The non-classical ArsR-family repressor PyeR ( PA 4354 ) modulates biofilm formation in Pseudomonas aeruginosa

Received 16 February 2012 Revised 30 June 2012 Accepted 16 July 2012 BIOMERIT Research Centre, Department of Microbiology, University College Cork, Cork, Ireland National Laboratory of Plant Engineering and Protein Genetic Engineering, College of Life Science, Peking University, Beijing, PR China Marine Biotechnology Centre, Environmental Research Institute, University College Cork, Cork, Ireland Department of Microbiology, University College Cork, Cork, Ireland


INTRODUCTION
ArsR-family transcriptional repressors are widespread among bacteria and are functionally linked to the detoxification of diverse metals and metalloids (Busenlehner et al., 2003).The metalloregulatory repressor ArsR was the first described member of this large protein family, which was initially identified in Escherichia coli as a plasmidmediated, arsenic-responsive regulator controlling expression of an arsenic resistance operon (San Francisco et al., 1990).ArsR was subsequently characterized at the molecular level, revealing its function as a dimeric autorepressor, which dissociated from its promoter on binding of arsenic or antimony (Shi et al., 1996).Several features of ArsR, including dimerization and metal(loid)-associated derepression, were found to hold true for diverse ArsR orthologues, such as the cyanobacterial SmtB and CadC of Staphylococcus aureus, which regulate metal resistance genes, including metallothionins and metal efflux machinery, in response to toxic metals such as cadmium, zinc and lead (Busenlehner et al., 2003;Huckle et al., 1993).As such, this represents the classical paradigm whereby ArsR regulators allow bacteria to sense and respond to diverse metal(loid)s at the transcriptional level.As both toxic and essential metal(loid)s may be detrimental to bacterial survival, ArsR-family regulators are implicated in the adaptation of bacteria to fluctuating metal(loid) concentrations in their natural environments (Busenlehner et al., 2003;Summers, 2009).
Many pathogenic bacteria encode ArsR-family repressors in their genomes, including the opportunistic human pathogen Pseudomonas aeruginosa, which contains four such genes in its genome (Arunkumar et al., 2009;Cai et al., 1998;Campbell et al., 2007;Liu et al., 2009;Stover et al., 2000;Janga & Pe ´rez-Rueda, 2009;Summers, 2009).These include a classical arsenic-responsive ArsR repressor with homology to ArsR of E. coli (PA2277) and three uncharacterized ArsR-family proteins (PAO279, PAO547 and PA4354) (Cai et al., 1998;Stover et al., 2000).The aim of this study was to characterize the novel ArsR-family regulator encoded by PA4354, which appears to be a non-classical ArsR-family regulator, as reflected by the fact that PA4354 is encoded within a putative operon with an uncharacterized major facilitator superfamily (MFS) transporter and the old yellow enzyme (OYE)-family reductase XenB (Pak et al., 2000;van Dillewijn et al., 2008).Previously, we showed that PA4354 is directly regulated by the LysR-type transcriptional regulator MexT (Tian et al., 2009a, b).Here we characterize this non-classical PA4354 regulator at the molecular level and investigate its role in mediating key pathogenic traits.

