Antibiotic Inducibility of the mexXY Multidrug Efflux Operon of Pseudomonas aeruginosa: Involvement of the MexZ Anti-Repressor ArmZ

Thomas Hay; Sebastien Fraud; Calvin Ho-Fung Lau; Christie Gilmour; Keith Poole

doi:10.1371/journal.pone.0056858

Abstract

Expression of the mexXY multidrug efflux operon in wild type Pseudomonas aeruginosa is substantially enhanced by the ribosome-targeting antimicrobial spectinomycin (18-fold) and this is wholly dependent upon the product of the PA5471 gene. In a mutant strain lacking the mexZ gene encoding a repressor of mexXY gene expression, expression of the efflux operon increases modestly (5-fold) and is still responsive (18-fold) to spectinomycin. Spectinomycin induction of mexXY expression in the mexZ mutant is, however, independent of PA5471 suggesting that PA5471 functions as an anti-repressor (dubbed ArmZ for anti-repressor MexZ) that serves only to modulate MexZ's repressor activity, with additional gene(s)/gene product(s) providing for the bulk of the antimicrobial-inducible mexXY expression. Consistent with PA5471/ArmZ functioning as a MexZ anti-repressor, an interaction between MexZ and ArmZ was confirmed using a bacterial 2-hybrid assay. Mutations compromising this interaction (P68S, G76S, R216C, R221W, R221Q, G231D and G252S) were identified and localized to one region of an ArmZ structural model that may represent a MexZ-interacting domain. Introduction of representative mutations into the chromosome of P. aeruginosa reduced (P68S, G76S) or obviated (R216C, R2211W) antimicrobial induction of mexXY gene expression, rendering the mutants pan-aminoglycoside-susceptible. These data confirm the importance of an ArmZ-MexZ interaction for antimicrobial-inducible mexXY expression and intrinsic aminoglycoside resistance in P. aeruginosa.

Citation: Hay T, Fraud S, Lau CH-F, Gilmour C, Poole K (2013) Antibiotic Inducibility of the mexXY Multidrug Efflux Operon of Pseudomonas aeruginosa: Involvement of the MexZ Anti-Repressor ArmZ. PLoS ONE 8(2): e56858. https://doi.org/10.1371/journal.pone.0056858

Editor: Willem van Schaik, University Medical Center Utrecht, The Netherlands

Received: November 26, 2012; Accepted: January 17, 2013; Published: February 18, 2013

Copyright: © 2013 Hay et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by an operating grant from Cystic Fibrosis Canada (http://www.cysticfibrosis.ca). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Multidrug efflux systems of the 3-component Resistance-Nodulation-Division (RND) family are significant contributors to intrinsic and acquired antimicrobial resistance in a number of Gram-negative bacteria [1], [2], including Pseudomonas aeruginosa [3]. P. aeruginosa expresses several RND type multidrug efflux systems of which four, MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM, are significant determinants of multidrug resistance in lab and clinical isolates [4], [5]. MexXY-OprM is, however, somewhat unique in P. aeruginosa in providing resistance to the aminoglycoside class of antimicrobials [6]–[8] and in being inducible by many of its substrate antimicrobials [9].

The MexXY-OprM system is comprised of a cytoplasmic membrane antibiotic-proton antiporter (MexY), an outer membrane porin (OprM) and a periplasmic membrane fusion protein that joins the membrane-associated components together (MexX) [10]. The MexXY components are encoded by an operon under the control of an adjacent repressor gene, mexZ [10], [11], while OprM, which functions as the outer membrane component of several multidrug efflux systems in P. aeruginosa [2], is encoded by the 3^rd gene of an additional multidrug efflux operon, mexAB-oprM [12]. Only the mexXY operon is antimicrobial-inducible, with only those agents known to target the ribosome promoting mexXY expression [9], [13], [14], and this is compromised by so-called ribosome protection mechanisms [13], suggesting that the MexXY efflux system is recruited in response to ribosome disruption or defects in translation. Consistent with this, mutations in fmt (encoding a methionyl-tRNA-formyltransferase) [15], folD (involved in folate biosynthesis and production of the formyl group added to initiator methionine) [15], and the ribosomal protein genes rplA [16], rplY [17], the rplU-rpmA operon [18], all of which are expected to negatively impact protein synthesis, increase expression of mexXY. Upregulation of mexXY by antimicrobials [14] or mutations (fmt/folD [15], rplY [17] and rplU-rpmA [18]) is dependent upon a gene, PA5471, encoding a conserved hypothetical protein. Expression of PA5471 is also promoted by ribosome-disrupting antimicrobials [14] and fmt/folD [15] or rplU-rpmA [18] mutations via a translational attenuation mechanism [19].

MexXY expression, while uncommon as a mechanism of aminoglycoside resistance in most clinical strains of P. aeruginosa, is the predominant mechanism of resistance to these agents in lung isolates of infected cystic fibrosis (CF) patients [6]–[8], with mutations in mexZ common in pan-aminoglycoside-resistant CF isolates expressing mexXY [6], [20]–[23]. Indeed, mexZ has been identified as the most commonly mutated gene in P. aeruginosa CF isolates [24]. Recently, an interaction between MexZ and PA5471 has been reported, with PA5471 apparently modulating the repressor activity of MexZ [25]. We show here that the requirement for PA5471 for antimicrobial induction of mexXY expression is limited to wild type cells expressing the MexZ repressor, suggesting that PA5471 functions solely as an MexZ anti-repressor in the antimicrobial induction of mexXY. Further, we identify a putative MexZ-binding region in PA5471 and demonstrate that the interaction of this anti-repressor, dubbed ArmZ, with MexZ is key to the antimicrobial inducibility of this efflux system and its promotion of aminoglycoside resistance in P. aeruginosa.

