprN is positively controlled by a local activator, MexT. In addition, other several genes such as mexS (Sobel et al., 2005a), mvaT (Westfall et al., 2006), ampR (Balasubramanian et al., 2012), mxtR (Zaoui et al., 2012), brlR (Liao et al., 2013), parR (Wang et al., 2013), nmoR [START_REF] Vercammen | Pseudomonas aeruginosa LysR PA4203 regulator NmoR acts as a repressor of the PA4202 nmoA gene, encoding a nitronate monooxygenase[END_REF] and PA2449 [START_REF] Lundgren | Gene PA2449 is essential for glycine metabolism and pyocyanin biosynthesis in Pseudomonas aeruginosa PAO1[END_REF] are also known to influence, either positively or negatively, expression of the efflux operon. NfxC mutants have been identified among isolates from cystic fibrosis (CF) and non-CF patients [START_REF] Terzi | The effects of active efflux pumps on antibiotic resistance in Pseudomonas aeruginosa[END_REF][START_REF] Wolter | Multiple genotypic changes in hypersusceptible strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients do not always correlate with the phenotype[END_REF].

However, the regulatory pathways by which mexEF-oprN is activated in some clinical resistant mutants remain to be clarified. The observation that nitrosative (Fetar et al., 2011) or disulfide (Fargier et al., 2012) stresses induce operon expression provides some clues on its physiological role. Lines of evidence suggest that MexEF-OprN is one of the mechanisms that P. aeruginosa sets up in response to redox stressors (Fargier et al., 2012).

This manuscript is divided in two main chapters dealing with: (i) the regulation of operon mexEF-oprN in clinical nfxC mutants and (ii) induction of mexEF-oprN by electrophilic stress. Each chapter begins with a literature review and presents the results obtained on the topic during the doctoral training. The first chapter provides a general description of regulation of the four major RND efflux operons of clinical importance in P. aeruginosa. A special focus is made on contribution of MexEF-OprN to multidrug resistance phenotype in clinical isolates and on the role of mutations in operon dysregulation. The second chapter presents data of literature on induction of some of the RND efflux systems of P. aeruginosa by oxidative stress. We then describe how MexEF-OprN can be induced by toxic electrophiles through the action of a novel regulator named CmrA.

The manuscript ends with a general conclusion and give research perspectives and finally the last section describes all materials and methods used all along this work. Once assembled, the tripartite complex is able to promote the transport of amphiphilic and lipophilic molecules from the periplasm or the inner membrane, to the extracellular environment (Figure 1). Rather polyspecific, most RND pumps are able to extrude a wide variety of substrates such as antibiotics, biocides, dyes, detergents, organic solvents, aromatic hydrocarbons and homoserine lactones (Poole and Srikumar, 2001;Schweizer, 2003).

II. Regulation of operon mexEF-oprN in clinical nfxC mutants

RND pumps play an important role in natural and acquired resistance to antibiotics of Pseudomonas aeruginosa [START_REF] Nikaido | Structure and mechanism of RND-type multidrug efflux pumps[END_REF]. So far, 12 operons coding for RND-type efflux systems have been detected in the genome of this species [START_REF] Lee | Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial[END_REF][START_REF] Stover | Pseudomonas aeruginosa contains multiple glyoxalase Iencoding genes from both metal activation classes[END_REF] (Table 1). While all these pumps have been experimentally proved to accommodate antibiotic molecules and to confer some degree of resistance when overexpressed, only a minority of them appears to have a real impact in the clinical context.

Efflux Systems of Clinical Importance

As stated before, RND efflux systems can be involved in natural (baseline) susceptibility to antibiotics and/or can contribute to acquisition of higher resistance levels when overexpressed upon mutations [START_REF] Nikaido | Structure and mechanism of RND-type multidrug efflux pumps[END_REF]. Of the 12 RND-type transporters encoded by P. aeruginosa, only four have been shown to be associated with multidrug resistance phenotypes in clinical isolates, namely MexAB-OprM, MexXY/OprM, MexCD-OprJ and MexEF-OprN (Li et al., 2015;Lister et al., 2009).

MexAB-OprM

The efflux system MexAB-OprM is a major contributor to natural resistance of P. aeruginosa because of its constitutive, though growth-phase dependent expression, and its polyspecificity [START_REF] Evans | The MexA-MexB-OprM multidrug efflux system of Pseudomonas aeruginosa is growth-phase regulated[END_REF][START_REF] Li | Role of mexA-mexB-oprM in antibiotic efflux in Pseudomonas aeruginosa[END_REF][START_REF] Yang | Inactivation of MuxABC-OpmB transporter system in Pseudomonas aeruginosa leads to increased ampicillin and carbenicillin resistance and decreased virulence[END_REF]. Inactivation of any of its three components (MexA, MexB or OprM) results in a strong decrease in the minimal inhibitory concentrations (MICs) of antibiotics exported by the pump (i.e. β-lactams or fluoroquinolones) [START_REF] Li | Role of mexA-mexB-oprM in antibiotic efflux in Pseudomonas aeruginosa[END_REF]. The very broad substrate specificity of transporter MexA includes several antibiotic classes [START_REF] Köhler | Multidrug efflux in intrinsic resistance to trimethoprim and sulfamethoxazole in Pseudomonas aeruginosa[END_REF]Li et al., 1994aLi et al., , 1994b[START_REF] Li | Role of mexA-mexB-oprM in antibiotic efflux in Pseudomonas aeruginosa[END_REF][START_REF] Mistry | High-level pacidamycin resistance in Pseudomonas aeruginosa is mediated by an opp oligopeptide permease encoded by the opp-fabI operon[END_REF][START_REF] Schweizer | Intrinsic resistance to inhibitors of fatty acid biosynthesis in Pseudomonas aeruginosa is due to efflux: application of a novel technique for generation of unmarked chromosomal mutations for the study of efflux systems[END_REF], dyes [START_REF] Li | Contributions of MexAB-OprM and an EmrE homolog to intrinsic resistance of Pseudomonas aeruginosa to aminoglycosides and dyes[END_REF], antiseptic molecules such as triclosan [START_REF] Schweizer | Intrinsic resistance to inhibitors of fatty acid biosynthesis in Pseudomonas aeruginosa is due to efflux: application of a novel technique for generation of unmarked chromosomal mutations for the study of efflux systems[END_REF], organic solvents [START_REF] Li | Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance[END_REF][START_REF] Li | Role of the multidrug efflux systems of Pseudomonas aeruginosa in organic solvent tolerance[END_REF], and quorum sensing molecules or inhibitors [START_REF] Minagawa | RND type efflux pump system MexAB-OprM of Pseudomonas aeruginosa selects bacterial languages, 3-oxo-acyl-homoserine lactones, for cell-to-cell communication[END_REF][START_REF] Moore | Active efflux influences the potency of quorum sensing inhibitors in Pseudomonas aeruginosa[END_REF][START_REF] Pearson | Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals[END_REF] (Table 1).

The mexAB-oprM operon expression is tightly controlled by several regulators. MarR-like repressor MexR, whose gene is located upstream and is divergently transcribed from mexAB-oprM, ensures a local regulation of the operon (Poole et al., 1996a). However, other regulatory proteins such as ArmR (depending on NalC) [START_REF] Cao | MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719[END_REF], NalD (Morita et al., 2006a;[START_REF] Sobel | Mutations in PA3574 (nalD) lead to increased MexAB-OprM expression and multidrug resistance in laboratory and clinical isolates of Pseudomonas aeruginosa[END_REF], MexT (Maseda et al., 2004), the two-component system RocS1/2-A2 (Sivaneson et al., 2011), AmpR (Balasubramanian et al., 2012), BrlR (Liao et al., 2013), CpxR [START_REF] Tian | CpxR activates MexAB-OprM efflux pump expression and enhances antibiotic resistance in both laboratory and clinical nalB-type isolates of Pseudomonas aeruginosa[END_REF], and PA3225 [START_REF] Hall | PA3225 is a transcriptional repressor of antibiotic resistance mechanisms in Pseudomonas aeruginosa[END_REF]) are also involved in the complex regulation of the pump (Figure 2).

Several types of mutants (nalB, nalC and nalD) respectively harboring inactivating mutations in genes mexR, nalC and nalD have been characterized [START_REF] Cao | MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719[END_REF]Poole et al., 1996a;[START_REF] Sobel | Mutations in PA3574 (nalD) lead to increased MexAB-OprM expression and multidrug resistance in laboratory and clinical isolates of Pseudomonas aeruginosa[END_REF]. They can be found in the clinical setting and exhibit significant resistance to most MexAB-OprM substrates (e.g. carbenicillin MIC from 4-to 8-fold higher than a WT strain) (Campo [START_REF] Esquisabel | Mechanisms of resistance in clinical isolates of Pseudomonas aeruginosa less susceptible to cefepime than to ceftazidime[END_REF][START_REF] Llanes | Clinical strains of Pseudomonas aeruginosa overproducing MexAB-OprM and MexXY efflux pumps simultaneously[END_REF][START_REF] Quale | Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates[END_REF]. However, the role of the other genes listed above in MexAB-OprM derepression remains unclear in strains recovered from infections or colonizations. Mutations in these three repressors have been reported in nalB, nalD, and nalC mutants, respectively. Two direct activators of mexAB-oprM have been described, namely CpxR and BrlR (this latter being active exclusively in biofilms). Finally, the products of four other genes would have an indirect negative (mexT, rocS1/2-A2, PA3225) or positive (ampR) impact on mexAB-oprM expression.

MexXY/OprM

Like MexAB-OprM, the efflux pump MexXY/OprM plays a significant role in wild-type resistance phenotype of P. aeruginosa to antibiotics. Most of strains harbor a chromosomallylocated operon composed of only two genes, mexX and mexY (Mine et al., 1999). The outer membrane component OprM required to form a functional efflux pump is provided by the mexAB-oprM locus [START_REF] Aires | Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides[END_REF]. Remarkably and for unclear reasons, strains belonging to PA7 lineage possess a three-gene operon, mexXY-oprA, encoding an OMP named OprA sharing 47% of sequence identity with OprM [START_REF] Morita | Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7[END_REF]. The MexXY/OprM and MexXY/OprA pumps exhibit very close substrates profiles that include aminoglycosides, macrolides and tetracyclines [START_REF] Aires | Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides[END_REF]Masuda et al., 2000a) (Table 1).

Expression of mexXY/oprA is maintained at low-basal levels in wild-type strains by a TetRlike regulator, MexZ, whose gene resides upstream the operon [START_REF] Aires | Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides[END_REF]. In addition to MexZ, transcription of the efflux operon can be influenced by several genes such as armZ [START_REF] Yamamoto | Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa[END_REF], rplU-rpmA [START_REF] Lau | Reduced expression of the rplU-rpmA ribosomal protein operon in mexXY-expressing pan-aminoglycosideresistant mutants of Pseudomonas aeruginosa[END_REF], suhB [START_REF] Shi | SuhB is a novel ribosome associated protein that regulates expression of MexXY by modulating ribosome stalling in Pseudomonas aeruginosa[END_REF], and PA2572- The three types of MexXY-overproducing mutants identified so far (agrZ, agrW1 and agrW2)

show a 2-to 8-fold increased resistance to the pump substrates as compared with wild-type strains [START_REF] Li | Antimicrobial drug efflux pumps in Pseudomonas aeruginosa. In efflux-mediated antimicrobial resistance in bacteria[END_REF]. All of them can be isolated from CF and non-CF patients (El' Garch et al., 2007;[START_REF] Hocquet | Relationship between antibiotic use and incidence of MexXY-OprM overproducers among clinical isolates of Pseudomonas aeruginosa[END_REF][START_REF] Muller | A two-component regulatory system interconnects resistance to polymyxins, aminoglycosides, fluoroquinolones, and β-lactams in Pseudomonas aeruginosa[END_REF][START_REF] Vogne | Role of the multidrug efflux system MexXY in the emergence of moderate resistance to aminoglycosides among Pseudomonas aeruginosa isolates from patients with cystic fibrosis[END_REF] though agrZ mutants are predominant in the CF lung [START_REF] Feliziani | Mucoidy, quorum sensing, mismatch repair and antibiotic resistance in Pseudomonas aeruginosa from cystic fibrosis chronic airways infections[END_REF][START_REF] Smith | Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients[END_REF]. The role of PA2572-PA2573 and AmgRS as cause of MexXY-dependent drug resistance remains uncertain in clinical isolates.

MexCD-OprJ

The MexCD-OprJ efflux system is thought to be quiescent in wild-type strains, as inactivation of its encoding operon, mexCD-oprJ, does not affect the susceptibility of P. aeruginosa to antibiotics (Poole et al., 1996b;[START_REF] Srikumar | Inner membrane efflux components are responsible for beta-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa[END_REF]. On the other hand, overproduction of the pump in so-called nfxB mutants is responsible for a significant increase in MICs of antibiotics such as chloramphenicol (4-fold), quinolones/fluoroquinolones (8-fold), and fourth-generation cephalosporins (8-fold) (Masuda et al., 2000b;Poole et al., 1996b). Other cytotoxic molecules listed in Operon mexCD-oprJ is regulated by two so-called "local repressors", NfxB and EsrC, whose genes are located upstream and downstream from the operon, respectively (Figure 4).

Additionally, mexCD-oprJ transcription can be influenced by VqsM, an AraC-type transcriptional regulator involved in regulation of virulence factors and quorum sensing molecules [START_REF] Liang | Molecular mechanisms of master regulator VqsM mediating quorum-sensing and antibiotic resistance in Pseudomonas aeruginosa[END_REF], by AlgU, a sigma factor mediating the bacterial response to envelope stress [START_REF] Fraud | MexCD-OprJ multidrug efflux system of Pseudomonas aeruginosa: involvement in chlorhexidine resistance and induction by membrane-damaging agents dependent upon the AlgU stress response sigma factor[END_REF], and by the product of gene PA2572 (McLaughlin et al.,

2012).

MexCD-OprJ upregulating mutants isolated in the clinical context, for instance from urinary tract infections, harbor disrupted nfxB genes [START_REF] Shigemura | Association of overexpression of efflux pump genes with antibiotic resistance in Pseudomonas aeruginosa strains clinically isolated from urinary tract infection patients[END_REF]. Interestingly, the pump has been found to be co-produced along with MexAB-OprM or MexXY in fluoroquinoloneand/or carbapenem-resistant clinical isolates, suggesting additional effects on resistance to shared substrates such as fluoroquinolones [START_REF] Li | Antimicrobial drug efflux pumps in Pseudomonas aeruginosa. In efflux-mediated antimicrobial resistance in bacteria[END_REF].

MexEF-OprN

This efflux system is expressed at very low levels in wild-type strains grown under standard laboratory conditions and thus does not significantly contribute to intrinsic resistance to antibiotics or biocides (Köhler et al., 1997). In nfxC-type mutants, the pump is overproduced and provides P. aeruginosa with a higher resistance to a rather limited number of substrates including chloramphenicol, fluoroquinolones/quinolones, trimethoprim and tetracycline (Li and Plésiat, 2016) (Table 1). The nfxC mutants exhibit some additional phenotypic traits listed in Table 2, such as a tolerance to carbapenems (i.e. imipenem), an increased susceptibility to various β-lactams (i.e. ticarcillin) and to aminoglycosides (i.e. gentamicin), which are not related to MexEF-OprN activity but to downregulation of gene oprD and of operons mexAB-oprM and mexXY, respectively [START_REF] Fukuda | nfxC-type quinolone resistance in a clinical isolate of Pseudomonas aeruginosa[END_REF]Köhler et al., 1997).

These mutants are also strongly defective in production of some extracellular virulence factors (Köhler et al., 2001). This lower pathogenicity would result from lower, effluxmediated intracellular concentrations of quorum sensing molecules in the mutants [START_REF] Lamarche | MexEF-OprN efflux pump exports the Pseudomonas quinolone signal (PQS) precursor HHQ (4-hydroxy-2-heptylquinoline)[END_REF]Olivares et al., 2012) and/or repression of quorum-sensing-related genes (Tian et al., 2009a). Based on the capacity of the strain to express virulence traits: (1) pyocyanin, (2) elastase, (3) biofilm, (4) rhamnolipids and (5) swarming motility.

Based on laboratory data

The pump MexEF-OprN is encoded by a three-gene operon, the regulation of which is quite different from those of the other RND efflux systems present in P. aeruginosa, in that it involves a LysR-type "local" activator, named MexT (Köhler et al., 1999). The expression of mexEF-oprN can be modulated by several other genes including mexS (Sobel et al., 2005a),

mvaT

The LysR-type Activator MexT

MexT, a LysR-type transcriptional LTTRs are functionnally active as tetramers. They regulate expression of target genes through a particular mechanism. DNA-footprinting experiments using several of these regulators have revealed the existence of two binding sites upstream from target promoters, namely a regulatory binding site (RBS) and an activation-binding site (ABS). These two DNA regions, formerly named "Nod-box", are not occupied at the same time in absence of co-inducer.

Indeed, the apo-form has less affinity to bind DNA than the co-inducer-bound form (Maddocks and Oyston, 2008). When a co-inducer is present, the affinity of the LTTR for DNA is increased and thus both sites are occupied. The formation of the functional tetramer and its binding to the regulatory sequences provoke a bending of DNA allowing the stabilization of RNA polymerase and thereby triggering gene expression (Figure 7) (Maddocks and Oyston, 2008). Following the general mode of action of LTTRs, MexT binds a consensus sequence (ATC-N 9 -GAT-N 7 -ATC-N 9 -GAT) upstream from operon mexEF-oprN, consistent with the presence of two "Nod-boxes" in the intergenic region between mexT and mexE (Köhler et al., 1999;[START_REF] Maseda | Transcriptional regulation of the mexEF-oprN multidrug efflux pump operon by MexT and an unidentified repressor in nfxC-type mutant of Pseudomonas aeruginosa[END_REF]Tian et al., 2009a). Specific amino acid substitutions in LTTRs have been reported to constitutively activate some of these regulators without the need of co-inducer, as exemplified by BenM from Acinectobacter baylyi and CysB from Salmonella enterica serovar Typhimurium (Colyer and Kredich, 1996;Craven et al., 2009). Single point mutations have also been identified in MexT from nfxC mutants of P. aeruginosa, but their impact on protein functionality has remained undetermined so far (Llanes et al., 2011).

Oxidoreductase MexS

Most of nfxC mutants selected in vitro harbor disruptive mutations in another gene, named mexS, located close to, and transcribed divergently from mexT (Figure 5) (Köhler et al., 1999;Sobel et al., 2005a). Inactivation of mexS triggers the MexT-dependent overexpression of mexEF-oprN which in turn results in a typical NfxC resistance phenotype ( ). Compared to missense mutations (9.4% of strains in average), indels and nonsense mutations appear to be rather infrequent (1.7%) among the mexS mutants (Table 3).

Other regulators of mexEF-oprN

So far, upregulation of operon mexEF-oprN in clinical strains has only been associated with mutations occurring in genes mexT and mexS (Llanes et al., 2011). However, according to the results of experiments performed on in vitro mutants, at least four repressors (MvaT, AmpR, MxtR and PA2449) and three activators (BrlR, ParRS and NmoR) can also influence operon expression (Li and Plésiat, 2016) (Figure 5).

MvaT is a member of Histone-like Nucleoid Structuring proteins (H-NS) family, that negatively controls about 150 virulence-related genes in P. aeruginosa [START_REF] Castang | High-order oligomerization is required for the function of the H-NS family member MvaT in Pseudomonas aeruginosa[END_REF]. A deletion mutant, PAO1ΔmvaT, was found to exhibit a modest 5-to 7-fold increase in mexEF-oprN transcription relative to wild-type strain PAO1, suggesting an indirect effect of this global regulator on pump activity (Westfall et al., 2006).

AmpR, a LTTR regulator, is known to differentially modulate the expression of target genes depending on which ligand is bound to it [START_REF] Balcewich | Crystal structure of the AmpR effector binding domain provides insight into the molecular regulation of inducible AmpC beta-lactamase[END_REF]. AmpR regulates the expression of gene bla ampC encoding for the intrinsic β-lactamase AmpC [START_REF] Kong | Pseudomonas aeruginosa AmpR is a global transcriptional factor that regulates expression of AmpC and PoxB β-Lactamases, proteases, quorum sensing, and other virulence factors[END_REF].

The repression this regulator exerts on mexEF-oprN is likely indirect (Balasubramanian et al., 2012). Finally, gene PA2449 encodes a TyrR-like member of the Enhancer Binding Proteins (EBP) family known to stabilize the interaction of sigma factor RpoN with DNA, thereby enhancing expression of target genes. Interestingly, a deletion mutant, PAO1ΔPA2449, appeared to strongly overexpress the efflux operon (139-fold), but its antibiotic susceptibility was not investigated further; thus limiting conclusions on the role PA2449 could play in resistance [START_REF] Lundgren | Gene PA2449 is essential for glycine metabolism and pyocyanin biosynthesis in Pseudomonas aeruginosa PAO1[END_REF].

Homologue of ArcB from

BrlR belongs to the MerR-like family of regulators and is exclusively active in the biofilm mode of life (Liao et al., 2013). Supporting a role for BrlR as activator of mexEF-oprN, a 200fold decrease in gene mexE transcripts was scored in biofilms of deletion mutant PAO1ΔbrlR compared to biofilms of wild-type strain. Confirming these data, direct binding of BrlR to the upstream region of mexEF-oprN could be established by several approaches including chromatin immunoprecipitation (ChIP), streptavidin bead-pulldown and electrophoretic mobility shift assays (EMSA) (Liao et al., 2013).

The two-component regulatory system (TCS) ParRS has been reported by our laboratory to positively control the expression of another pump-encoding operon, namely mexXY [START_REF] Muller | A two-component regulatory system interconnects resistance to polymyxins, aminoglycosides, fluoroquinolones, and β-lactams in Pseudomonas aeruginosa[END_REF]. Activation of mexEF-oprN by ParRS is expected to be indirect through a negative impact on mexS expression (Wang et al., 2013).

The last activator of pump MexEF-OprN described so far, NmoR, is a LTTR having a role in the defense against nitro molecules. A PAO1ΔnmoR mutant showed a decreased expression (20-fold) of operon mexEF-oprN and other MexT-dependent genes, but whether NmoR directly binds to the promoter region of the operon remains to be determined [START_REF] Vercammen | Pseudomonas aeruginosa LysR PA4203 regulator NmoR acts as a repressor of the PA4202 nmoA gene, encoding a nitronate monooxygenase[END_REF]. P seudomonas aeruginosa is a notorious cause of acute and chronic infections in vulnerable patients. The ability of this environmental Gram-negative bacterium to produce a broad range of virulence factors (1) and to become resistant to multiple antimicrobial agents is considered a key to its success in the hospital setting. When overexpressed upon mutation, several efflux systems belonging to the resistance-nodulation-cell division (RND) family of drug transporters are able to decrease the susceptibility of the pathogen to structurally unrelated antibiotics (2). One of these systems, named MexEF-OprN, is quiescent in wildtype strains grown under standard laboratory conditions. Its contribution to the intrinsic resistance of P. aeruginosa is therefore minimal. In contrast, in so-called nfxC mutants, stable overproduction of the pump results in a significant increase in the MICs (4-to 16-fold) of chloramphenicol, trimethoprim, and fluoroquinolones (3). Compared with the susceptibility of wild-type strains, typical nfxC mutants exhibit a hypersusceptibility to some antipseudomonal ␤-lactams (penicillins, cephalosporins) and aminoglycosides, a phenotype possibly due to the impaired activity of two other RND pumps, namely, MexAB-OprM and MexXY/ OprM (4). Furthermore, this typical NfxC phenotype includes a decreased susceptibility to carbapenems, linked to the downregulation of the oprD gene, which codes for the specific porin OprD, allowing the facilitated diffusion of these antibiotics into the cell (3).

Results

Amino Acid Substitutions Account for Most MexS Alterations in Clinical nfxC

Mutants of Pseudomonas aeruginosa

In P. aeruginosa, while most RND pumps have their expression modulated by repressors (5), transcription of the mexEF-oprN operon is controlled by a LysR-type activator, MexT, encoded by an adjacent gene (6). In some drug-susceptible laboratory strains of the PAO1 lineage, mexT is inactivated by an 8-bp insert (7). Spontaneous excision of this intragenic fragment restores the open reading frame of mexT with the concomitant overexpression of mexEF-oprN and the development of the typical NfxC phenotype (6). In other strains, mexEF-oprN transcription is triggered by mutations in another gene, mexS, which is divergently transcribed from mexT and encodes an oxidoreductase (8). In any case, a functional MexT is mandatory for the in vitro selection of MexEF-OprN-overproducing mutants. This regulator has been reported to increase mexS expression (6), even if the consensus nod-box DNA sequence, considered the binding site of MexT, remains to be identified in the promoter region of mexS (9). To explain the MexS/MexT-dependent regulation of mexEF-oprN, it has been postulated that MexS is involved in the detoxification of some endogenously produced MexT-activating molecule(s) (10,11). In this scenario, if it is not processed by MexS, the toxic metabolite(s) would be exported out of the cell by MexEF-OprN as a rescue mechanism.

In clinical strains, nfxC mutations are difficult to characterize because of polymorphic variations in the MexS and MexT protein sequences (http://pseudomonas.com). Moreover, data suggest that still uncharacterized pathways might influence mexEF-oprN expression (12). Supporting this notion, in vitro mutants with alterations in the mvaT, ampR, or mxtR gene have been reported to overexpress mexEF-oprN and to exhibit a multidrug resistance phenotype (13)(14)(15). However, the relevance of such mutations in clinical strains awaits confirmation.

In vitro-selected nfxC mutants were found to be deficient in the production of several quorum-sensing-dependent virulence factors ( 16) without an apparent loss of fitness (17). The mutants derived from reference strain PAO1 typically produce less pyocyanin, rhamnolipids, and elastase than the wild-type parents (3,16) and less type III secretion system (T3SS) effector toxin ExoS (18). This phenotype was attributed to (i) reduced intracellular levels of the Pseudomonas quinolone signal (PQS), caused by a shortage of a metabolic precursor (kynurenine or 4-hydroxy-2-heptylquinoline [HHQ]) exported by the pump (17,19), and (ii) MexT acting as a global regulator and indirectly impairing the T3SS in an MexEF-OprN-independent way (18). Information about the rates and traits of nfxC mutants in cystic fibrosis (CF) patients (20,21) and non-CF patients (12,(22)(23)(24) remains scarce. As a plausible explanation, the low virulence of these mutants would be detrimental to their survival in the host or in the hospital setting and would account for their infrequent isolation from clinical samples. Alternatively, these mutants would be phenotypically and genetically distinct from their in vitro counterparts (i.e., they would keep some degree of pathogenicity or persistence) and thus would be underrecognized. In this study, we show that most clinical nfxC mutants have mild de-fects in MexS and are still able to produce substantial amounts of virulence factors.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. The reference strains and cloning plasmids used in this study are listed in Table 1. Twenty-two clinical nfxC mutants collected between May 2012 and May 2013 at the University Hospital of Besançon, Besançon, France (see Table S1 in the supplemental material), and 7 drug-susceptible strains of P. aeruginosa collected from surface waters (PE1, PE1346, PE1361, PE1393, PE1423, PE1446, and PE1450) were also investigated. All the bacterial cultures were grown in Mueller-Hinton broth (MHB) with adjusted concentrations of Ca 2ϩ (range, 20 to 25 mg liter Ϫ1 ) and Mg 2ϩ (range, 10 to 12.5 mg liter Ϫ1 ) (Becton Dickinson and Company, Cockeysville, MD) or on Mueller-Hinton agar (MHA; Bio-Rad, Marnes-la-Coquette, France). Escherichia coli transformants were selected on MHA containing 50 g ml Ϫ1 kanamycin (a marker of the vector pCR-Blunt), 15 g ml Ϫ1 tetracycline (a marker of the vector mini-CTX1), or 50 g ml Ϫ1 streptomycin (a marker of the vector pKNG101). Recombinant plasmids were introduced into P. aeruginosa strains by triparental matings and mobilization with broad-host-range vector pRK2013 in E. coli HB101 as a helper strain (25). Transconjugants were selected on Pseudomonas isolation agar (PIA; Antibiotic susceptibility testing. The MICs of selected antibiotics were determined by the standard serial 2-fold dilution method in MHA with an inoculum of 10 4 CFU per spot, according to CLSI recommendations (26). Growth was assessed visually after 18 h of incubation at 37°C.

RT-qPCR experiments. Specific gene expression levels were measured by real-time quantitative PCR (RT-qPCR) after reverse transcription, as described previously (27). Briefly, 2 g of total RNA was reverse transcribed with ImProm-II reverse transcriptase as specified by the manufacturer (Promega, Madison, WI). The amounts of specific cDNA were assessed in a Rotor Gene RG6000 instrument (Qiagen, Courtaboeuf, France) by using a QuantiFast SYBR PCR green kit (Qiagen). When primers were not already published, the primers used for amplification were designed from the gene sequences available in the Pseudomonas Genome Database, version 2, by using primer3 software (http://bioinfo.ut.ee /primer3-0.4.0/) (see Table S2 in the supplemental material). For each strain, the mRNA levels of the target genes were normalized to those of the rpsL housekeeping gene and expressed as a ratio to the level in wild-type reference strain PA14. Mean gene expression values were calculated from two independent bacterial cultures, each of which was assayed in duplicate. Strain PA14⌬S was used as a positive control for mexE gene overexpression. As shown in preliminary experiments, all mexE transcript levels Ն20-fold above the mexE transcript level of PA14 were associated with a decreased susceptibility (Ն2-fold) of the strains to MexEF-OprN substrate antibiotics and considered significant.

Virulence factor analysis. Biofilm production was assessed by measuring bacterial adhesion to 96-well polystyrene plates (28). Cultures were incubated in triplicate in MHB medium overnight at 30°C and washed twice with 200 l of distilled water to eliminate planktonic bacteria. Attached bacteria were colored by 1% (wt/vol) crystal violet and solubilized by 99% (vol/vol) ethanol. Attachment was evaluated at 600 nm and considered negative when the optical density (OD) was Ͻ1, as previously reported (19).

Swarming motility was tested on freshly prepared M8 medium (42.2 mM Na 2 HPO 4 , 22 mM KH 2 PO 4 , 7.8 mM NaCl, pH 7.4) supplemented with 2 mM MgSO 4 , 0.5% casein, 0.5% agar, and 1% glucose (29). After 15 min of incubation of the plates at 37°C, 5 l of culture (7.5 ϫ 10 5 CFU) was spotted onto the medium surface, with strain PA14 being used as a positive control. The formation of dendrites after 24 h of culture at 37°C was considered a positive result, while a steady spot was considered negative.

Elastase activity was assessed by using MHA plates supplemented with 4 mg ml Ϫ1 elastin-Congo red (Sigma-Aldrich, St. Louis, MO) and inoculated with 5-l volumes of bacterial suspension (7.5 ϫ 10 5 CFU). Enzymatic degradation of the substrate formed clear halos around the culture spots after 48 h of incubation at 37°C (30). The absence of a visible halo was considered a negative result.

Rhamnolipid production was appreciated using a hemolysis assay. Briefly, after 18 h of growth at 37°C in agitated MHB (A 600 ϭ 6.7 Ϯ 0.4), bacterial supernatants containing rhamnolipids were collected and mixed with defibrinated horse blood diluted 1/100 (vol/vol) in phosphate-buffered saline. After 30 min of incubation at room temperature, the mixture was centrifuged for 10 min at 950 ϫ g. The concentration of hemoglobin in the supernatants was determined spectrophotometrically at 405 nm. OD values were expressed as the percent hemolysis relative to the complete hemolysis achieved with Triton X-100 (by definition, 100%). The results presented are mean values from two independent experiments. Hemolytic activity was considered to be significantly reduced when it was less than 50% of that for the control.

Finally, pyocyanin assays were carried out on culture supernatants after 18 h of growth at 37°C in a specific broth [120 mM Tris HCl, pH 7.2, 0.1% tryptone, 20 mM (NH 4 ) 2 SO 4 , 1.6 mM CaCl 2 , 10 mM KCl, 24 mM sodium citrate, 50 mM glucose] (A 600 ϭ 1.6 Ϯ 0.2). The pigment was extracted from the cultures with chloroform (1 volume) and mixed with 0.1 M HCl (0.06 volume) before spectrophotometric measurement at 520 nm (31). Results are mean values from two independent experiments. Pyocyanin production was considered to be significantly reduced when it was less than 50% of that of reference strain PA14 grown under the same conditions.

The virulence factor production of individual clinical strains was rated by a global score ranging from 0 to 5, which corresponds to the number of positive or significant results obtained by each of the 5 assays mentioned above, the result of each of which was given a value of 1 if it was positive or significant.

Construction of deletion mutants from strain PA14. Single mexS and mexT deletion mutants were constructed by using overlapping PCRs and recombination events, as described by Kaniga et al. (32). First, the 5= and 3= regions flanking mexS (417 and 433 bp, respectively) and mexT (408 and 453 bp, respectively) were individually amplified by PCR with specific primers (see Table S2 in the supplemental material) under the following conditions: 3 min of denaturation at 98°C followed by 30 cycles of amplification, each of which was composed of 10 s at 98°C, 30 s at 60°C, and 30 s at 72°C, and a final extension step of 7 min at 72°C. The resultant amplicons were used as the templates for overlapping PCRs with external pairs of primers to generate the mutagenic DNA fragments. The reaction mixtures contained 1ϫ iProof HF master mix, 3% dimethyl sulfoxide, and 0.5 M each primer (Bio-Rad). The amplified products were cloned into plasmid pCR-Blunt according to the manufacturer's instructions (Invitrogen, Carlsbad, CA) and next subcloned as BamHI/ApaI fragments into the suicide vector pKNG101 in E. coli CC118pir (32). The recombinant plasmids were transferred into P. aeruginosa by conjugation and selected on PIA containing 2,000 g ml Ϫ1 streptomycin. The excision of the undesired pKNG101 sequence was performed by plating transformants on M9 minimal medium plates containing 5% (wt/vol) sucrose and 1% (wt/vol) glucose. Negative selection on streptomycin was carried out to confirm the loss of the plasmid. The allelic exchanges were confirmed by PCR. Nucleotide sequencing experiments confirmed deletion of 826 bp in mexS and 929 bp in mexT, yielding strains PA14⌬S and PA14⌬T, respectively.

Chromosomal complementation with full-length mexS and mexT. A search for mutations in the mexS and mexT genes, as well as in the mexS-mexT and mexT-mexE intergenic regions, was performed with 43 clinical strains by using the primers listed in Table S2 in the supplemental material. The mutated mexS and mexT genes along with their respective promoter regions were amplified from purified genomic DNA by PCR. The resulting DNA fragments were cloned into plasmid pCR-Blunt and next ligated to BamHI/HindIII-linearized plasmid mini-CTX1 (33). The recombinant plasmids were then transferred from E. coli CC118 to P. aeruginosa strains PA14⌬mexS or PA14⌬mexT by conjugation with subsequent selection on PIA plates containing 200 g ml Ϫ1 tetracycline, to allow their chromosomal insertion into the attB site. Chromosomal integration was confirmed by PCR and sequencing.

RESULTS AND DISCUSSION

Wild-type genes mexS and mexT. Strains PAO1 (3,8,34), PA14 (10), and PAK (35) have alternatively been used as wild-type reference strains in studies on the MexEF-OprN efflux pump. However, the mexS and mexT genes in these strains show a nonsilent sequence polymorphism whose impact on the functionality of the encoded proteins, MexS and MexT, respectively, remained to be clarified. For instance, in addition to carrying an 8-bp intragenic fragment inactivating mexT (7), most of the laboratory strains of the PAO1 lineage differ from PA14 (or PAK) by an aspartic acid residue (D) instead of an asparagine (N) at position 249 (D 249 ) in MexS (http://pseudomonas.com).