METHODS
Strains and plasmids.The strains and plasmids used in this study are listed in Table 1.All experiments were performed in P. aeruginosa strain PAO1, except the biofilm experiment, which was also performed in strain UCBPP-PA14 (PA14).All primers were designed based on the PAO1 genome sequence (NC_002516.2) and are shown in Table 2.All strains were routinely cultured in Luria-Bertani (LB) broth at 37 uC with shaking at 150 r.p.m.Antibiotics were added to cultures where required: for E. coli, kanamycin (25 mg ml 21 ), tetracycline (10 mg ml 21 ) and chloramphenicol (20 mg ml 21 ); for P. aeruginosa, tetracycline (20-50 mg ml 21 ), gentamicin (20-50 mg ml 21 ) and streptomycin (100 mg ml 21 ).
All plasmids were constructed in E. coli S17lpir, which carries the transfer genes of the broad-host-range INcP-type plasmid RP4 integrated into its chromosome.Biparental mating was used to conjugate plasmids into recipient strains.In brief, overnight cultures of donor and recipient strains were washed in fresh media and transferred to VSWP 0.025 mm filters (Millipore), which were placed on LB agar to reduce growth and thereby increase conjugation efficiency.
Microcolony formation assay.Artificial sputum medium (ASM) was prepared as described by Sriramulu et al. (2005).ASM (1 ml) was inoculated to OD 600 0.05 with an overnight culture of P. aeruginosa PA14 in 24-well cell-culture plates.IPTG was added at 1 mM for all pME6032-derivative plasmids.Plates were then incubated for 72 h at 37 uC at 150 r.p.m.Three independent biological replicates were performed.
Generation of deletion mutants.Disruption of PA4354 and PA4355 was achieved using the gene replacement vector pEX18Tc, as previously described (Tian et al., 2009b).Regions flanking the PA4354 ORF were amplified using primer sets p5FD54 and p5RD54 (to amplify the PA4354 59 flanking region) and p3FD54 and p3RD54 (to amplify the PA4354 39 flanking region).Chimeric PCR was employed to link the PA4354 59 and 39 flanking regions.Primers incorporating HindIII, XhoI and KpnI restriction sites were used to generate a 1.8 kb HindIII-KpnI fragment with a XhoI site between the PA4354 59 and 39 flanking regions into which the excised FRTcontaining gentamicin resistance cassette from pPS856 was ligated (Hoang et al., 1998).For generation of the PA4355 deletion construct, a 59 flanking region was amplified with primers p5FD55 and p5RD55 and a 39 flanking region was amplified with primers p3FD55 and p3RD55 to generate a chimeric 1.8 kb HindIII-BamHI fragment with an intermittent SalI site into which the pPS856 gentamicin cassette was introduced.The pEX18Tc PA4354 and PA4355 deletion constructs were transformed into E. coli S17lpir and subsequently transferred to PAO1 by conjugation following routine cloning procedures (Sambrook, 2001).The gentamicin resistance cassette was excised from the PAO1 chromosome using plasmid pFLP3 (Hoang et al., 1998).Successful disruption of target genes was verified in each strain by PCR amplification and DNA sequencing using primers which flanked the deleted PA4354 (p5D54 and p3D54) or PA4355 (p5D55 and p3D55) ORFs.
Flow-cell experiments.Bacteria were grown in M63 medium throughout the flow-cell experiments (Sambrook, 2001).Flow-cell chambers with individual channel dimensions of 164640 mm were used.The flow system was assembled and prepared as described by Pamp et al. (2008).The flow chambers were inoculated by injecting 350 ml of overnight culture diluted to OD 600 0.001 into each flow channel using a small syringe.After inoculation, the flow channels were left without flow for 1 h, after which medium flow was started using a Watson Marlow 205S peristaltic pump.Mean flow velocity in the flow chambers was 0.2 mm s -1 , corresponding to laminar flow with a Reynolds number of 0.02.The flow-cell biofilm system was incubated at 30 uC throughout the experiment.Bacteria were stained using 5 mM SYTO 9 (Invitrogen).Visualization of the biofilm was performed using a Zeiss LSM5 confocal laser scanning microscope equipped with 488 nm laser lines to excite SYTO 9. Three independent biological replicates were performed.
Generation of the PyeR (PA4354) overexpression construct.To generate plasmid pME6032-PA4354, the PA4354 ORF was amplified using primers pF54.O and pR54.O, which contain incorporated EcoRI and KpnI restriction sites, respectively.This fragment was PCRpurified using the QIAquick PCR purification kit (Qiagen), digested and subsequently ligated directly into the pME6032 multiple cloning site downstream of the Ptac promoter (Heeb et al., 2000).The T4 DNA ligase and all restriction enzymes used in this study were purchased from Roche Applied Sciences.
Analysis of pyeR (PA4354) promoter activity in response to diverse metals.Induction of pyeR in the presence of diverse metals was investigated in a plate-based assay using the pyeR-lacZ promoter fusion pMP-PA4354p as a reporter in PAO1.Overnight cultures of PAO1 harbouring pMP-PA4354p were diluted to OD 600 0.125, and a bacterial lawn was inoculated onto M9 agar plates containing 0.4 % glucose, 40 mg X-Gal ml 21 and 100 mg streptomycin ml 21 .Filter paper discs containing metal salts including ZnSO 4 , V 2 SO 4 , MnCl 2 , MoO 4 Na 2 , NaAsO 2 , CdCl, CuSO 4 , K 2 TeO 3 , CoCl 2 , NiCl 2 , FeCl 3 , CrCl 3 , C 8 H 4 K 2 O 12 Sb 2 , AgNO 3 , Pb(NO 3 ) 2 , AuCl 3 , BiNO 3 , Na 2 O 3 Se and HgCl 2 were placed on the centre of each inoculated plate, which was incubated at 37 uC for 16-24 h and examined for induction of expression from the pyeR-lacZ reporter fusion, as indicated by an increase in blue pigmentation in the agar resulting from induction of the lacZ gene and the breakdown of X-Gal.Concentrations of each of the metal salts are given in Table S1 available with the online version of this paper.The ability of metals, for which induction was observed, to activate expression from the pMP-PA4354p reported fusion was also investigated in PAO1DPA4354 and PAO1DmexT deletion backgrounds.The diameter of the zones of inhibition around the 6 mm filter paper disc was also measured, to establish inhibitory properties of the metal salts.