Materials and Methods

Bacterial strains, plasmids, and growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1. All bacterial strains were cultured at 37°C in Luria (L-) broth or on L-agar, unless otherwise indicated, with antimicrobials added as necessary. Plasmid pEX18Tc and its derivatives were selected with 10 (in E. coli) or 50 (in P. aeruginosa) µg/ml tetracycline. Plasmid pMS604 and derivatives were maintained in E. coli with 10 µg/ml tetracycline. Plasmid pDP804 and derivatives were maintained in E. coli with 100 µg/ml ampicillin. Plasmid pSF004, a pMS604 derivative carrying the mexZ gene, was constructed by cloning a polymerase chain reaction (PCR)-generated product amplified from P. aeruginosa K767 chromosomal DNA using primers MSmexZ-F (5′-GACTCTGCAGGTGGCCAGGAAAACCAA-3′; PstI site underlined) and MSmexZ-R (5′-GACTCAGCTGTCAGGCGTCC-GCCAGCAA-3′; PvuII site underlined). The 50-µl PCR reaction mixture contained 1 µg of chromosomal DNA, 0.2 µM of each primer, 0.2 mM each deoxynucleoside triphosphate (dNTP), 10% (vol/vol) dimethyl sulphoxide (DMSO), 1× Pfu polymerase buffer and 1.25 U Pfu DNA polymerase (Promega, Madison, WI). Following an initial denaturation step at 95°C for 3 min, the mixture was subjected to 30 cycles of 95°C for 45 sec, 60°C for 45 sec and 72°C for 2 min, before finishing with a 5-min incubation at 72°C. The mexZ-containing PCR product was gel-purified (see below), digested with PstI and PvuII and cloned into PstI-PvuII-restricted pMS604. PA5471 (renamed armZ) was cloned into pDP804 on a PCR product that was generated with primers DP5471-F (5′-GACTCTCGAGATGGGCAACTACATCAAG-3′; XhoI site underlined) and DP5471-R (5′-GACTAGATCTTCATCGGCAGCACTCCCC-3′; BglII site underlined) in a reaction mixture formulated and processed as for mexZ. The armZ-containing PCR product was digested with XhoI and BglII and cloned into XhoI-BglII-restricted pDP804 to yield plasmid pSF005. armZ was also cloned into plasmid pEX18Tc on a PCR product generated with primers EX18armZ-F (5′-CGATGGTACCCCGGCAAGGAGTCCTTCATGACCTT-3′; KpnI site underlined) and EX18armZ-R (5′-CGATCTGCAGGCGCTCCGCGCCGATGAA-3′; PstI site underlined). The reaction mixture was formulated as above except that primers and DMSO were included at 0.6 µM and 5% (vol/vol), respectively, and 1 U Phusion High Fidelity DNA polymerase (Promega, Madison, WI) was employed in 1× Phusion HF buffer. Following an initial denaturation step at 98°C for 30 sec, the mixture was subjected to 30 cycles of 98°C for 30 sec, 68°C for 30 sec and 72°C for 1 min, before finishing with a 7-min incubation at 72°C. The armZ-containing PCR product was digested with KpnI and PstI and cloned into KpnI-PstI-restricted pEX18Tc to yield plasmid pTH008.

Download:

Table 1. Bacterial strains and plasmids.

https://doi.org/10.1371/journal.pone.0056858.t001

DNA methods

Standard protocols were used for restriction endonuclease digestions, ligations, transformations, and agarose gel electrophoresis, as previously described [26]. Plasmid DNA was extracted from E. coli using the Fermentas GeneJET Plasmid Miniprep Kit or the Qiagen Plasmid Midi Kit according to protocols provided by the manufacturers. Chromosomal DNA was extracted from P. aeruginosa using the Qiagen DNeasy Blood & Tissue Kit according to a protocol provided by the manufacturer. PCR products and restriction endonuclease digestion products requiring purification were purified using the Promega Wizard SV Gel and PCR Clean-Up System (Promega Corp., Madison, WI) according to a protocol provided by the manufacturer. CaCl₂-competent E. coli [26] and electrocompetent P. aeruginosa [27] cells were prepared as previously described. Oligonucleotide synthesis was performed by Integrated DNA Technologies (Coralville, Iowa), and nucleotide sequencing was performed by ACGT Corporation (Toronto, Canada).

Two-hybrid assay for ArmZ-MexZ interaction

To assess a potential ArmZ-MexZ interaction and the possible negative impact of armZ mutations on that interaction, E. coli SU202 harbouring the pMS604::mexZ plasmid pSF004 together with plasmid pDP804 derivatives carrying wild type or mutated armZ was cultivated overnight in L-broth containing tetracycline and ampicillin, diluted 1∶49 into the same medium containing in addition 5 mM isopropyl-thio-β-D-galactopyranoside (IPTG), and grown to mid-log phase before being assayed for β-galactosidase activity as described previously [28]. E. coli strain SU202 harbours a chromosomal lacZ gene engineered to contain a hybrid lexA operator sequence in the promoter region to which a heterodimer only of the LexA_WT-LexA₄₀₈ DNA-binding domains encoded by pDP804 and pMS604, respectively, can bind. pDP804- and pMS604-encoded LexA lack the natural dimerization domains of this protein, but fusion of the individual LexA DNA-binding domains encoded by these vectors to proteins that do interact can promote their dimerization and, ultimately, binding to the lexA hybrid operator upstream of lacZ in SU202, effectively repressing lacZ expression [29]. Thus, an interaction between ArmZ and MexZ encoded by pDP804 and pMS604, respectively, should promote dimerization of the LexA DNA-binding domains of these vectors and repression of lacZ, observable as lack of or reduction in β-galactosidase activity. As well, armZ mutations that compromise this interaction would interfere with LexA dimerization and lacZ repression, thereby increasing β-galactosidase activity. Production of ArmZ and MexZ proteins (as LexA fusions) was confirmed in all cases following immunoblotting of whole cell protein extracts with anti-LexA antibodies as described previously [30].

Screening hydroxylamine-mutagenized armZ for mutations abrogating ArmZ-MexZ interaction

The armZ-carrying pDP804 derivative pSF005 was mutagenised by a 50-min treatment with hydroxylamine at 70°C as described [31]. Twenty µl of the 225-µl mutagenesis mixture was treated with Tris-HCl (100 mM; to inactivate the hydroxylamine) and plasmid DNA was recovered (using the Promega Wizard SV Gel and PCR Clean-Up System) for transformation into E. coli SU202 harbouring the mexZ-carrying pMS604 derivative, pSF004. Transformants carrying both plasmids were selected on L-agar containing ampicillin and tetracycline, and supplemented with IPTG (5 mM) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; 80 µg/ml). Colonies that appeared blue on these agar plates (i.e. β-galactosidase-positive), indicative of a lack of or reduced ArmZ-MexZ interaction, were recovered, increased β-galactosidase activity confirmed [28] and the pDP804-resident armZ genes sequenced.