Because the MexS-D 249 protein was considered either functional (7) or nonfunctional (35), we deleted mexS in both PA14 and PAO1 and compared the effects of this deletion on mexEF-oprN expression and antibiotic resistance. In PA14, suppression of mexS (strain PA14⌬S) resulted in a strong increase in mexE transcription (427-fold) and in a 16-to 32-fold higher resistance to MexEF-OprN substrates, as in typical nfxC mutants (Table 2). As expected, complementation of PA14⌬S with the PA14 mexS allele (PA14⌬S PA14 ) restored the drug-susceptible phenotype. In contrast, the PAO1 mexS allele had virtually no impact on the resistance levels of strain PA14⌬S (strain PA14⌬S PAO1 ) and failed to reverse the overexpression of mexE, whose transcripts remained 45-fold more abundant in PA14⌬S in comparison with PA14 mexS allele. Consistent with PAO1 producing inactive MexS-D 249 and MexT peptides, spontaneous excision of the extra 8-bp sequence inserted in mexT is known to trigger MexEF-OprN production in this strain, with MexT recovering its functionality in a nonfunctional MexS background (36). Confirming that MexS-N 249 (and not MexS-D 249 ) is functional, analysis of 7 drug-susceptible strains of P. aeruginosa collected from surface waters (PE1, PE1346, PE1361, PE1393, PE1423, PE1446, and PE1450) showed that the genomes of all of them encoded MexS-N 249 together with an active MexT (without any insertion in the mexT gene) (data not shown). Based on these results, we therefore used strain PA14 instead of PAO1 in further experiments to investigate the functionality of MexS and MexT from clinical nfxC mutants.

Selection of clinical nfxC mutants. We screened a collection of 221 clinical isolates of P. aeruginosa exhibiting a reduced suscep- (6). The ciprofloxacin and imipenem concentrations used in the screening were equal to the MIC values for reference strain PA14 (0.12 g ml Ϫ1 and 1 g ml Ϫ1 , respectively; Table 2). As resistance to these antibiotics may also be due to other efflux pumps (e.g., MexXY/OprM, MexCD-OprJ, MexAB-OprM) as well as other mechanisms (e.g., fluoroquinolone target alterations, mutational loss of porin OprD), the levels of the mexE transcripts were determined in all the strains by RT-qPCR. Forty-three (19.5%) of the 221 selected isolates were found to significantly overexpress mexE (Ն20-fold) compared with the level of mexE expression by PA14 (data not shown). According to available clinical data, these 43 nfxC mutants were involved in the colonization or infection of 17 patients (from 1 to 12 isolates per patient) admitted to various medical and surgical units of University Hospital of Besançon, Besançon, France (see Table S1 in the supplemental material). Most of these patients (12/17) were treated with antibiotics prior to the isolation of the nfxC mutant strains, including 7/12 treated with fluoroquinolones known to easily select nfxC mutants (22,37). Sequencing of the mexS and mexT genes (data not shown) allowed us to identify the redundant mutants in individual patients and to eventually retain 22 strains (1, 2, or 3 different strains per patient) for further investigations (see Table S1 in the supplemental material).

Drug susceptibility of clinical nfxC mutant isolates. The level of overexpression of the mexE gene was found to vary greatly among the 22 clinical mutants (from 20-to 825-fold the level of expression by PA14; Table 2). These elevated values were associated with an increased resistance of the strains (except strain 1711) to the MexEF-OprN substrates chloramphenicol (MIC range, 2to 32-fold the MIC for PA14) and ciprofloxacin (MIC range, 2-to 512-fold the MIC for PA14), though the possibility that additional mechanisms may have influenced the drug MICs cannot be excluded. For unclear reasons, one strain, 1711, turned out to be more susceptible (2-fold) to chloramphenicol than PA14, despite the strong upregulation of its mexE gene (825-fold). As indicated in Table 2, 20/22 strains exhibited a 2-to 16-fold decrease in susceptibility to imipenem compared with that of PA14, possibly due to the MexT-dependent downregulation of specific porin OprD (6), a mutational loss of OprD, and/or carbapenemase production (38). Finally, the reported hypersusceptibility of typical in vitro nfxC mutants to the MexAB-OprM substrate ticarcillin and to the MexXY(OprM) substrate amikacin (4) was observed in only 7 strains and 1 strain, respectively, suggesting that this hypersusceptible phenotype either arises in specific genetic backgrounds, such as the PAO1 and PA14 backgrounds, or is masked in most clinical nfxC strains by additional mechanisms. It should be noted that because of this phenotypic variability, MexEF-OprN-overproducing mutants may be difficult to recognize in the medical laboratory unless molecular biology techniques are used.

Amino acid variations in the MexT regulator. MexT needs to be functional to activate mexEF-oprN operon expression in nfxC mutants (6). Concordant with this, DNA sequencing revealed that 20/22 strains (91%) produced a MexT protein identical to that of PA14 (Table 3). Interestingly, 2/22 strains (9%) harbored mexT genes with point mutations resulting in one (G 258 D; strain 0810) or two (Y 138 D and G 258 D; strain 1510) amino acid substitutions in the effector-binding domain of MexT. In these isolates, the sequence of mexS, as well as the sequences of the mexS-mexT and mexT-mexE intergenic regions, was identical to that of PA14 (Table 2). To investigate the impact of the G 258 D substitution and the Y 138 D plus G 258 D substitutions on MexT activity, we complemented PA14⌬T with the mexT alleles from strains 0810 and 1510. The expression of the mexE gene, the drug resistance, and the virulence factor score of PA14⌬T were unaffected by the complementation (data not shown), indicating that neither Y 138 D nor G 258 D influences MexT activity, as the mutational activation of MexT would have induced mexEF-oprN expression in a functional MexS background. Also, these results indirectly imply that still unknown mutations are involved in the NfxC phenotype of isolates 0810 and 1510.

Impact of alterations in mexS on resistance and virulence. Experimental results from our university laboratory (unpublished data) and of other research groups (8) indicate that most (77% and 66%, respectively) nfxC mutants selected in vitro on MexEF-OprN substrates, such as ciprofloxacin and chloramphenicol, harbor nucleotide deletions or insertions in the mexS gene that are predicted to result in inactive MexS peptides. In the present study, intriguingly, only 13.6% (n ϭ 3/22) of the strains turned out to carry such indels in mexS, whereas 45.5% (n ϭ 10/22) exhibited point mutations resulting in one (n ϭ 9 strains) or two (n ϭ 1 strain) amino acid substitutions in the MexS oxidoreductase. The remaining 41% (n ϭ 9/22) harbored a PA14-like, wild-type MexS (Table 2). The latter 9 strains were found to produce a MexT identical to that of PA14 (n ϭ 7) or harbor the nonsignificant amino acid variations Y 138 D and G 258 D (n ϭ 2; see above). As the mexS-mexT and mexT-mexE intergenic regions were 100% identical between the 9 strains and PA14, these results unambiguously demonstrate that mutations in still unknown loci (other than mexS and mexT) are able to upregulate mexEF-oprN expression in clinical strains.

Because amino acid substitutions may have less dramatic effects on MexS activity than disruption of the mexS gene, we cloned the 13 mutated mexS alleles in plasmid mini-CTX and complemented mutant PA14⌬S by chromosomal insertion of the cloned genes into the attB site. RT-qPCR experiments showed that all the transconjugants except one (complemented with MexS-V 104 A from strain 1307) significantly overexpressed the efflux operon (Table 3). The mexE mRNA levels were significantly correlated (Spearman's rho ϭ 0.96, P Ͻ 0.01) with the ciprofloxacin MICs (Fig. 1A). As expected, complementation with indel-carrying mexS alleles (from strains 1709, 1711, and 0607) failed to decrease the expression levels of mexE or the MICs of chloramphenicol (2,048 g ml Ϫ1 ) and ciprofloxacin (4 g ml Ϫ1 ) (compared with those for PA14⌬S; Table 3). Wild-type levels of resistance to imipenem, ticarcillin, and amikacin were also not restored in the null mutant upon complementation. Similar results were obtained with MexS-L 263 Q (from strain 2609), supporting the notion that this mutation is strongly detrimental to MexS activity. As an indication that the remaining mutations (except the well-tolerated variation V 104 A from strain 1307) partially compromise but do not abolish MexS activity, complementation of PA14⌬S with other MexS variants reduced the level of mexE expression from 1.9-to 20.3-fold and its level of resistance to both chloramphenicol and ciprofloxacin from 2-to 8-fold (Table 3). This was accompanied by the restoration of wild-type susceptibility to imipenem, ticarcillin, and amikacin in 1 (strain 2310 with MexS-F 253 L), 8, and 0 complemented mutants, respectively. Upon complementation with MexS-F 253 L, gene oprD expression was increased to wild-type levels (0.9-fold that of PA14), while mexE expression was strongly repressed (20.3-fold that of PA14), providing further evidence that F 253 L only weakly affects MexS activity. The mRNA levels of the oprD gene in PA14⌬S were not impacted or were only marginally impacted by expression of disrupted mexS genes or alleles encoding amino acid variations other than V 104 A and F 253 L, consistent with the unchanged resistance of PA14⌬S to imipenem upon complementation. The hypersusceptibility of in vitro nfxC mutants to some ␤-lactam antibiotics, such as ticarcillin, has been proposed to result from nfxC-dependent repression of the mexAB-oprM operon (39). As shown in Table 3, inactivation of MexS by indels or L 263 Q failed to restore wild-type ticarcillin susceptibility in transcomplemented strain PA14⌬S (for which the ticarcillin MIC was 2-fold lower than that for PA14), while less severe alterations did. Nevertheless, mexB expression was not significantly different among the transcomplemented mutants, with the level of expression by the mutants ranging from 0.6-to 1.4-fold that by PA14, which suggests the existence of more complex interplays between MexAB-OprM and MexEF-OprN in nfxC mutants, as already evoked (4). None of the mexS alleles except those encoding the V 104 A substitution was able to increase the levels of mexY expression and amikacin MICs up to wild-type levels in PA14⌬S (Table 3). However, the slight effect (a 2-fold increase in the MIC) was visible for strains with alleles with mutations resulting in mild defects but was absent for strains with alleles with mutations resulting in severe defects.

The virulence traits of the transcomplemented PA14⌬S mutants varied greatly according to the different mexS alleles. As for PA14⌬S, mutations leading to the complete inactivation of MexS and strong mexE upregulation (in alleles from strains 2609, 1709, 1711, and 0607) were associated with a low virulence score of 1 (Fig. 1B). Biofilm formation, swarming motility (Fig. 2), hemolytic activity, as well as pyocyanin production remained strongly impaired in the transcomplemented mutants (Table 3). Complementation with the other alleles (from strains 2310, 2505, 3005, 0911, 1009, 0801, 1409, and 2311) partially restored the wild-type virulence traits in PA14⌬S, yielding scores of 4 and 5. However, the level of pyocyanin production remained low in most of these complemented mutants (from 0.2-to 0.8-fold that of PA14⌬S PA14 ) and showed no evident correlation with mexE expression levels, as was observed in mutants PA14⌬S 0801 and PA14⌬S 1409 in Table 3.

Consistent with our previous conclusions, the mexS allele encoding the well-tolerated substitution V 104 A provided PA14⌬S with a wild-type phenotype of resistance and virulence (Fig. 2), indicating that mexEF-oprN overexpression in strain 1307 is due to mexS-independent (and also mexT-independent) genetic events.

FIG 1

Correlation between levels of mexE expression, resistance, and virulence factor production in strain PA14⌬S complemented with mexS alleles from 13 clinical isolates. The expression levels of the mexEF-oprN operon, as assessed by RT-qPCR of the mexE gene, are expressed as the ratios to the level of mexEF-oprN expression by wild-type strain PA14 (set at a value of 1, by definition). Ciprofloxacin (Cip) MICs (diamonds) are expressed as ratios relative to the ciprofloxacin MIC for PA14. The virulence factor scores (circles) were determined by the number of positive results by 5 different tests (biofilm formation, swarming motility, elastase production, hemolytic activity, and pyocyanin production), as indicated in Table 3. The negative and positive controls, strains PA14 and PA14⌬S, respectively, are indicated. The relationships between the variables mexE expression and ciprofloxacin MIC (Spearman's rho ϭ 0.96, P Ͻ 0.01) and the variables mexE expression and virulence factor scores (Spearman's rho ϭ Ϫ0.87, P Ͻ 0.01) were found to be significant. Other regulatory genes in nfxC mutants. As reported above, 10/22 clinical strains (strains 2502, 1206, 0708, 0309, 2607, 0712, 0608, 1307, 0810, and 1510) appeared to produce functional MexS and MexT proteins. Since mutations in genes coding for the global regulators MvaT and AmpR have been reported to activate the mexEF-oprN operon in in vitro-selected nfxC mutants (13,14), we carried out sequencing experiments, which eventually failed to reveal alterations in these genes. Again, these results clearly indicate that other loci are implicated in pump MexEF-OprN overproduction in the clinical setting.

Conclusion. This study provides an insight into the genetic events leading to MexEF-OprN overproduction in clinical nfxC isolates. The hypothesis of preferential selection of partially derepressed MexEF-OprN mutants rather than fully derepressed ones in vivo is reinforced by our observation that mexE expression was lower in most clinical nfxC mutants (mean, 205-fold Ϯ 187-fold that of wild-type strain PA14; median, 179-fold that of wild-type strain PA14) than in comparator strain PA14⌬S (427-fold that of wild-type strain PA14) (Table 2). None of the amino acid variations found in the mexT product (2/22 isolates, 9%) proved to be significant, a result consistent with the observation that LysR regulators are rarely constitutively activated by mutations (e.g., BenM in Acinetobacter baylyi and CysB in Salmonella enterica serovar Typhimurium) (40,41). In contrast, single point mutations in the MexS oxidoreductase (9/22, 40.9%) represent a significant cause of MexEF-OprN upregulation in clinical P. aeruginosa strains. Consistent with these findings, a decrease in ciprofloxacin MICs from 2-to 4-fold was observed in 7 clinical nfxC mutants upon complementation with a plasmid-borne copy of mexS from strain PA14 (see Table S3 in the supplemental material). Of note, another mutation in MexS (A 155 V) leading to multidrug resistance has recently been reported in a clinical isolate (42). Our results demonstrate that most MexEF-OprN-overproducing clinical strains either have a wild-type, PA14-like MexS (10/22, 45.5%) or are only partially deficient in MexS activity (8/22, 36.3%). Mutants harboring these mutations resulting in presumed mild defects display resistance and virulence traits intermediate between those of wild-type strains and strongly defective MexS mutants (4/22, 18.2%), which could account for their emergence in vivo. However, analysis of our clinical strains gave contrasting results (see Table S4 in the supplemental material), reinforcing the idea that the virulence of P. aeruginosa is multifactorial and factors other than those tested in this study may well contribute to the pathogenicity of strongly deficient mexS mutants, some of which were still able to cause infections. The MexEF-OprN overproducers studied here had similar growth rates (data not shown). Finally, this work indirectly demonstrates that still unknown regulators are involved in the activation of mexEF-oprM in 10/22 (45.5%) clinical nfxC mutants. We are currently trying to determine such regulatory pathways. MexS of PA14 is functional (N249) and considered as WT contrary to that of PAO1-UW (D249) (www.pseudomonas.com). b MexT of PA14 is functional and considered as WT, contrary to that of PAO1-UW (+8bp 118) (www.pseudomonas.com). c Virulence factors are in bold when positive or considered significant. d Results from each test (biofilm/1, swarming/1, elastase/1, haemolysis/1, pyocyanin/1). Swarming motility and biofilm production are correlated with rhamnolipid production.

Constitutive Activation of MexT by Amino Acid Substitutions found in Clinical Isolates of Pseudomonas aeruginosa

In a previous study on the role of MexEF-OprN in low-level resistance to ciprofloxacin, two non-CF clinical isolates of P. aeruginosa (strains 4177 and 4088) had been found to overexpress mexEF-oprN while having intact mexS genes (Llanes et al., 2011). Both strains harbored amino acid substitutions in MexT. A retrospective analysis of MexEF-OprNoverproducing CF isolates also revealed that one of them (strain 10-12) displayed a G 257 A change in MexT (Llanes et al., 2013). Finally, screening of our lab collection (Richardot et al., 2016) led to the identification of two other isolates, 0810 and 1510, harboring one and two amino-acid substitutions in MexT, respectively (Table 4). To determine whether these sequence variations could have an impact on mexEF-oprN expression, by constitutively activating MexT, we first reassessed the expression of mexE in all the selected bacteria.

Confirming that all of them were nfxC mutants, their transcripts levels were from 20-to 112fold higher than that of wild-type strain PA14. These results were consistent with the higher resistance of these clinical strains to MexEF-OprN substrates (Table 4). To get an insight into the impact of the aforementioned variations on MexT activity, we next deleted mexT gene in strain PA14 and re-complemented this strain with individual mutated alleles. For each complemented strain, we measured the expression of mexE and determine the susceptibility to MexEF-OprN substrates. Gene replacement with mexT alleles from strains 4177, 0810, and 1510 had virtually no impact on mexE expression and MIC values (Table 5). In contrast, alleles from strains 4088 and 10-12 were associated with a strong overexpression of mexE (189-and 110-fold, respectively), and increased resistance to ciprofloxacin (16x, 8x), chloramphenicol (32x, 16x), and trimethoprim (≥ 32x, 16x) (Table 5). If MexT conforms to the usual mode of action of LysR transcriptional regulators (LTTRs) (Maddocks and Oyston, 2008), it should be inactive in its monomeric conformation and should be active when forming oligomers upon binding of its cognate co-inducer(s).

Consistent with this, it was shown that under reducing conditions, MexT forms an inactive monomer while oxidative conditions lead to an active tetrameric regulator (Fargier et al., 2012). Given that strains 4088 and 10-12 both harbor a substitution of Glycine residue-257, we thought that this position might be necessary for MexT oligomerization and thus, that changing this residue into a Serine or Alanine would generate a constitutively active form of MexT. To ascertain this hypothesis, we performed a bacterial two-hybrid experiment based on activity reconstitution of enzyme adenylate cyclase (AC) from Bordetella pertussis (Battesti and Bouveret, 2012). This enzyme is composed of two subunits T18 and T25 which, when physically separated, do not present any enzymatic activity. Reconstitution of AC activity can be achieved if two proteins, in our case two monomers of MexT, interact with each other and bring closer the two subunits. Production of a functional AC activates transcription factor CRP, and subsequently cAMP/CRP-upregulated genes such as those of operons maltose and lactose. The activity of these operons can be visualized by growth on reporter plates (MC Maltose and MH X-Gal) and can be quantified by measuring β-galactosidase activity (Battesti and Bouveret, 2012).

In accordance with a previous work (Fargier et al., 2012), our results showed that the wildtype MexT from strain PA14 is under a monomeric form as neither the reporter plates nor the β-galactosidase activity turned out to be positive (17 ± 1.66 Miller units) (Table 6). In contrast, the mutated MexT variants from strains 4088 and 10-12 gave positive results with both tests ( a : plasmids pUT18 and pKNT25 for which the tag is at the C-terminus of the recombinant protein were used in this experiment. b : Mean values were calculated from five independent bacterial cultures each assayed in triplicate.

As no crystal structure is available for MexT, we used Raptor X server A short article is currently being prepared to publish these results (draft in appendix).

III. Induction of mexEF-oprN by Electrophilic Stress

1 Background: activation of RND efflux systems in response to oxidative stress 1.1 Overview Some RND efflux systems provide P. aeruginosa with a significant resistance to antibiotics when overproduced upon mutations affecting regulatory genes [START_REF] Li | Antimicrobial drug efflux pumps in Pseudomonas aeruginosa. In efflux-mediated antimicrobial resistance in bacteria[END_REF].

Nevertheless, under physiological conditions the primary role of RND systems may not be to export antimicrobials, as these molecules are often present at very low or undetectable concentrations in most of natural environments [START_REF] Fruci | Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria[END_REF]. Indeed, evidence that at least some of these pumps are induced by environmental signals suggest that they are part of specific responses enabling bacteria to adapt to various stresses (Poole, 2014) (Table 7). unbalance between production and removal of oxygen-derived free radicals [START_REF] Betteridge | What is oxidative stress?[END_REF]. This type of stress has long been considered as exclusively linked to ROS formation.

However, other subcategories (nitrosative, electrophilic and disulfide) were identified further

1.2.1

Operon mexAB-oprM Exposure of P. aeruginosa PAO1 to H 2 O 2 or cumene peroxide results in overexpression of operon mexAB-oprM, suggesting that at least one of the regulatory genes of this operon senses these reactive species. Peroxide activation of mexAB-oprM is in part dependent on local repressor MexR [START_REF] Chen | The Pseudomonas aeruginosa multidrug efflux regulator MexR uses an oxidation-sensing mechanism[END_REF]. Indeed, in vitro experiments showed that the purified MexR protein is unable to repress operon expression when an inter-monomer disulfide bond is formed between cystein residues -30 and -62, upon peroxide treatment [START_REF] Chen | The Pseudomonas aeruginosa multidrug efflux regulator MexR uses an oxidation-sensing mechanism[END_REF][START_REF] Chen | Structural insight into the oxidation-sensing mechanism of the antibiotic resistance of regulator MexR[END_REF]) (Figure 11). Redox-dependent inactivation of MexR is a good example of thiol-based oxidation sensing by which a bacterium protects itself against oxidative stressors including some antibiotics. Moreover, a transcriptomic analysis of strain PAO1 treated with environmental pollutant pentachlorophenol (PCP) revealed that mexAB-oprM was overexpressed under these stressing conditions (4.3-fold after 13h of treatment) [START_REF] Muller | Transcriptome analysis reveals that multidrug efflux genes are upregulated to protect Pseudomonas aeruginosa from pentachlorophenol stress[END_REF]. Later on, bacterial exposure to other chlorinated phenols was found to have similar effects [START_REF] Muller | Chlorinated phenol-induced physiological antibiotic resistance in Pseudomonas aeruginosa[END_REF]. While regulator NalC, which controls the expression of anti-repressor ArmR, was first hold responsible for this induction of mexAB-oprM expression [START_REF] Ghosh | Chlorinated phenols control the expression of the multidrug resistance efflux pump MexAB-OprM in Pseudomonas aeruginosa by interacting with NalC[END_REF], another study demonstrated that the operon was still upregulated upon PCP exposure in absence of ArmR [START_REF] Starr | Pentachlorophenol induction of the Pseudomonas aeruginosa mexAB-oprM efflux operon: involvement of repressors NalC and MexR and the antirepressor ArmR[END_REF]. It was thus proposed that chlorinated phenols generate an oxidative stress in P. aeruginosa, with subsequent inactivation of redox-sensor MexR leading to mexAB-oprM derepression [START_REF] Chen | The Pseudomonas aeruginosa multidrug efflux regulator MexR uses an oxidation-sensing mechanism[END_REF][START_REF] Starr | Pentachlorophenol induction of the Pseudomonas aeruginosa mexAB-oprM efflux operon: involvement of repressors NalC and MexR and the antirepressor ArmR[END_REF]. Whether peroxide and chlorinated phenols can promote MexAB-OprM-dependent antibiotic resistance has not been established.

1.2.2

Operon mexXY Production of MexXY proteins is also induced by oxidative stress (Masuda et al., 2000b). Indeed, exposure of strain PAO1 to H 2 O 2 is associated with increased transcript levels of mexXY and stronger resistance to MexXY/OprM substrates such as amikacin (4-fold), tobramycin (4-fold), gentamicin (2-fold), and paromomycin (2-fold) [START_REF] Fraud | Oxidative stress induction of the MexXY multidrug efflux genes and promotion of aminoglycoside resistance development in Pseudomonas aeruginosa[END_REF]. This induction depends on ArmZ, a repressor that binds and sequester the negative regulator (MexZ) of operon mexXY [START_REF] Yamamoto | Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa[END_REF]. However, the molecular mechanism by which peroxide induces gene armZ expression is still unresolved [START_REF] Fraud | Oxidative stress induction of the MexXY multidrug efflux genes and promotion of aminoglycoside resistance development in Pseudomonas aeruginosa[END_REF].

Since the pump MexXY/OprM is activated by ArmZ when P. aeruginosa is exposed to ribosome-targeting antibiotics (Morita et al., 2006b), one could assume that the inducing effects of ROS on ArmZ also result from their deleterious action on the ribosomal machinery

1.3

Induction of mexEF-oprN by non-classical oxidative stress Operon mexEF-oprN expression remains at basal levels in cells exposed to ROS (Fetar et al., 2011), but is increased upon nitrosative and disulfide stresses (Fargier et al., 2012;Fetar et al., 2011). Several lines of evidence suggest that some stress elicitors are able to activate MexT, through a still unknown mechanism (Figure 12). In line with this, it was reported that mexEF-oprN is upregulated (12.9-fold) when bacteria are in contact with airways epithelial cells, which is believed to cause a kind of oxidative stress to P. aeruginosa (Frisk et al., 2004).

1.3.1

Nitrosative stress During host colonization and/or infection, P. aeruginosa is exposed to nitric oxide (NO·), a reactive nitrogen species (RNS) that is mostly produced by macrophages and that causes a nitrosative stress in bacterial cells [START_REF] Poole | Stress responses as determinants of antimicrobial resistance in Pseudomonas aeruginosa: multidrug efflux and more[END_REF]. Other RNS include peroxinitrite (ONOO -) and nitrogen dioxide (NO 2 ·), two species known to be highly reactive and able to alter lipids, thiols, amino acids and DNA, though these reactions occur at relatively slow rates in comparison with ROS reactions [START_REF] Fang | Antimicrobial reactive oxygen and nitrogen species: concepts and controversies[END_REF][START_REF] O'donnell | Nitration of unsaturated fatty acids by nitric oxide-derived reactive nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide, and nitronium ion[END_REF]. Operon mexEF-oprN transcription is inducible by nitrosating agent S-nitrosoglutathione (GSNO) (not quantified), by (NO·)-generating agent diethylenetriamine NONOate (DETA) (not quantified) and by nitro-containing antibiotic chloramphenicol (3-fold increase, measured by β-galactosidase activity), with no evidence that these inductions do impact MICs of antibiotic substrates of MexEF-OprN (Fetar et al., 2011).

1.3.2

Disulfide stress The perturbation of thiol/disulfide (-SH/S-S) balance inside the cell is referred to disulfide stress [START_REF] Masip | The many faces of glutathione in bacteria[END_REF][START_REF] Smirnova | Glutathione in bacteria[END_REF]. Such a stress occurs when thiolreactive molecules, such as quinones, accumulate intracellularly [START_REF] Liebeke | Depletion of thiol-containing proteins in response to quinones in Bacillus subtilis[END_REF]. It has pleiotropic effects on bacterial physiology as it can modify the ratio of reduced to oxidized glutathione (GSH/GSSG). Reduced glutathione (GSH) is a protein-reducing molecule that prevents formation of aberrant disulfide bonds in cytoplasmic proteins; as such it plays an important role in bacterial protection against osmotic, acid, electrophilic, chlorinated and oxidative stresses [START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. ). Supporting the notion that diamide is a substrate of the pump, disruption of gene mexT, mexE, or mexF was found to increase the susceptibility of P. aeruginosa to the elicitor. However, disruption of oprN that codes for the outer membrane component of the efflux system turned out to have minimal effects on diamide MICs, suggesting that proteins MexEF preferentially use another exit channel to extrude the inhibitor (Fargier et al., 2012).

The same study showed that the purified MexT protein was capable to form an oligomer in vitro when treated with oxidized glutathione (GSSG), and that increasing concentrations of GSH increased formation of the monomeric form at the expense of oligomers (Figure 13). Of note, it was reported that under oxidizing conditions, MexT binds to the promoter region of PA4881, a MexT-regulated gene of unknown function (Fargier et 8).

Electrophile sensing

In order to implement an adapted response to specific stress situations, bacteria need to detect the presence or accumulation of stressing molecules. Stress sensing usually involves one or several transcriptional regulators that are de facto the initial elements of the bacterial response. Such regulators directly interact with stressors or are activated by cognate membrane sensors (Table 8) (Lee and Park, 2017). 16).

In E. coli, the glyoxalase system is the most efficient mechanism for detoxification of GO and MG. It consists of two glutathione (GSH)-dependent enzymes, namely glyoxalases I and II, and one GSH-independent enzyme, glyoxalase III (Kosmachevskaya et al., 2015; Lee and Park, 2017) (Figure 17). Glyoxalase I (Glo1) is encoded by gene gloA. Thanks to its S-Dlactoylglutathione lyase activity, the enzyme catalyzes formation of GSH adducts, S-2hydroxyethylglutathion (SHG) and S-D-lactoylglutathion (SLG) with GO and MG, respectively [START_REF] Thornalley | The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life[END_REF]. These adducts can modulate the activity of two potassium efflux pumps KefB and KefC which will be described below [START_REF] Ferguson | Protection of the DNA during the exposure of Escherichia coli cells to a toxic metabolite: the role of the KefB and KefC potassium channels[END_REF]MacLean et al., 1998). P. aeruginosa differs from E. coli as it harbors three glyoxalase I genes in its chromosome (gloA1, gloA2, and gloA3), but the differential activities of their products remain unknown [START_REF] Sukdeo | Pseudomonas aeruginosa contains multiple glyoxalase I-encoding genes from both metal activation classes[END_REF]. Glyoxalase II (Glo2) is a hydroxyacylglutathione hydrolase that converts SHG and SLG into glycolic acid and D-lactate, respectively with concomitant release of GSH (Figure 17) (Lee and Park, 2017). Finally, glyoxalase III catalyzes reduction of GO and MG through a single-step reaction, without the need of any cofactor (Lee and Park, 2017).

The second pathway of electrophile detoxification involves NAD(P)H-dependent enzymes (Figure 16). In E. coli, several enzymes, for the most aldehyde reductases, are able to reduce various electrophiles into less toxic alcohols (Lee and Park, 2017). Remarkably, YqhD can convert MG, GO and CNA in vitro (Lee et al., 2010;Visvalingam et al., 2013). However, its role in vivo in the metabolism of MG is minor compared to that of the glyoxalase system (Lee et al., 2010(Lee et al., , 2013b)). Actually, YqhD is of major importance in protection of E. coli from electrophiles derived from lipid peroxidation, such as acetaldehyde, malondialdehyde, propanaldehyde, butanaldehyde, and acrolein) [START_REF] Pérez | Escherichia coli YqhD exhibits aldehyde reductase activity and protects from the harmful effect of lipid peroxidation-derived aldehydes[END_REF].

1.4.3

Protective mechanisms against electrophiles Electrophiles are highly toxic for bacteria (Ferguson et al., 1998a). Survival of E. coli to these agents depends on multiple mechanisms including activation of K + efflux systems KefB and KefC and intervention of sigma factor RpoS in DNA protection [START_REF] Ferguson | Protective mechanisms against toxic electrophiles in Escherischia coli[END_REF].

As stated before, Glo1 produces GSH-adducts in the presence of GO or MG (Lee and Park, 2017; [START_REF] Thornalley | The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life[END_REF]. These adducts are activators of the KefB and KefC pumps [START_REF] Elmore | Activation of potassium efflux from Escherichia coli by glutathione metabolites[END_REF][START_REF] Ferguson | Activation of potassium channels during metabolite detoxification in Escherichia coli[END_REF]. In the absence of electrophiles, the binding of reduced glutathione (GSH) to these systems make them adopt a closed conformation which prevents the leakage of potassium outside the cell (Figure 18 The rapid efflux of potassium by these systems is coupled with an influx of protons likely independent from the transport activity of KefB/C [START_REF] Ferguson | Protective mechanisms against toxic electrophiles in Escherischia coli[END_REF]. The acidification of cytoplasm (decrease of 0.4-0.6 units upon exposure to MG) that results from H + import would reduce the reactivity of electrophiles by decreasing their interaction with macromolecules (Ferguson et al., 1998a). Confirming the important role of cytoplasm acidification in RES response, weak acids were found to protect E. coli from MG [START_REF] Booth | Bacterial production of methylglyoxal: a survival strategy or death by misadventure?[END_REF][START_REF] Ferguson | Protective mechanisms against toxic electrophiles in Escherischia coli[END_REF]Ferguson et al., 1998a). However, though potassium efflux systems are crucial in bacterial survival to some electrophiles such MG, they are dispensable in the defense against iodoacetate and chlorodinitrobenzene [START_REF] Ferguson | Protective mechanisms against toxic electrophiles in Escherischia coli[END_REF]Ferguson et al., 1998a;[START_REF] Muraoka | Crystal structure of a full-length LysR-type transcriptional regulator, CbnR: unusual combination of two subunit forms and molecular bases for causing and changing DNA bend[END_REF]. It is interesting to note that in P. aeruginosa overproduction of RND pumps has also been proposed to result in cytoplasm acidification (decreased of about 0.6 units) [START_REF] Olivares Pacheco | Metabolic compensation of fitness costs is a general outcome for antibiotic-resistant Pseudomonas aeruginosa mutants overexpressing efflux pumps[END_REF] but the impact of this on electrophile detoxification has not been investigated.

Finally, the observation that stationary-phase cells of E. coli are more resistant to electrophiles than fast-growing bacteria led to the hypothesis that sigma factor RpoS has a protective role against these reactive species. Indeed, expression of several genes of RpoS regulon is induced in stationary phase. The resistance to electrophiles could involved protein Dps that binds to and would protect DNA from damages (Ferguson et al., 1998b). However, again the mode of action of RpoS as well as Dps needs to be clarified. KEYWORDS Pseudomonas aeruginosa, efflux, MexEF-OprN, CmrA, electrophilic stress, efflux pumps P seudomonas aeruginosa, a Gram-negative pathogen of major clinical importance, is notorious for its ability to develop a high level of resistance to multiple antibiotics and cause hard-to-treat infections (1). When upregulated upon mutations in regulatory genes, RND (resistance nodulation cell division) efflux pumps contribute substantially to multiresistance in clinical isolates (2). One of these systems, MexEF-OprN, is able to export fluoroquinolones, trimethoprim (TMP), and chloramphenicol (CHL) (3). This tripartite pump is regulated by MexT, a LysR-like activator, whose gene (mexT) is located upstream from operon mexEF-oprN (4). The stable overproduction of MexEF-OprN in so-called nfxC mutants increases the resistance levels for all the pump substrates by 2to 16-fold, while the MICs of carbapenems increase from 2-to 4-fold as a result of concurrent MexT-dependent downregulation of porin OprD (3). The reduced expression of RND pumps MexAB-OprM and MexXY/OprM observed in these mutants has been proposed to account for the paradoxical hypersusceptibility to penicillins, cephalosporins, and aminoglycosides (5). In addition, nfxC mutants are deficient in the production of some extracellular virulence factors, such as pyocyanin, elastase, and rhamnolipids, and exhibit reduced activity of the type III secretion system compared to its activity in wild-type strains (6,7). Such impaired virulence involves the pump itself, which is thought to export some precursors of the quorum-sensing signal molecule PQS (Pseudomonas quinolone signal) (8). Additionally, it also involves the action of MexT, acting as a global regulator of gene expression (9).

Results

Toxic Electrophiles Induce Expression of the

Most nfxC mutants harbor disruptive mutations in gene mexS, predicted to encode a quinone oxidoreductase (10). Suppression of MexS activity in P. aeruginosa activates the regulator MexT, which in turn triggers the transcription of the operon mexEF-oprN. We recently showed that in the clinical setting, most of the MexEF-OprN-overproducing mutants studied either harbored single-amino-acid substitutions in MexS or contained wild-type copies of mexS and mexT genes (11). Furthermore, it appeared that none of the other genes previously reported to upregulate mexEF-oprN expression in vitro (ampR, mvaT, parRS, mxtR, and brlR [12][13][14][15][16]) was mutated in these isolates, indirectly suggesting the existence of additional regulatory loci controlling mexEF-oprN expression.