b-Galactosidase assays.Expression of pyeR (PA4354), as measured from pMP190 fusion constructs, was measured by a b-galactosidase assay, and promoter activity was calculated and expressed as Miller units (Miller, 1972).Three independent biological replicates were performed.
Site-directed mutagenesis.To introduce mutations into the PA4354 promoter region, the PA4354 promoter fragment of pMP-PA4354p was first amplified using previously described primers with incorporated XbaI and KpnI restriction sites (Tian et al., 2009a).This fragment was cloned into the pCR2.1-TOPOcloning vector (Invitrogen) and site-specific mutations within the putative PA4354 autoregulatory motif were introduced as described by Fisher & Pei (1997).The site-directed mutations A91C and T92G (relative to the PA4353 stop codon) were introduced using primers pFsdm54 and pRsdm54 to amplify the entire pCR2.1-TOPOvector harbouring the PA4354 upstream region.The amplified plasmid was transformed into E. coli DH5a and the presence of the introduced mutations was verified by DNA sequencing.Once verified, the mutated promoter region was excised and ligated into the XbaI and KpnI restriction sites of pMP190 to yield the mutated promoter-lacZ fusion construct pMP-PA4354pMa.The ability of PA4354 (as expressed from the pME6032-PA4354 overexpression construct) to repress activity from the mutated promoter region of pMP-PA4354pMa was assessed in PAO1 by b-galactosidase assay (Miller, 1972).
His-tag purification of PyeR (PA4354).The PA4354 ORF was amplified using primers pFH54 and pRH54 and cloned into the multiple cloning site of pET28a to yield pET28aPA4354H6C, which expressed a C-terminal His-tagged PA4354 protein.This construct was transformed into the E. coli expression host strain BL21-CodonPlusBL21(DE3)-RIPL (Merck) and grown at 37 uC with shaking at 150 r.p.m. in 200 ml LB medium containing kanamycin (50 mg ml 21 ) until OD 600 1 was reached.At this point, 1 mM IPTG was added to the culture to induce expression of the His-tagged PA4354 protein.After 4 h, cells were harvested by centrifugation at 5000 g at 4 uC and stored overnight at 270 uC.Cell pellets were subsequently thawed and resuspended in CelLytic B II buffer (Sigma Aldrich) (10 ml per gram of cell paste) with 5 mg DNase ml 21 and 200 ml Protease Inhibitor Cocktail (Sigma) per gram cell paste.Soluble protein was extracted in accordance with the manufacturer's instructions.Protein extract was applied to a Poly-Prep Chromatography Column (Bio-Rad) containing 1 ml of HIS-Select Nickel Affinity Gel (Sigma).The gel was washed with 2 ml sterile deionized water and equilibrated with 5 ml wash buffer (100 mM HEPES, pH 7.5, 10 mM imidazole).Following washing and equilibration steps, the crude protein extract (10 ml) was passed through the column by gravity flow.Protein which bound to the resin was washed twice with 10 ml wash buffer and purified His-tagged PA4354 protein was eluted in wash buffer containing 250 mM imidazole.The homogeneity of the purified PA4354 protein was verified by SDS-PAGE.Purified protein aliquots were either frozen in 20 % (v/v) glycerol at 270 uC or used promptly for electromobility shift assays (EMSAs).Protein concentrations were determined by the Bio-Rad protein assay.
EMSA.Purified PA4354 protein was incubated with an IR-labelled PA4354 promoter fragment generated with DY-682 IR-labelled primers pF54emsa and pR54emsa (Eurofins MWG Operon).EMSA was set up with 20 ml reaction volumes containing varying concentrations of purified PA4354 protein (10-2000 nM) in the presence of 10 fmol of labelled promoter DNA in EMSA binding buffer {20 mM HEPES, pH 7.6, containing 30 mM KCl, 5 mM (NH 4 ) 2 SO 4 , 1 mM EDTA, 1 mM DTT, 0.2 % (w/v) Tween 20 and 5 mg ml ) and CyeR (NP_602237.1), using MAFFT version 6, and the resulting alignment was analysed and edited using BioEdit (Hall, 1999;Katoh & Toh, 2008).To determine whether PA4354 and homologous repressors exhibited conserved autoregulatory binding sites in their promoters, the upstream regions of 57 putative PA4354 orthologues were retrieved from the Database of Prokaryotic Operons (DOOR) and analysed for the presence of conserved motifs (Mao et al., 2009).Upstream sequences of approximately 200 bp retrieved from each identified PA4354-like regulator were searched using the Multiple Em for Motif Elicitation (MEME) algorithm for the presence of conserved motifs using the following parameters: motif occurrence, one per sequence; ¢6 optimum motif width ¡50 (Bailey & Elkan, 1994).To determine the distribution of this putative PA4354 binding motif in the PAO1 genome the Motif Alignment and Search Tool (MAST) was employed (Bailey & Gribskov, 1998).