Generation of armZ missense mutants

Selected armZ missense mutations were engineered into P. aeruginosa strain K767 following their creation in the armZ-carrying pEX18Tc plasmid, pTH008, and mobilization into strain K767. The mutations were introduced into pTH008 using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, La Jolla, CA) according to the manufacturer's instructions. Mutations were confirmed by sequencing and the mutant pTH008 derivatives were mobilized into P. aeruginosa K767 from E. coli S17-1 as before [32]. P. aeruginosa harbouring chromosomal inserts of these pTH008 derivatives were selected on L-agar containing tetracycline (50 µg/ml) and chloramphenicol (5 µg/ml; to counter-select donor E. coli), and subsequently patched onto L-agar containing sucrose (10% [wt/vol]). Sucrose-resistant colonies were then screened on paromomycin (128 µg/ml; ½ MIC for K767) to identify colonies that were paromomycin-sensitive and, so, likely to harbour the armZ mutations. The armZ genes of these were sequenced following their amplification using primers armZ767-F (5′-CGATCTGCAGGGCTTCGGTCTGCCCCCCGGATCTA-3′) and armZ767-R (5′-CGATGGATCCACAGCCGCTCGAACGCCTTCGCCAC-3′) and Phusion High Fidelity DNA polymerase. Reaction mixtures were formulated as above for construction of plasmid pTH008, with the exception that DMSO was included at 10% (vol/vol), and were subjected to an initial denaturation step at 98°C for 2.5 min, followed by 30 cycles of 98°C for 30 sec, 66.5°C for 30 sec and 72°C for 30 sec, before finishing with a 7-min incubation at 72°C.

Antimicrobial susceptibility testing

The susceptibility of bacterial strains to various antimicrobials was assessed using the two-fold serial dilution technique in 96-well microtiter plates as previously described [33].

Quantitative real-time PCR

RNA was prepared from log phase cells grown in L-broth without or with ¼ MIC of spectinomycin (128 µg/ml; added 90 min prior to harvesting) as described previously [18]. RNA conversion to cDNA and assessment of mexX expression using quantitative real time PCR (qRT-PCR) was carried out as described [18].

Protein structure modelling

The ArmZ model was developed by threading the ArmZ sequence primarily onto the crystal structure of an RtcB homolog protein (PH1602-extein protein) from Pyrococcus horikoshii (PDB code: 1UC2, chain A) using the SWISS-MODEL program [34]. The model was visually inspected and subjected to energy minimization using GROMOS [35].

Results and Discussion

The requirement for PA5471 (ArmZ) for drug-inducible mexXY expression is MexZ-dependent

Previous studies have demonstrated that the induction of mexXY expression in response to ribosome-disrupting antimicrobials is dependent upon the PA5471 gene product [14]. In agreement with this, induction of mexXY by a model ribosome-disrupting compound, spectinomycin, the agent observed to most strongly induce this efflux operon (C.H.F. Lau, unpublished), was wholly compromised in a ΔPA5471 mutant (Fig. 1; compare P. aeruginosa strains K767 and K2413). Given that PA5471 was itself inducible by the same ribosome-targeting agents as mexXY and that cloned PA5471 was able to promote mexXY expression in the absence of antimicrobials, it was reasoned that antimicrobial induction of mexXY resulted from antimicrobial induction of PA5471, which functions as a simple anti-repressor to block MexZ repression of mexXY and, so, promote expression of this efflux operon [14]. When examining the impact of a mexZ knockout on mexXY expression, however, it was noted that loss of this repressor enhanced mexXY expression only 5-fold (Fig. 1; see K1525), much less than the ca. 18-fold increase in mexXY expression that was afforded by spectinomycin exposure (Fig. 1). This result is in agreement with an earlier study which showed that a mutation causing hyperexpression of PA5471 only modestly enhanced mexXY expression, and less than was observed in antimicrobial-exposed cells [19]. Clearly, then, drug induction of mexXY involves more than drug inducible production of PA5471 which then operates as a MexZ anti-repressor. Indeed, exposure of the ΔmexZ mutant strain K1525 to spectinomycin markedly enhances mexXY expression, to levels seen for spectinomycin-exposed wild type strain K767 (Fig. 1), indicating that loss of MexZ repression contributes only modestly to spectinomycin induction of mexXY expression. Moreover, elimination of PA5471 in the ΔmexZ mutant had no effect on spectinomycin-inducible mexXY expression, in contrast to its negative impact in a MexZ⁺ strain, K767 (Fig. 1), indicating that PA5471 was required for antimicrobial-inducible mexXY expression only when the MexZ repressor was present. This was consistent with with PA5471 serving only as a MexZ anti-repressor, with additional gene(s)/gene product(s) responsible for maximal drug-inducible efflux gene expression. For this reason we have named PA5471 armZ (antirepressor MexZ).

Download:

Figure 1. Influence of ArmZ on spectinomycin-inducible mexXY expression.

mexX expression was assessed in the indicated P. aeruginosa strains in the absence (−) or presence (+) of spectinomycin (SPC) using quantitative RT-PCR. The armZ and mexZ status (−, absent; +, present) of the strains is highlighted. Expression was normalized to rpoD and is reported relative (fold change) to the wild-type P. aeruginosa PAO1 strain K767 not exposed to spectinomycin. Values represent the mean ± SEM from at least three independent determinations, each performed in triplicate.

https://doi.org/10.1371/journal.pone.0056858.g001

Identification and localization of mutations compromising the ArmZ-MexZ interaction