The present study was thus undertaken to decipher the complex regulation and to gain insight into the physiological function of this efflux system. Analysis of in vitro mutants derived from reference strain PA14 has led to the identification of a novel regulator of MexEF-OprN, CmrA, that responds to electrophilic stress.

RESULTS AND DISCUSSION

In vitro selection of chloramphenicol-resistant mutants overproducing MexEF-OprN. A previous study on multidrug-resistant clinical strains of P. aeruginosa showed that the active efflux system MexEF-OprN can be constitutively upregulated in mutants producing intact MexS and MexT proteins (11). In order to identify novel regulators of this pump, we carried out the selection of spontaneous mutants overproducing MexEF-OprN from reference strain PA14 on agar plates supplemented with chloramphenicol at 128, 256, and 512 g ml Ϫ1 . In contrast to most isolates of the PAO1 lineage, PA14 (chloramphenicol MIC of 64 g ml Ϫ1 ) harbors functional mexS and mexT genes, which makes it suitable for such experiments (11). Thirty resistant colonies were randomly selected under each condition (90 colonies) and further screened by the disk diffusion method for increased resistance to ciprofloxacin (CIP) and imipenem compared to that of the parent strain PA14 (data not shown). Typical nfxC mutants indeed exhibit cross-resistance to chloramphenicol, fluoroquinolones, and carbapenems (3). Reverse transcription-quantitative PCR (RT-qPCR) experiments revealed that 40 subselected mutants significantly overexpressed mexE (from 34-to 116-fold more than in PA14; data not shown). Of these 40 mutants, 24 harbored indels disrupting gene mexS (mutation rate of 2.5 ϫ 10 Ϫ7 ), and 12 produced single-amino-acid variants of protein MexS (1.4 ϫ 10 Ϫ7 ). Interestingly, the remaining four mutants appeared to have intact mexS and mexT genes (2.5 ϫ 10 Ϫ8 ). We focused our attention on the latter mutants, named PJ01, PJ02, PJ03, and PJ04 (collectively referred to as PJ mutants below). All of them exhibited an nfxC-type resistance profile, characterized by 8-to 16-fold increases in the MICs of ciprofloxacin, chloramphenicol, and trimethoprim relative to those of PA14 (Table 1). Interestingly, these resistance levels were somewhat lower than those of the nfxC control strain PA14ΔmexS. Other features of nfxC strains were also less pronounced in PJ mutants, such as the resistance to imipenem and the hypersusceptibility to the MexXY substrate gentamicin, although the MICs of aztreonam (a MexAB-OprM substrate) were identical (Table 1). Likewise, the production of virulence factors, such as pyocyanin, biofilm formation, and swarming motility, was less compromised in these mutants than in PA14ΔmexS (see Fig. S1 in the supplemental material). Consistent with these observations, all these phenotypic traits were associated with relative abundances of mexE transcripts that were 1.5-to 3.4-fold lower than in PA14ΔmexS. The decreases in the relative expression of genes oprD, mexB, and mexY were also com-paratively smaller in the PJ mutants (Table 1). Because of all the genetic and phenotypic differences described above, this new type of MexEF-OprN-upregulated mutants was dubbed nfxC2.

A novel gene implicated in regulation of mexEF-oprN. Sequencing of genes mexT, mvaT, ampR, and mxtR, known to influence in vitro the expression of the operon mexEF-oprN (12,13,15), did not reveal any mutations in the PJ mutants. Therefore, three of them (PJ01, PJ03, and PJ04) were submitted to whole-genome sequencing. Alignment of the sequence reads from PJ01 (n ϭ 1,041,118), PJ03 (n ϭ 1,290,598), and PJ04 (n ϭ 1,443,653) with the strain UCBPP-PA14 genome revealed the existence of three single-nucleotide polymorphisms (SNPs) in each of the mutants tested (Table S1). In an interesting way, it appeared that all of them harbored different SNPs in the same gene, PA14_ 38040. These results were confirmed by PCR and Sanger sequencing. Compared to the sequence of PA14, the mutations were predicted to generate amino acid substitutions A 68 V (a change of A to V at position 68), L 89 Q, and N 214 K in the encoded products of PJ01, PJ03, and PJ04, respectively (Table 1). Subsequent sequencing of the last mutant, PJ02, also revealed an H 204 L substitution in the same protein. According to the GenBank database, gene PA14_38040 codes for an as-yetuncharacterized transcriptional regulator of the AraC family. This gene was named cmrA (for chloramphenicol resistance Activator), referring to the conditions of selection of PJ mutants. Interestingly, it turned out that the relative expression of gene cmrA was upregulated in all PJ mutants (from 53-to 120-fold that of PA14), indicating that CmrA likely activates its own gene transcription (Table 1).

Role of CmrA in antibiotic resistance. To confirm the potential implication of CmrA in mexEF-oprN activation and, thus, the resistance phenotype of PJ mutants, we deleted the coding sequence of cmrA in both PJ01 and PA14, yielding PJ01ΔcmrA and PA14ΔcmrA, respectively. The MICs of MexEF-OprN substrates (ciprofloxacin, chloramphenicol, and trimethoprim) and of carbapenems (imipenem) were restored to wildtype levels in PJ01ΔcmrA but remained unchanged for PA14ΔcmrA (Table 1). These results clearly demonstrated that CmrA activates the efflux operon in mutant PJ01 but does not contribute to the intrinsic resistance of P. aeruginosa to the drugs tested. Moreover, deletion of cmrA in PJ01 restored the wild-type susceptibility to ␤-lactams (aztreonam) and to aminoglycosides (gentamicin), cancelling the negative impact of overproduced MexEF-OprN on efflux pumps MexAB-OprM and MexXY, respectively. To confirm these data, we inserted a single copy of the cmrA allele from PJ01 into the chromosome of PA14ΔcmrA (to yield PA14ΔcmrA PJ01 ). As expected, PA14ΔcmrA PJ01 displayed a resistance profile similar to that of PJ01, whereas the control strain complemented with the wild-type cmrA allele, PA14ΔcmrA PA14 , remained fully susceptible to all the antibiotics tested (Table 1). PA14ΔcmrA PJ02 , PA14ΔcmrA PJ03 , and PA14ΔcmrA PJ04 were phenotypically similar to PA14ΔcmrA PJ01 (data not shown).

Finally, as shown by the results in Table 2, overexpression of a plasmid-borne copy of the PJ01 cmrA allele [PA14(pJN105::cmrA PJ01 )] was associated with increased resistance to ciprofloxacin (8-fold), chloramphenicol (8-to 16-fold), and to a lesser extent, trimethoprim (2-to 4-fold), concomitant with a 20-to 50-fold increase in mexE transcripts. Overexpression of the wild-type allele cmrA in PA14 had no significant effects on the susceptibility of the strain to antibiotics [see PA14(pJN105::cmrA PA14 ) in Table 2], reinforcing the idea that the CmrA peptide produced by PJ01 is under an activated conformation. According to the structure modeling of CmrA provided by the RaptorX Web server ( 17), the amino acid substitutions A 68 V and L 89 Q reside in the putative N-terminal ligand-binding domain, while H 204 L and N 214 K are located in the C-terminal DNA-binding domain of the protein (Fig. 1). Altogether, these findings showed that CmrA is a novel regulator of the AraC family, able to trigger directly or indirectly the expression of mexEF-oprN when constitutively activated by single-aminoacid substitutions located in two different functional domains of the protein. a Values obtained without and with 0.5% arabinose, used as an inducer of gene expression.

b Expressed as the ratio to the value for PA14(pJN105). c CIP, ciprofloxacin; CHL, chloramphenicol; TMP, trimethoprim. Characterization of cmrA locus. The cmrA gene is highly conserved among P. aeruginosa strains. Homologs were found in PAO1 (PA2047, 99% sequence identity), PACS2 (AOK_RS09840, 98%), and LESB58 (PALES_30281, 98%). However, in strain PAO1 (18), a potential start codon (ATG PAO1 ) has been mapped 57 nucleotides upstream from that of PA14 (ATG PA14 ), leading to a translated peptide of 329 amino acids instead of 310 (Fig. 2A). In silico analysis of the upstream region of both ATGs failed to show any putative ribosome binding site (RBS) sequence, which could have helped us to define the correct start codon of cmrA. We thus carried out 5=-RACE (rapid amplification of 5= cDNA ends) experiments to identify the transcription start site (TSS) of the gene. Sequencing analysis of the 5=-RACE product revealed that the cmrA TSS is a guanine residue located 19 bp downstream from the proposed ATG PAO1 , a result that contradicts this annotation but validates that of PA14, with the TSS (ϩ1) located 38 bp upstream from ATG PA14 (Fig. 2A).

Analysis of the putative sigma factor binding motifs (SFBMs) of PA14 (sigmulome) (19) proved to be useful to predict the location of the cmrA promoter (P cmrA ). Hence, comparison of the DNA region upstream from cmrA TSS with the PA14 sigmulome highlighted the presence of two overlapping SFBMs for RpoN (Ϫ80 bp) and RpoS (Ϫ75 bp) (Fig. 2A). cis complementation of strain PA14ΔcmrA with a fragment containing cmrA preceded by the full-length RpoN/RpoS binding region (Ϫ152 bp upstream from the TSS, Fig. 2B) successfully generated an nfxC2 resistance phenotype, while complementation with a shorter fragment lacking the RpoN/RpoS SFBMs (Ϫ68 bp upstream from the TSS) had no effect (data not shown). These data provide evidence that the RpoN/RpoS region is necessary to activate the expression of cmrA (19). Whether cmrA is under the control of RpoN and/or RpoS remains to be determined.

MexT-and MexS-dependent upregulation of the operon mexEF-oprN in nfxC2 mutants. Because the upregulation of mexEF-oprN requires a functional regulator, MexT, in mexS mutants (5), we examined whether this also applies to cmrA mutants. Inactivation of mexT in PJ01 (PJ01ΔmexT) (Table 1) restored a wild-type profile, thereby demonstrating MexT-dependent activation of MexEF-OprN in nfxC2 mutants. In agreement with these data, the transcript levels of mexE were strongly reduced in the absence of mexT (Ͼ380-fold), while those of genes oprD, mexB, and mexY increased significantly, from 2.7-to 5-fold (Table 1).

Finally, as the inactivation of mexS is the main mutational cause of mexEF-oprN overexpression in both in vitro (10) and clinical strains (11), we deleted this gene in PJ01 to see whether this would result in a stronger activation of the efflux operon and, thus, higher resistance levels. Surprisingly, loss of mexS in mutant PJ01ΔmexS totally abolished mexEF-oprN overexpression and restored a PA14-like resistance phenotype (Table 1), a result that underlines a functional link between MexS and CmrA or between MexS and one or several genes under the control of CmrA. Again, as for PJ01ΔmexT, the phenotypic changes noted in PJ01ΔmexS relative to PJ01 were associated with significant variations in the expression of genes mexE, oprD, mexB, and mexY. Similar results were obtained with the other mutants, PJ02ΔmexS, PJ03ΔmexS, and PJ04ΔmexS (data not shown).

Identification of genes regulated by CmrA. To better understand the physiological role of CmrA and to identify the genes under its control, we performed a transcriptomic analysis by RNA-seq of cmrA mutant PJ01 in comparison with parental strain PA14. Since MexT regulates the expression of 143 genes in P. aeruginosa (20), we analyzed the transcriptome of mutant PJ01ΔmexT as well. This allowed us to identify, by subtraction, genes whose expression is exclusively regulated by CmrA. Data analysis showed that 53 genes were differentially expressed between mutant PJ01 and strain PA14, 26 of them being upregulated and 27 downregulated in PJ01 (threshold fixed at 3.0-fold) (GEO accession number GSE86211) (see Fig. S2). Comparison of strains PA14 and PJ01ΔmexT allowed the identification of 42 genes under the control of MexT and only a small set of 11 CmrA-dependent upregulated genes clustering into three genetic loci (Table 3). The overexpression of these 11 genes was confirmed by RT-qPCR. Altogether, our data showed that CmrA is a transcriptional activator influencing the expression of a few genes, including cmrA itself (Table 3). Reminiscent of the putative function of MexS, the four most activated CmrA-dependent genes (homologs of PA2048, PA1881, PA1880, and PA2275 in PAO1) are predicted to encode oxidoreductases (a quinol monooxygenase, an oxidoreductase, an aldehyde dehydrogenase, and an alcohol dehydrogenase, respectively). Furthermore, it appeared that CmrA also regulates the expression of two genes coding for uncharacterized transcriptional regulators (PA2276 and PA1879) and the determinant of the arsenic/antimony response regulator ArsR (PA2277) (21). Whether CmrA directly regulates all these genes remains to be determined.

PA2048 is responsible for the nfxC2 phenotype. Single inactivation of the CmrAdependent genes in mutant PJ01 revealed that none of them was implicated in mexEF-oprN overexpression except PA2048, the deletion of which restored a wild-type phenotype of resistance to the selected antibiotics (Table S2). PA2048 is adjacent to and divergently transcribed from cmrA (Fig. 2).

Because a long intergenic region of 581 bp between cmrA and PA2048 was mentioned in the annotated genomes of PAO1 and PA14 (18), we carried out 5=-RACE experiments to localize the TSS of PA2048. Our results confirmed that the transcript of PA2048 begins 315 nucleotides upstream from ATG PA2048 as determined in the PA14 sigmulome (19). This long untranslated region (UTR) contains two inverted repeat sequences of 10 nucleotides with a spacer of 91 nucleotides (Fig. 2, IR-N91), the function of which remains unknown. ATG PA2048 is preceded by a potential Shine-Dalgarno sequence (AGGAGG) of 8 bp upstream from the gene coding sequence.

PA2048 codes for a small hypothetical protein of 98 amino acids, sharing conserved domains with YgiN from Escherichia coli (NCBI pblast). Sequence alignment with Clustal Omega (22) showed 40% similarity between YgiN (protein accession number ABV07440) and PA2048 (protein accession number WP_003088696) (Fig. S3). YgiN was initially described as a small protein of 104 residues having orthologs in other bacterial species, such as Bacillus subtilis, Synechocystis sp., Mycobacterium tuberculosis, Neisseria gonorrhoeae, and Rhodococcus erythropolis, but whose function was completely unknown (23). Subsequent analysis of the crystal structure of YgiN revealed a folding similar to that of monooxygenase ActVA-Orf6 from Streptomyces coelicolor, an enzyme that uses quinols as substrates (24). Consistent with this, in vitro experiments demonstrated that the purified YgiN protein was able to oxidize menadiol into menadione without the need of any cofactor, data that allowed the classification of this enzyme as a quinol monooxygenase (24).

Quinol monooxygenases are usually functionally coupled with oxidoreductases that catalyze inverse reactions (quinones to quinols) and so participate in a quinone redox cycle, as previously illustrated by MdaB-YgiN and YdhR-YdhS in E. coli (24,25). Later on, Adams and Jia showed that the coordinate activity of MdaB-YgiN allows bacterial cells to resist polyketide antibiotics, such as adriamycin and tetracycline (26). It was proposed that this enzymatic couple might function primarily to protect E. coli cells from polycyclic quinones by recycling them until they are conjugated and exported out of the cell (26). YgiN would also be involved in cell respiration by transferring electrons to molecular oxygen when cytochrome oxidases are deficient (27). Interestingly, our results suggest that, in P. aeruginosa, the activities of PA2048 (a putative quinol monooxygenase) and MexS (a putative quinone oxidoreductase) are functionally linked, perhaps to adjust the redox state of the respiratory quinone pool when bacteria face some stressful conditions. In support of this assumption, we found that mexS expression was increased from 2.3-to 5.6-fold in the PA2048-upregulated PJ mutants compared with its expression in PA14 (Table 1). To our knowledge, quinol monooxygenases have never been studied so far in Pseudomonas species and the physiological role of PA2048 remains to be explored. Since PA2048 was the most upregulated gene (199-fold) of the PJ01 transcriptome (Table 3), a plasmid-borne copy of this gene (including its 5= UTR) was overexpressed in strain PA14 to determine whether the nfxC2 phenotype results exclusively from the activity of the encoded protein. Indeed, overexpression of PA2048 in PA14(pJN105:: PA2048) strongly increased mexE transcripts, as well as the MICs of the pump substrates, without a significant change in the cmrA expression level (Table 2). These data clearly confirmed that CmrA is not directly responsible for the activation of mexEF-oprN in mutant PJ01 but exerts an indirect effect on the pump through the product of PA2048.

The CmrA pathway is activated by electrophilic stress. To screen for inducers of the CmrA pathway, we constructed a transcriptional fusion between PA2048 and the luxCDABE operon. Gene PA2048 was selected for the lux fusion because its expression is highly augmented in response to CmrA activation (i.e., as in mutant PJ01), and its encoded product is key in the regulatory cascade that finally triggers production of MexEF-OprN via MexS and MexT. Gene PA2048 was fused to the lux reporter and inserted into the chromosomes of PA14 and PJ01. As expected, PJ01::PA2048-lux emitted 10-fold more luminescence than PA14::PA2048-lux at the mid-log phase of growth (data not shown).

A first set of experiments using known MexEF-OprN substrates (ciprofloxacin, trimethoprim, and chloramphenicol) failed to demonstrate any induction of the lux reporter in strain PA14 (data not shown). The same negative results were obtained when disulfide stress (diamide) (28), oxidative stress (H 2 O 2 , paraquat, and dimethyl sulfoxide [DMSO]), and nitrosative stress (S-nitrosoglutathione [GSNO] and 2-n-heptyl-4-hydroxyquinoline N-oxide [HQNO]) (29) elicitors were added at subinhibitory concentrations to the bacterial cultures (data not shown). Finally, we found that three CmrA-regulated proteins (PA2048, PA2275, and PA2276) share Ն40% amino acid similarity with E. coli enzymes implicated in the response to electrophilic stress (YgiN, YqhD, and YqhC, respectively) (30,31). This cellular stress is characterized by an imbalance between the formation and the removal of reactive electrophilic species (RES) containing ␣,␤-unsaturated carbonyl (32) or other electrophilic groups (33). For this reason, strain PA14::PA2048-lux was challenged with increasing concentrations of three toxic electrophilic molecules: glyoxal (GO), methylglyoxal (MG) (33), and cinnamaldehyde (CNA) (34). Interestingly, the PA2048-lux-dependent luminescence increased with subinhibitory concentrations of GO (12 to 200 g ml Ϫ1 ) (Fig. 3A), MG (10 to 100 g ml Ϫ1 ), and CNA (70 to 280 g ml Ϫ1 ) (Fig. S4).

To confirm these results, the impacts of electrophilic stress on mexE and PA2048 expression were then assessed by RT-qPCR. As shown by the results in Fig. 3B, both genes were rapidly and strongly activated 15 min after the addition of 200 g ml Ϫ1 GO to the bacterial cultures. The same was observed for cmrA, whose mRNA levels increased 67-fold over those of the untreated control (data not shown). Confirming our bioluminescence data, the transcript amounts of mexE and PA2048 started to decline 45 min after the initiation of electrophilic stress. Finally, induction of the MexEF-OprN efflux system upon GO exposure was phenotypically confirmed in strain PA14 with antibiograms on Mueller-Hinton agar (MHA), showing antagonistic interactions between (i) disks containing MexEF-OprN antibiotic substrates (chloramphenicol, ciprofloxacin, and trimethoprim) and (ii) a disk loaded with GO (Fig. 3C). As anticipated, no evidence of antagonism was visible with the negative-control strain PA14ΔmexEF-oprN. Identical results were obtained when the double-disk tests were performed with MG and CNA (data not shown).

Differential activation of detoxification mechanisms by electrophilic stress elicitors. In E. coli, GO and MG are rapidly sequestered under the form of glutathione adducts and transformed into nontoxic metabolites (glycolic acid and lactate, respectively) via enzymes of the glyoxalase system (32,33,35). Suggesting the existence of a similar detoxification pathway, two glyoxalase genes (36) turned out to be significantly overexpressed in strain PA14 when treated with GO (gloA2 and gloA3) or MG (gloA3) (Table S3). It should be noted that activation of the glyoxalase genes is not CmrA dependent, as their expression remains unchanged in mutant PA14ΔcmrA treated with either GO or MG (data not shown). Conversely, CNA exposure failed to activate the glyoxalase pathway, while it dramatically triggered the expression of another gene, PA2275 (2,873-fold compared to its expression in untreated cells). In comparison, GO and MG treatments had smaller effects on PA2275 transcription levels (17-and 7-fold, respectively) (Table S3). The PA2275-encoded product shares 40% sequence similarity with YqhD from E. coli, an enzyme promoting the degradation of CNA into the less-toxic cinnamic alcohol (31,37). Altogether, these results corroborate the hypothesis that the transient activation of CmrA by GO, MG, and CNA is due to the rapid degradation of these molecules into metabolites lacking electrophilic properties, via different bacterial enzymes.

MexEF-OprN efflux pump protects bacteria from cinnamaldehyde. Since MexEF-OprN is overproduced in response to electrophilic stress, we wondered whether the pump offers protection against the agents able to elicit such a stress. Surprisingly, the resistance levels to GO (MIC of 512 g ml Ϫ1 ), MG (256 g ml Ϫ1 ), and CNA (512 g ml Ϫ1 ) were not different between strain PA14 and mutant PA14ΔmexEF-oprN, consistent with the idea that this efflux system does not contribute to the intrinsic resistance toward any of these products. Alternatively, efflux of the elicitors might be masked by the effects of more efficient detoxification mechanisms, such as those described above. To test this hypothesis, time-kill experiments were performed with bactericidal concentrations of GO (1,000 g ml Ϫ1 ), MG (500 g ml Ϫ1 ), and CNA (1,000 g ml Ϫ1 ) on FIG 3 Response of P. aeruginosa to electrophilic stress. (A) The bioluminescence of strain PA14::PA2048-lux was monitored at defined time points after exposure to increasing concentrations of glyoxal (GO) and is expressed as the ratio of relative light units (RLU) to bacterial density (A 600 ). Nontreated bacteria were used as the control (CTRL). Results are mean values Ϯ standard deviations from three independent experiments. (B) Expression levels of genes mexE and PA2048 were determined by RT-qPCR in strain PA14 exposed to 200 g ml Ϫ1 GO and compared with those of a nontreated control (CTRL). Results are mean values of four determinations from two independent experiments. (C) Induction of pump MexEF-OprN with glyoxal was assessed by a double-disk antagonism test using strain PA14 and negative-control strain PA14ΔmexEF-oprN. Paper disks were loaded with 8,000 g glyoxal (GO), 5 g ciprofloxacin (CIP), 1,000 g chloramphenicol (CHL), or 240 g trimethoprim (TMP). Antagonism is visible between GO and all the tested MexEF-OprN substrates in strain PA14 (left) but not in mutant PA14ΔmexEF-oprN (right).

bioluminescent strains PA14-lux, PA14-lux-ΔmexEF-oprN, and PA14-lux-ΔcmrA. While no differences were observed between the killing curves of the three strains exposed to GO or MG (data not shown), deletion of mexEF-oprN or cmrA was associated with a slight though reproducible sensitization of bacteria to CNA killing that impaired bacterial regrowth 45 min after drug exposure (Fig. 4). Since mutants PA14-lux-ΔmexEF-oprN and PA14-lux-ΔcmrA behaved similarly, the simplest explanation for these results is that the CmrA-dependent activation of MexEF-OprN allows surviving bacteria to better resist CNA (but not GO or MG) through the potentiation of detoxification mechanisms.

Conclusion. This work describes a novel regulator of the AraC family, named CmrA, which when activated by single point mutations or electrophilic stressors is able to upregulate the expression of mexEF-oprN via MexS and MexT. The cmrA mutants (dubbed nfxC2) exhibit the same, albeit less pronounced, multidrug resistance phenotype as prototypal nfxC mutants. Consistent with lower expression of the operon mexEF-oprN and, probably, lower levels of activation of the regulator MexT, nfxC2 mutants are less compromised than their nfxC counterparts in quorum-sensingdependent production of virulence factors (6).

CmrA influences the expression of a very small set of nonessential genes all predicted to catalyze redox reactions on as-yet-undetermined substrates. The overexpression of one of these genes, PA2048, coding for a presumed quinol monooxygenase, is sufficient to trigger MexEF-OprN production. However, our observation that MexEF-OprN is overproduced only when the putative quinone oxidoreductase MexS is functional in nfxC2 mutants indicates that both enzymes are functionally linked, at least under specific physiological or stress conditions. Whether these two enzymes act coordinately or sequentially to maintain the redox state of respiratory quinones in stressed cells is currently being investigated. Therefore, the present work reinforces the conclusions reached by previous investigators on MexT being a redox-responsive regulator (28), though it introduces a new player, PA2048, in the activation pathway of MexT and MexEF-OprN. Interestingly, while MexT activation requires a functional MexS in nfxC2 mutants, it is triggered by the loss of this enzyme in nfxC strains (Fig. 5). The fact that PA2048 is the only CmrA-regulated gene whose deletion abolishes MexEF-OprN activation in nfxC2 mutants suggests that the metabolites of putative quinol monooxygenase PA2048 are directly or indirectly processed by MexS to form the ligand(s) that, in fine, will cause the oligomerization of MexT, as suggested previously (28). The absence of exogenous stress in the CmrA mutants PJ01 to PJ04 demonstrates that the substrates of PA2048 are endogenous molecules. Furthermore, this strongly suggests that the mutations that drive the enzyme overproduction generate an imbalance in the redox state of preexisting compounds that MexS tends to compensate (i.e., note that gene mexS is overexpressed in the PJ mutants). Whether the MexS metabolites are effluxed by MexEF-OprN or only serve as a signal to activate the pump as part of a global response of defense remains to be elucidated.

Reactive electrophilic species (RES) are compounds containing ␣,␤-unsaturated carbonyl or other electrophilic groups. These highly toxic molecules interact with proteins, nucleic acids, lipids, and carbohydrates, generating pleiotropic cellular effects (31,38). They also affect the redox state of the cell via their interaction with redox cofactors, such as glutathione and NAD(P)H (33). As such, the electrophilic stress can be considered a subcategory of oxidative stress, as is the case with disulfide (28) and nitrosative (29) stresses. Even if all these different subcategories of stress result in Redox-dependent oligomerization of MexT then triggers production of the pump MexEF-OprN and active efflux of still-undetermined endogenous products. In nfxC2 mutants (bottom left), regulator CmrA is activated as a result of gain-of-function mutations in gene cmrA. Among the 11 genes positively regulated by CmrA, PA2048 codes for a putative quinol monooxygenase. Concomitant activation of PA2048 and MexS is assumed to generate oxidized metabolites, the accumulation of which would modify the cellular redox state. As in MexS-deficient mutants, these changes upregulate the production of MexEF-OprN via MexT. Finally, upon electrophilic stress (bottom right), reactive electrophilic species (RES), such as glyoxal and methylglyoxal, induce the glyoxalase detoxification system (GloA2 and GloA3) and CmrA-dependent expression of MexEF-OprN. We propose that P. aeruginosa uses the efflux pump MexEF-OprN as a defense mechanism in response to toxic electrophilic stressors encountered in its environment.

MexS/MexT-dependent upregulation of the efflux system MexEF-OprN, only electrophiles were found to specifically activate the CmrA pathway, highlighting a distinctive feature of these molecules despite their multiple cellular targets (Fig. 5) (31). Since MexEF-OprN does not provide P. aeruginosa with meaningful protection against harmful electrophiles, such GO and MG, our hypothesis is that the physiological damage generated by such agents mimics a stress the bacterium has to face when adapting to other challenging conditions, perhaps those encountered during infection. It should be noted that all the electrophiles tested were able to upregulate MexEF-OprN via CmrA, PA2048, and MexS. Thus, these results unambiguously show that MexT is activated not by gross changes in the redox state of the cytoplasm but by specific ligand molecules, likely produced by MexS. In contrast to GO and MG, cinnamaldehyde (CNA) seems to be a substrate of MexEF-OprN. This natural substance present in the cinnamon stem bark is well known for its antimicrobial properties against various fungi and bacteria, including P. aeruginosa (39). As an inducer and a substrate of the MexEF-OprN pump, CNA would be the first example of a plant substance against which P. aeruginosa has evolved an efflux-based defense triggered by electrophilic stress.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. The reference strains, derived mutants, and plasmids used in this study are listed in Table S4 in the supplemental material. All the bacterial cultures were grown in Mueller-Hinton broth (MHB) with adjusted concentrations of Ca 2ϩ (from 20 to 25 g ml Ϫ1 ) and Mg 2ϩ (from 10 to 12.5 g ml Ϫ1 ) (Becton Dickinson and Company, Cockeysville, MD) or on Mueller-Hinton agar (MHA) (Bio-Rad, Marnes-la-Coquette, France). Spontaneous mutants overproducing the efflux pump MexEF-OprN were selected on MHA supplemented with chloramphenicol (128, 256, or 512 g ml Ϫ1 ). Escherichia coli transformants were selected on MHA containing 50 g ml Ϫ1 kanamycin (marker for vectors pCR-Blunt and pCR2.1), 15 g ml Ϫ1 tetracycline (plasmids mini-CTX1 and miniCTXlux), 50 g ml Ϫ1 streptomycin (pKNG101), or 10 g ml Ϫ1 gentamicin (pJN105 and pUC18T-mini-Tn7Tlux-Gm). Recombinant plasmids were introduced into P. aeruginosa strains by triparental mating and mobilization with the broad-host-range vector pRK2013 using E. coli HB101 as a helper strain (40). Transconjugants were selected on Pseudomonas isolation agar (PIA; Becton Dickinson and Company) supplemented with 200 g ml Ϫ1 tetracycline, 2,000 g ml Ϫ1 streptomycin, or 10 g ml Ϫ1 gentamicin, as required. Excision of pKNG101 was obtained by subculture on M9 minimal medium (8.54 mM NaCl, 25.18 mM NaH 2 PO 4 , 18.68 mM NH 4 Cl, 22 mM KH 2 PO 4 , 2 mM MgSO 4 , pH 7.4) supplemented with 5% sucrose and solidified with 0.8% agar.

Antibiotic susceptibility testing. The MICs of selected antibiotics were determined by the standard serial 2-fold dilution method in MHA with inocula of 10 4 CFU per spot, according to CLSI recommendations (41). Growth was visually assessed after 18 h of incubation at 37°C. Spontaneous nfxC mutants developing on solid medium were screened for their resistance to both ciprofloxacin and imipenem by measuring the inhibition zones around Bio-Rad disks (loaded at 5 g and 10 g, respectively) on MHA plates incubated for 18 h at 37°C (42).

Virulence factor analysis. Biofilm formation was assessed in 96-well polystyrene plates after coloration of adherent bacteria with 1% (wt/vol) crystal violet (43). Swarming motility, which is characterized by the formation of bacterial dendrites on low-agar culture medium, was assayed on freshly prepared M8 medium (42.2 mM Na 2 HPO 4 , 22 mM KH 2 PO 4 , 7.8 mM NaCl, pH 7.4) supplemented with 2 mM MgSO 4 , 0.5% casein, 0.5% agar, and 1% glucose, as previously described (44). Finally, pyocyanin production was evaluated in the supernatants of 18-h cultures at 37°C in a specific broth [12 mM Tris HCl, pH 7.2, 0.1% tryptones, 20 mM (NH 4 ) 2 SO 4 , 1.6 mM CaCl 2 , 10 mM KCl, 24 mM sodium citrate, and 50 mM glucose] (absorbance [A 600 ] ϭ 1.6 Ϯ 0.2). The blue pigment was extracted with 1 volume of chloroform (45). Virulence assays were all repeated twice with independent cultures of strains PA14 and PA14ΔmexS as controls.

RT-qPCR experiments. Specific gene expression levels were measured by quantitative PCR after reverse transcription (RT-qPCR), as described previously (46). Briefly, 2 g of total RNA was reverse transcribed with ImProm-II reverse transcriptase as specified by the manufacturer (Promega, Madison, WI). The amounts of specific cDNA were quantified on a Rotor-Gene RG6000 instrument (Qiagen, Courtaboeuf, France) by using the QuantiFast SYBR green PCR kit (Qiagen). When not already published, the nucleotidic primers used for gene amplifications were designed from the sequences available in the Pseudomonas Genome Database, version 2, using primer3 Web software (Table S5) (47). For each strain, the mRNA levels of target genes were normalized to that of housekeeping gene rpsL and are expressed as the ratios to the transcript levels of strain PA14. Mean gene expression values were calculated from two independent bacterial cultures, each assayed in duplicate. Strain PA14ΔmexS was used as a positive control for the overexpression of gene mexE. As shown in previous experiments (11), transcript levels of mexE Ն20-fold those of PA14 are associated with a Ն2-fold increase in resistance to MexEF-OprN substrates, and such levels were considered significant.

SNP identification. Single-nucleotide polymorphisms (SNPs) between strain PA14 and its derived mutants PJ01, PJ03, and PJ04 were identified with the Ion Torrent technology (Life Technologies, CA). Briefly, genomic DNA of each strain was extracted and purified by using the PureLink genomic DNA minikit (Life Technologies). Ion Torrent libraries were prepared from 100 ng of each DNA preparation (Qubit 2.0 fluorometer; Invitrogen) using dedicated equipment (Veriti thermocycler and E-Gel iBase; Invitrogen). Alignment of the sequence reads (about 200 bp in length) of PJ01, PJ03, and PJ04 with the UCBPP-PA14 genome (NCBI accession number NC_008463.1) was performed using BioNumerics version 7.1 and led to the identification of potential sequence variations (SNPs). Identification of an SNP was considered reliable if the coverage was Ն20-fold and its percentage was Ն29%. Sequence variations were verified on both strands by capillary sequencing on an Applied Biosystems 3130 GA apparatus (Applied Biosystems, Life Technologies; Courtaboeuf, France) after PCR amplification with the proper primers.

Identification of gene transcriptional start sites (؉1). Rapid amplification of 5= cDNA ends (5=-RACE) was performed to identify the transcriptional start site (TSS) of cmrA with the 5=-RACE system, as recommended by Invitrogen. Briefly, total RNA from two independent cultures of cmrA-overexpressing mutant PJ01 was extracted at mid-log phase of growth (A 600 ϭ 1) using the RNeasy plus minikit (Qiagen) and was treated with RNase-free DNase (Qiagen). Five micrograms of total RNA was subjected to first-strand cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen) and the specific primer GSP1-PA2047 (Table S5). The presence of cDNA was checked by PCR using nested gene-specific primers GSP2-PA2047 (reverse) and GSP3-PA2047 (forward) (Table S5). A homopolymeric tail was then added to the 3= cDNA using terminal deoxynucleotidyl transferase (TdT) and dCTP. PCR amplification was carried out using GSP2-PA2047, a deoxyinosine-containing abridged anchor primer (AAP), and poly(C)-tailed cDNA as the template. The 5=-RACE product was cloned into the pCR2.1 vector and further sequenced to allow the identification of the TSS of cmrA. The verification of PA2048 TSS, previously identified by Schulz et al., (19), was performed under the same conditions using specific primers GSP1-PA2048, GSP2-PA2048, and GSP3-PA2048 (Table S5).