RESULTS
PA4354 (PyeR) encodes a novel, non-classical member of the ArsR family PA4354 is predicted to be an ArsR-family transcriptional repressor, as it contains the conserved helix-turn-helix domain found in ArsR-family regulators in its primary sequence.However, PA4354 does not harbour any of the previously characterized metal(loid)-binding sequence motifs associated with classical ArsR regulators, as determined by bioinformatic analysis using the ExPASy search tool (Campbell et al., 2007;Osman & Cavet, 2010).Moreover, it exhibits a unique 13 amino acid region in its primary sequence, which does not align with previously characterized ArsR-family repressors and distinguishes PA4354 from other ArsR repressors (Fig. 1).Orthologues of PA4354, containing the non-classical features, are found in many micro-organisms among diverse bacterial genera, including Pseudomonas, Rhizobium, Bordetella and Burkholderia.
To further investigate whether PA4354 is a metal(loid)responsive regulator, we examined if diverse metal(loid)s which contains the PA4354-promoter region upstream of a promoterless lacZ-reporter plasmid, was used (Tian et al., 2009a).Approximately 50 % of the metals inhibited growth of P. aeruginosa (Table S1).Interestingly, PA4354 expression was induced in the presence of lead and tellurite, but not by the other metals tested (Table S1).However, this induction was apparently independent of PA4354, as disruption of PA4354 did not abrogate the observed differential changes.This suggests that although lead and tellurite positively influence expression from the PA4354 promoter, they are not cognate metal(loid) inducers of PA4354 in the classical sense of the direct binding and de-repression observed for metal(loid)-responsive ArsR repressors.Moreover, tellurite also inhibited the growth of P. aeruginosa, suggesting that it would induce PA4354 expression at a sub-inhibitory concentration.In addition, deletion of PA4354 had no impact on the level of resistance to inducing metal(loid)s, as the MIC levels of lead and tellurite, 0.125 and 0.06 mM, respectively, were unchanged upon deletion of PA4354.This is not surprising, as PA4354 is encoded within a putative operon with an uncharacterized MFS transporter and the OYE-family reductase XenB, and not with metal(loid) resistance genes, unlike classical members of the metalresponsive ArsR family (Pak et al., 2000;van Dillewijn et al., 2008).
Given that PA4354 is located in a putative operon with the OYE protein xenB we propose the gene name PyeR (Pseudomonas yellow enzyme regulator) for PA4354, in accordance with the study of Ehira et al. (2010), where an ArsR-family repressor was observed to regulate expression of an OYE-family enzyme in Corynebacterium glutamicum.By extension, the MFS transporter PA4355 was named PyeM (Pseudomonas yellow enzyme MFS transporter).Hereafter, PA4354 and PA4355 are referred to as PyeR and PyeM, respectively.Comparative analysis of PyeR orthologues revealed that many ArsR-family regulators (COG0640) are also associated with MFS transporters (COG0477) and OYE-family enzymes (COG1907).This is reflected by a strong association score between COG0640 and both COG0477 (0.824) and COG1902 (0.671) in the STRING database (Szklarczyk et al., 2011).This suggests the existence of a subgroup of ArsR-family repressors with homology to PyeR that regulate expression of MFS transporters and OYEfamily enzymes, as opposed to classical ArsR-family regulators, which regulate expression of genes associated with metal(loid) resistance (e.g.COG0798, COG1393, COG1230).