A previous study using a yeast two-hybrid approach demonstrated an interaction between MexZ and ArmZ [25]. In attempting to confirm these results we employed a bacterial 2-hybrid system [29] in which the armZ and mexZ genes were cloned in-frame to coding sequences for the DNA-binding domain of LexA on plasmids pDP804 and pMS604, respectively, and introduced into E. coli SU202 carrying a chromosomal lacZ gene under the control of a LexA operator. LexA binding to its operator and subsequent repression of lacZ in this strain requires prior dimerization of the LexA-binding domains encoded by pDP804 and pMS604, necessitating interaction of the ArmZ and MexZ sequences fused to the LexA DNA-binding domains of these vectors. As such, lack of or reduced β-galactosidase activity is a measure of the ArmZ-MexZ interaction. The two-hybrid vectors also contain sequences encoding Jun and Fos zipper motifs (which are known to interact) fused to lexA, such that E. coli SU202 carrying these vectors demonstrates substantial repression of lacZ (Fig. 2A). The unaltered vectors thus provide a positive control for the system, although the Jun and Fos zipper-encoding sequences will be disrupted upon cloning of armZ and/or mexZ sequences, making lacZ repression dependent upon the ArmZ-MexZ interaction. Initially, no evidence for an interaction was obtained (no decrease in β-galactosidase activity was observed in E. coli SU202 harbouring pMS604 and pDP804 derivatives with both mexZ and armZ relative to one or the other alone; data not shown). When these experiments were carried out using SU202 cells exposed to IPTG (to enhance expression of the mexZ and armZ genes cloned into pMS604 and pDP804, respectively), however, a reduction in β-galactosidase activity was observed relative to SU202 carrying the mexZ plasmid only (Fig. 2A). Apparently, the relatively lower levels of the armZ and mexZ products expressed from pMS604 and pDP804 in the absence of IPTG were insufficient for the assay to detect an interaction. This was in contrast with an earlier study of another anti-repressor in P. aeruginosa, ArmR, which targets the MexR repressor of the mexAB-oprM multidrug efflux operon and whose interaction with MexR was confirmed using the same bacterial 2-hybrid assay without IPTG induction [30]. This suggests that the relative affinity of ArmZ for MexZ is low, requiring higher protein levels to see it in the 2-hybrid assay, though it may be enhanced in vivo by the effects of ribosome perturbation. Consistent with a somewhat weaker ArmZ-MexZ interaction, only a 3-fold reduction β-galactosidase activity was observed when mexZ and armZ were both present in E. coli SU202, in contrast with the >100-fold reduction seen previously for armR and mexR [30]. Nonetheless, these data confirm an interaction between ArmZ and MexZ, consistent with ArmZ functioning as a MexZ anti-repressor.

Download:

Figure 2. MexZ-ArmZ interaction.

A MexZ-ArmZ interaction and the impact of armZ mutations this interaction was assessed using a bacterial 2-hybrid assay, in which the interaction leads to repression of lacZ expression and reduced β-galactosidase activity in the reporter E. coli strain SU202. (a) The β-galactosidase activity of E. coli SU202 harbouring pMS604 derivatives expressing wild type (WT) or no (−) MexZ together with DP804 derivatives expressing wild type (WT), mutant (amino acid substitutions highlighted) or no (−) ArmZ is indicated. The results shown are mean ± SEM from at least three independent determinations, each performed in triplicate. (b) Western immunoblot of whole cell extracts of E. coli SU202 harbouring pMS604 derivatives expressing wild type MexZ and pDP804 derivatives expressing wild type and mutant ArmZ developed with anti-LexA antibodies.

https://doi.org/10.1371/journal.pone.0056858.g002

To assess the details of the ArmZ-MexZ interaction, including identification of residues or regions of ArmZ important for or involved in this interaction, mutations in armZ compromising this interaction were selected using the two-hybrid system mentioned above. Thus, armZ-carrying pDP804 was mutagenized and introduced into pMS604::mexZ-carrying E. coli SU202, and LacZ⁺ colonies (i.e., blue colonies) were selected on L-agar containing X-Gal and IPTG since a defect in the ArmZ-MexZ interaction was expected to obviate lacZ repression in the SU202 reporter strain. Following confirmation that putative mutant ArmZ proteins (as fusions to LexA) defective in the MexZ interaction were expressed (to eliminate further study of mutations compromising ArmZ production or stability, which would also compromise the interaction with MexZ and obviate lacZ repression in SU202), armZ genes were sequenced and the genes carrying single point mutations were saved for further study. Seven mutant armZ genes producing stable ArmZ (-LexA) products (Fig. 2B) carried single mutations (P68S, G76S, R216C, R221W, R221Q, G231D and G252S) and were shown in β-galactosidase assays to be defective in MexZ interaction (i.e., the β-galactosidase activity increased relative to that of wild-type ArmZ) (Fig. 2A). Using a model for ArmZ constructed by threading the ArmZ sequence through the structure of the PH1602 intein from Pyrococcus horikoshii (PDB code: 1UC2), a homologue with 43% overall similarity (28% identity), these resides were mapped to a region of ArmZ within or in proximity to a large α-helix on one side of the protein, which may thus be or contribute to the MexZ interacting domain (Fig. 3). An ArmZ-interacting domain has also been proposed for MexZ, located within the C-terminal region of the protein [36], one of two regions where mexZ mutations tend to cluster in CF isolates of P. aeruginosa [24]. Unlike the mutations that occur in the second region, within the N-terminally-located DNA-binding helix-turn-helix where they compromise MexZ binding to target DNA [36], mutations in the C-terminal domain have a minimal impact on DNA binding [36]. In linking these latter mexZ mutations to mexXY derepression one possibility is that they enhance ArmZ binding to and modulation of the repressor activity of MexZ.

Download:

Figure 3. Location of mutations in ArmZ that compromise its interaction with MexZ.