High-throughput RNA sequencing (RNA-seq) library construction. Total RNA extracts were obtained in triplicate from exponential cultures (A 600 ϭ 1) of strains PA14, PJ01, and PJ01ΔmexT at 37°C. Bacterial cells were collected by centrifugation in RNAprotect bacterial reagent (Qiagen) and disrupted with 0.15-to 0.60-mm ceramic beads in a TissueLyzer II (Qiagen) according to the manufacturer's instructions. Total RNA was then purified from bead-beaten samples with the RNeasy plus 96 kit (Qiagen). The concentration and purity of the RNA extracts were assessed by RiboGreen measurement (Quant-iT RiboGreen RNA reagent and kit; Invitrogen) and by using an Agilent 2100 Bioanalyzer system (Agilent Technologies), respectively. Depletion of rRNA from those RNA samples was performed with the Ribo-Zero rRNA removal reagents (bacteria) from Epicentre (Madison, WI). Libraries were then constructed using the TruSeq stranded-mRNA high-throughout (HT) sample preparation kit from Illumina (San Diego, CA). The final libraries were quantified with PicoGreen fluorescent dye (Quant-iT PicoGreen double-stranded DNA [dsDNA] assay kit; Invitrogen), showing yields of 200 to 800 ng per sample. Qualitative analysis was done using the Agilent high-sensitivity DNA assay. High-throughput sequencing was performed by Microsynth (Balgach, Switzerland) on an Illumina NextSeq 500 platform using V2 chemistry.

RNA-seq data analysis. RNA-seq data analysis was performed by Genostar (Montbonnot Saint Martin, France). Reads were mapped on 5,892 annotated coding sequences (CDS) of strain PA14 using CLC Genomic Workbench 7.5 software. Transcript abundance and differential-expression results between the three replicates of each sample (PA14/PJ01, PA14/PJ01ΔmexT, and PJ01/PJ01ΔmexT) were determined with the Cufflinks and Cuffdiff algorithms (48). A difference in gene expression was considered significant when the false-discovery rate (q value) was Յ0.05 and the expression ratio was Յ0.3or Ն3.0-fold. The transcriptomic data have been deposited in NCBI's Gene Expression Omnibus (49) and are accessible through GEO Series accession number GSE86211.

Construction of PA14-derived deletion mutants. Single-deletion mexS, mexT, cmrA, PA2048, and mexEF-oprN mutants were constructed using overlapping PCRs and recombination events as described by Kaniga et al. (50). First, the 5= and 3= regions flanking mexS (417 and 433 bp, respectively), mexT (408 and 453 bp, respectively), cmrA (418 and 445 bp, respectively), PA2048 (451 and 457 bp, respectively), and mexEF-oprN (467 and 452 bp, respectively) were each amplified by PCR with specific primers (Table S5) under the following conditions: 3 min of denaturation at 98°C, followed by 30 cycles of amplification, each composed of 10 s at 98°C, 30 s at 60°C, and 30 s at 72°C, with a final extension step of 7 min at 72°C. The resultant amplicons were used as templates for overlapping PCRs with external pairs of primers to generate the mutagenic DNA fragments. The reaction mixtures contained 1ϫ iProof high-fidelity (HF) master mix, 3% DMSO, and 0.5 M each primer (Bio-Rad). The amplified products were cloned into plasmid pCR-Blunt according to the manufacturer's instructions (Invitrogen) and then subcloned as BamHI/XbaI fragments into the suicide vector pKNG101 in E. coli CC118pir (50). The recombinant plasmids were next transferred to P. aeruginosa (PA14 or PJ01) by conjugation and selected on PIA containing 2,000 g ml Ϫ1 streptomycin. The excision of undesired pKNG101 sequence was obtained by plating transformants on M9 plates containing 5% (wt/vol) sucrose. Negative selection on streptomycin was carried out to confirm the loss of the plasmid in transconjugants. The allelic exchanges were verified by PCR. Nucleotide-sequencing experiments confirmed the deletion of 826 bp in mexS, 929 bp in mexT, 997 bp in cmrA, 442 bp in PA2048, and 6,039 bp in mexEF-oprN, yielding strains P01JΔmexS, PJ01ΔmexT, PA14ΔcmrA, PJ01ΔcmrA, PJ01ΔPA2048, and PA14ΔmexEFN, respectively.

Chromosomal complementation with wild-type or mutated cmrA. Wild-type (from PA14) and mutated (from PJ01, PJ02, PJ03, and PJ04) cmrA alleles, along with their promoter regions, were amplified by PCR by using genomic DNA as the template. The resulting amplicons, once digested with BamHI and HindIII, were inserted into linearized plasmid mini-CTX1 (51). The resultant recombinant plasmids were then transferred from E. coli CC118 to P. aeruginosa strain PA14ΔcmrA by conjugation, with subsequent selection on PIA plates supplemented with 200 g ml Ϫ1 tetracycline to allow their chromosomal insertion into the attB site (51). Chromosomal integration of the cloned alleles was confirmed by PCR and sequencing.

Overexpression of cmrA and PA2048 from the araBAD promoter. To study the impact of cmrA overexpression on the phenotype of P. aeruginosa, a wild-type copy of the gene was PCR amplified using specific primers (Table S5). The amplicon was cloned into vector pCR-Blunt and then subcloned as an EcoRI fragment into arabinose-inducible expression vector pJN105 (52). The new construct was introduced by electroporation into strain PA14, yielding PA14(pJN105::cmrA PA14 ), and selected on gentamicin at 10 g ml Ϫ1 . A positive control with the mutated allele from PJ01 [PA14(pJN105::cmrA PJ01 )] and a negative control harboring pJN105 alone [PA14(pJN105)] were generated in parallel. Gene PA2048 was cloned in pJN105 under the same conditions as cmrA (see above) after amplification with specific primers (Table S5) and was then electrotransferred into PA14 to yield PA14(pJN105::PA2048). The transformants were finally analyzed for their resistance phenotypes (MICs of ciprofloxacin [CIP], chloramphenicol [CHL], and trimethoprim [TMP]) and the relative expression levels (by RT-qPCR) of genes cmrA, PA2048, and mexE in the absence and presence of the inducer arabinose (0.5%).

Construction of a bioluminescent reporter of the CmrA pathway. To evaluate the activation of the CmrA pathway under various challenging conditions, a transcriptional fusion between gene PA2048 and the operon luxCDABE was constructed. For this, a 1,822-bp genomic fragment of strain PA14 carrying cmrA was amplified by using specific primers (Table S5). The amplicon was cloned into vector pCR-Blunt and then subcloned as an EcoRI fragment into plasmid miniCTX-lux (53). The new construct was introduced into strain PA14 by conjugation, with subsequent selection of transconjugants on PIA supplemented with 200 g ml Ϫ1 tetracycline. In parallel, the same plasmid construct was transferred into strain PJ01 as a positive control of PA2048::lux overexpression, as this mutant constitutively produces an activated form of CmrA.

Bioluminescence induction assays. Induction of PA2048-lux expression was measured in real time during the exponential-growth phase. Briefly, overnight cultures of luminescent strains were diluted into fresh MHB to yield an A 600 of 0.01. Bacteria were incubated with shaking (250 rpm) at 37°C for 4 h (A 600 ϭ 0.1) prior to the addition of the following stressors at the indicated concentrations: ciprofloxacin (0.01 g ml Ϫ1 ), chloramphenicol (20 g ml Ϫ1 ), trimethoprim (12 g ml Ϫ1 ), diamide (8 mM), H 2 O 2 (50 M), paraquat (25 M), dimethyl sulfoxide (0.5%), S-nitrosoglutathione (0.125 g ml Ϫ1 ), 2-n-heptyl-4hydroxyquinoline N-oxide (25 g ml Ϫ1 ), glyoxal (12, 50, 100, and 200 g ml Ϫ1 ), methylglyoxal (10, 50, and 100 g ml Ϫ1 ), and cinnamaldehyde (70, 140, and 280 g ml Ϫ1 ). The activity of the PA2048-lux fusion was monitored in white 96-well assay plates (Corning, NY), using a Synergy H1 microplate reader (Biotek Instruments, Winooski, WI) with the gain set at 150, read height set at 7 mm, and integration time of 1 s. In parallel, the bacterial densities were measured by their A 600 in 96-well microtest plates (Sarstedt, Nümbrecht, Germany). The activity of the reporter, expressed as the ratio of bioluminescence (relative light units [RLU]) to bacterial density (A 600 ), was measured over a 6-h time course.

Killing experiments with cinnamaldehyde. Strain PA14 and its mutants PA14ΔmexEF-oprN and PA14ΔcmrA were first rendered constitutively bioluminescent using the pUC18T-mini-Tn7T-lux-Gm plasmid as described by Damron et al. (54). Overnight cultures were then diluted into fresh MHB to yield an A 600 of 0.1. Bacteria were incubated with shaking (250 rpm) at 37°C for 2.5 h (A 600 ϭ 0.8) prior to the addition of cinnamaldehyde at 1,000 g ml Ϫ1 . The bioluminescence of strains was measured as described above.

Induction of MexEF-OprN by disk diffusion tests. The inducing activity of glyoxal (GO), methylglyoxal (MG), and cinnamaldehyde (CNA) on mexEF-oprN expression was indirectly investigated by double-disk antagonism tests with PA14 and PA14ΔmexEF-oprN on MHA. Disks loaded with 8,000 g GO, 8,000 g MG, or 10,000 g CNA were deposited onto the surface of seeded MHA plates at the appropriate distance from disks containing ciprofloxacin (5 g), chloramphenicol (1,000 g), or trimethoprim (240 g). Flattening of the inhibition zone around an antibiotic disk in the direction of the GO, MG, and/or CNA disk(s) was interpreted as an increase in resistance induced by the aldehyde(s).

Accession number(s).

The whole sequence of cmrA, including its 5= UTR, is accessible through the GenBank database under accession number KX274690. The transcriptomic data of our RNA-seq experiments is accessible through the GEO database under accession number GSE86211.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AAC .00585-17.

SUPPLEMENTAL FILE 1, PDF file, 0.8 MB.

Electrophilic Stress Induce Expression of RND Efflux Pumps in Pseudomonas aeruginosa

Our work on CmrA revealed that exposure of strain PA14 to subinhibitory concentrations of toxic electrophiles induces expression of mexEF-oprN through CmrA-dependent upregulation of PA2048 [START_REF] Juarez | Toxic electrophiles induce expression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa through a novel transcriptional regulator[END_REF]. It was suggested that the pump MexEF-OprN itself could protect P. aeruginosa against RES by exporting them or their metabolites outside the cell To test this hypothesis, we performed antibiograms by using wild-type strain PA14 and a Mueller-Hinton agar medium supplemented with subinhibitory concentrations of glyoxal (GO), methylglyoxal (MG) and cinnamaldehyde (CNA), respectively. RT-qPCR experiments were also carried out after 15 min of electrophile exposure to determine expression levels of genes mexB, mexY and mexE (Table 9). Confirming our previous results, exposure to electrophiles (GO, MG, CNA) reinforced resistance of PA14 to chloramphenicol and imipenem, and strongly induced mexE expression (172-, 31-, and 233-fold, respectively) (Table 9). By contrast, α-oxoaldehydes GO and MG increased resistance to MexXY/OprM substrates gentamicin and tobramycin with concomitant derepression of gene mexY (7.7-and 6.0-fold), while cinnamaldehyde (CNA) had similar effects on susceptibility to MexAB-OprM substrates aztreonam and ticarcillin, and mexB activity (9.4-fold) (Table 9).

Time-kill experiments comparing deletion mutant PA14ΔmexEF-oprN to its wild-type parent demonstrated that MexEF-OprN, to some extent, is able to protect P. aeruginosa from CNA [START_REF] Juarez | Toxic electrophiles induce expression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa through a novel transcriptional regulator[END_REF]. Curiously, it was observed that CNA MIC (512 µg/mL) was identical for strain PA14 and its mutant. It was thus hypothesized that pumps MexAB-OprM and/or MexXY/OprM could perhaps compensate for the absence of MexEF-OprN and contribute to CNA resistance.

To test this assumption and better understand the role of MexAB-OprM in the response to CNA, we deleted genes mexAB in PA14 and PA14ΔmexEF-oprN, and determined the susceptibility of the two strains in MIC and time kill experiments. Deletion of gene oprM that also provides the outer membrane component of the MexXY/OprM system will be carried out in another project (Alexandre Tetard, 2018).

Our results showed that susceptibility of strain PA14 to CNA remains unaffected unless both pumps, MexAB-OprM and MexEF-OprN, are inactivated (Figure 19A). Actually, the double deletion prevents adaptive regrowth after CNA exposure (Figure 19B). Altogether, these data suggest that MexAB-OprM and MexEF-OprN are able to export CNA, and that one pump can compensate for the other one if impaired. RT-qPCR experiments demonstrated in parallel that operon mexAB-oprM expression is strongly induced upon CNA challenge. The regulatory pathway involved in this induction is about to be studied in the laboratory.

IV. Conclusion and Perspectives

Overexpression of mexEF-oprN in clinical strains

The role of several RND efflux pumps in natural and acquired resistance of P. aeruginosa to antibiotics and antiseptics is now well established [START_REF] Aires | Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides[END_REF]Li et al., 2015;Poole, 2004;Poole and Srikumar, 2001;Schweizer, 2003). Furthermore, evidence accumulates that the efflux system MexEF-OprN contributes to drug resistance in some clinical strains (Li and Plésiat, 2016). The so-called nfxC mutants that stably overproduce this pump have long been . Thus, the most reliable technique for characterization of nfxC strains remains RT-qPCR. However, the threshold of mexE expression depends on which strain is used as reference. For instance, our characterization of 55 in vitro selected nfxC mutants showed that this threshold is 20-fold and 100-fold above the baseline levels of strains PA14 and PAO1, respectively. This difference lies in the fact that mexT is inactivated by an 8 bp sequence in most isolates of the PAO1 lineage, while this gene is functional in PA14 [START_REF] Stover | Pseudomonas aeruginosa contains multiple glyoxalase Iencoding genes from both metal activation classes[END_REF]Winsor et al., 2016). Thus, according to the threshold used, RT-qPCR results may lead to misinterpretations.

In this context, we carried out the characterization of a collection of 22 non-CF clinical isolates overexpressing the mexE gene (from 21-to 404-fold relative to PA14). It became rapidly evident that most nfxC clinical strains are phenotypically different from in vitro selected nfxC mutants as they rarely exhibit the typical hypersusceptibility to β-lactams (7/22 isolates) or aminoglycosides (0/22). Furthermore, severe deficiency in virulence factor production which is characteristic of in vitro nfxC mutants (Köhler et al., 2001;Sobel et al., 2005a) is rare in clinical counterparts (Richardot et al., 2016). In line with these observations, we found that 13/22 of nfxC clinical strains harbored mutations in gene mexS, but disruptive events were four times less frequent (3/13) than missense mutations (10/13). Expression levels of gene mexE were in general higher in the mexS knockout group (Figure 20 Other differences were noted between the nfxC strains in relation with the degree of presumed impairment of oxidoreductase MexS. We showed that the levels of mexE expression were roughly correlated with the MICs of pump substrates and inversely related to different virulence traits. Since mexS knockout mutants are four times less prevalent than mexS missense mutants in the clinic, it is tempting to assume that disruption of mexS is too detrimental to P. aeruginosa to allow its persistence in vivo. Supporting this notion, the three clinical isolates (1709, 1711, and 0607) harboring disrupted mexS copies were responsible for host colonization and not infections. As previously noted in the lab (Richardot et al., 2016), the expression of gene mexT remains unchanged in nfxC mutants versus wild-type strains. The ratio of active/inactive MexT occurring in the cell upon nfxC mutations thus appears as the main factor determining levels of mexEF-oprN transcription and associated phenotypic traits.

Several authors (Fargier et al., 2012;Köhler et al., 1999;Sobel et al., 2005a) suggested that

MexS could play a central role in detoxification of noxious metabolites or xenobiotics which, in absence of the enzyme, would need to be extruded by MexEF-OprN. Since the physiological role of MexS is still unknown, it was quite difficult to set up assays to verify this hypothesis. Enzymatic activity of MexS remains to be characterized in vitro. In this perspective, we engineered two recombinant proteins tagged with 6x-His or MBP. In both cases, we checked their functionality in mutant PA14ΔmexS. As expected, the mutant recovered a wild-type susceptibility profile to antibiotics once complemented with one or the other protein. Both of them will soon be produced in large scale, will be purified and used to screen several potential substrates such as quinones [START_REF] Nordling | Medium-chain dehydrogenases/reductases (MDR). Family characterizations including genome comparisons and active site modeling[END_REF][START_REF] Persson | Medium-and short-chain dehydrogenase/reductase gene and protein families: the MDR superfamily[END_REF], by spectrophotometric [START_REF] Matthews | Methods of Enzymatic Analysis[END_REF] and/or fluorimetric assays [START_REF] Passonneau | Pyridine nucleotides. In enzymatic analysis, A Practical Guide[END_REF]. We hope that the purified products will be suitable to determine the crystal structure of MexS and to identify its functional domains. The amino acid substitutions found in clinical nfxC mutants will be helpful to find out residues critical for enzyme activity.

In addition to mexS mutations, we showed for the first time that specific single amino acid substitutions (G 257 S and G 257 A) in MexT are responsible for constitutive activation of this regulator and overexpression of operon mexEF-oprN (short-paper in preparation). Gain-offunction mutations in LTTRs had already been reported. For instance, substitutions T 149 M and T 149 P in LTTR CysB that controls expression of cysteine regulon in S. enterica, result in overexpression of genes cysK, cysP and cysJIH without the need of co-inducer N-acetyl-Lserine (Colyer andKredich, 1994, 1996). Furthermore, site-directed mutagenesis experiments demonstrated that substitutions R 156 H in CatM, and R 156 H/T 157 S in BenM, two LTTRs involved in aromatic compounds degradation in Acinetobacter baylyi, generate variants that no longer require inducers for activation (Craven et al., 2009). Based in our two-hybrid results, we hypothesize that the two MexT variants identified in this study form active oligomers. However, to ascertain this assumption we have the project to crystallize the wildtype and mutated MexT proteins to understand why mutations at position Gly-257 activate the regulator. This will be done in collaboration with the Crystallization laboratory of Paris Descartes University (Pr. Isabelle Broutin, UMR 8015 Laboratoire de Cristallographie et RMN Biologiques).

A novel pathway of mexEF-oprN activation and a novel type of mutants

Seven of the 22 nfxC clinical strains that we studied did not harbor mutations in proteins Moreover, it appeared that CmrA positively regulates a small set of 11 non-essential genes (in standard laboratory growth conditions). It remains to be determined whether CmrA directly binds to the regions upstream of all these genes. Production and purification of a 6x-His-CmrA is ongoing in our lab. Once the recombinant protein will be obtained in sufficient amounts, we will perform Electrophoretic Mobility Shift Assays (EMSA) by incubating the purified protein with DNA fragments of putative targets [START_REF] Hellman | Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions[END_REF] b Expressed as a ratio of wild-type strain PA14 c Based on the capacity of the strain to produce: (1) pyocyanin, (2) elastase, (3) biofilm, (4) rhamnolipids and to (5) swarm on semisolid surfaces. ND: not determined.

Compared with MexS deficient/null mutants, nfxC2 mutants do not seem to have an impaired virulence. The presumed pathogenicity of nfxC2 mutants should make them clinically relevant, especially as they are more resistant to major antibiotics such as fluoroquinolones.

Identification of such mutants in clinical samples remains to be done (see below). We are currently collaborating with the laboratory headed by Prof. Mustapha Si-Tahar (UMR 1100 "Infection Respiratoire et Immunité", Université François Rabelais, Tours) to evaluate their virulence in an animal model of acute pulmonary infection. Preliminary results showed that cmrA mutants caused the death of all infected mice at 39h post-inoculation while, confirming previous data, mexSmutants are avirulent (Figure 21). Additional experiments have been

CmrA, a new target to consider when screening multi-drug resistant strains

In one study, insertion of a transposon upstream from gene cmrA (PA2047) was hold responsible for an 8-fold increase in ciprofloxacin resistance [START_REF] Breidenstein | Complex ciprofloxacin resistome revealed by screening a Pseudomonas aeruginosa mutant library for altered susceptibility[END_REF].

Whether similar genetic events are relevant in clinical isolates is unlikely as we demonstrated that overexpressing a plasmid-borne copy of wild-type cmrA had no impact on PA14 drug susceptibility [START_REF] Juarez | Toxic electrophiles induce expression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa through a novel transcriptional regulator[END_REF]. More interestingly, missense mutations in cmrA were reported in 3.2% of a collection of 474 CF clinical strains, leading to S 45 L, E 61 D, S 266 N, Y 271 H, and A 293 V substitutions in regulator CmrA [START_REF] Marvig | Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis[END_REF]. The link between these changes and MexEF-OprN-dependent resistance to various antibiotics was not investigated, but it will be assessed by our group through site-directed mutagenesis and transcomplementation of the PA14ΔcmrA mutant.

Retrospective screening of three collections of P. aeruginosa housed by the lab revealed the presence of single amino acid variations in CmrA from five strains (T 5 I, L 7 F, R 29 E, E 42 D, G 188 S). However, these changes were non-significant since inactivation of gene cmrA did not modify the resistance phenotype of these strains. Thus, while nfxC and nfxC1 mutants have been characterized in the clinical setting (Llanes et al., 2011(Llanes et al., , 2013;;Richardot et al., 2016), nfxC2 strains still await to be identified.

Induction of RND pumps by electrophilic stress

Our work demonstrated that the CmrA-PA2048-MexEF-OprN pathway is part of the electrophile stress response in P. aeruginosa [START_REF] Juarez | Toxic electrophiles induce expression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa through a novel transcriptional regulator[END_REF]. In contrast to other oxidative stressors (Fargier et Henceforth, it would be interesting to consider if RND pumps are induced by natural electrophiles generated by the host during P. aeruginosa infection. Indeed, significant levels of malondialdehyde, an electrophile derived from lipid peroxidation, were detected in the lungs of patients suffering from asthma, bronchiectasis, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis. The average physiological concentration of this molecule was around 30 nM in 194 patients versus 15 nM in 14 healthy controls [START_REF] Bartoli | Malondialdehyde in exhaled breath condensate as a marker of oxidative stress in different pulmonary diseases[END_REF]. In another work on CF-patients (n=40), malondialdehyde reached 176 nM in plasma, 140 nM in exhaled breath condensate and 280 nM in sputum [START_REF] Antus | Comparison of airway and systemic malondialdehyde levels for assessment of oxidative stress in Cystic Fibrosis[END_REF].

Therefore, we have planned to investigate the response of P. aeruginosa to this quite unstable electrophile, in particular the impact of reported concentrations on mexEF-oprN, mexAB-oprM, and mexXY expressions in several clinical strains including PA14. Since the laboratory has access to sputa from CF patients, we will also measure malondialdehyde concentrations in these samples by HPLC/MS.

Novel antimicrobials are urgently needed to counter multidrug resistance in human pathogens, especially nosocomial species such as P. aeruginosa [START_REF] Silver | Two-component regulatory systems in Pseudomonas aeruginosa: an intricate network mediating fimbrial and efflux pump gene expression[END_REF][START_REF] Tacconelly | Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics[END_REF]. In this context, understanding the regulatory mechanisms leading to antibiotic resistance may in theory help to set up innovative therapeutic strategies. RND efflux systems are deplored for their contribution to intrinsic resistance with respect to development of new anti-Gram negatives; and as cause of poor clinical outcomes in line with emergence of resistant mutants. However, the contribution of such pumps to adaptive (non-mutational) resistance in vivo is largely unknown, as this transient phenomenon cannot be appreciated by classical in vitro drug susceptibility testing. The pump MexXY/OprM has been shown by our group to play a significant role in adaptive resistance to aminoglycosides [START_REF] Hocquet | Relationship between antibiotic use and incidence of MexXY-OprM overproducers among clinical isolates of Pseudomonas aeruginosa[END_REF]. Whether the highly inducible MexEF-OprN also enables P. aeruginosa to better resist some antibiotics in vivo warrants further investigations. 11 and12.

V. Materials and Methods

New England Biolabs

Abbreviations: Zeo R (zeocin resistance), Kan R (kanamycin resistance), Amp R (ampicillin resistance), Str R (streptomycin resistance), Tet R (tetracycline resistance), Gen R (gentamicin resistance).

Culture Media

All culture media used in this work are listed in the Table 13.

In Vitro Selection of Chloramphenicol Resistant Mutants

In vitro selection of mutants overproducing the MexEF-OprN efflux system was performed by plating about 10 8 CFU/mL of reference strain PA14 on MHA medium containing 128, 256 or 512 µg/mL of chloramphenicol. Colonies grown after 18 h of incubation at 37°C were replicated on the same selective medium before their characterization.

Determination of Antibiotic Sensitivity

Antibiograms

Antibiograms were performed using the agar diffusion technique according to recommendations of the "Comité de l'Antibiogramme de la Société Française de

Microbiologie" (CA-SFM-2013). Briefly, a bacterial suspension (0.2 McFarland) was diluted (10 -2 ) into cMHB. Next, 10 mL of this suspension were poured into a cMHA plate and then removed (≈1.5x10 6 CFU/mL). Disks (BioRad) with standard loads of antibiotics were added over the seeded plate once dried. Finally, the inhibition diameters were measured after 18 h of incubation at 37°C. Spontaneous nfxC mutants developing on solid media were screened for their resistance to both ciprofloxacin and imipenem, by measuring the inhibition zones around Bio-Rad disks (loaded with 5 µg and 10 µg, respectively).

Minimum Inhibitory Concentration (MIC)

The Minimum Inhibitory Concentrations (MICs) of selected antibiotics were determined by the standard serial 2-fold dilution method in cMHA with inocula of 10 4 CFU per spot, according to the CLSI recommendations (Clinical and Laboratory Standards Institute, 2015).

Growth was visually assessed after 18 h of incubation at 37°C. MICs of ciprofloxacin, chloramphenicol and trimethoprim were established to control the NfxC phenotype.

Virulence Factors Determination

For all virulence tests, results are presented as the mean of three independent experiments.

Swarming Mobility

Swarming mobility is due to the combined action of rhamnolipids, type IV pili and flagella, and was evaluated on a semi-solid surface. Positive swarming strains form macroscopic colony patterns characterized by the coordinate translocation of bacteria on the semi-solid medium, when grown under optimal conditions. Briefly, 5 µL of a bacterial suspension (0.2

McFarland) were spotted onto the surface of an M8-Swarm medium (Table 13). Swarming colonies were observed after an incubation of 24 h at 37°C.

Detection of Biofilm Formation by Adherence Test

Adherence being the first phase of biofilm production, this test measures the capacity of the strain to adhere to a plastic surface. Briefly, a bacterial suspension (0.3 McFarland) was diluted (10 -1 ) in cMHB. Next, 200 µL of diluted bacteria (≈9x10 7 CFU/ml) were incubated 24 h at 30°C into a polystyrene 96-well microplate. Each well was washed twice using 200 µL of distilled water to eliminate planktonic bacteria. Biofilm was colored using 200 µL of crystal-violet solution at 1% (w/v). After 15 min at room temperature, the wells were washed as indicated previously. Crystal-violet attached to sessile bacteria was solubilized with 2 x 200 µL ethanol at 99% (v/v). Finally, absorbance was read at 600 nm and calibrated with a negative control containing 400 µL of cMHB (Vasseur et al., 2007).

Pyocyanin Production

A bacterial suspension (0.2 McFarland) prepared with fresh colonies from sBAP was diluted 1:20 in 50 mL PYO broth. The medium was then incubated 24h at 37°C with shaking, and centrifuged 20 min at 4,100 rpm. Pyocyanin was extracted twice with 2.5 mL chloroform. The organic phase was collected and acidified with 5 mL HCl 0.2 M. Upon acidification, pyocyanin turned from deep blue to pink; its concentration was measured spetrophotometrically at an absorbance of 520 nm (Essar et al., 1990).

Elastase Activity

Elastase degrades elastin in connective tissues. Its activity was measured in bacteria grown aerobically at 37°C up to an A 600 of 1.8. Supernatants from 1 mL volumes of cultures were diluted 1:20 in E-buffer (Tris 0.1 M pH 7.4, and CaCl 2 1 mM) containing 4 mg/mL RedCongo-elastin (Sigma, France). After an overnight incubation at 37°C, the reaction mixtures were centrifuged 10 min at 14,000 g. The concentration of soluble dye RedCongo released from RedCongo-elastin complex by elastase was determined spectrophotometrically at A 495nm in supernatants.

Rhamnolipid Production

Rhamnolipid production was assessed by a hemolysis assay.

Cloning Techniques

DNA Digestion using Restriction Enzymes

Plasmids or PCR products were digested using the following restriction enzymes: BamHI, ApaI, HindIII, EcoRI, KpnI, XbaI, SacI, NdeI (Promega). All restriction reactions were performed following the supplier recommendations.

Agarose Gel Electrophoresis

The size of DNA fragments was analyzed by agarose gel electrophoresis. Briefly, 0.8% agarose gels were soaked in 1X TAE buffer (Tris-acetate 40 mM, EDTA 1 mM pH 8) and submitted at 100 V during 30 min. 1X SYBR-safe (Invitrogen, St. Aubin, France) was added to agarose gels in order to visualize DNA under UV-light using the UV ChemiDoc XRS system (BioRad).

2.3.3

Purification of DNA Fragments DNA fragments or digestion products were purified using the Wizard® SV Gel and PCR clean up system (Promega) following supplier recommendations.

DNA Ligation

DNA fragments were ligated into specific plasmids (see Table 12) using the T4 DNA ligase (Promega) and following the supplier recommendations. Ligation reaction was performed overnight at 15°C.

Bacterial Transformation

Heat-shock Transformation

Cloning Efficiency® DH5α Competent Cells (Invitrogen) were transformed with purified DNA following the supplier instructions. E. coli strains CC118, CC118λpir, DHM1 and ER2523 cells were first rendered competent by using the RbCl protocol [START_REF] Hanahan | Studies on transformation of Escherichia coli with plasmids[END_REF]. In both cases, 100 µL of competent cells were mixed with 10 ng plasmid and left 30 min at 0 o C, and then submitted to a heat-shock at 42°C for 40 s. After 2 min at 0 o C, 900 µL of cMHB were added to the cells. Bacteria were then incubated aerobically 1 h at 37°C with agitation, and finally were plated on appropriate selective plates.

Electroporation

P. aeruginosa strains were transformed with purified DNA by using the MicroPulser TM Electroporator (Bio-Rad). Briefly, 1 mL of an overnight culture in MHB was centrifuged 2 min at 16,000 g. The pellet was washed twice with 1 mL of 300 mM sucrose solution, and finally resuspended in 100 µL sucrose 300 mM. Electroporation was performed at 2.5 kV/cm, 200 Ω and 25 µF for 4.5 -5 ms, in presence of 20 ng of plasmid [START_REF] Choi | A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation[END_REF].

Bacterial Conjugation

Plasmid transfers from E. coli to P. aeruginosa were performed by triparental mattings with "helper" plasmid pRK2013 that carries mobilization genes (mob). Each strain was cultured overnight with shaking. Then, 50 µL of E. coli HB101(pRK2013) culture were mixed with 50 µL of donor culture (E. coli CC118 containing a mini-CTX1 recombinant plasmid or CC118λpir containing a pKNG101 recombinant plasmid). The mixture was spotted onto a cMHA plate and allowed to grow for 2 h at 37°C. 50µL of P. aeruginosa recipient strain, previously grown for 2 h at 42°C, were directly added onto the spot. After an overnight incubation at 37°C, the bacterial spot was resuspended in 3 mL of cMHB. Bacteria were incubated 1 h at 37°C and, finally plated onto selective PIA agar medium containing appropriate antibiotics.

Gene Inactivation using Overlapping PCR and Homologous Recombination

Gene inactivation was achieved by homologous recombination between chromosomal DNA and truncated PCR products. Briefly, the DNA sequences (ca 450 bp in length) flanking the target gene were amplified by PCR (see Table 14). A PCR overlapping the two amplicons was next performed to obtain a single fragment lacking the gene to delete. This fragment was cloned into vector pCR-Blunt and then sub-cloned into suicide vector pKNG101.

Chromosomal Complementation

Genes mexT and cmrA and their promoters were amplified by PCR (see Table 14) and cloned into vector Mini-CTX1 using the restriction enzymes BamHI and HindIII. This vector contains the coding regions for an integrase allowing insertion at the attB site of the bacterial chromosome (Hoang et al., 2000). Recombinant vectors were inserted into E. coli CC118 by heat-shock transformation and then transferred into P. aeruginosa strains by conjugation.

Transformants were selected on PIA plates supplemented with tetracycline 200 µg/mL. For the excision of undesirable vector sequences, electrocompetent cells of P. aeruginosa were prepared as above (see section 2.4.2) and electroshocked with 200 ng of purified pFLP2 plasmid coding for the Flp flippase [START_REF] Hoang | A broadhost-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants[END_REF]. Clones were selected in cMHA plates supplemented with ticarcillin 150 µg/mL and next plated onto M9-suc plates for the excision of pFLP2. Finally, excision of both plasmids was verified by plating the clones onto three different plates: the first, a cMHA plate without any antibiotics, the second supplemented with tetracyclin 200 µg/mL, and the third supplemented with ticarcillin 150 µg/mL.

Gene Overexpression using the araBAD Promoter

To study the impact of cmrA overexpression on the phenotype of P. aeruginosa, a wild-type copy of the gene was amplified by PCR using primers pJN-cmrA Fw and pJN-cmrA Rv (Table 14). The amplicon was cloned into vector pCR-Blunt, and then subcloned as an EcoRI fragment into the arabinose-inducible expression vector pJN105 (Newman and Fuqua, 1999).

The recombinant plasmid was introduced by electroporation into strain PA14 and selected on gentamicin 10 µg/mL, yielding PA14(pJN105::cmrA PA14 ). A positive control with the mutated allele from PJ01 [PA14 (pJN105::cmrA PJ01 )] and a negative control harboring pJN105 alone [PA14 (pJN105)] were generated in parallel.

Gene PA2048 was cloned as well in pJN105 under the same conditions (see above) after amplification with primers pJN-PA2048 Fw and pJN-PA2048 Rv (Table 14). It was then electrotransferred into PA14 to yield PA14(pJN105::PA2048). The transformants were finally analyzed for their resistance phenotype (MICs of ciprofloxacin, chloramphenicol, trimethoprim) and relative expression (by RT-qPCR) of genes cmrA, PA2048 and mexE, in the absence and in the presence of inducer arabinose (0.5%).

Luminescent Reporter of the CmrA-pathway

To evaluate the activation of the CmrA-pathway under various challenging conditions, a transcriptional fusion between gene PA2048 and operon luxCDABE was constructed (Figure 23). For this, the 1,822 bp genomic fragment of strain PA14 carrying genes cmrA and PA2048 was amplified by using primers cmrA-PA2048 Fw and cmrA-PA2048 Rv (Table 14). The amplicon was cloned into vector pCR-Blunt and then sub-cloned as an EcoRI fragment into plasmid miniCTX-lux (Becher and Schweizer, 2000). The new construct was introduced into strain PA14 by conjugation with subsequent selection of transconjugants on PIA supplemented with 200 µg/mL tetracycline. In parallel, the same recombinant plasmid was transferred into strain PJ01 as a positive control of PA2048::lux overexpression, as this mutant constitutively produces an activated form of CmrA.

2.4

Bioluminescence Induction Assays Induction of PA2048-lux expression was measured in real-time during the exponential growth phase. Briefly, overnight cultures of luminescent strains were diluted into fresh cMHB to yield an absorbance of A 600 = 0.01. Bacteria were incubated with shaking (250 rpm) at 37°C for 4 h (A 600 = 0.1) prior to the addition of the following stressors at indicated concentrations: ciprofloxacin (0.01 µg/mL), chloramphenicol (20 µg/mL), trimethoprim (12 µg/mL), diamide (8 mM), H 2 O 2 (50 µM), paraquat (25 µM), dimethyl sulfoxide (0.5 %), S-nitrosoglutathione (0.125 µg/mL), 2-n-heptyl-4-hydroxyquinoline-N-oxide (25 µg/mL), glyoxal (12, 50, 100 and 200 µg/mL), methylglyoxal (10, 50 and 100 µg/mL), and cinnamaldehyde (70, 140 and 280 µg/mL). The activity of the PA2048-lux fusion was monitored in white 96-well assay plates (Corning, NY USA) using a Synergy H1 microplate reader (Biotek Instruments, Winooski, USA), with a gain set at 150, a read height set at 7 mm and an integration time of one second.