PyeR has autoregulatory properties
As both classical and non-classical ArsR-family repressors generally function as autoregulatory proteins (Busenlehner et al., 2003), expression analysis of PyeR was performed to investigate the autoregulatory characteristics of PyeR.For this, the pMP-PA4354p vector, which contains the pyeRpromoter region upstream of a promoterless lacZ-reporter plasmid, was used.Expression of pyeR in PAO1 wild-type reached 395.3±19.4Miller units (mean±SD) during exponential growth (absorbance of 0.6-0.8 at 600 nm, equivalent to ~4 h of growth with a starting OD 600 of 0.02).By contrast, expression of the vector control (pMP190) over all conditions was 20.27±11.04Miller units.Overexpression of PyeR (pME6032-PA4354) resulted in repression of pyeR promoter activity, demonstrating that PyeR functions as an autorepressor (Fig. 2).In addition, pyeR promoter activity was elevated in a pyeR deletion mutant compared with wild-type cells and on overexpression of its known transcriptional activator MexT, which further substantiates the evidence that PyeR has autoregulatory properties (Table 3).

Identification of the PyeR autoregulatory motif
As orthologues of PyeR are present in diverse species, these sequences could be used to identify the putative autoregulatory site of PyeR.Upstream regions of PyeR orthologues were retrieved from the DOOR database (Mao et al., 2009).These sequences were aligned and searched for the presence of a conserved motif using the MEME algorithm (Bailey & Elkan, 1994).This identified a putative PyeR autoregulatory site which was highly conserved among all 57 retrieved upstream regions of PyeR orthologues.The putative PyeR binding motif was located downstream of the previously identified MexT binding site and overlaps a putative 210 region (Fig. 3).Apart from the upstream region of pyeR, no highly significant hits to the putative PyeR binding motif were identified within the PAO1 genome using the MAST algorithm, suggesting that this motif is not conserved outside the PyeR promoter region (Bailey & Gribskov, 1998).However, the absence of this motif does not rule out the possibility that PyeR binds to other motifs.Further analysis will be required to investigate this; for example, microarray analysis on a wild-type and pyeR mutant strain under the conditions in which PyeR exerts its own autoregulation.
To confirm the role of the identified PyeR binding motif in PyeR autorepression, site-directed mutagenesis of this region was performed.Mutation of conserved residues within the putative pyeR autoregulatory site (TATGC-N 8 -CGATA-N 5 -TCG-N 8 -CGA) resulted in increased expression from the pyeR promoter in the presence of the pyeR overexpressor (pME6032-PA4354) (Fig. 4).This demonstrated the importance of the identified cis-acting regulatory site in mediating PyeR autorepression.