The 3-dimensional model of ArmZ was created by threading the ArmZ amino acid sequence onto the crystal structure of the PH1602-extein protein from Pyrococcus horikoshii (PDB code: 1UC2, chain A).

https://doi.org/10.1371/journal.pone.0056858.g003

ArmZ mutations compromising its interaction with MexZ block drug-inducible mexXY expression

In order to assess the importance of ArmZ-MexZ interaction for drug-inducible mexXY expression in P. aeruginosa, representatives of the armZ mutations compromising its interaction with MexZ (P68S, G76S, R216C and R221W) were engineered into the chromosome of P. aeruginosa strain K767 and the impact on drug (i.e. spectinomycin) induction of mexXY was assessed. All mutations yielded a reduction in spectinomycin-induced mexXY expression, with the armZ (R216C) and armZ (R221W) mutations that occur within the aforementioned α-helix almost completely obviating spectinomycin induction of efflux gene expression (Fig. 4). Consistent with these results, the four mutants harbouring armZ mutations were pan-aminoglycoside susceptible, reminiscent of the armZ knockout strain K2413, with the armZ (R216C) and armZ (R221W) mutants the most susceptible overall (Table 2). The spectinomycin MICs in particular agree perfectly with the mexXY qRT-PCR data for all mutants. These results confirm that the predicted C-terminal α-helix of ArmZ is important for MexZ interaction and that such interaction is critical for drug-inducible mexXY expression. Still, there appears to be a large segment of ArmZ that is not involved in the interaction with MexZ, and ArmZ is considerably larger (379 amino acids) than other examples of anti-repressors that function only to interact with and modulate repressor binding to target DNA (e.g., ArmR is comprised of 52-amino acids [30] while the CarS protein that modulates the activity of a CarA repressor that controls expression of carotenoid biosynthetic genes in Myxococcus xanthus is an 111-amino acid protein [37]). Indeed, ArmZ is more reminiscent of larger anti-repressors such as NifL (519 amino acids) and AppA (450 amino acids), which modulate the activities of the NifA repressor of nitrogen fixation (nif) genes in Proteobacteria [38] and the PpsR repressor of photosynthesis genes in Rhodobacter sphaeroides [39], respectively. Significantly, these anti-repressors respond/bind to additional signals/cofactors that impact the interactions with their cognate repressors [38], [39], with the additional protein sequences responsible for cofactor binding/signal responding and ‘communicating’ with the repressor-binding domain. Thus, it is likely that ArmZ also responds to additional signals(s) likely related to and downstream of ribosome perturbation as discussed above, and these promote ArmZ interaction with MexZ in vivo. What these co-factors/signals might be is, at present, unknown. Interestingly, this represents the 3^rd example of multidrug efflux gene regulation in Pseudomonas involving an anti-repressor (the activity of the SrpS repressor of the srpABC solvent exporter operon in Pseudomonas putida is also modulated by an anti-repressor, SrpR [40]). The significance of this observation, if any, is unclear.

Download:

Figure 4. Impact of armZ mutations on spectinomycin-inducible mexXY expression.

mexX expression was assessed in P. aeruginosa strains expressing wild type (WT) or mutated (amino acid substitution highlighted) armZ genes in the absence (−) or presence (+) of spectinomycin (SPC) using quantitative RT-PCR. Expression was normalized to rpoD and is reported relative (fold change) to the wild-type P. aeruginosa PAO1 strain K767 not exposed to spectinomycin. Values represent the mean ± SEM from at least three independent determinations, each performed in triplicate.

https://doi.org/10.1371/journal.pone.0056858.g004

Download:

Table 2. Influence of armZ mutations on aminoglycoside resistance in P. aeruginosa.

https://doi.org/10.1371/journal.pone.0056858.t002

Acknowledgments

The authors thank Z. Jia for providing the ArmZ model.

Author Contributions

Conceived and designed the experiments: TH SF CHL CG KP. Performed the experiments: TH SF CHL CG. Analyzed the data: TH SF CHL KP. Wrote the paper: TH KP.