In parallel, the bacterial density was measured at A 600 in 96-well microtest plates (Sarstedt, Nümbrecht, Germany). The activity of the reporter, expressed as a bioluminescence (RLU) to bacterial density (A 600 ) ratio was measured over a 6 h time course.

mRNAs Quantitation using RT-qPCR 2.5.1 RNA Extraction

A 1:100 dilution of an overnight culture was grown at 37°C and 250 rpm until it has reached A 600 = 1.0 (≈1 x 10 8 UFC/mL). The extraction of total RNA was performed using the RNeasy Protect Bacteria Mini Kit (Qiagen) following the supplier's recommendations. Extracted

RNAs were treated with the RNase-free DNase Set (Qiagen) during 30 min in order to degrade DNA traces and then stocked at -80°C.

2.5.2

cDNA Synthesis by RT-PCR cDNA synthesis was performed using the Im-Prom-II TM RT System (Promega) and the random primers included in the kit using 2 µg of total RNA and following supplier's recommendations. A negative control with no enzyme was performed in order to detect DNA contamination. Extracted cDNAs were stocked at -20°C.

cDNA Amplification by qPCR

cDNA amplification and relative quantitation of gene expression were performed using the Rotor-Gene Q apparatus (Qiagen). Briefly, 120 ng of cDNA was amplified in a Master Mix (15 µL) containing: 1X QuantiFast SYBR green PCR Master Mix (Qiagen) and 1.5 mM of specific primers (Table 14). Cycling conditions started with 5 min of denaturation at 95°C and 40 cycles of: denaturation at 95°C for 5 sec, priming at 60°C for 10 sec and amplification at 72°C for 20 sec. Fluorescent information was acquired during the priming stage. Moreover, negative (no cDNA) and positive controls as well as standard samples (10 0 , 10 -1 and 10 -2 ) of the parental strain PA14 were included in each run. Relative quantitation of gene expression was calculated based on the standard curve created with the standard samples.

Relative Quantitation of Gene Expression

Gene expression was estimated using the relative standard curve method and using the expression of housekeeping genes rpsL encoding the ribosomal S12 protein and uvrD coding for DNA helicase II (Dumas et al., 2006) . This quantitation method uses the amplification efficacy (E) of the gene of interest (E interest ) and the standard gene (E rpsL ) as well as the CT (Cycle Threshold) values of both genes for the sample and the parental or reference strain.

= ∆ - ( ) ∆ - = - ⁄
This ratio was then compared to that obtained from the parental or reference strain. The values of relative gene expression were calculated using two independent experiments.

DNA Sequencing

Both, Sanger and Ion-Torrent® sequencing were performed by the platform "Séquençage et Analyse de fragments" belonging to the SFR-4234 located in Besançon, France.

3.1

Sanger Sequencing DNA sequencing was performed following the Sanger sequencing method. This technique is based on the selective incorporation of chain-terminating dideoxynucleotides by the DNA polymerase. The fragments were amplified using the automatic sequencer ABI Prism 3130 (Life Technologies. California, US) and analyzed using SnapGene software.

3.2.2

Extraction of Genomic DNA Genomic DNA (gDNA) of P. aeruginosa strain PA14 and its derived mutants PJ01, PJ03, and PJ04, was extracted using the PureLink TM DNA Mini Kit (Invitrogen) following the supplier's recommendations. Mutant PJ02 was not used for whole-genome sequencing because it was derived from the same experiment as PJ01.

3.2.3

Preparation of a Genomic DNA Library The initial stage in the workflow required for the Ion Torrent® Personal Machine TM (PGM TM ) is to generate a library of DNA fragments flanked by two adapters. The first step of this library generation is to digest gDNA into small fragments of approximately 200 bp with a restriction cocktail. Next, two adapters are ligated to the DNA fragments (Figure 25). The first adapter is a Universal Sphere Primer used to bind a molecule of DNA to an Ion Sphere.

The second adapter, biotin, will be used to purify the template. Ion Torrent libraries were prepared from 100 ng of each DNA preparation (Qubit 2.0 Fluorometer, Invitrogen) using a dedicated equipment (E-Gel iBase for purification of 200 bp fragments, and Veriti thermocycler for emulsion PCR, Invitrogen).

3.2.4

Template Preparation Proper library fragments are then clonally amplified by emulsion PCR. Briefly, fragments and empty Ion-Spheres are mixed together in order to create spheres containing one-single copy of a labeled fragment. Monoclonal spheres are then emulsified and aerosolized into droplets.

Finally, amplification is performed by emulsion PCR resulting in Ion-Spheres coated with several copies of the DNA template (Figure 26). These particles are used as template for the Ion Torrent® sequencing. Each sphere will contain one-single fragment, which will be amplified by emulsion PCR.

3.2.5

Sequencing Ion-Sphere particles coated with DNA template were applied to the Ion chip by pipetting.

Next, template was deposited in the wells by a short centrifugation step. Then, the chip was placed into the Ion PGM TM and dNTPs were sequentially added as explained above (Figure 27).

3.2.6

Data Analysis Alignment of the sequence reads (about 200 bp in length) of PJ01, PJ03 and PJ04 with the UCBPP-PA14 genome (NCBI accession number: NC_008463.1) was performed using Bionumerics version 7.1, and led to the identification of potential sequence variations (SNPs).

A SNP was considered reliable if the coverage was ≥ 20-fold and its percentage was ≥ 29%.

Sequence variations were verified on both strands by capillary sequencing on an Applied

Biosystem 3130 GA apparatus (Applied Biosystems, Life Technologies, Courtaboeuf, France) after PCR amplification with proper primers.

4

Identification of the Transcription Start Site using 5'-RACE

Overview

The Rapid Amplification of cDNA Ends (RACE) is a technique for the analysis of the untranslated regions (UTRs) of a messenger RNA (mRNA) at either 5'-or the 3'-end of the template (Figure 28). The principle of the technique is the amplification of a UTR using a defined internal site by anchored PCR. This technique requires two gene-specific primers (GSP1 and GSP2) downstream the sequence to be amplified. The 5'-UTR region usually contains a Ribosome Binding Site (RBS) and it may contain regulatory sequences; it is used to establish the Transcription Start Site (TSS) of a gene. The first step of 5'-RACE is the conversion of a specific mRNA into cDNA using reverse transcriptase and a gene-specific antisense oligonucleotide (GSP1) which maximizes the potential for complete extension to de 5'-end of the template. Next step is the purification of the cDNA from the mixture followed by TdT-tailing, which adds homopolymeric tails of cytosine to the 3' ends of the cDNA. The dC-tailed cDNA is then amplified by nested PCR using a second gene-specific primer (GSP2) and an adapter primer (Abriged Adapter Primer, AAP), which permit amplification from the homopolymeric tail. This allows amplification of unknown regions between GSP2 and the 5'end of the mRNA.

4.1

RNA extraction Total RNA used for 5'-RACE experiments was extracted as described above (See section 2.5.1).

cDNA synthesis using the GSP1 primer

The synthesis of cDNA was performed using the SuperScript TM II RT (Invitrogen) using the GSP1 primers listed in Table 14. Briefly, 5 µg of total RNA were used as template in a reaction mixture containing: 100 nM of GSP1 primer, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl 2 , 10 mM DTT, 400 µM of dNTPs and 200 U of reverse transcriptase. The reaction was performed at 42°C for 50 min followed by 15 min of termination at 70°C.

Remaining RNA was degraded using 1 µL of RNase mix (Invitrogen) at 37°C for 30 min. The presence of cDNA was controlled by nested PCR using GSP2 and GSP3 primers. Once total RNA is extracted, a reverse-transcription is performed using a reverse Gene Specific Primer (GSP1). The newly synthezised cDNA is depleted from RNA using a RNAse cocktail. Purified cDNA is next tailed with cytosines on the 3'-terminus using a terminal deoxynucleotidyl terminase (TdT). Tailed-cDNA is then amplified by nested PCR using a second reverse Gene Specific Primer (GSP2) and the adapter primer (Abriged Anchor Primer) allowing amplification from the dC-homopolymeric tail. The RACE product can be next cloned in different vectors for sequencing experiments and identification of the Transcription Start Site (TSS).

TdT Tailing of cDNA

Terminal deoxynucleotidyl transferase (TdT) generates a homopolymeric dC-tail, which creates the Abridged Anchor Primer (AAP) binding site on the 3'-end of the cDNA. An efficient tailing provides both: (i) homopolymeric tails long enough to allow the primer to anneal and (ii) sufficient dC-tailed cDNA to assure the correct amplification of the RACE product. Briefly, 10µL of purified cDNA were added to a reaction mixture containing: 10 mM Tris-HCl (pH 8.4), 25 mM KCl, 1.5 mM MgCl 2 , 200 µM dCTP and 1 U of TdT. The reaction was performed at 37°C for 10 min. TdT was inactivated at 65°C for 10 min.

Nested PCR, cloning and sequencing of the RACE product

The dC-tailed cDNA product was amplified by PCR using GSP2 and AAP primers (Table 14) as described above (see section 2.2). The RACE product was then cloned into the pCR2.1-TOPO vector following the supplier's recommendations (Invitrogen). Further sequencing of the resulting vector with M13 primers was performed to identify the 5'-UTR region.

5

Transcriptomic Analysis using RNA sequencing 5.1 Overview The Illumina, as the Ion Torrent technology, is another technique of NGS sequencing. It can be used to perform transcriptomic analysis also known as RNA sequencing (RNAseq).

RNAseq provides a comprehensive view of a cellular transcriptional profile at a given biological moment. This method uses total RNA, depleted from ribosomal RNA (rRNA), as template. The RNA is converted into cDNA by reverse transcription before the standard NGS library preparation (Figure 29). This technique uses the Sequencing By Synthesis (SBS) technology, which takes four fluorescently-labeled nucleotides to sequence the DNA clusters generated in the flow cell surface. During each sequencing cycle, a single labeled dNTP is added to the nucleic acid chain. The nucleotide label serves as a terminator for polymerization, at this moment the fluorescent dye is imaged to identify the base, which is then enzymatically cleaved to allow the incorporation of the next nucleotide. The final result is a great collection of images corresponding to nucleotide incorporation; this data is then transformed into sequence data to be analyzed.

5.1

Preparation of the cDNA library and sequencing Total RNA extracts were obtained in triplicate from exponential cultures (A 600nm = 1) of strains PA14, PJ01 and PJ01∆mexT at 37 o C. Bacterial cells were collected by centrifugation in RNA protect bacteria reagent (Qiagen) and disrupted with 0.15-0.60 mm ceramic beads in a TissueLyzer II (Qiagen) according to the manufacturer's instructions. Total RNA was then purified from beaded samples with RNeasyPlus 96 kit (Qiagen). Concentration and purity of the RNA extracts were assessed by RiboGreen measurement (Quant-iT RiboGreen RNA reagent and kit, Invitrogen) and an Agilent 2100 Bioanalyzer system (Agilent Technologies), respectively. Depletion of rRNA from those RNA samples was performed with the Ribo-Zero rRNA Removal reagents (Bacteria) from Epicentre (Madison, Wisconsin, USA). Libraries were then constructed using TruSeq Stranded mRNA HT Sample Prep kit from Illumina (San Diego, California, USA). The final libraries were quantified with Picogreen fluorescent dye (Quant-iT PicoGreen dsDNA assay kit, Invitrogen) and yielded between 200 and 800 ng per sample. Moreover, qualitative analysis was done using High Sensitivity DNA assay (Agilent). Finally, once reads are obtained, data are analyzed using a reference sequence, in our case the genome of strain PA14 (D).

Cluster Generation

For cluster generation, the library was loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. Each fragment was next amplified into distinct, clonal clusters through bridge amplification using adapterprimers.

Sequencing

The Illumina sequence technology uses a reversible terminator-based method that detects single bases as they are incorporated into DNA template strands. This technology takes four fluorescently-labeled nucleotides to sequence the tens of millions of clusters on the flow cell surface in parallel. During each sequencing cycle, a single labeled dNTP is added to the DNA chain. The nucleotide label serves as a terminator for polymerization. So, after each dNTP incorporation, the fluorescent dye is imaged to identify the base and then it is enzymatically cleaved to allow a new incorporation. Sequencing for RNAseq experiments was performed using the Illumina NextSeq 500 platform by Microsynth (Balgach, Switzerland).

Data Analysis

Data analysis of RNAseq was performed by Genostar (Grenoble, France). Reads were mapped on 5,892 annotated coding sequences (CDS) of strain PA14 using CLC Genomic Workbench 7.5 software. Transcripts abundance and differential expression results between the three replicates of each sample (PA14/PJ01, PA14/PJ01∆mexT and PJ01/PJ01∆mexT) were determined with Cufflinks and Cuffdiff algorithms (Wang et al., 2012). A difference in gene expression was considered significant when the q-value was ≤0.05 and when the expression ratio was ≤0.3-and ≥3.0-fold. The transcriptomic data have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through the GEO Series accession number GSE86211.

6

Protein-protein Interaction using Bacterial Two-Hybrid System 6.1 Overview The Bacterial Adenylate Cyclase Two Hybrid (BACTH) system is based on the reconstitution of a regulatory cascade depending on the secondary messenger cAMP, a molecule naturally produced by the family of adenylate cyclases (AC). The BACTH system uses the AC domain (CyaA) from Bordetella pertussis, the etiological agent of whooping cough. This domain, which catalyzes the conversion of ATP into cAMP, can be divided in two sub-units: a 25 kDa fragment called T25 and an 18 kDa called T18 (Figure 30). The two subunits of this enzyme are not active when they are physically separated, thus no cAMP is produced. This feature becomes practical in a cya -strain (no AC production). Under this context, two proteins of interest, X and Y, are fused with the two subunits T25 and T18 of the AC from B. pertussis. If X interacts with Y, the dimerization of chimeric proteins brings together both sub-units, restoring the AC activity and thus producing cAMP. On the other hand, if X does not interact with Y, there is no cAMP production.

Newly synthesized cAMP interacts with the Catabolite Activator Protein (CAP). The complex cAMP/CAP binds to specific promoters, regulating the expression of several genes, such as the maltose operon which is activated by this complex (Figure 30). The activation of the maltose operon can be detected by measuring the activity of the reporter gene lacZ (β-Galactosidase assay) or even more simple by culturing the strains on reporter plates (McConkey Maltose 1% or MHA X-Gal) (Battesti and Bouveret, 2012). cAMP is produced when both sub-units of the adenylate cyclase are bind together (A). When both subunits are physically separated, no cAMP is produced (B). The T25 and T18 subunits can be brought back together by the interaction of the proteins X and Y restoring the production of cAMP (C). When using cya -strains, the production of cAMP by the hybrid proteins induces the expression of the maltose operon, which is dependent of the cAMP/CAP complex (D). The activity of the maltose operon can be detected by measuring the activity of gene lacZ or even more simple by plating the clones on reporter plates (McConkey Maltose 1% or MH IPTG/X-Gal).

6.1

Cloning of gene mexT Dimerization of MexT protein of P. aeruginosa strains was studied using the BACTH system.

The entire coding sequence of gene mexT was amplified (see primers in Table 14) and bluntcloned into BACTH plasmids, pKNT25 and pUT18, previously digested with SmaI.

Recombinant plasmids, producing the hybrids MexT-T25 and MexT-T18 were then transferred into DHM1 (cya -) competent cells by heat-shock transformation as described above (see section 5.2.4.1).

6.1

Cloning of gene cmrA Putative dimerization of CmrA protein of P. aeruginosa strains was also studied. CmrA belongs to the AraC family of transcriptional regulators and is composed of 310 residues; it is predicted to possess two functional domains, a Ligand Binding Domain (LBD) (40 -191 residues) and a DNA Binding Domain (DBD) (198 -310 residues). For this approach, the whole gene was amplified by PCR using specific primers (Table 14) and cloned into the cloning vector pCR-Blunt. The region containing gene cmrA was then subcloned into the BACTH plasmids, pKT25 and pUT18C, as a BamHI-XbaI fragment. Recombinant plasmids were inserted into DHM1 cells as described above.

Hybrid Expression and Interaction Assay

For hybrid expression, strains containing both BACTH recombinant plasmids are cultured for 48 h at 30°C in selective cMHA plates containing kanamycin 50 µg/mL (for plasmids pKT25 and pKNT25) and ampicillin 100 µg/mL (for plasmids pUT18 and pUT18c). Colonies are grown overnight at 30°C with shaking (225 rpm) in selective cMHB. The next day, bacterial solutions are prepared to an A 600 =1 (10 9 CFU/mL) and 5 µL of each culture are dropped on MHTH and MCTH (see section 5.1.1). Plates are cultured at 30°C for 24 h. Blue colonies on MHTH or red colonies on MCTH indicate AMPc production consecutive to interaction between the two proteins.

β-Galactosidase Assay in 96-well Arrays

The β-Galactosidase assay was performed using the same liquid cultures used for the Interaction Assay. Briefly, 50 µL of culture were transferred into a flat-bottom microtiter plate containing 150 µL of cMHB in order to measure the A 600 . In parallel, 200 µL of each culture were transferred into a glass tube containing 800 µL of Z buffer (NaH 2 PO 4 22 mM, Na 2 HPO 4 22 mM pH 7, KCl 5 mM, MgSO 4 0.5 mM and β-Mercaptoethanol 0.3%). Cells were lysed by adding 50 µL of SDS 0.01 % and 100 µL of chloroform were used to separate the lipids from the aqueous solution. 50 µL of lysed cells were then transferred into a 96-well flat-bottom microplate containing 150 µL of Z buffer stabilized at 28°C. Then, 40 µL of ONPG 0.4% were dispensed and the enzymatic reaction is carried out at 28°C for 20 min. The measures of A 420 , performed every 2 min, were carried out using the Synergy H1 microplate reader (BioTek. Colmar, France) and data was collected using the Gen5 Software v2.04.11. The relative β-Galactosidase activity was calculated using the formula above, where 200 is the dissociation factor of ONPG under these experimental conditions (Battesti and Bouveret, 2012).

- = 4 -4 - ⁄ 6 * * 7
Analysis of Metabolites by HPTLC Metabolites produced by P. aeruginosa strains, PA14, PA14ΔmexS and PJ01 were observed by High Performance Thin Layer Chromatography (HPTLC). Briefly, bacteria were grown overnight in 50 mL of cMHB at 37°C and 250 rpm. Bacteria were then pelleted and supernatants were filtered in tandem using two filters of 0.4 µm and 0.2 µm pore diameter.

Organic extraction was performed in 5 times using 1 mL of chloroform (for a total of 5 mL). Organic fractions were pooled and dried overnight in a chemical hood and were suspended in 100 µL of chloroform. Ten microliters of each sample were then deposited on a HPTLC silica gel 60 F 254 (Merck) and separation was performed using a mobile phase methanoldichloromethane (10:90) for 30 -35 min. Spots were first observed using UV-light (250 nm) and then a second revelation using vanillin 1% in H 2 SO 4 was performed by spraying and heating for 15 min at 100°C. Relative expression of gene mexE, as determined by RT-qPCR (6), was found to be higher (from 20-to 112-fold) in these bacteria than in wild-type reference strain PA14 (Table I).

In addition, Minimal Inhibitory Concentrations (MIC) experiments (8) confirmed that all of the isolates were more resistant to CIP (from 0.5 to 8 µg mL -1 ), CHL (from 128 to 2,048 µg mL -1 ) and TMP (from 512 to >2,048 µg mL -1 ) than PA14 (0.125, 64, and 64 µg mL -1 respectively) (Table I).

To investigate the relevance of the observed amino acid changes in MexT, we first deleted gene mexT from PA14 as described elsewhere (6). The mutated alleles from clinical strains were then transferred by conjugation using MiniCTX1-derived recombinant plasmids (9), and were inserted into the chromosome of mutant PA14ΔmexT.

Complementation of this mutant with alleles from strains 4177, 0810, and 1510 had no impact on mexE transcription and MICs values (Table I). In contrast, MexT variants from strains 4088 and 10-12 triggered mexE expression 189-and 110-fold above the baseline level, respectively. As expected, this was associated with an increased resistance of PA14ΔmexT4088 and PA14ΔmexT10-12 to CIP (16x, 8x, respectively), CHL (32x, 16x), and TMP (≥ 32x, 16x) as compared to PA14ΔmexTPA14 (Table I). These results suggested that these two latter MexT variants were under a constitutively active conformation, able to upregulate pump MexEF-OprN. They also pointed to the importance of residue G257 in MexT activation as both variants harbored a single-amino acid substitution at this position (G257S and G257A).

It had been previously shown that, under oxidative conditions, MexT forms an active oligomer while under reducing conditions, it remains as an inactive monomer (10). This is in accordance with the usual mode of action of LTTRs in which an active tetramer is formed after a cognate co-inducer has bound to the inactive monomers (11). To determine whether variants from strains 4088 and 10-12 spontaneously form oligomers (i.e., in absence of ligand), a bacterial two-hybrid (BACTH) assay (12) was performed in strain DHM1 (cya -) of Escherichia coli, with plasmids pUT18 and pKNT25 that respectively code for T18 and T25 subunits of CyaA adenylate cyclase. This assay, which has been set up to study protein-protein interactions (12) is based on reconstitution of the adenylate cyclase activity and cAMP synthesis in E. coli. BACTH experiments confirmed that in the absence of cognate ligand MexTPA14 occurs a monomer, as no signal of oligomerization was observed either by using reporter plates or by measuring β-Gal activity (17 ± 1.66

Miller units) (Table II). In contrast, MexT variants from strains 4088 and 10-12 yielded positive results (Table II), suggesting that they can form oligomers.

To get an insight into what effects substitutions G257S and G257A may have on MexT oligomerization, we mapped these mutations on a three-dimensional dimeric LTTR model. As the crystal structure of MexT has not been determined yet, we used the dimeric structure of DntR from Burkholderia spp, another LTTR that shares 66% of sequence similarity with MexT, according to Clustal Ω results (13). In DntR, position 257 is occupied by a phenylalanine residue within the co-inducer binding domain. Interestingly, Phe-257 residues of DntR monomers face each other at the interphase of the dimer (Figure 1), suggesting that they could play a role in dimer stabilization. Nevertheless, the structural changes caused by amino acid substitutions at position 257 on MexT oligomerization will have to be confirmed once the crystal structure of this regulator is available.

Gain-of-function mutations in LTTRs had already been reported for Salmonella enterica serovar Typhimurium (14,15) and Acinetobacter baylyi (16). In S. enterica, gene cysB encodes a LTTR controlling expression of the cysteine regulon. It was found that spontaneous mutants harboring substitutions T149M and T149P in CysB overexpressed genes cysK, cysP and operon cysJIH in the absence of co-inducer N-acetyl-L-serine (14,15). In A. baylyi, LTTRs CatM and BenM regulate aromatic compounds degradation. The ability of these regulators to become constitutively active was studied by site-directed mutagenesis. As a result, substitutions R156H in CatM and R156H + T157S in BenM yielded mutants that did not require inducers such as benzoate and cis,cis-mucoate to activate the catabolic pathway (16). The present study is the first one to report on MexT-dependent mutational activation of efflux pump MexEF-OprN in antibiotic resistant clinical isolates of P. aeruginosa. This observation comes in complement of another study showing that some multidrug resistant strains of P. aeruginosa upregulate the intrinsic ß-lactamase AmpC through a gain-of-function mutation (G154R) in related LTTR AmpR (17). Altogether these data highlight the role that LTTRs may play in the emergence of multidrug resistance in this highly adaptive pathogen. b : Average values were calculated from five independent bacterial cultures each assayed in triplicate.

c : Muller-Hinton plates containing 40 µg mL -1 X-Gal (revealing cAMP production in blue), 50 µg mL -1 kanamycin and 100 µg mL -1 ampicillin.

d : MacConkey plates containing 1% maltose (revealing cAMP production in red), 50 µg mL -1 kanamycin, and 100 µg mL -1 ampicillin. Pseudomonas aeruginosa is a Gram negative opportunistic pathogen, responsible for several nosocomial infections in immunocompromised patients, and the main cause of mortality and morbidity of patients suffering from cystic fibrosis. Treatment of P. aeruginosa infections turns to be difficult due to its natural resistance to antibiotics, increased in part by the overproduction of RND efflux pumps capable to export antibiotics out of the cell. Amongst these systems, MexEF-OprN exports several antibiotics such as fluoroquinolones, chloramphenicol and trimethoprim. This efflux pump is quiescent in wild-type strains but it is highly produced in nfxC mutants, making them resistant to MexEF-OprN substrates. In addition, these mutants are characterized by their concomitant resistance to carbapenems and their low-virulence profile. MexEF-OprN is encoded by a three-gene operon, mexEF-oprN, whose transcription is activated by MexT, a member of the LysR family of transcriptional regulators. In the clinical context, nfxC mutants being poorly described, we evaluated their prevalence and characterized the genetic events responsible for mexEF-oprN overexpression. A collection of 221 clinical isolates from the University Hospital of Besançon exhibiting a reduced susceptibility to ciprofloxacin and imipenem was screened. We found that 19.5% of these strains overexpressed mexEF-oprN and further characterization of the 22 non-redundant mutants showed that only 13.6% of these mutants harbored a disrupted mexS gene. Moreover, 40.9% of nfxC clinical strains harbored missense mutations in mexS conducing to the substitution of a single amino-acid residue in the encoding protein. Interestingly, these mutations were associated to moderate effects on resistance and virulence factor production while disruptive mutations produced highly resistant but completely non-virulent strains. For the 45.5% of remaining strains, we failed to identify genetic mutations, which could explain mexEF-oprN overexpression; this indirectly suggested that there might be additional regulatory loci controlling the expression of this operon.

We thus studied chloramphenicol resistant mutants selected in vitro derived from reference strain PA14 and found a new class of MexEF-OprN overproducers, which we called nfxC2, harboring gainof-function mutations in a so-far uncharacterized gene, PA14_38040 (hereafter called cmrA) coding for an AraC transcriptional regulator. In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the expression of mexEF-oprN through MexS and MexT, resulting in a multi-drug resistant phenotype without altering virulence factor production. Transcriptomic experiments showed that CmrA positively regulates the expression of 11 genes, including PA14_38020, which is required for the MexS/MexT-dependent activation of mexEF-oprN. Gene PA14_38020 is predicted to code a quinol monooxygenase sharing conserved domains with YgiN of Escherichia coli, which was reported to be involved in the response of the bacterium to electrophiles. Interestingly, exposure of strain PA14 to sub-inhibitory concentrations of toxic electrophiles (glyoxal, methylglyoxal or cinnamaldehyde) strongly activates the CmrA-pathway and upregulates mexEF-oprN sufficiently to provoke the resistance to the pump substrates. Finally, we found that the same exposure to electrophiles is capable to activate two other RND pumps, MexAB-OprM and MexXY/OprM. The regulatory pathways conducing to activation of these two efflux operons will be elucidated at the laboratory.

v 6 . 1

 61 Cloning of gene cmrA ...................................................................................................................... 6.2 Hybrid Expression and Interaction Assay ........................................................................................ 6.3 β-Galactosidase Assay in 96-well Arrays ........................................................................................ 7 ANALYSIS OF METABOLITES BY HPTLC ............................................................................................... 8 MBP PROTEIN FUSION AND PURIFICATION ........................................................................................... 8.1 Overview .......................................................................................................................................... 8.2 Cloning and Production of MBP-tagged proteins ............................................................................ VI. APPENDIX ................................................................................................................................... VII. REFERENCES .............................................................................................................................. vi vii

Figure 1 :

 1 Figure 1: General structure and function of a RND efflux system and its coding genes ...........

Figure 2 :

 2 Figure 2: Regulation of mexAB-oprM operon ..........................................................................

Figure 3 :

 3 Figure 3: Regulation of meXY operon ......................................................................................

Figure 4 :

 4 Figure 4: Regulation of mexCD-oprJ operon ...........................................................................

Figure 5 :

 5 Figure 5: Regulation of mexEF-oprN operon ..........................................................................

Figure 6 :

 6 Figure 6: Subunit structure of LysR-type transcriptional regulator CbnR ...............................

Figure 7 :

 7 Figure 7: Schematic representation of LTTR mode of action ..................................................

Figure 8 :

 8 Figure 8: Crystal structure of the DntR dimer ..........................................................................

Figure 9 :

 9 Figure 9: Subcategories of oxidative stress ..............................................................................

Figure 10 :

 10 Figure 10: Efflux pump encoding operons induced by classical oxidative stress ....................

Figure 11 :

 11 Figure 11: Crystal structure of non-oxidized and oxidized MexR protein ..............................

Figure 12 :

 12 Figure 12: Induction of operon mexEF-oprN by non-classical oxidative stresses ...................

Figure 13 :

 13 Figure 13: Effect of redox environment on MexT ...................................................................

Figure 14 :

 14 Figure 14: Chemical structure of common Reactive Electrophilic Species (RES) ..................

Figure 15 :

 15 Figure 15: Formation of RES and cellular damage ..................................................................

Figure 16 :

 16 Figure 16: Catabolic pathways of electrophiles .......................................................................

Figure 17 :

 17 Figure 17: Biochemical reactions catalyzed by the glyoxalase system....................................

Figure 18 :

 18 Figure 18: Activation of KefB/C potassium channels by glutathione-adducts ........................

Figure 19 :

 19 Figure 19: Antimicrobial activity of cinnamaldehyde on P. aeruginosa .................................

Figure 20 :

 20 Figure 20: Relative expression of gene mexE from clinical and complemented strains according to the type of mutations found in mexS .................................................................

Figure 21 :

 21 Figure 21: Survival of BALB/cJRj mice infected with different strains of P. aeruginosa ....

Figure 22 :

 22 Figure 22: Gene inactivation by overlapping PCR and homologous recombination .............

Figure 23 :Figure 24 :

 2324 Figure 23: Representation of the luminescent reporter used to evaluate the activation of the CmrA pathway. ......................................................................................................................

Figure 25 :

 25 Figure 25: Preparation of gDNA library ................................................................................

Figure 26 :

 26 Figure 26: Template preparation ............................................................................................

Figure 27 :

 27 Figure 27: Ion Torrent® Sequencing ....................................................................................

Figure 28 :

 28 Figure 28: Overview of the 5' RACE procedure ...................................................................

Figure 29 :

 29 Figure 29: Overview of Illumina sequencing for RNAseq experiments ................................

Figure 30 :

 30 Figure 30: Rational of the BACTH technique based on T25 and T18 domains and the reconstitution of adenylate cyclase activity ...........................................................................

Figure 31 :

 31 Figure 31: Workflow of MBP-protein production and purification. .....................................

  to the Resistance Nodulation cell Division (RND) family are encoded in Gram negative species by chromosomal genes organized in operons (Li and Nikaido, 2009; Nikaido, 1998). These multimeric systems form nanomolecular machineries spanning across the two (inner and outer) membranes of the cell; they derive energy, for substrate export, from the proton motive force (Nikaido, 1996; Poole, 2000). The general structure of these efflux systems consists in a tripartite assembly containing a periplasmic membrane fusion protein (MFP), an outer membrane factor (OMF) and a trans-membrane transporter (RND) (Lister et al., 2009).

Figure 1 :

 1 Figure 1: General structure and function of a RND efflux system and its coding genes. MFP: membrane fusion protein, RND: resistance nodulation cell division, OMF: outer membrane factor. Lister et al., 2009

Figure 2 :

 2 Figure 2: Regulation of mexAB-oprM operon. Self-regulated local repressor MexR ensures basal expression of operon mexAB-oprM in wild-type strains by binding as a homodimer to the distal promoter. A second repressor, NalD, directly binds as a monomer to the proximal promoter of the mexAB-oprM operon. The third repressor, NalC, has an indirect effect on mexAB-oprM expression as it represses the expression of gene armR, the product of which acts as an anti-repressor of MexR.Mutations in these three repressors have been reported in nalB, nalD, and nalC mutants, respectively. Two direct activators of mexAB-oprM have been described, namely CpxR and BrlR (this latter being active exclusively in biofilms). Finally, the products of four other genes would have an indirect negative (mexT, rocS1/2-A2, PA3225) or positive (ampR) impact on mexAB-oprM expression.

  (McLaughlin et al., 2012) as well as by activity of two-component systems ParRS (Muller et al., 2011) and AmgRS (Lau et al., 2013) (Figure 3).

Figure 3 :

 3 Figure 3: Regulation of meXY operon. Acting as a local repressor, MexZ binds as a homodimer to the promoter region upstream of mexXY. Gene armZ encodes an anti-repressor that interacts with and inhibits MexZ. The product of gene suhB or defects in ribosomal proteins encoded by genes rplU and rpmA, result in overproduction of ArmZ and subsequent overexpression of mexXY. The operon can also be indirectly activated by mutations occurring in operons parRS and amgRS that code for two-component regulatory systems ParRS and AmgRS, respectively. Finally, the products of genes PA2572-PA2573 have been reported to indirectly repress mexXY by still unknown mechanisms. To date, three types of MexXYoverproducing mutants have been characterized among clinical isolates, namely agrZ mutants (harboring mutations in mexZ), agrW1 mutants (exhibiting alterations in various ribosomal proteins or 23S rRNA) and agrW2 mutants (with mutations activating ParRS).

Figure 4 :

 4 Figure 4: Regulation of mexCD-oprJ operon. Acting as a local repressor, NfxB binds as a tetramer to the DNA region ahead of mexCD-oprJ. The product of esrC gene also acts as a local repressor of the operon. Other proteins such as VqsM and AlgU would positively affect the expression of mexCD-oprJ, while the product of gene PA2572 seems to have an indirect impact. Only nfxB mutants harboring a disrupted nfxB gene have been recognized in the clinical setting so far.

(

  Westfall et al., 2006), ampR (Balasubramanian et al., 2012), mxtR (Zaoui et al., 2012), brlR (Liao et al., 2013), parR (Wang et al., 2013), nmoR (Vercammen et al., 2015) and PA2449 (Lundgren et al., 2013) (Figure 5).

Figure 5 :

 5 Figure 5: Regulation of mexEF-oprN operon. Operon mexEF-oprN is the sole RND-type operon of P. aeruginosa known to be controlled by an activator, named MexT. The MexT-dependent expression of mexEF-oprN can be triggered by mutational inactivation or impairment of a putative oxido-reductase, MexS, whose gene resides upstream mexT. The twocomponent system ParRS would interact with MexS and indirectly activate mexEF-oprN. The products of genes nmoR and brlR (only active in biofilm) are also thought to have a positive impact on mexEF-oprN expression, while genes mvaT, ampR, mxtR, and PA2449 would have negative effects.

  regulator (LTTR) of 305 residues, is essential to operon mexEF-oprN expression (Köhler et al., 1999) (Figure 5). However, in addition to its role in the control of efflux activity in P. aeruginosa, MexT is involved in direct or indirect, positive or negative regulation of about 143 genes. For instance, this protein acts as a repressor of porin OprD encoding gene and PQS (Pseudomonas Quinolone Signal) biosynthesis operon pqsABCDE (Tian et al., 2009b). Of note, a sequence polymorphism or mutations in gene mexT have been reported to cause inactivation of the regulator in some PAO1-like strains sub-cultured in research labs (Henrichfreise et al., 2007; Köhler et al., 1999; Luong et al., 2014; Maseda et al., 2000; Olivas et al., 2012; Tian et al., 2009b; Vercammen et al., 2015). Usually composed of 330 residues, LTTRs are global regulators that positively or negatively regulate expression of target genes (Maddocks and Oyston, 2008). The family is highly conserved and ubiquitous among bacteria, with orthologous members identified in Archaea and eukaryotic organisms (Pérez-Rueda and Collado-Vides, 2001). Most of these regulators are activated upon binding of specific ligands some of which can be intermediates of biosynthetic or catabolic pathways (Maddocks and Oyston, 2008). The canonical structure of a LTTR (Figure 6) contains an N-terminal helixturn-helix (HTH) motif for DNA binding, and a C-terminal co-factor binding domain composed of two distinct α/β subdomains (RD-I and RD-II). These subdomains are connected by a hinge which is thought to accommodate co-inducer molecules (Maddocks and Oyston, 2008; Muraoka et al., 2003; Stec et al., 2006).