PyeR binds directly to its own upstream promoter region
EMSA was used to demonstrate unequivocally that PyeR binds directly to its own promoter.A His-tagged PyeR expression construct was generated (pET28a-pyeRH6C) and the PyeR repressor was subsequently purified by nickel affinity column chromatography.As expected, the purified PyeR protein caused a mobility shift in the presence of an IR-labelled DNA probe spanning the PA4353-pyeR intergenic region (Fig. 5).In contrast, no shift was observed in the presence of a non-specific DNA probe amplified from the PA4881 upstream region, which does not contain a PyeR autoregulatory motif (Fig. 5).This demonstrates the direct and specific binding of PyeR to its own upstream promoter region, consistent with the autoregulatory function of the PyeR autorepressor.

pyeR is co-transcribed with pyeM and xenB
To investigate whether the predicted pyeR-pyeM-xenB operon was expressed as a tri-cistronic transcript, RT-PCR was employed to determine the nature of the RNA transcript(s).For this, cDNA was synthesized from RNA isolated from PAO1 (pME6032-mexT), which harbours a MexT overexpression construct to drive induction of pyeR-pyeM-xenB.Primers flanking the intergenic regions between pyeR and pyeM as well as between pyeM and xenB were used to verify that the tri-cistronic transcript was expressed.Amplification of the pyeR-pyeM and pyeM-xenB intergenic regions from reverse-transcribed cDNA confirmed the cotranscription of pyeR, pyeM and xenB (Fig. 6).

The pyeR-pyeM-xenB operon exhibits complex regulation
PyeR has previously been demonstrated to be directly regulated by MexT, which has been shown to regulate key Table 3. Influence of pyeR deletion and mexT overexpression on pyeR promoter activity Values are mean±SD of three independent biological replicates.

Plasmid
Exponential* StationaryD *Observed b-galactosidase activity in exponential phase cultures (OD 600 0.4-0.7).DObserved b-galactosidase activity in stationary phase cultures (OD 600 .2.5, samples taken at 24 h).pathogenic traits in P. aeruginosa, such as biofilm formation, expression of the type III secretion system, phenazine production and antibiotic resistance (Tian et al., 2009a, b).As pyeR promoter activity is observed in wildtype cells, in which MexT is not expressed, it is likely that other regulatory proteins are also involved in the regulation of pyeR (Tian et al., 2009b).To investigate this further, the expression of pyeR in a MexT deletion background was assessed.Deletion of MexT had no impact on pyeR promoter activity (256±30 Miller units) in PAO1 as the expression level was comparable with PAO1 wild-type (287±15 Miller units), implicating other regulators in the modulation of pyeR expression.Furthermore, expression of pyeR declined to approximately 80 Miller units in stationary phase, whereas the empty vector control (pMP190) did not reach above 60 Miller units at any time point, with a mean over all time points of 15.97±14.22Miller units (Fig. 7).This stationary phase repression occurred irrespective of whether pyeR was deleted or whether MexT was overexpressed, ruling out a role for either regulator in pyeR downregulation during stationary phase (Table 3).This further suggests that other regulatory elements are involved in the regulation of pyeR, which may integrate this regulator into several signal transduction pathways.Further research will focus on the elucidation of other elements that are involved in the regulation of pyeR.

PyeR plays a role in biofilm formation
As PyeR is a direct regulatory target of MexT, which downregulates several virulence-related phenotypes, we hypothesized that the PyeR repressor might influence MexT-linked phenotypes.Neither deletion of pyeR nor overexpression of pyeR had any impact on antibiotic resistance or virulence-related phenotypes, including expression of the type III secretion system, pyocyanin production and early attachment (data not shown).However, flow cell biofilm experiments showed that the architecture of 3-dayold biofilms of the pyeR mutant differed significantly from that of PAO1 wild-type (Fig. 8).The mushroom-shaped microcolony structures were larger and more abundant in  the mutant compared with the wild-type biofilm (Fig. 8).To investigate whether PyeM and/or XenB are also involved in biofilm formation, a pyeM out-of-frame deletion mutant was constructed that resulted in loss of both pyeM and xenB transcript.Flow cell experiments showed that there was no difference in biofilm architecture between PAO1 wild-type and this mutant (data not shown), suggesting that only PyeR plays a role in biofilm formation.
To assess further the role of PyeR in biofilm formation, a tight microcolony assay was performed.Deletion of pyeR resulted in loss of tight microcolony formation in ASM (Fig. S1a, b).This phenotype was restored by complementation with a plasmid expressing pyeR in trans (Fig. S1c, d).Moreover, a similar phenotype was observed in another P. aeruginosa model strain, PA14, as a mutant with a mariner transposon in PA4354 (PA14 PA4354 : : Tm) (Liberati et al., 2006) was unable to form a tight microcolony (Fig. S1d, e).Taking the above data together, PyeR is implicated in biofilm formation, which provides a novel function for this non-classical ArsR-family transcriptional regulator.