References

1. Poole K (2005) Efflux-mediated antimicrobial resistance. J. Antimicrob. Chemother 56: 20–51.
- View Article
- Google Scholar
2. Poole K (2004) Efflux-mediated multiresistance in Gram-negative bacteria. Clin. Microbiol. Infect 10: 12–26.
- View Article
- Google Scholar
3. Poole K (2003) Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms. In: Paulsen IT, Lewis K, editors. Microbial multidrug efflux. Norwich: Horizon Scientific Press. pp. 273–298.
4. Poole K (2005) Aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother 49: 479–487.
- View Article
- Google Scholar
5. Poole K (2004) Efflux pumps. In: Ramos JL, editor. Pseudomonas, Vol. I. Genomics, life style and molecular architecture. New York: Kluwer Academic/Plenum Publishers. pp. 635–674.
6. Henrichfreise B, Wiegand I, Pfister W, Wiedemann B (2007) Resistance mechanisms of multiresistant Pseudomonas aeruginosa strains from Germany and correlation with hypermutation. Antimicrob. Agents Chemother 51: 4062–4070.
- View Article
- Google Scholar
7. Poole K (2011) Pseudomonas aeruginosa: resistance to the max. Front. Microbio 2 65.
- View Article
- Google Scholar
8. Vettoretti L, Plesiat P, Muller C, El'Garch F, Phan G, et al. (2009) Efflux unbalance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob. Agents Chemother 53 1987–1997.
- View Article
- Google Scholar
9. Masuda,N, Sakagawa E, Ohya S, Gotoh N, Tsujimoto H, et al. (2000) Contribution of the MexX-MexY-OprM efflux system to intrinsic resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother 44 2242–2246.
- View Article
- Google Scholar
10. Aires JR, Köhler T, Nikaido H, Plesiat P (1999) Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob. Agents Chemother 43 2624–2628.
- View Article
- Google Scholar
11. Matsuo Y, Eda S, Gotoh N, Yoshihara E, Nakae T (2004) MexZ-mediated regulation of mexXY multidrug efflux pump expression in Pseudomonas aeruginosa by binding on the mexZ-mexX intergenic DNA. FEMS Microbiol. Lett 238 23–28.
- View Article
- Google Scholar
12. Mine T, Morita Y, Kataoka A, Mitzushima T, Tsuchiya T (1999) Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa. Antimicrob. Agents Chemother 43 415–417.
- View Article
- Google Scholar
13. Jeannot K, Sobel ML, El'Garch F, Poole K, Plesiat P (2005) Induction of the MexXY efflux pump in Pseudomonas aeruginosa is dependent on drug-ribosome interaction. J. Bactereiol 187 5341–5346.
- View Article
- Google Scholar
14. Morita Y, Sobel ML, Poole K (2006) Antibiotic inducibility of the MexXY multidrug efflux system of Pseudomonas aeruginosa: involvement of the antibiotic-inducible PA5471 gene product. J. Bacteriol 188 1847–1855.
- View Article
- Google Scholar
15. Caughlan RE, Sriram S, Daigle DM, Woods AL, Buco J, et al. (2009) Fmt bypass in Pseudomonas aeruginosa causes induction of MexXY efflux pump expression. Antimicrob. Agents Chemother 53 5015–5021.
- View Article
- Google Scholar
16. Westbrock-Wadman S, Sherman DR, Hickey MJ, Coulter SN, Zhu YQ, et al. (1999) Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability. Antimicrob. Agents Chemother 43 2975–2983.
- View Article
- Google Scholar
17. El'Garch F, Jeannot K, Hocquet D, Llanes-Barakat C, Plesiat P (2007) Cumulative effects of several nonenzymatic mechanisms on the resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob. Agents Chemother 51: 1016–1021.
- View Article
- Google Scholar
18. Lau CHF, Fraud S, Jones M, Peterson SN, Poole K (2012) Reduced expression of the rplU-rpmA ribosomal protein operon in mexXY-expressing pan-aminmoglycoside-resistant Pseudomonas aeruginosa. Antimicrob. Agents Chemother 56: 5171–5179.
- View Article
- Google Scholar
19. Morita Y, Gilmour C, Metcalf D, Poole K (2009) Translational control of the antibiotic inducibility of the PA5471 gene required for mexXY multidrug efflux gene expression in Pseudomonas aeruginosa. J. Bacteriol 191: 4966–4975.
- View Article
- Google Scholar
20. Islam S, Jalal S, Wretlind B (2004) Expression of the MexXY efflux pump in amikacin-resistant isolates of Pseudomonas aeruginosa. Clin. Microbiol. Infect 10: 877–883.
- View Article
- Google Scholar
21. Islam S, Oh H, Jalal S, Karpati F, Ciofu O, et al. (2009) Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Clin. Microbiol. Infect 15: 60–66.
- View Article
- Google Scholar
22. Llanes C, Hocquet D, Vogne C, Benali-Baitich D, Neuwirth C, et al. (2004) Clinical strains of Pseudomonas aeruginosa overproducing MexAB-OprM and MexXY efflux pumps simultaneously. Antimicrob. Agents Chemother 48: 1797–1802.
- View Article
- Google Scholar
23. Vogne C, Aires JR, Bailly C, Hocquet D, Plesiat P (2004) Role of the multidrug efflux system MexXY in the emergence of moderate resistance to aminoglycosides among Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrob. Agents Chemother 48: 1676–1680.
- View Article
- Google Scholar
24. Smith EE, Buckley DG, Wu Z, Saenphimmachak C, Hoffman LR, et al. (2006) Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc. Natl. Acad. Sci. U S A 103: 8487–8492.
- View Article
- Google Scholar
25. Yamamoto M, Ueda A, Kudo M, Matsuo Y, Fukushima J, et al. (2009) Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa. Microbiol 155: 3312–3321.
- View Article
- Google Scholar
26. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd ed, vol. 3. Cold Spring Harbour: Cold Spring Harbor Laboratory Press.
27. Choi KH, Kumar A, Schweizer HP (2005) A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Methods 64: 391–397.
- View Article
- Google Scholar
28. Miller JH (1992) A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbour: Cold Spring Harbour Laboratory Press.
29. Dmitrova M, Younes-Cauet G, Oertel-Buchheit P, Porte D, Schnarr M, et al. (1998) A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. Mol. Gen. Genet 257: 205–212.
- View Article
- Google Scholar
30. Daigle DM, Cao L, Fraud S, Wilke MS, Pacey A, et al. (2007) Protein modulator of multidrug efflux gene expression in Pseudomonas aeruginosa. J. Bacteriol 189: 5441–5451.
- View Article
- Google Scholar
31. Garinot-Schneider C, Pommer AJ, Moore GR, Kleanthous C, James R (1996) Identification of putative active-site residues in the DNase domain of colicin E9 by random mutagenesis. J. Mol. Biol 260: 731–742.
- View Article
- Google Scholar
32. Sobel ML, McKay GA, Poole K (2003) Contribution of the MexXY multidrug transporter to aminoglycoside resistance in Pseudomonas aeruginosa clinical isolates. Antimicrob. Agents Chemother 47: 3202–3207.
- View Article
- Google Scholar
33. Jo JT, Brinkman FS, Hancock RE (2003) Aminoglycoside efflux in Pseudomonas aeruginosa: involvement of novel outer membrane proteins. Antimicrob. Agents Chemother 47: 1101–1111.
- View Article
- Google Scholar
34. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195–201.
- View Article
- Google Scholar
35. Christen M, Hünenberger PH, Bakowies D, Baron R, Bürgi R, et al. (2005) The GROMOS software fort biomolecular simulation: GROMOS05. J. Comput. Chem 26: 1719–1751.
- View Article
- Google Scholar
36. Alguel Y, Lu D, Quade N, Sauter S, Zhang X (2010) Crystal structure of MexZ, a key repressor responsible for antibiotic resistance in Pseudomonas aeruginosa. J Struct Biol 172: 305–310.
- View Article
- Google Scholar
37. Whitworth DE, Hodgson DA (2001) Light-induced carotenogenesis in Myxococcus xanthus: evidence that CarS acts as an anti-repressor of CarA. Mol. Microbiol 42: 809–819.
- View Article
- Google Scholar
38. Martinez-Argudo I, Little R, Shearer N, Johnson P, Dixon R (2004) The NifL-NifA system: a multidomain transcriptional regulatory complex that integrates environmental signals. J. Bacteriol 186: 601–610.
- View Article
- Google Scholar
39. Masuda S, Bauer CE (2002) AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides. Cell 110: 613–623.
- View Article
- Google Scholar
40. Sun X, Zahir Z, Lynch KH, Dennis JJ (2011) An antirepressor, SrpR, is involved in transcriptional regulation of the SrpABC solvent tolerance efflux pump of Pseudomonas putida S12. J. Bacteriol 193: 2717–2725.
- View Article
- Google Scholar
41. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, et al.. (1992) Short protocols in molecular biology, 2nd ed. New York: John Wiley & Sons, Inc.
42. Simon R, Priefer U, Puehler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology 1: 784–791.
- View Article
- Google Scholar
43. Masuda N, Ohya S (1992) Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa. Antimicrob. Agents Chemother 36: 1847–1851.
- View Article
- Google Scholar
44. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212: 77–86.
- View Article
- Google Scholar

[ref1] 1. Poole K (2005) Efflux-mediated antimicrobial resistance. J. Antimicrob. Chemother 56: 20–51.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Poole K (2004) Efflux-mediated multiresistance in Gram-negative bacteria. Clin. Microbiol. Infect 10: 12–26.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Poole K (2003) Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms. In: Paulsen IT, Lewis K, editors. Microbial multidrug efflux. Norwich: Horizon Scientific Press. pp. 273–298.