Figure 6 :

 6 Figure 6: Subunit structure of LysR-type transcriptional regulator CbnR. In Ralstonia eutropha, CbnR initiates chlorocathecol degradation. In red, the N-terminus contains the DNA binding domain (DBD). In blue, the C-terminus harbors the co-factor binding domain composed of two subdomains RD-I and RD-II connected by a hinge. The linker between the two domains is represented in yellow (Muraoka et al., 2003).

Figure 7 :

 7 Figure 7: Schematic representation of LTTR mode of action. The dimeric conformation of the apo-form of the LTTR usually binds the regulatory binding site (RBS) with high affinity, and the activation binding site (ABS) with low affinity (A); once the co-inducer is produced at relevant levels, the affinity of the co-inducer-bound LTTR is increased; binding of two dimers to regulatory sequences creates a bending of the DNA (B); the functional tetramer stabilizes RNA polymerase and triggers the expression of the target gene (C); based on Maddocks and Oyston, 2008. A three-dimensional structure of the functional tetramer of a LTTR is represented (D) (Muraoka et al., 2003).

  Escherichia coli, MxtR is an orphan sensor kinase encoded by gene PA3271 in P. aeruginosa. Via its action on MexT, this sensor represses biosynthesis of PQS and the pump MexEF-OprN (Zaoui et al., 2012).

a

  Expressed as a ratio relative to that of wild-type reference strain PA14. nfxC mutants (the values for which are in bold) have a mexE transcript level of Ն20. b CHL, chloramphenicol; CIP, ciprofloxacin; IMP, imipenem; MEM, meropenem; TIC, ticarcillin; AMK, amikacin. c The results for virulence factors are in bold when they are positive or considered significant. OD 600 , OD at 600 nm. d Hemolytic activity and pyocyanin production were measured on stationary-phase cultures after 18 h of growth (see Materials and Methods for details). e The score was determined from the results of each test (biofilm formation, swarming motility, elastase production, hemolytic activity, pyocyanin production), positivity by each of which was given a value of 1. Of note, swarming motility and biofilm formation are correlated with rhamnolipid production. f aa, amino acid. g WT, wild type. h ND, not detected. i The strain tolerated substitutions in MexS. Clinical nfxC Mutants of P. aeruginosa April 2016 Volume 60 Number 4 aac.asm.org 2307 Antimicrobial Agents and Chemotherapy on August 4, 2016 by INIST-CNRS BiblioVie http://aac.asm.org/ Downloaded from

FIG 2

 2 FIG 2 Swarming motility of PA14⌬S complemented with different mutated mexS alleles from clinical isolates. Swarming motility was evaluated as the capacity to give rise to dendrite-like patterns. The patterns for strain PA14 and PA14⌬S PA14 (positive controls) (A and C, respectively) and PA14⌬S (negative control) (B) are indicated. (F) Inactivation leading to an aberrant MexS protein (in PA14⌬S 1711 , for example) abolished the swarming. In most cases, substitutions in MexS, for example, V 104 A (D), F 185 L (G), and D 44 E (H), did not affect the ability of the bacteria to swarm; however, the L 263 Q substitution led to an almost complete loss of motility (E).

  [START_REF] Källberg | Templatebased protein structure modeling using the RaptorX web server[END_REF] to obtain a prediction based on DntR (35% sequence identity with MexT), a LTTR sensor of nitrotoluene isomers in Burkholderia spp.[START_REF] Smirnova | Development of a bacterial biosensor for nitrotoluenes: the crystal structure of the transcriptional regulator DntR[END_REF]. The prediction showed that MexT has the classical conformation of LTTRs with its co-inducer binding domain at the Nterminus and its DNA-binding domain at the C-terminus of the protein. According to this prediction, Gly-257 is positioned in the external phase of the co-inducer binding domain, at the end of an α-helix. The location of this residue suggests its involvement in dimer/oligomer formation and regulator activation. As a dimer structure has experimentally been determined for DntR(Devesse et al., 2011), we examined the location of Phe-257 (Gly-257 in MexT) to check out whether this position could participate in intermolecular interactions between the two monomers. As represented in Figure 8, Phe-257 residues of DntR monomers face each other in the dimeric model. Based on these data, one can assume that change of Gly-257 into Ser or Ala allows MexT to adopt an active conformation more easily. However, this will be confirmed once the crystal structure of MexT (either in a monomeric or dimeric form) is available. An ongoing collaboration with Pr. Isabelle Broutin at university Paris Descartes (UMR 8015, Laboratoire de Cristallographie et RMN Biologiques) is expected to provide such structural details.

Figure 8 :

 8 Figure 8: Crystal structure of the DntR dimer. Crystal structure of DntR dimer from Burkholderia spp (PDB 2Y7K) was experimentally determined previously (Devesse et al., 2011). The two monomers are indicated in yellow and brown and Phenylalanine-257 is highlighted in red.

(Figure 9 )

 9 [START_REF] Bild | The interdependence of the reactive species of oxygen, nitrogen, and carbon[END_REF][START_REF] Green | Bacterial redox sensors[END_REF]; Vázquez-Torres, 2012). In any case, intracellular accumulation of reactive species is deleterious to proteins, DNA, and lipids.

Figure 9 :

 9 Figure 9: Subcategories of oxidative stress. Formation of superoxide anion (O 2 -·) can be achieved by environmental signals or by aerobic metabolism. Superoxide can react with different species inside the cell creating several subcategories of oxidative stress. AGEs, advanced glycation end-products; H 2 O 2 , hydrogen peroxide; NO·, nitric oxide; NO 2 ·, nitrogen dioxide; OH·, hydroxyl radical; ONOO-, peroxinitrite; SOD, superoxide dismutase. Expression of several RND-type efflux systems involved in antibiotic resistance can be affected by one or more of the oxidative stress subcategories (Table 7) (Fruci and Poole, 2016; Poole, 2012b, 2012a, 2014). MexAB-OprM (Chen et al., 2008; Ghosh et al., 2011; Muller et al., 2007, 2015; Starr et al., 2012), and MexXY/OprM (Fraud and Poole, 2011) are upregulated by classical oxidative stress, while MexEF-OprN production is increased upon nitrosative (Fetar et al., 2011) and disulfide (Fargier et al., 2012) stresses.

1 . 2

 12 Efflux operons induced by classical oxidative stressThis stress results from production of ROS such as superoxide (O 2 -·), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH·). To neutralize these harmful reactive species, P. aeruginosa and other bacteria produce basal levels of ROS-scavenger enzymes, such as superoxide dismutase (SOD) and catalase[START_REF] Imlay | Cellular defenses against superoxide and hydrogen peroxide[END_REF]. However, when ROS levels are increased under stressful conditions, other detoxification mechanisms are set up to protect the cell, including production of additional detoxifying enzymes and antioxidant molecules (e.g., ascorbate)[START_REF] Ezraty | Oxidative stress, protein damage and repair in bacteria[END_REF][START_REF] Fruci | Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria[END_REF][START_REF] Imlay | Pathways of oxidative damage[END_REF].

Figure 10 :

 10 Figure 10: Efflux pump encoding operons induced by classical oxidative stress. Two efflux operons are known to respond to peroxides (classical oxidative stress), namely mexAB-oprM and mexXY. Redox activation of operon mexAB-oprM is dependent on redox-sensing repressor MexR. The second operon, mexXY, is indirectly activated by peroxides via antirepressor ArmZ. AG, aminoglycosides; BL, β-lactams; CHL, chloramphenicol; FQ, fluoroquinolones; ML, macrolides; TMP, trimethoprim.Derepression of efflux pumps is coordinated with regulation of other genes via regulators able to sense, directly or indirectly, the presence of ROS or to monitor the redox state of cell[START_REF] Ezraty | Oxidative stress, protein damage and repair in bacteria[END_REF][START_REF] Green | Bacterial redox sensors[END_REF]). Since P. aeruginosa is constantly exposed to ROS in both natural-and host-environments, RND systems play an important role in the response to oxidative stress (Poole, 2014) (Figure10).

Figure 11 :

 11 Figure 11: Crystal structure of non-oxidized and oxidized MexR protein. MexR is a local repressor of mexAB-oprM operon. (A) The reduced monomer (blue) is capable to bind the upstream region of mexAB-oprM operon and thus to repress operon expression. Upon peroxide exposure (pink), the formation of a disulfide bond between cystein residues -30 and -62 causes severe steric clashes with the DNA backbone, thereby preventing MexR binding to the mexAB-oprM promoter. Adapted from Chen et al., 2010.

(

  [START_REF] Fraud | Oxidative stress induction of the MexXY multidrug efflux genes and promotion of aminoglycoside resistance development in Pseudomonas aeruginosa[END_REF][START_REF] Fruci | Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria[END_REF] Jeannot et al., 2005).

Figure 12 :

 12 Figure 12: Induction of operon mexEF-oprN by non-classical oxidative stresses. The efflux pump MexEF-OprN is quite unique in P. aeruginosa as it is the only RND system known to be induced by non-classical oxidative stresses. Nitrosative or disulfide stress elicitors activate the global regulator MexT and thus operon mexEF-oprN. However, the exact mechanism by which MexT gets activated remains unknown. CHL, chloramphenicol; DA, diamide; DETA, diethylenetriamine NONOate; GSNO, s-nitrosoglutathione; IST, isothiocyanate.Nitrosative upregulation of operon mexEF-oprN is dependent on MexT(Köhler et al., 1999).Among the many MexT-regulated genes identified by transcriptomic analysis(Tian et al., 2009b), xenB is of particular interest because of its role in the removal of nitro groups from xenobiotics such as nitroglycerine and trinitrotoluene, in Pseudomonas spp.[START_REF] Blehert | Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases[END_REF][START_REF] Fuller | Transformation of RDX and other energetic compounds by xenobiotic reductases XenA and XenB[END_REF][START_REF] Pak | Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase B from Pseudomonas fluorescens I-C[END_REF]. This link between MexT, MexEF-OprN and xenB has led to the hypothesis that the pump contributes to the detoxification of nitrated products formed upon nitrosative stress(Fetar et al., 2011; Poole, 2012b).

Figure 13 :

 13 Figure 13: Effect of redox environment on MexT. In accordance with LTTRs mode of action, MexT is under an inactive monomeric form when reduced glutathione (GSH) is predominant in the medium. In contrast, when the concentration of oxidized glutathione (GSSG) increases, MexT forms oligomers and becomes active. Exposure of P. aeruginosa strain PA14 to disulfide stress elicitors diamide (Fargier et al., 2012) and isothyocyanate compound iberin (Tan et al., 2014) is associated with an increased expression of MexT-dependent genes including operon mexEF-oprN (180-and 129-fold, respectively). Supporting the notion that diamide is a substrate of the pump, disruption of

  al., 2012; Tian et al., 2009b).

1 . 4

 14 Bacterial response to electrophilic stress Although the bacterial stress response to classical oxidative or nitrosative stress has been extensively studied, research on electrophilic stress is somewhat scarce (Lee and Park, 2017). Electrophilic stress is defined as an unbalance between the formation and degradation of reactive electrophilic species (RES), which are compounds containing α,β-unsaturated carbonyl or other electrophilic groups (Lee and Park, 2017). This subcategory of oxidative stress is also known as carbonyl stress as the most studied reactive species are compounds containing two carbonyl groups, the α-oxoaldehydes such as glyoxal (GO) and methylglyoxal (MG) (Booth et al., 2003; Kosmachevskaya et al., 2015) (Figure 14). Induction of RND pumps by electrophilic stressors has not been reported thus far. Moreover, in one study exposure of strain PAO1 to MG was found to have no impact on multi-drug resistance of P. aeruginosa (Hayashi et al., 2014). Like oxidative stress, electrophilic stress can be generated by endogenous sources such as the oxidative degradation of glucose, lipid peroxidation, and DNA oxidation (Kosmachevskaya et al., 2015) (Figure 15). Interestingly, Escherichia coli and several Pseudomonas spp. (not P. aeruginosa) harbor a chromosomal gene (mgsA) encoding a methylglyoxal synthase, suggesting an important role for MG in intracellular signaling at low concentrations (Booth et al., 2003; Tötemeyer et al., 1998; Winsor et al., 2016). Under stress conditions, RES can affect the cell redox potential at two levels: (i) by directly perturbing the electron transport chains and promoting ROS formation or (ii) by depleting redox cofactors like glutathione (GSH) and NAD(P)H (Ferguson et al., 1998a; Kosmachevskaya et al., 2015; Lee et al., 2013a). Additionally, RES react with amino groups of lysine, cysteine, arginine, and histidine residues to form Advanced Glycated-End products (AGEs) which compromise protein activities (Jacobs and Marnett, 2010) (Ferguson et al., 1998a), as well as with the guanosine residues present in DNA or other nucleotide-containing molecules to form adducts such as carboxymethyl-and carboxyethyl-guanosine that affect gene expression and cellular homeostasis (Marnett et al., 2003) (Figure 15).

Figure 14 :

 14 Figure 14: Chemical structure of common Reactive Electrophilic Species (RES). RES are chemical compounds containing α-unsaturated carbonyl (GO or MG) or α,β-electrophilic groups (double bond in the case of CNA).

Figure 15 :

 15 Figure 15: Formation of RES and cellular damage. Formation of Reactive Electrophilic Species (RES) results from different cellular processes such as oxidation of sugars and DNA, and from lipid peroxidation. Accumulation of RES has pleiotropic effects (depletion of cofactors, alteration of electron transport chains (ETC), macromolecular damage of proteins and nucleotides) that impact cellular homeostasis.Though RES are ubiquitous, highly reactive molecules acting on a broad spectrum of biological targets, description of bacterial responses to these stressors is scarce. Escherichia coli remains the model organism to understand cellular adaptation to electrophilic stress, as several studies have analyzed the mechanisms by which this bacterium senses, detoxifies, and protects itself against RES(Ferguson et al., 1998a; Kosmachevskaya et al., 2015; Lee and Park, 2017). Such mechanisms have never been studied in P. aeruginosa. However, several genes carried by the genome of this microorganism are homologous to genes involved in RES response in E. coli (Table8).

Figure 16 :

 16 Figure 16: Catabolic pathways of electrophiles. Glyoxal (GO) and methylglyoxal (MG) are two major Reactive Electrophilic Species (RES) that can be metabolized by two mechanisms: the glyoxalase system (in blue) and the NAD(P)Hdependent enzymes system (in red). AldA, aldehyde dehydrogenase; AKRs, aldo-keto reductases; GloI, GloII and GloIII, glyoxalases I, II and III; GSH, glutathione; SHG, S-2-hydroxyethylglutathione; SLG, S-D-lactoylglutathione.

Figure 17 :

 17 Figure17: Biochemical reactions catalyzed by the glyoxalase system. Spontaneous reaction between methylglyoxal (MG) and reduced glutathione (GSH) forms a hemithioacetal which is substrate for glyoxalase I (Glo1, in yellow); this enzyme produces the GSH-adduct S-D-lactoylglutathione (SLG). Glyoxalase II (Glo2, in blue) takes SLG as substrate to form Dlactate enabling the turnover of GSH. Finally, glyoxalase III (Glo3, green) can directly reduce MG into D-lactate without any cofactor.[START_REF] Honek | Bacterial glyoxalase I enzymes: structural and biochemical investigations[END_REF]

  left panel)[START_REF] Kilfoil | Regulation of ion channels by pyridine nucleotides[END_REF]. Upon electrophile exposure, the GSH-adducts (e.g. SLG) produced by Glo1 bind to KefB/C and provoke the opening of the channel with subsequent efflux of K +[START_REF] Ferguson | Protective mechanisms against toxic electrophiles in Escherischia coli[END_REF][START_REF] Kilfoil | Regulation of ion channels by pyridine nucleotides[END_REF]) (Figure18right panel).

Figure 18 :

 18 Figure 18: Activation of KefB/C potassium channels by glutathione-adducts. The KefB and KefC channels are activated by glutathione-adducts produced by glyoxalase I. In the absence of electrophiles, the channels are under a closed conformation due to binding of reduced glutathione (GSH) (left panel). When electrophiles accumulate in the cell, glyoxalase I produces glutathione-adducts such as S-D-lactoylglutathione (SLG), that bind and open the KefB/C channels provoking the release of K + from the cell. Efflux of K + is coupled with influx of protons, with subsequent acidification of cytosol. Adapted from Kilfoil et al., 2013.

  Multi-Drug Efflux Pump MexEF-OprN in Pseudomonas aeruginosa Through a Novel Transcriptional Regulator, CmrA Paulo Juarez, Katy Jeannot, Patrick Plésiat and Catherine Llanes Antimicrobial Agents and Chemotherapy. 2017 May 15; 61(8): e00585-17. Doi: 10.1128/AAC.00585-17 Toxic Electrophiles Induce Expression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas aeruginosa through a Novel Transcriptional Regulator, CmrA Paulo Juarez, a Katy Jeannot, a,b Patrick Plésiat, a,b Catherine Llanes a Laboratoire de Bactériologie, UMR CNRS 6249 Chrono-Environnement, Université Bourgogne Franche-Comté, Besançon, France a ; Centre National de Référence de la Résistance aux Antibiotiques, Centre Hospitalier Régional Universitaire de Besançon, Besançon, France bABSTRACT The multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa. However, in so-called nfxC mutants, mutational alteration of the gene mexS results in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA (for chloramphenicol resistance activator). In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprN through MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN. PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli. Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosa to the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.

FIG 1

 1 FIG 1 RaptorX prediction of CmrA structure. A three-dimensional structure of the regulator CmrA was modeled using the RaptorX Web server (http://raptorx.uchicago.edu/). The putative N-terminal ligandbinding domain (from amino acid position 40 to 191) was predicted based on ToxT from Vibrio cholerae (PDB 3GBG; P ϭ 5.96eϪ4), while the putative C-terminal DNA-binding domain (from 198 to 310) is based on AdpA from Streptomyces griseus (PDB 3W6V; P ϭ 3.45eϪ5). The amino acid substitutions found in PJ mutants are highlighted by blue spots.

FIG 2

 2 FIG 2 Genetic environment of gene cmrA. (A) Gene annotations are those available in GenBank for strain PA14 (RefSeq accession number NC_008463.1). Homologs in strain PAO1 are indicated in brackets. The DNA sequence upstream from cmrA is shown below the schematic. Different positions were assigned to the start codon of cmrA in genomic maps of PA14 (PA14_38040, 933 bp; boldface and underlined) and PAO1 (PA2047, 990 bp; underlined). Two putative overlapping RpoN and RpoS binding motifs are highlighted in gray. The transcription start sites (TSS, ϩ1) of cmrA and PA2048 were mapped by 5=-RACE at Ϫ38 bp and Ϫ315 bp, respectively. The 5= UTR of PA2048 contains a 111-bp region of unknown function, bordered by two 10-bp inverted repeats (IR-N91). (B) Complementation experiments in mutant PA14ΔcmrA were carried out to clarify the role of the factor binding sites in cmrA expression. A full-length DNA fragment from mutant PJ01, carrying cmrA and the RpoN/RpoS binding sites (Ϫ152 bp upstream from the TSS), conferred an nfxC2 resistance phenotype on PA14ΔcmrA, while a short version lacking the RpoN/RpoS binding sites (Ϫ68 bp) did not. The whole sequence of cmrA and its 5= UTR is accessible through the GenBank database (accession number KX274690).

FIG 4

 4 FIG4 Bactericidal activity of cinnamaldehyde on P. aeruginosa. Bioluminescent strain PA14-lux and its derived mutants PA14-lux-ΔcmrA and PA14-lux-ΔmexEF-oprN were cultured to mid-log phase and then challenged with 1,000 g ml Ϫ1 cinnamaldehyde. Bioluminescence (RLU) was recorded every 30 min and used as an indicator of cell survival. RLU values are mean values Ϯ standard deviations from three independent experiments. A bioluminescence threshold was established with sterile MHB (dotted line).

FIG 5

 5 FIG5 Schematic representation of activation pathways of MexEF-OprN in P. aeruginosa. In wild-type strains (top left), such as strain PA14, regulators MexT and CmrA remain quiescent because of redox homeostasis. In so-called nfxC mutants (top right), mutational alteration of putative quinone oxidoreductase MexS is thought to result in intracellular accumulation of some redox-active MexS substrate(s). Redox-dependent oligomerization of MexT then triggers production of the pump MexEF-OprN and active efflux of still-undetermined endogenous products. In nfxC2 mutants (bottom left), regulator CmrA is activated as a result of gain-of-function mutations in gene cmrA. Among the 11 genes positively regulated by CmrA, PA2048 codes for a putative quinol monooxygenase. Concomitant activation of PA2048 and MexS is assumed to generate oxidized metabolites, the accumulation of which would modify the cellular redox state. As in MexS-deficient mutants, these changes upregulate the production of MexEF-OprN via MexT. Finally, upon electrophilic stress (bottom right), reactive electrophilic species (RES), such as glyoxal and methylglyoxal, induce the glyoxalase detoxification system (GloA2 and GloA3) and CmrA-dependent expression of MexEF-OprN. We propose that P. aeruginosa uses the efflux pump MexEF-OprN as a defense mechanism in response to toxic electrophilic stressors encountered in its environment.

(

  [START_REF] Juarez | Toxic electrophiles induce expression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa through a novel transcriptional regulator[END_REF]. Since electrophiles generate a subcategory of oxidative stress (Lee and Park, 2017), we wondered whether these molecules could upregulate other RND pumps known to respond to oxidative stress, such as MexAB-OprM and MexXY/OprM[START_REF] Fraud | Oxidative stress induction of the MexXY multidrug efflux genes and promotion of aminoglycoside resistance development in Pseudomonas aeruginosa[END_REF][START_REF] Starr | Pentachlorophenol induction of the Pseudomonas aeruginosa mexAB-oprM efflux operon: involvement of repressors NalC and MexR and the antirepressor ArmR[END_REF].

Figure 19 :

 19 Figure19: Antimicrobial activity of cinnamaldehyde on P. aeruginosa. (A) Susceptibility of strain PA14 and its derived mutants to cinnamaldehyde (CNA 10 µg) was measured by disk diffusion method. (B) Bioluminescent strain PA14 and its derived mutants were cultured to mid-log phase and then challenged with 1,000 µg mL -1 cinnamaldehyde. Luminescence (RLU) was measured in a plate reader and was used as indicator of cell survival. The curve is a representation of one experiment. A luminescence threshold was determined with sterile MHB (dotted line).

  considered as rare or absent from the clinical setting (Quale et al., 2006; Walsh and Amyes, 2007; Wolter et al., 2004; Xavier et al., 2010). However, a growing number of publications now report on identification of such mutants in CF and non-CF patients (Jalal et al., 2000; Llanes et al., 2011; Oh et al., 2003; Terzi et al., 2014), though the underlying causes of pump dysregulation most often remain uncharacterized. One of the reasons why nfxC mutants are difficult to detect among clinical strains is that their resistance phenotype to chloramphenicol, trimethoprim, fluoroquinolones, and carbapenems is often masked by more common mechanisms including overproduction of pump(s) MexAB-OprM or (and) MexXY/OprM, and loss of porin OprD (Llanes et al., 2013; Richardot et al., 2015)

  , left panel). Complementation experiments with various mexS mutated alleles in PA14ΔmexS confirmed this observation (Figure 20, right panel). It should be noted that while other investigators reported that missense mutations in mexS are more prevalent than disruptive mutations among clinical strains (as summarized in Table 3 page 20), the correlation with mexEF-oprN expression had not been established until this study.

Figure 20 :

 20 Figure 20: Relative expression of gene mexE from clinical and complemented strains according to the type of mutations found in mexS. Adapted from Richardot et al, 2016

  MexS and MexT. This result confirmed previous conclusions about the existence of one or more additional regulatory pathways for MexEF-OprN(Kumar and Schweizer, 2011;[START_REF] Maseda | Transcriptional regulation of the mexEF-oprN multidrug efflux pump operon by MexT and an unidentified repressor in nfxC-type mutant of Pseudomonas aeruginosa[END_REF]. Characterization of in vitro selected mutants led to the discovery of CmrA, a novel AraC-type transcriptional regulator which when activated by specific single amino acid substitutions is able to indirectly upregulate mexEF-oprN expression[START_REF] Juarez | Toxic electrophiles induce expression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa through a novel transcriptional regulator[END_REF].

  initiated to delete the gene encoding potent cytotoxin ExoU, to allow a better discrimination between wild-type PA14 and nfxC2 mutants (da Cunha et al., 2015; Juan et al., 2017; Shaver and Hauser, 2004).

Figure 21 :

 21 Figure 21: Survival of BALB/cJRj mice infected with different strains of P. aeruginosa. Male mice from 8 -10 weeks were inoculated intranasally (5 x 10 5 UFC) and their survival was followed up to 4 days post-infection (Results obtained by UMR 1100, unpublished data).

  al., 2012; Fetar et al., 2011;[START_REF] Fraud | Oxidative stress induction of the MexXY multidrug efflux genes and promotion of aminoglycoside resistance development in Pseudomonas aeruginosa[END_REF][START_REF] Starr | Pentachlorophenol induction of the Pseudomonas aeruginosa mexAB-oprM efflux operon: involvement of repressors NalC and MexR and the antirepressor ArmR[END_REF], electrophiles enhance the resistance of the bacterium to various antibiotics[START_REF] Juarez | Toxic electrophiles induce expression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa through a novel transcriptional regulator[END_REF]. We showed that in addition to MexEF-OprN, two other systems (MexAB-OprM and MexXY/OprM) are strongly upregulated when bacteria are exposed to GO, MG and CNA. Because of the strong toxicity of these molecules, a high efflux activity is probably needed to quickly remove these agents, some toxic metabolites or damaged components, from the cell. The regulatory pathways by which MexAB-OprM and MexXY/OprM are activated by RES are currently studied in our lab (Master's project of Alexandre Tetard).

  Recombinant pKNG plasmids were transferred from E. coli to P. aeruginosa by triparental mattings and selection on PIA plates supplemented with 2,000 µg/mL streptomycin. The transconjugants were subcultured on M9-Suc medium to force the excision of the inserted plasmids. Finally, negative selection on streptomycin confirmed the loss of episomal plasmids while PCR/sequencing experiments confirmed the allelic exchanges. The complete inactivation strategy is illustrated in Figure22.

Figure 22 :

 22 Figure 22: Gene inactivation by overlapping PCR and homologous recombination Inactivation of gene cmrA; based on Muller et al., 2011.

Figure 23 :

 23 Figure 23: Representation of the luminescent reporter used to evaluate the activation of the CmrA pathway.

  Amplicon Sequencing takes part of the Next Generation Sequencing (NGS) technologies. This approach couples a semiconductor chip, capable of directly translating chemical signals into digital information. Each micro-well of the Ion Torrent sequencing chip contains approximately 10 6 copies of a DNA molecule previously amplified by emulsion PCR. The machine sequentially floods the chip with one nucleotide after another. If one nucleotide is incorporated into the DNA molecule in a micro-well, one proton (H + ) is released. The pH modification is detected in each specific well by the ion sensor (Figure 24). If the next nucleotide that floods the chip is not incorporated, no voltage change is recorded, and no base is called. Sequencing of the DNA template is performed in seconds and this enables very short run times.

Figure 24 :

 24 Figure 24: Schematic representation of a single well of an Ion Torrent sequencing chip. The well harbors Ion Spheres particles containing DNA template. When a nucleotide incorporates, a proton (H+) is released and the pH of the well changes (∆pH). A sensing layer detects this change of charges (∆Q) and translates the chemical signal into a digital signal (∆V).

Figure 25 :

 25 Figure 25: Preparation of gDNA library. Genomic DNA is digested and fragments of around 200 bp are used for library generation. Two adapters, sphere primer (red) and biotin (green), are next ligated to each end of DNA fragments. This will allow the fragment to bind to an Ion Sphere and to be purified from the matrix.

Figure 26 :

 26 Figure 26: Template preparation. Library generated fragments are bond to Ion Spheres by complementation of adapters.Each sphere will contain one-single fragment, which will be amplified by emulsion PCR.

Figure 27 Ion

 27 Figure 27 Ion Torrent® Sequencing. Amplification of monoclonal spheres by emulsion PCR creates the template for sequencing. The template is applied to an Ion Semiconductor Sequencing Chip by pipetting. The chip is then charged into the sequencing apparatus.

Figure 28 :

 28 Figure 28: Overview of the 5' RACE procedure.Once total RNA is extracted, a reverse-transcription is performed using a reverse Gene Specific Primer (GSP1). The newly synthezised cDNA is depleted from RNA using a RNAse cocktail. Purified cDNA is next tailed with cytosines on the 3'-terminus using a terminal deoxynucleotidyl terminase (TdT). Tailed-cDNA is then amplified by nested PCR using a second reverse Gene Specific Primer (GSP2) and the adapter primer (Abriged Anchor Primer) allowing amplification from the dC-homopolymeric tail. The RACE product can be next cloned in different vectors for sequencing experiments and identification of the Transcription Start Site (TSS).

4. 3 S

 3 .N.A.P. Column Purification of cDNA S.N.A.P. column purification of cDNA was performed following the supplier's recommendations (Invitrogen). Purified cDNA was eluted using 30 µL of preheated sterilized distilled water.

Figure 29 :

 29 Figure 29: Overview of Illumina sequencing for RNAseq experiments. Total RNA is extracted from bacterial cell using a classical extraction protocol, which is follow by depletion from ribosomal RNA and other non-coding RNAs. Depleted mRNAs are next retro-transcribed to create a cDNA library which is tagged using sequencing adapters to create the sequencing library (A). The sequencing library is then attached to the flow cell by complementation with the surface-specific adapter where single fragments are next amplified to generate monoclonal clusters (B). Illumina sequencing uses a reversible terminator-based method allowing the detection of a single base as they are incorporated into DNA template strands; sequential addition of fluorescent-labeled nucleotides allow the detection of nucleotide incorporation in real time (C).Finally, once reads are obtained, data are analyzed using a reference sequence, in our case the genome of strain PA14 (D).

Figure 30 :

 30 Figure 30: Rational of the BACTH technique based on T25 and T18 domains and the reconstitution of adenylate cyclase activity.cAMP is produced when both sub-units of the adenylate cyclase are bind together (A). When both subunits are physically separated, no cAMP is produced (B). The T25 and T18 subunits can be brought back together by the interaction of the proteins X and Y restoring the production of cAMP (C). When using cya -strains, the production of cAMP by the hybrid proteins induces the expression of the maltose operon, which is dependent of the cAMP/CAP complex (D). The activity of the maltose operon can be detected by measuring the activity of gene lacZ or even more simple by plating the clones on reporter plates (McConkey Maltose 1% or MH IPTG/X-Gal).

  The maltose-binding protein (MBP) vectors allow the production and purification of a protein encoded by a cloned gene by fusing it to MBP (encoded by malE). This method uses the strong, inducible tac promoter and the malE translation initiation signal to give high-level production of the recombinant protein. Then, isolation of the fusion protein is achieved by affinity purification for MBP with amylose resin.First, the gene of interest is cloned in-frame with gene malE into one of the MBP vectors, pMALc5x for cytoplasmic proteins or pMALp5x for periplasmic proteins. Both MBP vectors contain a small sequence encoding the recognition site of the specific protease factor Xa, consisting of four amino acids. This protease site allows the target protein to be cleaved from the MBP-Xa protein and to be purified for further analysis (Figure31).

Figure 31 :

 31 Figure 31 : Workflow of MBP-protein production and purification.

  pUT18 (ampicillin R ) and pKNT25 (kanamycin R ) for which the tag is at the C-terminus of the recombinant protein were used in this experiment. Full length alleles of mexT (915-bp) were cloned using primers TH-MexT Fw (CCATGAACCGAAACGACCTGCG) and TH-MexT Rv (AGAGACTGTCCGGATCGCCGA).

Figure 1 :

 1 Figure 1: Crystal structure of DntR dimer. Crystal structure of DntR dimer from Burkholderia spp (PDB 5ae5) (13). One of the monomers is colored in function of its constituting domains (green for the co-inducer binding domain, blue for the DNA binding domain, and orange for the loop linking the two domains). The second monomer is in yellow. Phenylalanine-257 residues of the two monomers are highlighted in red.

Table 1 :

 1 Substrate specificities and regulatory genes of characterized RND efflux pumps of Pseudomonas aeruginosa .........................................................................................................

Table 2 :

 2 Characteristics of a conventional nfxC mutant selected in vitro ................................

Table 3 :

 3 Non-exhaustive list of mexS mutations in clinical strains of P. aeruginosa .............

Table 4 :

 4 Characterization of clinical nfxC mutants harboring single-amino acid substitutions in MexT ....................................................................................................................................

Table 5 :

 5 Effect of mutated alleles of mexT from clinical isolates on gene expression and drug susceptibility. ............................................................................................................................

Table 6 :

 6 Bacterial two-hybrid experiments for modified MexT ..............................................

Table 7 :

 7 Mechanisms of antibiotic resistance induced by stress in P. aeruginosa ..................

Table 8 :

 8 Electrophilic stress response in E. coli and homologous genes in P. aeruginosa .....

Table 9 :

 9 Effect of electrophiles on drug susceptibility and gene expression in strain PA14 ...

Table 10 :

 10

Genotypic and phenotypic features of MexEF-OprN overproducers .................... Table 11: Bacterial strains ...................................................................................................... Table 12: Plasmids ................................................................................................................. Table 13: Culture Media ........................................................................................................ Table 14: Primers used in this work ....................................................................................... x

Table 1

 1 can also be actively exported by MexCD-OprJ(Li and Plésiat, 2016). The paradoxical hypersusceptibility of nfxB mutants to aminoglycosides and β-

lactams (except those cited above) would result from operons mexXY and mexAB-oprM being downregulated to counterbalance mexCD-oprJ overexpression (Gotoh et al., 1998; Jeannot et al., 2008; Li et al., 2000). In addition, MexCD-OprJ overproducers exhibit impaired fitness and virulence (Linares et al., 2005; Martínez-Ramos et al., 2014; Stickland et al., 2010), reinforcing the notion of the strong impact that this pump may have on P. aeruginosa physiology.

Table 2 : Characteristics of a conventional nfxC mutant selected in vitro

 2

	Strain	Relative gene expression a		Resistance (fold change in MICs) b	Virulence
		mexE oprD mexB mexY	CIP CHL TMP IMP TIC GEN	score c
	WT	1x	1x	1x	1x	1x	1x	1x	1x	1x	1x	5/5
	nfxC ≥100x -10x -10x	-5x	32x 32x	16x	4x 0.5x 0.25x	1/5

a Expressed as a ratio to wild-type strain PA14 b CIP, ciprofloxacin; CHL, chloramphenicol; IMP, imipenem; TIC, ticarcillin; GEN, gentamicin c

Table 2 )

 2 (Sobel et al., 2005a).