DISCUSSION
PyeR is a novel ArsR-type transcriptional regulator from P. aeruginosa, which exhibits classical features such as a conserved helix-turn-helix domain, negative autoregulation and transcription as part of an operon.However, it does not exhibit metal(loid) perception or resistance characteristics associated with classical ArsR-family regulators.This is an emerging theme within the ArsR family, where several members have now been demonstrated to be involved in processes unrelated to metal(loid) perception or resistance (Ehira et al., 2010;Ellermeier et al., 2006;Gristwood et al., 2011;Gueune ´et al., 2008;Guimara ˜es et al., 2011).For example, the SdpR repressor of Bacillus subtilis, which regulates cannibalistic behaviour during sporulation, dissociates from its promoter via sequestration at the cell membrane by a cell membrane immunity protein (Ellermeier et al., 2006).The HlyU repressor of Vibrio vulnificus activates expression of its target by acting as an antirepressor of a histone-like nucleoid structuring protein, which negatively regulates expression of the rtxA1 toxinencoding gene (Liu et al., 2009).As such, PyeR belongs to a growing subgroup of the non-classical ArsR family of transcriptional regulators linked to diverse physiological phenotypes rather than specifically to metal resistance.
The ArsR-family repressor CyeR of Corynebacterium glutamicum has been shown to be induced by oxidative and thiol-specific stress rather than the presence of metals and also regulates the OYE-family enzyme Cye1, which has  homology XenB (Ehira et al., 2010).This is interesting, as the two metals that were found to induce expression of pyeR-pyeM-xenB are implicated in oxidative stress.Moreover, transcriptome analysis has shown upregulation of pyeR transcript in response to hydrogen peroxide (Palma et al., 2004).Tellurium has been shown to induce a thiolspecific stress response in Pseudomonas pseudoalcaligenes, and the pathotoxicity of lead to eukaryotic organisms is attributed to toxicity-induced oxidative stress (Tremaroli et al., 2009;Verstraeten et al., 2008).As metal(loid)mediated induction of expression from the pyeR promoter was observed in both mexT and pyeR deletion backgrounds, it is likely that the pyeR-pyeM-xenB operon is regulated by another unidentified regulator in response to metal(loid)-induced changes in the cellular redox environment, independently of both MexT and PyeR.
Although XenB is known to catalyse the reductive removal of nitro groups from electrophilic pollutants, including 2,4,6-trinitrotoluene and nitroglycerine, these are unlikely to reflect the natural substrates of XenB (Pak et al., 2000).As a member of the OYE family, XenB is likely to be involved in the reduction of a,b-unsaturated carbonyl compounds, which may emerge from metabolism or be encountered in the environment (Breithaupt et al., 2009;Trotter et al., 2006;Williams & Bruce, 2002).The fact that pyeR promoter activity is observed in exponentially growing cells suggests that the substrate(s) of the pyeR-pyeM-xenB operon are compounds endogenous to P. aeruginosa.Such molecules or their breakdown products could represent the specific ligands recognized by PyeR.Thus, it now appears probable that several ArsR-family regulators modulate gene expression in response to nonmetalloid inducing molecules.However, formal validation of this hypothesis awaits the identification of the first non-metalloid ArsR-binding ligand.Just as work on metal(loid)-perceptive ArsR regulators has allowed the identification of motifs associated with specific metal(loid)s, the identification of non-metal(loid) ligands would greatly increase our understanding of ArsR-family repressor ligand interactions.PyeR lacks any previously determined metal-binding motifs in its protein sequence and appears not to play a direct role in metal perception, although the identified unique 13 amino acid region in PyeR (and its many orthologues) may have significance in defining the interaction of PyeR with a non-metal(loid) ligand.
Although non-classical ArsR-family transcriptional regulators are linked to many physiological processes, this study is believed to be only the second report to implicate the ArsR family of transcriptional regulators in biofilm formation.The BigR repressor of Xylella fastidiosa and Agrobacterium tumifaciens has been shown to negatively influence biofilm formation and the expression of genes involved in hydrogen sulphide resistance (Barbosa & Benedetti, 2007).Similarly to PyeR, deletion of the BigR target gene, bhl, had no effect on biofilm formation (Barbosa & Benedetti, 2007;Guimara ˜es et al., 2011).This may be due to functional redundancy and interplay among biofilm-associated genes or to regulatory effects of BigR on as-yet-unidentified targets, as may also be the case for PyeR.The high-throughput biofilm screen in which PyeR was independently identified was performed in ASM.This medium closely resembles the sputum found in the lungs of cystic fibrosis patients, in whom P. aeruginosa is the main cause of morbidity and mortality (Sriramulu et al., 2005).As PyeR is essential for tight microcolony formation in this medium, this indicates that PyeR might be a key player in biofilm formation in vivo.