[ref4] 4. Poole K (2005) Aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother 49: 479–487.
View Article
Google Scholar

[9] View Article

[10] Google Scholar

[ref5] 5. Poole K (2004) Efflux pumps. In: Ramos JL, editor. Pseudomonas, Vol. I. Genomics, life style and molecular architecture. New York: Kluwer Academic/Plenum Publishers. pp. 635–674.

[ref6] 6. Henrichfreise B, Wiegand I, Pfister W, Wiedemann B (2007) Resistance mechanisms of multiresistant Pseudomonas aeruginosa strains from Germany and correlation with hypermutation. Antimicrob. Agents Chemother 51: 4062–4070.
View Article
Google Scholar

[13] View Article

[14] Google Scholar

[ref7] 7. Poole K (2011) Pseudomonas aeruginosa: resistance to the max. Front. Microbio 2 65.
View Article
Google Scholar

[16] View Article

[17] Google Scholar

[ref8] 8. Vettoretti L, Plesiat P, Muller C, El'Garch F, Phan G, et al. (2009) Efflux unbalance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob. Agents Chemother 53 1987–1997.
View Article
Google Scholar

[19] View Article

[20] Google Scholar

[ref9] 9. Masuda,N, Sakagawa E, Ohya S, Gotoh N, Tsujimoto H, et al. (2000) Contribution of the MexX-MexY-OprM efflux system to intrinsic resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother 44 2242–2246.
View Article
Google Scholar

[22] View Article

[23] Google Scholar

[ref10] 10. Aires JR, Köhler T, Nikaido H, Plesiat P (1999) Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob. Agents Chemother 43 2624–2628.
View Article
Google Scholar

[25] View Article

[26] Google Scholar

[ref11] 11. Matsuo Y, Eda S, Gotoh N, Yoshihara E, Nakae T (2004) MexZ-mediated regulation of mexXY multidrug efflux pump expression in Pseudomonas aeruginosa by binding on the mexZ-mexX intergenic DNA. FEMS Microbiol. Lett 238 23–28.
View Article
Google Scholar

[28] View Article

[29] Google Scholar

[ref12] 12. Mine T, Morita Y, Kataoka A, Mitzushima T, Tsuchiya T (1999) Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa. Antimicrob. Agents Chemother 43 415–417.
View Article
Google Scholar

[31] View Article

[32] Google Scholar

[ref13] 13. Jeannot K, Sobel ML, El'Garch F, Poole K, Plesiat P (2005) Induction of the MexXY efflux pump in Pseudomonas aeruginosa is dependent on drug-ribosome interaction. J. Bactereiol 187 5341–5346.
View Article
Google Scholar

[34] View Article

[35] Google Scholar

[ref14] 14. Morita Y, Sobel ML, Poole K (2006) Antibiotic inducibility of the MexXY multidrug efflux system of Pseudomonas aeruginosa: involvement of the antibiotic-inducible PA5471 gene product. J. Bacteriol 188 1847–1855.
View Article
Google Scholar

[37] View Article

[38] Google Scholar

[ref15] 15. Caughlan RE, Sriram S, Daigle DM, Woods AL, Buco J, et al. (2009) Fmt bypass in Pseudomonas aeruginosa causes induction of MexXY efflux pump expression. Antimicrob. Agents Chemother 53 5015–5021.
View Article
Google Scholar

[40] View Article

[41] Google Scholar

[ref16] 16. Westbrock-Wadman S, Sherman DR, Hickey MJ, Coulter SN, Zhu YQ, et al. (1999) Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability. Antimicrob. Agents Chemother 43 2975–2983.
View Article
Google Scholar

[43] View Article

[44] Google Scholar

[ref17] 17. El'Garch F, Jeannot K, Hocquet D, Llanes-Barakat C, Plesiat P (2007) Cumulative effects of several nonenzymatic mechanisms on the resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob. Agents Chemother 51: 1016–1021.
View Article
Google Scholar

[46] View Article

[47] Google Scholar

[ref18] 18. Lau CHF, Fraud S, Jones M, Peterson SN, Poole K (2012) Reduced expression of the rplU-rpmA ribosomal protein operon in mexXY-expressing pan-aminmoglycoside-resistant Pseudomonas aeruginosa. Antimicrob. Agents Chemother 56: 5171–5179.
View Article
Google Scholar

[49] View Article

[50] Google Scholar

[ref19] 19. Morita Y, Gilmour C, Metcalf D, Poole K (2009) Translational control of the antibiotic inducibility of the PA5471 gene required for mexXY multidrug efflux gene expression in Pseudomonas aeruginosa. J. Bacteriol 191: 4966–4975.
View Article
Google Scholar

[52] View Article

[53] Google Scholar

[ref20] 20. Islam S, Jalal S, Wretlind B (2004) Expression of the MexXY efflux pump in amikacin-resistant isolates of Pseudomonas aeruginosa. Clin. Microbiol. Infect 10: 877–883.
View Article
Google Scholar

[55] View Article

[56] Google Scholar

[ref21] 21. Islam S, Oh H, Jalal S, Karpati F, Ciofu O, et al. (2009) Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Clin. Microbiol. Infect 15: 60–66.
View Article
Google Scholar