Gene mexS (1020 bp) is predicted to encode a putative quinone oxidoreductase belonging to the Medium Chain Dehydrogenases-Reductases superfamily (MCDR)

(Winsor et al., 2016)

. Most of bacterial MCDR enzymes are active as tetramers and composed of two domains, namely an N-terminal catalytic domain and a C-terminal co-factor binding domain, which likely utilizes NAD(P)H as co-factor (Nordling et al., 2002; Persson et al., 2008). According to several investigators, MexS could be involved in detoxification of toxic secondary metabolites, that would behave as MexT co-inducers when accumulating in the cell (Fargier et al., 2012; Köhler et al., 1999; Sobel et al., 2005a). In clinical strains of P. aeruginosa, gene mexS is subject to sequence variations most of which have an unknown impact on mexEF-oprN expression (Henrichfreise et al., 2007; Klockgether et al., 2013; Marvig et al., 2015; Smith et al., 2006) (Llanes et al., 2011; Richardot et al., 2016

Table 3 : Non-exhaustive list of mexS mutations in clinical strains of P. aeruginosa

 3

	Patients	Strain screening	Sequencing method	n a	Frameshift mutations Prevalence Nature	Missense mutations Prevalence Amino acid substitutions	Gene mexE expression	References
	CF	Sequentially collected	WGS/Sanger	91	1.1%	Δ6bp	7.6%	R 47 C, A 52 D, P 93 S,	ND	(Smith et al., 2006)
		isolates						D 102 Y, T 177 A, G 244 S,
								F 312 C
	CF and	Multi-resistant isolates	Sanger	22	ND	ND	9.1%	G 68 S, F 273 I	ND	(Henrichfreise et al.,
	non-CF									2007)
	Non-CF	Fluoroquinolone	Sanger	85	1.2%	+3bp	10.6%	K 17 T, E 54 G, A 75 V,	From 128-to	(Llanes et al., 2011)
		resistant isolates						G 78 S, T 152 A, A 175 V,	2,281-fold †
								E 181 D, C 269 Y, S 289 T,
								V 308 I, V 318 I, R 332 W
	CF	Sequentially collected	WGS	474	3.1%	ND	10.9%	G 39 S, P 59 L, P 94 S,	ND	(Marvig et al., 2015)
		isolates						T 119 A, G 224 D, G 238 S,
								G 275 A, A 291 V, H 321 Y
	Non-CF	Ciprofloxacin and	Sanger	221	1.4%	Δ8bp,	8.6%	D 44 E, S 60 F, S 60 P,	From 29-to	(Richardot et al., 2016)*
		imipenem resistant				ΔC 293 ,		V 73 A, V 104 A, A 166 P,	825-fold ‡
		isolates				Δ30bp		F 185 L, C 245 G, N 249 D,
								F 253 L, L 263 Q,
	a number of strains used in the study
	WGS: whole genome sequencing
	ND: not determined
	† compared to PAO1 reference strain, threshold equal to100-fold minimum
	‡ compared to PA14 reference strain, threshold equal to 20-fold minimum

*Single-amino acid substitutions proven to be cause of mexEF-oprN overexpression, by chromosomal complementation in PA14ΔmexS: this work.

Multidrug-resistant mutants of Pseudomonas aeruginosa that overproduce the active efflux system MexEF-OprN (called nfxC mutants) have rarely been characterized in the hospital setting. Screening of 221 clinical strains exhibiting a reduced susceptibil- ity to ciprofloxacin (a substrate of MexEF-OprN) and imipenem (a substrate of the negatively coregulated porin OprD) led to the identification of 43 (19.5%) nfxC mutants. Subsequent analysis of 22 nonredundant mutants showed that, in contrast to their in vitro-selected counterparts, only 3 of them (13.6%) harbored a disrupted mexS gene, which codes for the oxidoreductase MexS, whose inactivation is known to activate the mexEF-oprN operon through a LysR-type regulator, MexT. Nine (40.9%) of the clini- cal nfxC mutants contained single amino acid mutations in MexS, and these were associated with moderate effects on resistance and virulence factor production in 8/9 strains. Finally, the remaining 10 (45.5%) nfxC mutants did not display mutations in any of the regulators known to control mexEF-oprN expression (the mexS, mexT, mvaT, and ampR genes), confirming that other loci are responsible for pump upregulation in patients. Collectively, these data demonstrate that nfxC mutants are probably more frequent in the hospital than previously thought and have genetic and phenotypic features somewhat different from those of in vitro-selected mutants.

TABLE 1

 1 Bacterial strains and plasmids used in this study Becton, Dickinson and Company) supplemented with 200 g ml Ϫ1 tetracycline or 2,000 g ml Ϫ1 streptomycin, as required. Excision of pKNG101 was obtained by selection on M9 minimal medium (8.54 mM NaCl, 25.18 mM NaH 2 PO 4 , 18.68 mM NH 4 Cl, 22 mM KH 2 PO 4 , 2 mM MgSO 4 , pH 7.4) supplemented with 5% sucrose and 0.8% agar.

	Strain or plasmid	Relevant characteristics

TABLE 2

 2 Genotypes and resistance profiles of nfxC mutants

						Clinical nfxC Mutants of P. aeruginosa
		Sequence		mexE transcript	MIC (g ml Ϫ1 ) d
	Strain	MexS or mexS a	MexT or mexT b	level c	CHL	CIP	IMP	TIC	AMK
	Reference strains
	PA14	WT	WT	1	64	0.12	1		2
	PA14⌬S	⌬809 bp (bp 1-809)	WT	427	2,048	2	2		0.5
	PA14⌬S PA14	WT	WT	1.9	64	0.12	1		2
	PA14⌬S PAO1	N 249 D	WT	87	2,048	2	2		1
	PA14⌬T	W T	⌬883 bp (bp 32-915)	0.4	64	0.12	1		2
	PA14⌬T PA14	WT	WT	6.2	64	0.12	1		2
	PAO1	N 249 D	ϩ8 bp (at bp 118)	0.2	16	0.12	1		8
	PAO7H	N 249 D	WT	265	2,048	2	4		4
	Clinical strains with no mutation
	in mexS and mexT
	2502	WT	WT	35	256	0.25	8		2
	1206	WT	WT	41	512	0.5	2		4
	0708	WT	WT	53	256	0.25	1		4
	0309	WT	WT	28	128	1	1		2
	2607	WT	WT	39	256	0.5	2		4
	0712	WT	WT	325	2,048	2	4		16
	0608	WT	WT	25	256	0.25	2		8
	Clinical strains with mutations
	in mexS
	1307	V 104 A	WT	29	256	16	2		8
	2310	F 253 L	WT	183	1,024	0.5	4		8
	2505	D 44 E	WT	212	512	64	16		4
	3005	S 60 F	WT	259	2,048	1	4		4
	0911	F 185 L	WT	133	1,024	0.5	4		2
	1009	V 73 A ϩ L 270 Q	WT	312	1,024	32	8		8
	0801	C 245 G	WT	81	256	0.5	4		64
	1409	A 166 P	WT	179	1,024	2	4		2
	2311	S 60 P	WT	455	1,024	1	4		2
	2609	L 263 Q	WT	534	2,048	1	4		2
	1709	⌬8 bp (bp 710-718)	WT	552	2,048	2	4		2
	1711	⌬C 293	WT	825	32	1	2		8
	0607	⌬30 bp (bp 927-956)	WT	556	512	1	8		2
	Clinical strains with mutations
	in mexT
	0810	WT	G 258 D	254	512	8	4		2
	1510	WT	Y 138 D ϩ G 258 D	20	256	0.25	16		1
	a MexS (339 aa) of PA14 is functional (N 249 ) and is considered the wild type (WT), contrary to PAO1-UW (D 249 ) (www.pseudomonas.com). aa, amino acid.
	b MexT (304 aa) of PA14 is functional and is considered the wild type, contrary to PAO1-UW (ϩ8 bp [starting at bp 118]) (www.pseudomonas.com).
	c Expressed as a ratio to that of wild-type reference strain PA14. nfxC mutants (the values for which are in bold) have a transcript level of Ն20.
	d CHL, chloramphenicol; CIP, ciprofloxacin; IMP, imipenem; TIC, ticarcillin; AMK, amikacin.

TABLE 3

 3 Genotypes and phenotypes of PA14⌬S complemented with mutated mexS alleles from clinical isolates

	MIC (mg ml Ϫ1
	Transcript level a
	MexS sequence	(339 aa f )
		Strain

Table S1 : Origin of the 22 nfxC clinical strains of Pseudomonas aeruginosa.

 S1 Strains 1206 and 0708 were included in the study because they exhibited different serotypes.

	Strains Patient	Dates of	Hospital Units	Samples	Infection /	Antibiotic courses c
		numbers a	isolation			Colonization
	Isolates with no mutation in mexS and mexT
	2502	17	02/25/13 Hepatology	Urine	Infection	ß-Lact, MCL, FQ
	1206	2	06/12/12 Neurosurgery	Tracheal aspirate	ND b	ß-Lact, OXZ, FQ
	0708	2	08/07/12 Neurosurgery	Urine	ND b	ß-Lact, OXZ, FQ
	0309	8	09/03/12 Haematology	Bronchial aspirate Infection	ß-Lact, MCL
	2607	6	07/26/12 Intensive Care	Peritoneal fluid	Infection	ß-Lact, CBP, FQ, AG
	0712	4	07/13/12 Vascular	Fascia of scarpa	Colonization	No antibiotic
				Surgery
	0608	7	08/06/12 Haematology	Mouth	Colonization	ß-Lact,FQ
	Isolates with mutations in mexS
	1307	5	07/13/12 Pneumology	Sputum	ND b	PMX
	2310	13	10/23/12 Intensive Care	Urine	Infection	ß-Lact
	2505	1	05/25/12 Thoracic	Mediastinal fluid	Infection	ß-Lact
				Surgery
	3005	2	05/30/12 Neurosurgery	Tracheal aspirate	ND b	ß-Lact, OXZ, FQ
	0911	14	11/09/12 Gynecology	Breast scare	Colonization	No antibiotic
	1009	9	09/10/12 Intensive Care	Tracheal aspirate	Infection	SUL
	0801	16	01/08/13 Pneumology	Sputum (CF)	Colonization	PMX, FQ
	1409	10	09/14/12 Nephrology	Bone biopsy	Infection	ß-Lact, AG
	2311	14	11/23/12 Oncology	Breast scare	Colonization	No antibiotic
	2609	11	09/26/12 Otolaryngology	Cervical wound	Colonization	No antibiotic
	1709	11	09/17/12 Otolaryngology	Cervical wound	Colonization	No antibiotic
	1711	15	11/17/12 Neurosurgery	Urine	Colonization	No antibiotic
	0607	3	07/06/12 Urologic	Wound	Colonization	No antibiotic
				Surgery
	Isolates with mutations in mexT
	0810	12	10/08/12 Intensive Care	Tracheal aspirate	Infection	CBP, GLP
	1510	12	10/15/12 Intensive Care	Tracheal aspirate	Infection	CBP, GLP

a patients 2, 11, 12 and 14 harbored more than one isolate, but with a same sequence of mexS or mexT b ND : not determined. c before the isolation of P. aeruginosa. ß-Lact: non-carbapenem ß-lactams, CBP: carbapenems, AG: aminoglycosides, FQ: fluoroquinolones, MCL: macrolides, OXZ: oxazolidinones, PMX: polymyxins, SUL: sulfamides, GLP: glycopeptides.

Table S2 : Primers used for DNA sequencing, gene cloning, and RT-qPCR.

 S2

	Primer	Sequence (5' --> 3')	Source
	Sequencing
	Seq-mexS-Ch1	GAACAGGATCAGCAGGTTCA	this study
	Seq-mexS-Ch2	CCACCGGGGTGAGTACCT	this study
	Seq-mexS-Ch3	GTCTCGGCTTCGAACTGG	this study
	Seq-mexS-Ch4	GGTGAAATCCATCAGGCAGT	this study
	Seq-mexS-Ch5	GCAAGCTGGTGCTGTATGG	this study
	Seq-mexS-Ch6	GAAGGCGACTTCGTCTGG	this study
	Seq-mexS-Ch8	TCGAACTGTCCCTTTGCTCT	this study
	seqmexT-1	CTATTGATGCCGAACCTGCT	(Llanes et al. 2011)
	seqmexT-2	AATAGTCGTCGAGGGTCAGC	(Llanes et al. 2011)
	seqmexT-3	TGATGAAAACGGATCACTCG	(Llanes et al. 2011)
	seqmexT-4	GGGAACTAATCGAACGACGA	(Llanes et al. 2011)
	Seq-mexT-mexE-Ch1	AAGCGCAAGGTGGTCCTG	this study
	MvaT-1	CGCGGTTTACTTACAGTTTCG	(Llanes et al. 2011)
	MvaT-2	AACGCTATTCGCTGGAGACT	(Llanes et al. 2011)
	Inactivation of mexS and mexT
	Inac-MexS-Ch1	GACAGGTGGGCGAAGATTT	this study
	Inac-MexS-Ch2	ATCCATCCATCACGGGGTGAATAACCT	this study
	Inac-MexS-Ch3	CGTGATGGATGGATTTCACCGGTCATC	this study
	Inac-MexS-Ch4	CGGCGAGATGTATGTGGTG	this study
	Inac-MexT-Ch1	AGCACATCCTTCCAGCTCAC	this study
	Inac-MexT-Ch2	ATAAGCCGAACACGATCAGCAGGTTCA	this study
	Inac-MexT-Ch3	CGTGTTCGGCTTATTCCATCGAAAGCA	this study
	Inact-MexT-Ch4	GTCGATCTGGAACAGCAGGT	this study
	Complementation of mexS and mexT
	Int-mexS-P1	CGGGGATCCGGGGCATAGGATCACTGACA	this study
	Int-mexS-P2	GCCAAGCTTGGTCAACGATCTGTGGATCTG	this study
	Int-mexS-Ch2	GCCAAGCTTCGAACTGTCCCTTTGCTCTC	this study
	Int-mexT-P1	CGGGGATCCCATCACGGGGTGAATAACCT	this study
	Int-mexT-P2	GCCAAGCTTCGATCGATTTTCCCGTTG	this study
	RT-qPCR
	rpsL3	CAACTATCAACCAGCTGGTG	(Dumas et al. 2006)
	rpsL5	CTGTGCTTTGCAGGTTGTG	(Dumas et al. 2006)
	mexE4	CCAGGACCAGCACGAACTTCTTGC	(Dumas et al. 2006)
	mexE5	CGACAACGCCAAGGGCGAGTTCACC	(Dumas et al. 2006)
	MexSP1	CAAGGGCGTCAATGTCATCC	this study
	MexSP2	GACCGGTGAAATCCATCAGG	this study
	MexT1	ATCTGAACCTGCTGATCGTG	this study
	MexT2	GTCCGGTACGGACGAACA	this study

Table S3 : Complementation of mexS-mutated clinical strains with the wild-type allele. Clinical strains Sequence CIP MICs (µg ml -1 ) after complementation by a MexS (339 aa) a

 S3 (-) clinical strains for which transformation with plasmid pMEQR1 failed or was not possible because of high gentamicin resistance (selection marker of pMEQR1).

			None	pME6001 b	pMEQR1 c
	1307	V104A	16	16	8
	2310	F253L	1	1	0.25
	2505	D44E	64	-	-
	3005	S60F	1	1	0.5
	0911	F185L	1	1	0.25
	1009	V73A+ L270Q	32	-	-
	0801	C245G	0.5	-	-
	1409	A166P	1	1	0.5
	2311	S60P	1	1	0.5
	2609	L263Q	2	2	0.5

a : b : Broad-host range vector pME6001 conferring gentamicin resistance (Blumer et al. 1999). c : Plasmid pME6001 carrying the wild-type mexS gene (Llanes et al. 2011).

Table S4 : Genotype and virulence factors of clinical nfxC mutants.

 S4

	Strains	Sequence			Virulence factors c
		MexS (339 aa) a	MexT (304 aa) b	Biofilm production (OD600)	Swarming motility (+/-)	Elastase activity (mm)	Haemolytic activity (%)	Pyocyanin production (%)	Virulence score (/5) d
	Reference strains
	PA14	WT	WT	2.6	+	18	72	100
	PA14∆S	∆809bp 1-809	WT	0.4	-	12	22	13
	PA14∆SPA14	WT	WT	2.7	+	18	79	92
	PA14∆SPAO1	N249D	WT	0.9	-	13	55	13
	PA14∆T	WT	∆883bp 32-915	1.9	+	16	78	86
	PA14∆TPA14	WT	WT	2	+	18	76	87
	PAO1	N249D	+ 8bp 118	0.5	+	16	77	168
	PAO7H	N249D	WT	0.6	-	0	50	17
	Clinical strains with no mutation in mexS and mexT
	2502	WT	WT	0.5	+	15	62	54
	1206	WT	WT	0.5	+	15	69	87
	0708	WT	WT	0.5	-	0	29	24
	0309	WT	WT	0.4	+	16	63	90
	2607	WT	WT	0.5	-	15	54	15
	0712	WT	WT	0.3	+	15	42	13
	0608	WT	WT	1.3	+	17	74	168
	Clinical strains with mutations in mexS
	1307	V104A	WT	2.2	-	0	20	13
	2310	F253L	WT	1.2	-	11	69	172
	2505	D44E	WT	1.3	-	0	60	12
	3005	S60F	WT	0.5	+	16	68	13
	0911	F185L	WT	0.3	+	16	57	100
	1009	V73A+ L270Q	WT	1.7	-	0	29	11
	0801	C245G	WT	0.1	-	0	15	12
	1409	A166P	WT	0.3	+	16	66	149
	2311	S60P	WT	0.5	+	16	64	11
	2609	L263Q	WT	1.8	-	11	58	12
	1709	∆8bp 710-718	WT	0.4	-	13	47	12
	1711	∆C 293	WT	0.3	+	11	41	245
	0607	∆30bp 927-956	WT	0.5	-	0	47	11
	Clinical strains with mutations in mexT
	0810	WT	G258D	1.4	-	0	28	16
	1510	WT	Y138D+ G258D	1.4	-	0	37	11
	a

Table 4 : Characterization of clinical nfxC mutants harboring single-amino acid substitutions in MexT

 4

	Strains	MexT substitutions (304 aa)	Transcripts levels of mexE a	MICs (µg mL -1 ) b CIP CHL TMP	References
	PA14	WT	1	0.12	64	64	F. Ausubel
	4177	R 166 H	20	0.25 1024	1024	(Llanes et al., 2011)
	4088	G 257 S	112	1	2048 >2048	(Llanes et al., 2011)
	10-12	G 257 A	56	2	64	>2048	(Llanes et al., 2013)
	0810	G 258 D	32	8	1024	1024	(Richardot et al., 2016)
	1510 Y 138 H + G 258 D	21	2	256	512	(Richardot et al., 2016)

a : Expressed as the ratio to the value for wild-type strain PA14. Mean values were calculated from two independent bacterial cultures each assayed in duplicate. b : CIP, ciprofloxacin; CHL, chloramphenicol; TMP, trimethoprim.

Table 5 : Effect of mutated alleles of mexT from clinical isolates on gene expression and drug susceptibility.

 5

	Strain	MexT substitution (304 aa)	Transcript levels of mexE a	MICs (µg mL -1 ) b CIP CHL TMP
	PA14	WT	1	0.12 64	64
	PA14ΔmexT	-	0.4	0.12 32	32
	PA14ΔmexT PA14	WT	2.2	0.12 64	64
	PA14ΔmexT 4177	R 166 H	3.2	0.12 64	64
	PA14ΔmexT 4088	G 257 S	189	2	2,048 >2,048
	PA14ΔmexT 10-12	G 257 A	110	1	1,024 1,024
	PA14ΔmexT 0810	G 258 D	1.9	0.12 64	64
	PA14ΔmexT 1510	Y 138 H + G 258 D	6.3	0.25 64	64

a : Expressed as the ratio to the value for wild-type strain PA14. Mean values were calculated from two independent bacterial cultures each assayed in duplicate. b : CIP, ciprofloxacin; CHL, chloramphenicol; TMP, trimethoprim. In bold: significant overexpression of gene mexE and increased resistance to MexEF-OprN substrates

Table 6 )

 6 , suggesting that they are capable to form active oligomers. Of note, our results suggest that MexT 4088 generates a stronger interaction between its subunits (296 ±14.31 Miller units) than MexT 10-12 (119 ± 5.36 Miller units), in agreement with mexE expression (189-fold and 110-fold for MexT 4088, MexT 10-12 , respectively) and resistance levels

to MexEF-OprN substrates (MICs increased from 16-to >32-fold for MexT 4088 and from 8-to 16-fold for MexT 10-12 ) (Table

5

).

Table 6 : Bacterial two-hybrid experiments for modified MexT

 6

	DHM1 strain containing BACTH plasmids a	Amino acid substitution	β-Gal Activity b (Miller Units)	MH X-Gal MC Maltose
	None	-	15 (±1.91)
	MexT PA14	WT	19 (±1.66)
	MexT 4088	G 257 S	296 (±14.31)
	MexT 10-12	G 257 A	119 (±5.36)

Table 7 : Mechanisms of antibiotic resistance induced by stress in P. aeruginosa

 7

	Stress	Sub-category	Resistance mechanism	Stress-responsive
	category			regulator
	Oxidative	Classical	-MexAB-OprM	MexR, NalC
			-MexXY/OprM	ArmZ
		Nitrosative	-MexEF-OprN	MexT
		Disulfide	-MexEF-OprN	MexT
	Envelope		-LPS modification, MexXY/OprM,	ParRS
			porin (OprD) downregulation
			-LPS modification	CprRS
			-Protection from misfolded proteins	AmgRS
			-MexCD-OprJ	AlgU
	Nutrient	Stringent response	-Antioxidant processes mediated by	Unknown
			ppGpp production
		Mg 2+ /Ca 2+ limitation	-LPS modification	PhoPQ, PmrAB
	Ribosomal		-MexXY/OprM	ArmZ
	Heat-shock		-Protection from misfolded proteins	RpoH, AsrA

Adapted from Fruci and Poole, 2016 and Poole, 2014

  These redox reactions inevitably generate ROS[START_REF] Bueno | Bacterial adaptation of respiration from oxic to microoxic and anoxic conditions: redox control[END_REF]. Of note, environmental factors such as ultraviolet light, air pollutants, heavy metals, and host-responses can modulate ROS formation[START_REF] Fang | Bacterial Stress Responses during Host Infection[END_REF][START_REF] Scandalios | Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses[END_REF]. Oxidative stress is thus defined as an

	Because of its oxidative metabolism, P. aeruginosa is constantly exposed to endogenously-
	produced, harmful molecular species able to provoke intracellular damage. Bacterial exposure
	to environmental signals can be stressful for the microorganism as well. One of the most
	important stresses bacteria have to cope with is caused by reactive oxygen species (ROS)

[START_REF] Betteridge | What is oxidative stress?[END_REF]

. Indeed, during cellular respiration, electrons resulting from oxidation of low-redox-potential electron donors (i.e. NADH), are sequentially transferred through the electron transport chains, up to finally reduce the high-redox-potential electron acceptor O 2 .

Table 8 : Electrophilic stress response in E. coli and homologous genes in P. aeruginosa

 8

	Response Mechanism	Genes in E. coli	Genes in P. aeruginosa *
	Electrophile sensing
		yqhC	PA2047, PA2276
		nemR	PA2196
		Crp	dnr
		Fnr	anr
		nsrR	-
	Detoxification of electrophiles
	GSH-dependent glyoxalase system gloA	gloA1, gloA2, gloA3
		gloB	gloB
	GSH-independent glyoxalase III	hchA	PA1135
	NAD(P)H-dependent enzymes	yqhD	PA2275
		yqhE	PA4167
		ygiN	PA2048
		yafB	-
		yghZ, yajO	PA1739
		yeaE	PA0804
		nemA	xenB, PA2932, PA1334, PA0840
		yhbO	pfpI, PA4171
		yajL	-
		elbB	PA5245
		gldA	-
	Protective mechanisms
	Cytoplasm acidification	kefB	kefB
		kefC	-
	DNA protection	rpoS	rpoS

*Amino acid sequence homology was considered significant when identity was ≥

30% Adapted from Lee and Park., 2017 and Ferguson et al., 1998

  Gene yqhC was discovered in GO-resistant mutants of E. coli that overproduced aldehyde reductase YqhD, an enzyme which catalyzes transformation of GO into the less toxic molecule 1,2-ethanediol (Figure16)(Lee et al., 2010). YqhC directly binds to the promoter region of operon yqhDE, the products of which are required for detoxification of GO, furfural, methylglyoxal and cinnamaldehyde (CNA)(Lee et al., 2010; Turner et al., 2011). However, it

is still unclear whether these electrophiles directly bind to YqhC as ligands or whether the regulator is capable to indirectly detect the presence of these reactive species through a redoxsensing mechanism (Lee and Park, 2017).

  ). Exposure of E. coli or the purified NemR protein to H 2 O 2 does not affect nemRA-gloA expression, suggesting that this regulator is sensitive to RES only(Lee et al., 2013b).The global transcriptional regulator CRP, which is activated by cAMP, modulates expression of more than 180 genes in E. coli[START_REF] Zheng | Identification of the CRP regulon using in vitro and in vivo transcriptional profiling[END_REF]. Screening of GO-resistant insertional mutants revealed that the CRP gene (named crp) was disrupted in some of them[START_REF] Lee | Screening for Escherichia coli K-12 genes conferring glyoxal resistance or sensitivity by transposon insertions[END_REF]. Consistent with this, inactivation of adenylate cyclase gene cya also turned out to confer resistance to GO and MG[START_REF] Lee | Screening for Escherichia coli K-12 genes conferring glyoxal resistance or sensitivity by transposon insertions[END_REF]. Actually, a number of genes involved in Genes fnr and nsrR that encode repressors of aldo-keto reductase YafB gene are other players in electrophile sensing(Lee and Park, 2017). YafB catalyzes reduction of GO and MG into the less toxic metabolites glycoaldehyde and acetol, respectively[START_REF] Kwon | Genomic rearrangements leading to overexpression of aldo-keto reductase YafB of Escherichia coli confer resistance to glyoxal[END_REF]. Fnr and NsrR are global regulators involved in redox-and nitrite-responses, respectively. Deletion of the Fnr and NsrR binding sites upstream from yafB as well as exposure of E. coli to GO were reported to increase yafB expression, suggesting that this induction could be linked to the presence of electrophiles themselves or to their effects on the redox state of cell. Bacterial detoxification of electrophiles (GO and MG) has extensively been studied. It involves both the glyoxalase system and NAD(P)H-dependent enzymes(Abdallah et al., 2016; Adams and Jia, 2005; Booth et al., 2003; Ferguson et al., 1998a; Kwon et al., 2012; Lee et al., 2010, 2013b, 2016; Ozyamak et al., 2013; Sukdeo and Honek, 2007) (Figure

	1.4.2	Detoxification of electrophiles

RES detoxification such as yqhC, yqhD, yafB, and gloA are dependent on cAMP/CRP

[START_REF] Lee | Screening for Escherichia coli K-12 genes conferring glyoxal resistance or sensitivity by transposon insertions[END_REF]

.

TABLE 1 Characterization of nfxC2 mutants Strain CmrA sequence variation Transcript level of a : MIC (g ml ؊1 ) of b :

 1 Expressed as the ratio to the value for wild-type reference strain PA14. Mean values were calculated from two independent bacterial cultures each assayed in duplicate. ND, not determined.

			cmrA	mexE	mexS	mexT	oprD	mexB	mexY	CIP	CHL	TMP	IPM	ATM	GEN
	PA14	WT	1	1	1	1	1	1	1	0.125	64	64	1		1
	PA14ΔmexS	WT	0.6	116	ND	0.9	0.1	0.1	0.2	4	2,048	1,024	4		0.25
	PA14ΔmexT	WT	1	0.4	1.1	ND	1	1	1	0.125	64	64	1		1
	PJ01	A 68 V	71	38	5.6	1.6	0.2	0.2	0.4	1	1,024	512	2		0.5
	PJ02	H 204 L	84	34	2.4	1.3	0.2	0.2	0.4	1	1,024	512	2		0.5
	PJ03	L 89 Q	53	74	2.3	1.6	0.2	0.1	0.4	2	2,048	1,024	2		0.5
	PJ04	N 214 K	120	62	2.8	1	0.2	0.1	0.4	2	2,048	1,024	2		0.5
	PA14ΔcmrA	-c	ND	0.5	1.1	0.6	1	1.1	0.9	0.125	64	64	1		1
	PJ01ΔcmrA	-	ND	0.6	1.6	1.1	1.5	1.1	1.2	0.125	64	64	1		1
	PA14ΔcmrA PA14	WT	1.8	1.7	1.9	0.9	1	0.8	0.9	0.125	64	64	1		1
	PA14ΔcmrA PJ01	A 68 V	68	29	6.1	2.7	0.2	0.1	0.4	1	512	256	2		0.5
	PJ01ΔmexT	WT	76	Ͻ0.1	1.3	ND	2.7	1	1.5	0.125	64	64	1		1
	PJ01ΔmexS	WT	70	Ͻ0.1	ND	1.9	1.3	0.8	1.7	0.125	64	64	1		1

a b CIP, ciprofloxacin; CHL, chloramphenicol; TMP, trimethoprim; IPM, imipenem; ATM, aztreonam; GEN, gentamicin. c -, strain lacks the gene.

TABLE 2

 2 Impact of overexpression of cmrA and PA2048

		Value without (with) arabinose for a :
		Transcript level b			MIC (g ml ؊1 ) c
	Transformant	cmrA	PA2048	mexE	CIP	CHL	TMP
	PA14(pJN105)	1 (1)	1 (1)	1 (1)	0.125 (0.125)	128 (128)	128 (128)
	PA14(pJN105::cmrA PA14 )	385 (Ͼ1,000)	4.5 (3.8)	1.1 (1.3)	0.125 (0.125)	128 (128)	128 (128)
	PA14(pJN105::cmrA PJ01 )	458 (Ͼ1,000)	35 (53)	20 (50)	1 (1)	1,024 (2,048)	256 (512)
	PA14(pJN105::PA2048)	1.6 (4.1)	135 (Ͼ1,000)	52 (270)	1 (2)	1,024 (2,048)	256 (512)

TABLE 3

 3 CmrA-dependent genes

Gene a PAO1 homolog a Name Fold change using b :

			RNA-seq	RT-qPCR	Predicted product
	PA14_40180 PA1881		115	130	Oxidoreductase
	PA14_40200 PA1880		104	152	Aldehyde dehydrogenase
	PA14_40210 PA1879		4	ND	Transcriptional regulator
	PA14_38010 PA2049		13	5	Metallophosphatase superfamily
					protein
	PA14_38020 PA2048		199	154	Quinol monooxygenase YgiN
	PA14_38040 PA2047	cmrA	41	74	AraC family transcriptional
					regulator CmrA
	PA14_38050 PA2046		9	13	Hypothetical protein
	PA14_35130 PA2277	arsR	3	ND	Transcriptional repressor ArsR
	PA14_35140 PA2276		21	22	AraC family transcriptional
					regulator
	PA14_35150 PA2275		62	137	Alcohol dehydrogenase
	PA14_35160 PA2274		9	10	Flavin-dependent monooxygenase

a Gene number from the Pseudomonas Genome Project (http://pseudomonas.com/). b Gene expression in PJ01ΔmexT relative to that in PA14.

TABLE S1

 S1 SNPs identified in the PJ mutants by whole genome sequencing

	Mutant	Gene	Encoded product	SNP	Coverage	Frequency %
	PJ01
		PA14_38040	AraC family transcriptional	C203	37	100
		PA14_43520	regulator Hypothetical protein	T ΔG62	35	36.4
		PA14_68750	Multiple drug resistance protein	3 ΔG24	38	29.6
	PJ03			1
		PA14_11100	Adhesive protein CupB5	ΔG21	22	95.5
		PA14_38040	AraC family transcriptional	08 T266	47	100
		PA14_55670	regulator Exodeoxyribonuclease V subunit	A ΔG35	22	50
	PJ04		β	2
		PA14_34320	DszC family monooxygenase	T1105	22	100
		PA14_38000	Hypothetical protein	C ΔG44	32	100
		PA14_ 38040	AraC family transcriptional	9 C642	47	100
			regulator	G

TABLE S3

 S3 Induction of the glyoxalase system coding genes and other CmrA-dependent genes by electrophilic molecules RT-qPCR assays were performed on culture samples collected after 15 min induction at 37°C. Genes whose expression was significantly activated are marked in bold. GO: glyoxal, MG: methylglyoxal, CNA: cinnamaldehyde.

	Condition a	Transcript levels b gloA1* gloA2* gloA3* gloB* cmrA PA2048 PA2275
	PA14 non-treated control	0.9	1.2	1.0	1.0	1.0	1.0	0.6
	PA14 treated with 200 µg mL -1 GO	0.9	2,367	30	1.1	67	155	17
	PA14 treated with 100 µg mL -1 MG	0.9	3.2	117	1.0	4.7	14	7.0
	PA14 treated with 280 µg mL -1 CNA	2.1	6.6	2.8	2.6	236	376	2,873

a b Expressed as a ratio to the value of PA14 at t0 *glyoxalase system coding genes

TABLE S5

 S5 Primers used for DNA sequencing, gene cloning and RT-qPCR

	Primer Gene sequencing mexS mexSCh1 mexSCh2 mexSCh3 mexSCh4 mexSCh5 mexSCh6 mexT seqmexT1 seqmexT2 seqmexT3 seqmexT4 ampR ampRC1 ampRC2 mvaT mvaT1 mvaT2 mxtR PA2047 cmrA iPA2047-PJ1 iPA2047-PJ2 iPA2047-PJ3 iPA2047-PJ4 mexS Inac-MexS-Ch1 Inac-MexS-Ch2 Inac-MexS-Ch3 Inac-MexS-Ch4 mexT Inac-MexT-Ch1 Inac-MexT-Ch2 Inac-MexT-Ch3 Inac-MexT-Ch4 mexEF-oprN iEFN-1 cmrA pJN-cmrA Fw pJN-cmrA Rv mxtR-PJ1 Gene inactivation PA2048	Sequence (5'→3') GAACAGGATCAGCAGGTTCA CCACCGGGGTGAGTACCT GTCTCGGCTTCGAACTGG GGTGAAATCCATCAGGCAGT GCAAGCTGGTGCTGTATGG GAAGGCGACTTCGTCTGG CTATTGATGCCGAACCTGCT AATAGTCGTCGAGGGTCAGC TGATGAAAACGGATCACTCG GGGAACTAATCGAACGACGA GTCGACCAGTGCCTTCAGGCGATCC CTCGAGAGCGAGATCGTTGCGGCACG CGCGGTTTACTTACAGTTTCG AACGCTATTCGCTGGAGACT CTGTGGGGTGTAAGGGTGAC CGGTGTTCAGGATCAGGCAAGCATTC GATCCTGAACACCGCCTAGGGGAAC TCTCGCTATTGCCGGTATTG GACAGGTGGGCGAAGATTT ATCCATCCATCACGGGGTGAATAACCT CGTGATGGATGGATTTCACCGGTCATC CGGCGAGATGTATGTGGTG AGCACATCCTTCCAGCTCAC ATAAGCCGAACACGATCAGCAGGTTCA CGTGTTCGGCTTATTCCATCGAAAGCA GTCGATCTGGAACAGCAGGT GCGCTAGTCTTGGGAAGGTT AAGGACAGGGCAAGCAG	Source (1) (1) (1) (1) (1) (1) (11) (11) (11) (11) (12) (12) (11) (11) This study This study This study This study (1) (1) (1) (1) (1) (1) (1) (1) This study This study
	pJN-PA2048 Fw	CCAGAACAAGGGGGACGGGC	This study
	pJN-PA2048 Rv	GCATGGCGGAATCTCCGTCA	This study
	RT-qPCR
	rpsL3	CAACTATCAACCAGCTGGTG	(13)
	rpsL5	CTGTGCTTTGCAGGTTGTG	(13)
	mexE4	CCAGGACCAGCACGAACTTCTTGC	(13)
	mexE5	CGACAACGCCAAGGGCGAGTTCACC	(13)
	MexSP1	CAAGGGCGTCAATGTCATCC	(1)
	MexSP2	GACCGGTGAAATCCATCAGG	(1)
	MexT1	ATCTGAACCTGCTGATCGTG	(1)
	MexT2	GTCCGGTACGGACGAACA	(1)
	oprDCh1	ACCAACCTCGAAGCCAAGTA	(14)
	oprDCh2	ACAGGATCGACAGCGGATAG	(14)
	mexY1A	TTACCTCCTCCAGCGGC	(15)
	mexY1B	GTGAGGCGGGCGTTGTG	(15)
	MexB1	ATGACCATCACCGTGACCTT	(16)
	MexB2	AGAGTGGGTCCTGGATGTTG	(16)
	PA2046Fw

Table 9 : Effect of electrophiles on drug susceptibility and gene expression in strain PA14

 9 Chloramphenicol is a good substrate for both MexAB-OprM and MexEF-OprN. Imipenem susceptibility is affected in MexEF-OprN overproducers, though this antibiotic is not a pump substrate. Determined on culture samples collected after 15 min of induction at 37°C and expressed as a ratio to the value at t 0 . Genes whose expression is significantly increased are indicated in bold. Results are mean values of four determinations from two independent experiments.