Fig. 2 .
Fig. 2. PyeR has autoregulatory properties.Induction of pyeR expression from the pMP-PA4354p promoter-lacZ fusion was determined in the presence of the PyeR overexpression construct pME6032-PA4354.Increasing concentrations of IPTG led to increased PyeR and resulted in decreased expression from the pyeR promoter.Data shown are mean values of three biological experiments; error bars, SD.

Fig. 3 .
Fig.3.A highly conserved motif was identified in the promoter region of the PyeR repressor.This motif was present downstream of the MexT binding site.Putative pyeR "35 and "10 sites are indicated by broken lines.A motif logo illustrating a position-specific scoring matrix for the PyeR binding site is illustrated above the matching nucleotide sequence in the pyeR upstream region.Highly conserved nucleotides are highlighted in grey and the nucleotides of the nod boxes within the MexT binding site are boxed.Conserved nucleotides within the PyeR binding motif which were subsequently targeted for sitedirected mutagenesis are indicated by black dots.

Fig. 5 .
Fig. 5. Direct binding of PyeR to the pyeR upstream region.Histag-purified PyeR causes a mobility shift in the presence of the pyeR upstream region (top row).No shift was observed with a nonspecific DNA target amplified from the PA4881 upstream region which lacks the PyeR binding site.The concentration of Histagged PyeR used in each assay is indicated above the gel image.

Fig. 6 .Fig. 4 .
Fig. 6.Confirmation of the pyeR-pyeM-xenB operon structure by RT-PCR.+, Positive control (PAO1 gDNA); ", negative control (cDNA synthesis reaction mixture to which no template was added); R, DNase-treated RNA isolated from PAO1(pME6032-mexT) before cDNA synthesis; C, cDNA synthesized from RNA isolated from PAO1(pME6032-mexT) using a specific primer complementary to the 39 end of the xenB transcript.The regions amplified in images A and B are indicated by solid black lines in the diagram of the pyeR-pyeM-xenB operon structure below the gel images.

Fig. 7 .
Fig. 7. PyeR expression is induced in exponential phase and declines at higher cell densities.&, Expression from the pMP-PA4354p lacZ reporter fusion (primary axis) measured in Miller units.#, Growth of PAO1 harbouring pMP-PA4354p represented by log(OD 600 ) at each time point (secondary axis).Data shown are mean values of three biological experiments; error bars, SD.

Table 1 .
Bacterial strains and plasmids used in this studyAbbreviations: Ap r , ampicillin resistance; Km r , kanamycin resistance; Tc r , tetracycline resistance; Cm r , chloramphenicol resistance; Str r , streptomycin resistance; Gm r , gentamicin resistance.

Table 2 .
Primers used in this studyPrimers were designed based on the PAO1 genome sequence (NC_002516.2).Underlined nucleotides indicate incorporated restriction sites.