[58] View Article

[59] Google Scholar

[ref22] 22. Llanes C, Hocquet D, Vogne C, Benali-Baitich D, Neuwirth C, et al. (2004) Clinical strains of Pseudomonas aeruginosa overproducing MexAB-OprM and MexXY efflux pumps simultaneously. Antimicrob. Agents Chemother 48: 1797–1802.
View Article
Google Scholar

[61] View Article

[62] Google Scholar

[ref23] 23. Vogne C, Aires JR, Bailly C, Hocquet D, Plesiat P (2004) Role of the multidrug efflux system MexXY in the emergence of moderate resistance to aminoglycosides among Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrob. Agents Chemother 48: 1676–1680.
View Article
Google Scholar

[64] View Article

[65] Google Scholar

[ref24] 24. Smith EE, Buckley DG, Wu Z, Saenphimmachak C, Hoffman LR, et al. (2006) Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc. Natl. Acad. Sci. U S A 103: 8487–8492.
View Article
Google Scholar

[67] View Article

[68] Google Scholar

[ref25] 25. Yamamoto M, Ueda A, Kudo M, Matsuo Y, Fukushima J, et al. (2009) Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa. Microbiol 155: 3312–3321.
View Article
Google Scholar

[70] View Article

[71] Google Scholar

[ref26] 26. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd ed, vol. 3. Cold Spring Harbour: Cold Spring Harbor Laboratory Press.

[ref27] 27. Choi KH, Kumar A, Schweizer HP (2005) A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Methods 64: 391–397.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref28] 28. Miller JH (1992) A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbour: Cold Spring Harbour Laboratory Press.

[ref29] 29. Dmitrova M, Younes-Cauet G, Oertel-Buchheit P, Porte D, Schnarr M, et al. (1998) A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. Mol. Gen. Genet 257: 205–212.
View Article
Google Scholar

[78] View Article

[79] Google Scholar

[ref30] 30. Daigle DM, Cao L, Fraud S, Wilke MS, Pacey A, et al. (2007) Protein modulator of multidrug efflux gene expression in Pseudomonas aeruginosa. J. Bacteriol 189: 5441–5451.
View Article
Google Scholar

[81] View Article

[82] Google Scholar

[ref31] 31. Garinot-Schneider C, Pommer AJ, Moore GR, Kleanthous C, James R (1996) Identification of putative active-site residues in the DNase domain of colicin E9 by random mutagenesis. J. Mol. Biol 260: 731–742.
View Article
Google Scholar

[84] View Article

[85] Google Scholar

[ref32] 32. Sobel ML, McKay GA, Poole K (2003) Contribution of the MexXY multidrug transporter to aminoglycoside resistance in Pseudomonas aeruginosa clinical isolates. Antimicrob. Agents Chemother 47: 3202–3207.
View Article
Google Scholar

[87] View Article

[88] Google Scholar

[ref33] 33. Jo JT, Brinkman FS, Hancock RE (2003) Aminoglycoside efflux in Pseudomonas aeruginosa: involvement of novel outer membrane proteins. Antimicrob. Agents Chemother 47: 1101–1111.
View Article
Google Scholar

[90] View Article

[91] Google Scholar

[ref34] 34. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195–201.
View Article
Google Scholar

[93] View Article

[94] Google Scholar

[ref35] 35. Christen M, Hünenberger PH, Bakowies D, Baron R, Bürgi R, et al. (2005) The GROMOS software fort biomolecular simulation: GROMOS05. J. Comput. Chem 26: 1719–1751.
View Article
Google Scholar

[96] View Article

[97] Google Scholar

[ref36] 36. Alguel Y, Lu D, Quade N, Sauter S, Zhang X (2010) Crystal structure of MexZ, a key repressor responsible for antibiotic resistance in Pseudomonas aeruginosa. J Struct Biol 172: 305–310.
View Article
Google Scholar

[99] View Article

[100] Google Scholar

[ref37] 37. Whitworth DE, Hodgson DA (2001) Light-induced carotenogenesis in Myxococcus xanthus: evidence that CarS acts as an anti-repressor of CarA. Mol. Microbiol 42: 809–819.
View Article
Google Scholar

[102] View Article

[103] Google Scholar

[ref38] 38. Martinez-Argudo I, Little R, Shearer N, Johnson P, Dixon R (2004) The NifL-NifA system: a multidomain transcriptional regulatory complex that integrates environmental signals. J. Bacteriol 186: 601–610.
View Article
Google Scholar

[105] View Article

[106] Google Scholar

[ref39] 39. Masuda S, Bauer CE (2002) AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides. Cell 110: 613–623.
View Article
Google Scholar

[108] View Article

[109] Google Scholar

[ref40] 40. Sun X, Zahir Z, Lynch KH, Dennis JJ (2011) An antirepressor, SrpR, is involved in transcriptional regulation of the SrpABC solvent tolerance efflux pump of Pseudomonas putida S12. J. Bacteriol 193: 2717–2725.
View Article
Google Scholar

[111] View Article

[112] Google Scholar

[ref41] 41. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, et al.. (1992) Short protocols in molecular biology, 2nd ed. New York: John Wiley & Sons, Inc.

[ref42] 42. Simon R, Priefer U, Puehler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology 1: 784–791.
View Article
Google Scholar

[115] View Article

[116] Google Scholar

[ref43] 43. Masuda N, Ohya S (1992) Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa. Antimicrob. Agents Chemother 36: 1847–1851.
View Article
Google Scholar

[118] View Article

[119] Google Scholar

[ref44] 44. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212: 77–86.
View Article
Google Scholar

[121] View Article

[122] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Bacterial strains, plasmids, and growth conditions

DNA methods

Two-hybrid assay for ArmZ-MexZ interaction

Screening hydroxylamine-mutagenized armZ for mutations abrogating ArmZ-MexZ interaction

Generation of armZ missense mutants

Antimicrobial susceptibility testing

Quantitative real-time PCR

Protein structure modelling

Results and Discussion

The requirement for PA5471 (ArmZ) for drug-inducible mexXY expression is MexZ-dependent

Identification and localization of mutations compromising the ArmZ-MexZ interaction

ArmZ mutations compromising its interaction with MexZ block drug-inducible mexXY expression

Acknowledgments

Author Contributions

References