Molecular Microbiology (2012) 83(3), 536–547 䊏
doi:10.1111/j.1365-2958.2011.07947.x First published online 3 January 2012
An orphan sensor kinase controls quinolone signal production via MexT in Pseudomonas aeruginosa mmi_7947 536..547
Caroline Zaoui,1 Jörg Overhage,1 Dagmar Löns,1 Ariane Zimmermann,1 Mathias Müsken,1,2 Piotr Bielecki,2 Christian Pustelny,1 Tanja Becker,1 Manfred Nimtz3 and Susanne Häussler1,2* 1 Chronic Pseudomonas Infection Research Group, Helmholtz Center for Infection Research, Braunschweig, Germany. 2 Department of Pathophysiology of Bacterial Biofilms, Twincore, Center for Clinical and Experimental Infection Research, a joint venture of the Helmholtz Center of Infection Research and the Hannover Medical School, Hannover, Germany. 3 Cellular Proteome Research Group, Helmholtz Center for Infection Research, Braunschweig, Germany.
Summary Pseudomonas aeruginosa employs both N-acylhomoserine lactone and 2-alkyl-4(1H)quinolone (AQ)-mediated interbacterial signalling for the orchestration of a genome-wide gene regulatory network. Despite the many advances that have been made in understanding the target genes of quorum sensing regulation, little is known on how quorum sensing systems are influenced by environmental cues. In this study, we show that AQ production is modulated by an orphan P. aeruginosa sensor kinase. Transcriptional studies of the sensor kinase (MxtR) mutant demonstrated that an induced expression of MexT, a LysR-type transcriptional regulator, largely determined the global transcriptional profile. Thereby, overexpression of the MexT-regulated MexEF-OprN efflux pump led to a delayed expression of the AQ biosynthetic genes and of AQ-dependent virulence factors. Furthermore, we demonstrated that autophosphorylation of MxtR was inhibited by ubiquinone, the central electron carrier of respiration in in vitro experiments. Our results elucidate on a mechanism by which P. aeruginosa senses environmental conditions and adapts by controlling the production of interbacterial AQ signal molecules. A regulatory function of a sensor Accepted 6 December, 2011. *For correspondence. E-mail susanne.
[email protected]; Tel. (+49) 511 220027215; Fax (+49) 220027 210.
© 2011 Blackwell Publishing Ltd
kinase may indicate that there is a pre-emptive role of adaptation mechanisms that are turned on under distinct environmental conditions and that are important for efficient colonization and pathogenesis.
Introduction Pseudomonas aeruginosa has emerged as one of the most important bacterial pathogens in acute nosocomial infections and it is the dominant cause of chronic infections in the cystic fibrosis lung (Govan and Deretic, 1996; Costerton, 2001). The opportunistic pathogen inhabits diverse terrestrial and aquatic niches and exhibits a unique capability to infect a broad range of hosts from plants to animals and humans (Rahme et al., 1995). The extreme capability to thrive in diverse environments is facilitated by one of the highest proportion of regulatory genes (8.4% of the genome) observed for a bacterial genome, which comprises an important number of genes involved in locomotion, attachment, transport and utilization of nutrients, antibiotic efflux and systems involved in sensing and responding to environmental changes (Stover et al., 2000). One widespread sensory transduction mechanism is the ‘two-component’ adaptive response system. Twocomponent systems are typically composed of a membrane-bound sensor kinase protein and a downstream response regulator protein. The sensor kinase protein mediates the detection of a particular environmental stimulus and transduces the signal to the response regulator protein. The response regulator in turn carries out an appropriate action in response to the stimulus (Beier and Gross, 2006; Mitrophanov and Groisman, 2008; Gooderham and Hancock, 2009). One of the best studied systems involved in mediating the response to changes in environmental oxygen levels is the ArcAB (for anoxic redox control) two-component system of Escherichia coli, consisting of the sensor kinase ArcB and the cognate response regulator ArcA (Gunsalus and Park, 1994; Sengupta et al., 2003). This system allows the facultative anaerobic bacteria to sense various respiratory growth conditions and to adapt their gene expression accordingly (Lynch and Lin, 1996). The ArcB sensor kinase autophosphorylates and transfers a phosphoryl group to ArcA, a DNA-binding protein. The ArcAB system
MexT-dependent quinolone signalling in P. aeruginosa 537
is most active under low oxygen conditions and least active under high oxygen conditions. There is evidence indicating that ArcB senses the redox status of the membrane-bound quinones, central electron carriers of respiration (Georgellis et al., 2001; Malpica et al., 2004). The ArcAB system has also been implicated in the pathogenesis of various other bacteria. ArcA mutants of both Hamophilus influenzae and Vibrio cholerae exhibited reduced lethality in mouse mortality studies (De SouzaHart et al., 2003; Sengupta et al., 2003); V. cholerae ArcA influences production of cholera toxin, which is essential for virulence; and in Salmonella enterica serovar Enteritidis, ArcA has been implicated in resistance to reactive oxygen and nitrogen intermediates (Lu et al., 2002). In the current study, we have identified a hybrid sensor kinase in P. aeruginosa that controls 2-alkyl-4(1H)quinolone (AQ) production, including the interbacterial signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone, referred to as the Pseudomonas quinolone signal (PQS) (Pesci et al., 1999) via the LysR-type transcriptional regulator MexT. Our results depict a novel mechanism of P. aeruginosa by which the bacterium senses environmental conditions and adapts by controlling the production of interbacterial AQ signal molecules and pyocyanin. This adaptation might be important for the co-ordination of gene expression profiles that are needed for efficient colonization and pathogenesis in different environments encountered within the host.