			Inhibition zone diameter b (mm)
	Conditions a	MexEF-OprN c	MexAB-OprM	MexXY/OprM		Transcript levels d
		CHL	IMP	ATM	TIC	GEN	TMN	mexE	mexB	mexY
	CTRL	30	34	31	28	29	33	1.5	1.2	1.4
	GO	17	30	30	30	25	27	172	2.4	7.7
	MG	21	33	30	29	25	26	31	1.4	6.0
	CNA	14	30	26	22	29	31	233	9.4	1.7

a : CTRL, non exposed control; GO, 200 µg/mL of glyoxal; MG, 100 µg/mL of methylglyoxal; CNA, 280 µg/mL of cinnamaldehyde b : Antibiotics used as markers of efflux pump activities. CIP, ciprofloxacin; IMP, imipenem; ATM, aztreonam; TIC, ticarcillin; GEN, gentamicin; TMN, tobramycin. Significant reduction of inhibition diameters is marked in bold. c : d :

  ;[START_REF] Hsieh | An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides[END_REF].Four different types of in vitro selected nfxC mutants have thus been characterized so far, namely (i) the well-known nfxC mutants harboring disruptive mutations in mexS(Sobel et al.,

2005a), (ii) the nfxC1 subclass with missense mutations in mexS (Richardot et al., 2016), (iii) the nfxC2 mutants with gain-of-function cmrA alleles (Juarez et al., 2017) and, (iv) the nfxC3 harboring gain-of-function mutations in mexT (unpublished data). All these mutants have different phenotypic features summarized in Table

10

.

Table 10 : Genotypic and phenotypic features of MexEF-OprN overproducers

 10

	Strain Genotype a	Frequency	Relative expression of gene mexE b	Ciprofloxacin MIC ratio	Virulence score c
	WT	-	-	1	1	5/5
	nfxC	mexS -	2.5 X 10 -7	>150x	16x	1/5
	nfxC1 nfxC2	mexS* cmrA +	1.4 X 10 -7 2.5 X 10 -8	40x -150x 20x -40x	8x -16x 4x -8x	4/5 5/5
	nfxC3	mexT +	1.9 X 10 -9	>110x	8x -16x	ND

a mexS -: disruptive mutations in mexS; mexS*: missense mutations in mexS; cmrA + : gain-of-function mutations in cmrA. mexT + : gain-of-function mutations in mexT.

Bacterial Strains and Plasmids

  All bacterial strains and plasmids used in this work are listed in Tables

	1	Microbiological Techniques
		1.1

Table 11 : Bacterial strains

 11

	Strain	Phenotype or genotype	Source or Reference
	Pseudomonas aeruginosa
	Reference strains
	PA14	Wild-type reference strain prototroph and sensitive to all	Liberati et al., 2006
		antibiotics. Functional MexS and MexT.
	Environmental strains
	591	Environmental strain serotype O:1	This study
	142951	Environmental strain serotype O:10	This study
	1053	Environmental strain serotype O:5	This study
	2140	Environmental strain serotype O:3	This study
	114793	Environmental strain serotype O:8	This study
	1033	Environmental strain serotype O:12	This study
	2531	Environmental strain serotype O:16	This study
	986-36	Environmental strain serotype O:6	This study
	201	Environmental strain serotype O:4	This study
	1281G	Environmental strain serotype O:13	This study
	2112	Environmental strain serotype O:11	This study
	1972G	Environmental strain serotype O:2	This study
	2910	Environmental strain non-agglutinable	This study
	2998x	Environmental strain poly-agglutinable	This study
	Clinical strains with amino acid substitutions in MexS
	1307	Clinical strain harboring the substitution V 104 A	Richardot et al.,
	2310	Clinical strain harboring the substitution F 253 L	Richardot et al.,
	2505	Clinical strain harboring the substitution D 44 E	Richardot et al.,
	3005	Clinical strain harboring the substitution S 60 F	Richardot et al.,
	0911	Clinical strain harboring the substitution F 185 L	Richardot et al.,
	1009	Clinical strain harboring the substitutions V 73 A + L 270 Q	Richardot et al.,
	0801	Clinical strain harboring the substitution C 245 G	Richardot et al.,
	1409	Clinical strain harboring the substitution A 166 P	Richardot et al.,
	2311	Clinical strain harboring the substitution S 60 P	Richardot et al.,
	2609	Clinical strain harboring the substitution L 263 Q	Richardot et al.,
	1709	Clinical strain harboring a deletion of 8 bp in mexS (710 -718)	Richardot et al.,
	1711	Clinical strain harboring a disrupted mexS gene (∆G 293 )	Richardot et al.,
	0607	Clinical strain harboring a deletion of 30 bp in mexS (927 -956)	Richardot et al.,
	Clinical strains with amino acid substitutions in MexT
	4088	Clinical strain harboring the substitution G 257 S	Llanes et al., 2011
	4177	Clinical strain harboring the substitution R 166 H	Llanes et al., 2011
	10-12	CF clinical strain harboring the substitution G 257 A	Llanes et al., 2013
	0810	Clinical strain harboring the substitution G 258 D	Richardot et al.,
	1510	Clinical strain harboring the substitutions Y 138 H + G 258 D	Richardot et al.,

Table 12 : Plasmids

 12

	Plasmids	Description

Table 13 : Culture Media

 13

	Medium Description	Application	Reference
	cMHA	Calibrated Muller-Hinton Agar. Nutritive	General bacterial growth. Can be	BioRad, Marnes-
		medium with adjusted calibrations of Ca 2+	supplemented with antibiotics	la-Coquette,
		and Mg 2+		France
	cMHB	Calibrated Muller-Hinton Broth. Nutritive	General bacterial growth. Can be	Becton
		broth with adjusted calibrations of Ca 2+	supplemented with antibiotics	Dickinson, Le
		and Mg 2+		Pont de Claix,
				France
	M8-	Minimal medium. NaCl 8 mM, NaH 2 PO 4	Test for swarming mobility	Köhler et al.,
	Swarm	42 mM, KH 2 PO 4 22 mM pH 7.4		2000
		supplemented with glucose 0.2%, MgSO 4
		2 mM, Casaminoacids 0.5% and agar
		0.5%
	M9-Suc	Minimal medium. NaCl 8.54 mM,	Used for excision of pKNG101	Kaniga et al.,
		NaH 2 PO 4 25.18 mM, NH 4 Cl 18.68 mM,	and pFLP2 plasmids	1991
		KH 2 PO 4 22 mM, MgSO 4 2 mM, agar 0.8%
		and sucrose 5%
	MCTH	McConkey agar base supplemented with	Bacterial two-hybrid medium.	Battesti and
		maltose 1%, kanamycin 50 µg/mL, and	Red/White selection	Bouveret, 2012
		ampicillin 100 µg/mL
	MHTH	Muller-Hinton plate supplemented with X-	Bacterial two-hybrid medium.	Battesti and
		Gal 40 µg/mL, kanamycin 50 µg/mL and	Blue/White selection	Bouveret, 2012
		ampicillin 100 µg/mL
	sBAP	Sheep Blood-Agar Plate. Sheep blood 5%,	Production of pyocyanin and	BioRad, Marnes-
		tryptone 0.1% (w/v), NaCl 8 mM, agar	hemolytic activity	la-Coquette,
		0.8%		France
	PYO	Tryptone broth. Tris-HCl 120 mM pH 7.2,	Production of pyocyanin	Essar et al., 1990
	broth	tryptone 0.1 % (w/v), (NH 4 ) 2 SO 4 20 mM,
		CaCl 2 1.6 mM, KCl 10 mM, glucose 50
		mM and sodium citrate 24 mM
	PIA	Pseudomonas isolation agar	Selective medium containing the	Becton
			Irgasan TM antibiotic that inhibits	Dickinson, Le
			gram-positive and gram-negative	Pont de Claix,
			bacteria other than P. aeruginosa	France

Table 14 : Primers used in this work

 14

	Name	Sequence (5'  3')	Reference
	Sequencing
	mexS
	Seq-MexS-Ch1	GAACACGATCAGCAGGTTCA
	Seq-MexS-Ch2	ACCGGGGTGAGTACCT
	Seq-MexS-Ch3	GTCTCGGCTTCGAACTGG	Richardot et al.,
	Seq-MexS-Ch4	GGTGAAATCCATCAGGCAGT	2016
	Seq-MexS-Ch5	GCAAGCTGGTGCTGTATGG
	Seq-MexS-Ch6	GAAGGCGACTTCGTCTGG
	mexT
	Seg-MexT1	TGATGAAAACGGATCACTCG
	Seg-MexT2 Seq-MexT1	GGGAACTAATCGAACGACGA CTATTGATGCCGAACCTGCT	Llanes et al., 2011
	Seq-MexT2	AATAGTCGTCGAGGGTCAGC
	ampR
	AmpRC1 AmpRC2	GTCGACCAGTGCCTTCAGGCGATCC CTCGAGAGCGAGATCGTTGCGGCACG	Bagge et al., 2002
	mvaT
	MvaT1 MvaT2	CGCGGTTTACTTACAGTTTCG AACGCTATTCGCTGGAGACT	Llanes et al., 2011
	mxtR
	mxtR.PJ_1	AAAACTCCGCTCCCATCAG
	mxtR.PJ_2	AGGACGATGCCTTTCAGTTG
	mxtR.PJ_3	GCAAGCAGGAGAACATCACC
	mxtR.PJ_4	AGCATGTCGTTGGAGACCAT
	mxtR.PJ_5	GTAATGCCGGACTCCTTCGT
	mxtR.PJ_6	CATCAGCAGGTCTTCCACCT
	mxtR.PJ_7 mxtR.PJ_8	GTACTGGAAACAGGCCAACC GTCCGGGTACTCGAACAGC	This study
	mxtR.PJ_9	AACATTACCCAGGGCATCAG
	mxtR.PJ_10	GGGCATCGTAGAGGTTGTTC
	mxtR.PJ_11	ACCGACCTGCTGGACATCT
	mxtR.PJ_12	GGCTTGGACAGGTAGTCGAG
	mxtR.PJ_13	GAGACCGGTACGCAATTGAT
	mxtR.PJ_14	GTCTACCATCGCCTGGAAGC
	cmrA PA2047
	AraC-PA2047-PJ1	GGATACTGTCGGGTTCTTGC
	AraC-PA2047-PJ2 AraC-PA2047-PJ3	CAGCAGCCGGTAGAAAATCT CGCCTGATCCATCTGCTG	This study
	AraC-PA2047-PJ4	CAAAGTCGTTCGCTGTGCT
	PA2449
	PA2449-PJ1	CCAGGCCGTACAATCGAC	This study

  Pseudomonas aeruginosa est un pathogène opportuniste à Gram-négatif, responsable d'infections nosocomiales chez des patients immunodéprimés. Il est également la principale cause de morbidité et de mortalité chez les patients atteints de mucoviscidose (CF). Les traitements utilisés contre P. aeruginosa peuvent être mis en échec en raison des nombreux mécanismes de résistance développés par la bactérie tels que les systèmes d'efflux RND, capables d'exporter les antibiotiques à l'extérieur de la cellule. Parmi ces systèmes, MexEF-OprN est très peu produit dans les souches sauvages mais il est surproduit chez les mutants appelés nfxC et conduit à une résistance aux fluoroquinolones, au chloramphénicol et au triméthoprime. Ces mutants ont également la particularité de résister de façon concomitante aux carbapénèmes et d'être peu virulents. Notons enfin que la pompe MexEF-OprN est codée par un opéron à trois gènes, mexEF-oprN, dont la transcription est activée par MexT, un régulateur appartenant à la famille LysR.Les mutants nfxC étant peu décrits dans le contexte clinique, nous avons évalué leur prévalence et caractérisé les événements génétiques conduisant à la surexpression de mexEF-oprN. A partir d'une collection de 221 souches cliniques isolées au CHRU de Besançon, et sélectionnées en raison de leur sensibilité diminuée à la ciprofloxacine et à l'imipénème, 19.5% surexprimaient mexEF-oprN. Nous avons par la suite caractérisé 22 souches non-redondantes et montré que seulement 13.6% d'entre elles possédaient des mutations inactivatrices dans le gène mexS alors que 40.9% avaient des mutations conduisant à la substitution d'un seul acide-aminé. Il est apparu que ces dernières mutations avaient des effets modérés sur les profils de résistance et de virulence alors que les mutations inactivatrices donnaient des hauts niveaux de résistance mais aucune virulence. Enfin, nous n'avons pas pu identifier de mutations génétiques pouvant expliquer la surexpression de mexEF-oprN des 45.5% de souches restantes, suggérant l'existence des mécanismes de régulation encore inconnus de cet opéron.Nous avons donc étudié des mutants résistants au chloramphénicol, sélectionnés in vitro à partir de la souche de référence PA14. Leur caractérisation nous a permis de découvrir un nouveau type de mutants surproducteurs de MexEF-OprN que nous avons appelé nfxC2. Tous possédaient des mutations gain-de-fonction sur le gène PA14_38040 (nommé cmrA) codant pour un régulateur de la famille AraC, jamais étudié auparavant. Chez les mutants nfxC2, l'expression de cmrA est augmentée, ainsi que celle de l'opéron mexEF-oprN et ceci, d'une façon MexS-et MexT-dépendante. De façon intéressante, ces mutations dans cmrA font apparaître un phénotype résistant sans toutefois altérer la virulence de la souche.Une analyse transcriptomique a montré que CmrA pouvait activer l'expression de 11 gènes parmi lesquels PA14_38020 apparaît comme étant nécessaire pour l'activation indirecte de mexEF-oprN. Ce gène code pour une quinol monooxygenase partageant des domaines conservés avec YgiN, une enzyme d'Escherichia coli qui participe à la réponse contre les électrophiles. D'ailleurs, l'exposition de la souche PA14 à des concentrations sub-inhibitrices d'électrophiles toxiques (glyoxal, méthylglyoxal et cinnamaldéhyde) active suffisamment la pompe MexEF-OprN pour générer un phénotype de résistance et ce, de façon CmrA-dépendante. Enfin, cette même exposition aux électrophiles active également deux autres pompes RND, à savoir MexAB-OprM et MexXY/OprM. Les voies de régulation conduisant à l'activation de ces deux opérons d'efflux seront étudiées prochainement au laboratoire.

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Acknowledgments

This manuscript is the result of the collaboration and support of several people that I would like to acknowledge. I would like to start by thanking the two reviewers of my work, Pr. Emile Van Schaftingen and Pr. Antonio Oliver. Thank you for accepting to read and to review my work. Your comments

ACKNOWLEDGMENTS

We thank Fabrice Poncet and Amandine Mariot (SFR FED 4234, Besançon, France) for DNA sequencing data and Loïs Andrey for technical assistance. We also thank the Centre National de Référence (CNR) de la Résistance aux Antibiotiques for the provision of clinical NfxC isolates. Steffi Rocchi contributed to statistical analyses.

This work was supported by a grant from the French Ministère de l'Enseignement Supérieur et de la Recherche.

We have no competing interests to declare.

ACKNOWLEDGMENTS

We are grateful to Xiyu Qian for her excellent technical assistance and to Benoît Valot (UMR 6249 Chrono-Environnement, France) and Emile Van Schaftingen (Institute de Duve, Brussels, Belgium) for helpful discussions. Romé Voulhoux (LISM Marseille, France) and Eric Morello (CEPR, Tours, France) provided plasmids pJN105 and pUC18Tmini-Tn7T-lux-Gm, respectively.

This work was supported with grants from the French Ministère de l'Enseignement Supérieur et de la Recherche, the Vaincre la Mucoviscidose Association, and the

ACKNOWLEDGMENTS

The authors thank Angélique Joriot for her technical assistance in DNA sequencing.

PA14-derived mexEF-oprN deletion mutant This study PJ01 PA14 spontaneous mutant harboring substitution A68V in CmrA This study PJ02 PA14 spontaneous mutant harboring substitution H204L in CmrA This study PJ03 PA14 spontaneous mutant harboring substitution L89Q in CmrA This study PJ04 PA14 spontaneous mutant harboring substitution N214K in CmrA This study PA14ΔcmrA PA14-derived cmrA deletion mutant This study PJ01ΔcmrA PJ01-derived cmrA deletion mutant This study PA14ΔcmrAPA14 PA14ΔcmrA complemented with wild-type cmrA from PA14 This study PA14ΔcmrAPJ01 PA14ΔcmrA complemented with mutated cmrA from PJ01 This study PJ01ΔmexT PJ01-derived mexT deletion mutant This study PJ01ΔmexS PJ01-derived mexS deletion mutant This study PJ01ΔPA2048 PJ01-derived PA2048 a deletion mutant This study PA14::PA2048-lux PA14 containing the luminescent reporter PA2048-lux This study PJ01::PA2048-lux PJ01 containing the luminescent reporter PA2048-lux This study PA14::lux Bioluminescent strain PA14 This study PA14::lux-ΔmexEF-oprN Bioluminescent mutant PA14ΔmexEF-oprN This study PA14::lux-ΔcmrA Bioluminescent mutant PA14ΔcmrA This study Escherichia coli DH5α F -ϕ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk -, mk + ) phoA supE44 thi-1 gyrA96 relA1 λ - AAAACTCCGCTCCCATCAG This study mxtR-PJ2 AGGACGATGCCTTTCAGTTG This study mxtR-PJ3 GCAAGCAGGAGAACATCACC This study mxtR-PJ4 AGCATGTCGTTGGAGACCAT This study mxtR-PJ5 GTAATGCCGGACTCCTTCGT This study mxtR-PJ6 CATCAGCAGGTCTTCCACCT This study mxtR-PJ7 GTACTGGAAACAGGCCAACC This study mxtR-PJ8 GTCCGGGTACTCGAACAGC This study mxtR-PJ9 AACATTACCCAGGGCATCAG This study mxtR-PJ10 GGGCATCGTAGAGGTTGTTC This study mxtR-PJ11 ACCGACCTGCTGGACATCT This study mxtR-PJ12 GGCTTGGACAGGTAGTCGAG This study mxtR-PJ13 GAGACCGGTACGCAATTGAT This study mxtR-PJ14 GTCTACCATCGCCTGGAAGC This study PA2449 PA2449-PJ1 CCAGGCCGTACAATCGAC This study PA2449-PJ2 GTTCGGTTCGTAGAGGGTCA This study PA2449-PJ3 CGCGAAGTGACATTCATGG This study PA2449-PJ4 TTGAGGCGGTAGAACAGGTC This study PA2449-PJ5 GCAACCTGGAGAAGATGGTC This study PA2449-PJ6 CAAGCAGGCGGACTTCAT This study PA2047 cmrA PA2047-PJ1 GGATACTGTCGGGTTCTTGC This study PA2047-PJ2 CAGCAGCCGGTAGAAAATCT This study PA2047-PJ3 CGCCTGATCCATCTGCTG This study PA2047-PJ4 CAAAGTCGTTCGCTGTGCT This study TCAGCTACACCGACGAACTG This study iEFN-2 ACGATGCGCGGAGTCAAGCTCAGAGAC This study iEFN-3 ACTCCGCGCATCGTGGCGATCTACC This study iEFN-4 CTGGTTCGCCGGCTATGT This study PA2048 iPA2048-1 GGAAACCTTCCCAAGACTAGC This study iPA2048-2 GAACAAGCTGCCTCCGATTACCAGTTG This study iPA2048-3 GAGGCAGCTTGTTCTGGCGTCTCAGC This study iPA2048-4 GCCGGGTAACTACCTGATCC This study PA2046 iPA2046-1 CAGGGCCAGCGCCTGCACGAG This study iPA2046-2 AAGCGCCTCCGGCGGGTACGGTTGCGCAAG This study iPA2046-3 CGTACCCGCCGGAGGCGCTTGGCGCGAGGG This study iPA2046-4 TCATTCGCCGGGCTTCGCCG This study PA1880-1881 iPA1880-81-1 TCTCTGCAGGGTCGTCCCCG This study iPA1880-81-2 GTGGTGCTCAGGCCTTGCCGGGCTTGCTCT This study iPA1880-81-3 CGGCAAGGCCTGAGCACCACCCAGGTCGGT This study iPA1880-81-4 GATGCCGCCCTCCATCTGCG This study PA2274 iPA2274-1 CCAGGTGATCCTTTCCACCG This study iPA2274-2 GTAACCCGCCGCCCGCCGGCGCCGACGGCCCG This study iPA2274-3 GCCGGCGGGCGGCGGGTTACTCCGGGTGAC This study iPA2274-4 CAACAGCAGCAGCTTGTCGA This study PA2275 iPA2275-1 GCGTCATAGCGGTAGGTTTC This study iPA2275-2 GTCGACGGTGTGTCCTCTTCGCAGGAT This study iPA2275-3 GACACACCGTCGACCAGGAAGGTCTC This study iPA2275-4 GGCTTGACCTCAAGTTTGCT This study 91 PA2276 iPA2276-1 TGCAGCCAGTCGCCGTTGGC This study iPA2276-2 CAGCCAAGGCCCGGCAGGATCGGGGGCGCT This study iPA2276-3 ATCCTGCCGGGCCTTGGCTGTTTCTCCAGG This study iPA2276-4 CGACCCGGCCGATGATCTCG This study Chromosomal complementation of PA2047 Int-PA2047-Fw1 CGGGGATCCATGACAGTCTGGCCTGTTGA This study Int-PA2047-Fw2 CGGGGATCCCGGATACTGTCGGGTTCTTG This study Int-PA2047-Fw3 CGGGGATCCCTGTTCCTGGCGAATGCT This study Int-PA2047-Rv GCCAAGCTTAAGGGACAGGGCAAGCAG This study 5' RACE cmrA GSP1-PA2047 GTGACCGAGACCACCAGGTA This study GSP2-PA2047 AATGCCTGAGCAGCGACT This study GSP3-PA2047 GCGCTAGTCTTGGGAAGGTT This study PA2048 GSP1-PA2048 CATGGCCGCTTCATCGCTCC This study GSP2-PA2048 GAACGCTGGCATGCTCTCTGC This study GSP3-PA2048 GGTTGCCTGCGCTACGAACT This study Overexpression with the araBAD promoter CGACAAGGCCTTCCTCTACA This study PA2046Rv AGGCATAGCTCAGGTCGAGA This study PA2047Fw AGATTTTCTACCGGCTGCTG This study PA2047Rv GCCGTAGTTCTGGTTGATCC This study PA2048Fw CATGGCCGCTTCATCGCTCC This study PA2048Rv GGTTGCCTGCGCTACGAACT This study PA2049Fw CTTCCAGCACTGGCACTACC This study PA2049Rv CAGGTCCTGCTGGAGTTCAC This study PA2274Fw GAACGCTGCTTCAGGAACTG This study PA2274Rv GTGAACGCCAGCAGGTGTAG This study 92 PA2275Fw GACCAGGTGATCCTTTCCAC This study PA2275Rv GTAGGGATTGAGGTCGTGCT This study PA2276Fw GTCCTGCACCATCAGGCTCC This study PA2276Rv TCGACCTTCCACCACAACTT This study PA1880Fw CTACCGGCCGATGTACTACC This study PA1880Rv GTGCCTTCCAGGATCGACT This study PA1881Fw GGTACGTTCCTGCGTGCT This study PA1881Rv ACTGCGGTACCTGGAGCTT This study Luminescent reporter PA2048-lux PA2048-2047 Fw GTTTCAGAACGGCCGCTCCT This study PA2048-2047 Rv GCGTTCCCCTAGGCGGTGTT This study

Funding : This work was supported with grants from the French "Ministère de l'Enseignement Supérieur et de la Recherche" and from the associations "Vaincre la

Mucoviscidose" and "Grégory Lemarchal".

In vitro mutants PA14ΔmexS PA14-derived mexS deleted mutant Richardot et al., PA14ΔmexT PA14-derived mexT deleted mutant Richardot et al., PA14ΔmexT 0810 PA14ΔmexT strain complemented with mexT from 0810 This study PA14ΔmexT 1510 PA14ΔmexT strain complemented with mexT from 1510 This study PA14ΔmexT 4088 PA14ΔmexT strain complemented with mexT from 4088 This study PA14ΔmexT 4177 PA14ΔmexT strain complemented with mexT from 4177 This study PA14ΔmexT 10-12 PA14ΔmexT strain complemented with mexT from 10-12 This study PA14ΔmexEF-oprN PA14-derived mexEF-oprN deleted mutant This study PA14ΔcmrA PA14-derived cmrA deleted mutant This study PA14ΔmexAB PA14-derived mexAB deleted mutant This study PA14ΔmexEF-oprNΔmexAB PA14-derived mexEF-oprN mexAB double deleted mutant This study PA14ΔcmrA PA14 PA14ΔcmrA complemented with WT cmrA from PA14 This study PA14ΔcmrA PJ01 PA14ΔcmrA complemented with mutated cmrA from PJ01 This study PA14ΔcmrA PJ02 PA14ΔcmrA complemented with mutated cmrA from PJ02 This study PA14ΔcmrA PJ03 PA14ΔcmrA complemented with mutated cmrA from PJ03 This study PA14ΔcmrA PJ04 PA14ΔcmrA complemented with mutated cmrA from PJ04 This study PJ01ΔmexS PJ01-derived mexS deleted mutant This study PJ02ΔmexS PJ02-derived mexS deleted mutant This study PJ03ΔmexS PJ03-derived mexS deleted mutant This study PJ04ΔmexS PJ04-derived mexS deleted mutant This study PJ01ΔmexT PJ01-derived mexT deleted mutant This study PJ01ΔmexEF-oprN PJ01-derived mexEF-oprN deleted mutant This study PJ01ΔcmrA PJ01-derived cmrA deleted mutant This study PJ02ΔcmrA PJ02-derived cmrA deleted mutant This study PJ03ΔcmrA PJ03-derived cmrA deleted mutant This study PJ04ΔcmrA PJ04-derived cmrA deleted mutant This study PJ01ΔPA2046 PJ01-derived PA2046 deleted mutant This study PJ01ΔPA2048 PJ01-derived PA2048 deleted mutant This study PJ01ΔPA1880-81 PJ01-derived PA1880and PA1881 deleted mutant This study PJ01ΔPA2274 PJ01-derived PA2274 deleted mutant This study PJ01ΔPA2275 PJ01-derived PA2275 deleted mutant This study PJ01ΔPA2276 PJ01-derived PA2276 deleted mutant This study PA14ΔIR91 PA14 strain deleted from the inversed repeated region between PA2047 and PA2048 (IR-N91) This study PJ01ΔIR91 PJ01 strain deleted from the inversed repeated region between PA2047 and PA2048 (IR-N91) This study PJ01ΔsoxR PJ01-derived soxR deleted mutant This study PJ01ΔPA2050 PJ01-derived PA2050 deleted mutant This study PA14::PA2048-lux PA14 containing the luminescent reporter PA2048-lux This study PJ01::PA2048-lux PJ01 containing the luminescent reporter PA2048-lux This study PA14-lux Bioluminescent strain PA14 This study PA14ΔmexS-lux Bioluminescent strain PA14ΔmexS This study PA14ΔmexEF-oprN-lux Bioluminescent strain PA14ΔmexEF-oprN This study PA14ΔmexAB Bioluminescent strain PA14ΔmexAB This study PA14ΔmexEF-oprNΔmexAB Bioluminescent strain PA14ΔmexEF-oprNΔmexAB This study PA14ΔcmrA-lux Bioluminescent strain PA14ΔcmrA This study PJ01-lux Bioluminescent strain PJ01 This study

Abbreviations

FUNDING INFORMATION

French Ministère de l'Enseignement Supérieur et de la Recherche provided funding to Charlotte Richardot.

Supplemental data

Grégory Lemarchal Association. The French national reference center for antibiotic resistance is financed by the Ministry of Health through the Santé publique France agency.

The authors declare no competing interests. Δ(ara-leu) araD ΔlacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am) recA1

81

(2)

CC118λpir

CC118 lysogenic for phage λpir (3) HB101 supE44 hsdS20(rB -mB -) recA13 ara-14 pro A2 lacY1 galK2 rpsL20 xyl-5 mtl-1 leuµB6 thi-1

Cloning vector for PCR products, lacZΔColE1 f1 ori, Amp R Kan R Invitrogen pRK2013

Helper plasmid for mobilization of non-self-transmissible plasmids, mob1, tra1 col E1, Kan R

pKNG101 Suicide vector oriR6K sacB insB, Str R (5) Mini-CTX1 Self-proficient integration vector, Ω-FRT-attP-MCS, ori, int oriT, Tet R (6) MiniCTX-lux Mini-CTX1 derived plasmid containing the luxCDABE operon, Tet R (7) pJN105

Broad host range vector carrying the araBAD promoter, Gen R (8) pUC18T-mini-Tn7T-lux-Gm Suicide vector for shuttling single copies of genes directly to the chromosome via a mini-Tn7 element; aacC1, oriT, P1 integron promoter driving expression of luxCDABE, Amp R Gen R

pTNS3 Helper plasmid encoding the Tn7 site-specific transposition pathway, Amp R (10)

a Homolog of PA14_38020 Abbreviations: Amp R (ampicillin resistance), Kan R (kanamycin resistance), Gen R (gentamicin resistance), Str R (streptomycin resistance), Tet R (tetracycline resistance), Zeo R (zeocin resistance)

Bacterial Growth

A bacterial suspension adjusted to an A 600nm of 0.1 in cMHB was cultured during 9 h at 37°C with agitation. Bacterial growth was measured every 30 min up to 8 hours by reading absorbance at A 600nm .

1.6

Killing Experiments Strain PA14 was rendered constitutively bioluminescent by using the pUC18T-mini-Tn7Tlux-Gm plasmid, as described by Damron et al., 2013. Once made bioluminescent, PA14 and its derived mutants PA14ΔmexEF-oprN and PA14ΔcmrA were cultured overnight and diluted into fresh cMHB to yield an A 600nm of 0.1. The strains were incubated with shaking (250 rpm) at 37°C 2.5 h (A 600nm of 0.8) prior to the addition of glyoxal at 1,000 µg/mL, methylglyoxal at 500 µg/mL or cinnamaldehyde at 1,000 µg/mL. Luminescence of surviving bacteria was monitored in white 96-well assay plates (Corning, NY USA) using a Synergy H1 microplate luminometer (Biotek Instruments, Winooski, USA), with a gain set at 150, a read height set at 7 mm and an integration time of one second. The threshold in Log 10 (RLU) was set at 1.69, using sterile cMHB.

Molecular Biology Techniques

Nucleic Acid Extraction

Genomic DNA Extraction

Bacterial genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Charbonnières-les-bains, France) following supplier recommendations.

Plasmid Extraction

Plasmid extraction and purification was performed using Wizard® plus SV Minipreps DNA purification system (Promega) following supplier recommendations.

DNA Amplification by PCR (Polymerase Chain Reaction)

Routine PCRs were performed in a Biometra T3 termocycler (Biolabs Scientific Instrument, Lausanne, Switzerland) using 2.5 U of MyTaq Red DNA polymerase (Bioline, London, UK) and following supplier recommendations. Briefly, 100 ng of DNA were amplified in a Master Mix (50µL) containing: gene specific primers 2µM (Table 14), 2.5 U of DNA polymerase and 10µL of 5x Reaction Buffer. PCR cycling conditions started with an initial denaturation at 95°C during 1 min followed by 30 cycles of: (i) 15 s of denaturation at 95°C (ii) 15 s of annealing using the optimal Tm of each primer and (iii) 10 s of extension at 72°C.

Cloning and Production of MBP-tagged proteins

Recombinant proteins MexS, CmrA and PA2048 tagged with the Mannose Binding Protein (MBP) were produced for further investigation. Briefly, coding sequences of genes mexS, cmrA and PA2048 were amplified using by PCR using specific primers (Table 14).

Amplicons were cloned into the production plasmid pMALc5X as BamHI-NdeI fragments Recombinant plasmids were transfered into ER2523 competent cells by heat-shock transformation as described above (see section 2.4.1).

For protein production, bacteria were grown to mid-log phase (A 600 = 0.8) in 10 mL of cMHB supplemented with ampicillin at 100 µg/mL at 37°C and 250 rpm. Bacteria culture was split in two sub-cultures adjusted to 10mL to evaluate protein production in inducing conditions (isopropyl-1-thio-β-D-galactoside, IPTG 0.3 mM) and non-inducing conditions. Subcultures were grown for three additional hours.

Protein production was checked by standard SDS-PAGE. Briefly, 500 µL of bacteria culture under inducing and non-inducing conditions were pelleted and suspended in 100 µL of Laemmli buffer (65.8 mM Tris HCl pH 6.8, 26.3% glycerol, 2.1% SDS, 0.01% bromophenol blue, and 1% β-mercaptoethanol). Five microliters of each sample was then loaded in a 4 -

VI. Appendix

Constitutive activation of MexT by amino acid substitutions results in

MexEF-OprN overproduction in clinical isolates of

TEXT (979 words)

Pseudomonas aeruginosa, an opportunistic pathogen of major clinical importance, is responsible for acute and chronic infections in vulnerable patients. Its intrinsic and/or acquired resistance to a wide range of antibiotics in part relies on constitutive or inducible production of several efflux systems belonging to the resistance-nodulation-cell division (RND) family of drug transporters (1). Amongst these systems, MexEF-OprN is able to export a rather short list of antimicrobials including ciprofloxacin (CIP), chloramphenicol (CHL), and trimethoprim (TMP). This efflux pump, which is quiescent in wild-type strains, is overproduced at high levels in nfxC mutants, making them more resistant (from 2-to 32-fold) to the pump substrates (2). MexEF-OprN production is regulated by MexT, a LysR-type transcriptional regulator (LTTR), whose gene (mexT) is located upstream of operon mexEF-oprN (3). All mutations that have been identified so far in clinical strains overproducing MexEF-OprN affect a gene, mexS, encoding a presumed quinone oxidoreductase, MexS (4)(5)(6). The present study reports on characterization of five nonclonal clinical mutants harboring a wild-type mexS gene (5-7). DNA sequencing experiments revealed that these strains contained missense mutations in mexT. Since the impact of these mutations on protein function was unknown, we sought to determine whether amino acid substitutions in regulator MexT can account for upregulation of mexEF-oprN operon and drug resistance.

Competing interests : Not declared.

Ethical approval : Not required. -: Deleted gene Significant overexpression of gene mexE (threshold fixed at 20-fold) and increase in resistance to MexEF-OprN substrates in indicated in bold type.

VII. References