Results Screen for AQ non-responsive mutants In this study, we aimed at detecting factors involved in AQ signalling in P. aeruginosa and set up a global screen for AQ non-responsive mutants. Therefore, we performed a transposon mutagenesis on the clinical P. aeruginosa small colony variant (SCV) 20265. This strain was chosen for the screen because it produces an intensive metallic iridescent sheen when streaked onto blood agar plates as a pre-requirement for the applicability of the assay. As depicted in Fig. 1, we spotted 2 ml of a P. aeruginosa SCV 20265 stationary phase culture methanol extract (containing high concentrations of AQs) onto the centre of bacterial lawns of individual transposon mutants and screened for the appearance of an iridescent metallic sheen on the surface of the lawns. It has been proposed that the iridescent metallic sheen is due to the iridescence of the AQ signal itself (D’Argenio et al., 2002). In the parental SCV 20265 the metallic sheen became visible at sites of high bacterial densities (at the margin of the plated lawns, where AQ concentrations are expected to be high) and at the site of the AQ inoculum (Fig. 1, left). Applying this screen for more
Fig. 1. Screen of SCV 20265 transposon mutants for their responsiveness towards the exogenous addition of AQs. The P. aeruginosa SCV 20265 (left) and a SCV 20265-PA3271::Tn5 mutant (right) were streaked as a lawn onto Columbia agar plates. Whereas the wild type produced an iridescent metallic sheen in regions of high cell densities and at the inoculum site of the AQ solution in the centre of the plate, the SCV 20265-PA3271::Tn5 mutant exhibited a delayed production of AQ at sites of high cell densities at the margin of the plate and a delayed response at the site of AQ inoculum.
than 5000 mutants, we identified 40 mutants that were affected in AQ production. Several of those harboured a transposon insertion within metabolic genes, the biosynthetic pqsA–E operon and in one mutant the transposon inserted within the PA3271 gene. This mutant was developing a delayed iridescent metallic sheen at the margins of the bacterial lawn and a delayed response to the exogenous addition of AQ extracts (Fig. 1, right) or of synthetic PQS (data not shown). Reduced production of AQ in mutants harbouring transposon insertions within PA3271 We next aimed at investigating the involvement of ORF PA3271 in AQ synthesis. We grew the SCV 20265 wild type and the SCV 20265-PA3271::Tn5 strain in BHI broth medium and monitored AQ production over time. As shown in Fig. 2A, although not impaired in growth (data not shown), SCV 20265-PA3271::Tn5 exhibited a reduced production of 2-heptyl-3-hydroxy-4(1H)-quinolone (C7PQS) and 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) as well as their immediate precursors 2-heptyl-4(1H)quinolone (C7-HHQ) and 2-nonyl-4(1H)-quinolone (C9HHQ) during the incubation period as determined by mass spectrometry. In order to rule out that a second mutation was contributing to the observed phenotype of the SCV 20265-PA3271::Tn5, we also analysed the transposon mutant ID9775 from the P. aeruginosa PAO1 Washington mutant library (PAO1-PA3271::Tn5; Jacobs et al., 2003) and the transposon mutant PA_21700 of the P. aeruginosa PA14 Harvard Medical School library (PA14-PA3271::MrT7; Liberati et al., 2006) for their ability to produce AQs as compared to their respective PA14 and PAO1 (Gallagher et al., 2002) wild types. As shown in Fig. 2B and C, although again not impaired in
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growth (data not shown), the PA3271::Tn strains exhibited a reduced production of AQs during the cultivation period. Pyocyanin and rhamnolipid production can be fully restored in PA14-PA3271::MrT7 by complementation with the parental gene We next complemented PA14-PA3271::MrT7 by introducing the PA14 wild-type gene encoded on the pME6032 vector (Heeb et al., 2000), and monitored pyocyanin and rhamnolipid production, both known to be positively influenced by the activity of the AQs. As depicted in Fig. 3, PA14-PA3271::MrT7 exhibited a clearly reduced expression of pyocyanin and rhamnolipid, unless complemented with the PA3271 gene in trans. Transcriptional profile of PAO1-PA3271::Tn5
Fig. 2. PA3271 dependent 2-alkyl-4(1H)-quinolone production in P. aeruginosa. Derivatives of 2-alkyl-4(1H)-quinolones from cultures of (A) SCV 20265 and SCV 20265-PA3271::Tn5, (B) PA14 and PA14-PA3271::MrT7 and (C) PAO1 and PAO1-PA3271::Tn5 at different time points of growth were analysed by GC-mass spectrometry. The intensity of 2-heptyl-3-hydroxy-4(1H)-quinolone (C7-PQS, green bars), 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS, yellow bars) and their immediate precursors 2-heptyl-4(1H)-quinolone (C7-HHQ, white bars), and 2-nonyl-4(1H)-quinolone (C9-HHQ, black bars) is displayed in arbitrary units. The experiment was carried out twice with three biological replicates each. The mean values of one representative experiment are shown, and error bars represent the standard deviation from the mean. Statistical significance is indicated by asterisks (one-tailed t-test, P-value of ⱕ 0.05).
With the aim to analyse the impact of PA3271 on the global P. aeruginosa transcriptional profile, we used the PAO1 P. aeruginosa GeneChip (Affymetrix) and determined the transcriptional profile of PAO1-PA3271::Tn5 as compared to that of a PAO1-reference strain that harboured a transposon insertion in the open reading frame of PA4684 (Jacobs et al., 2003). We choose this transposon mutant as the reference since ORF PA4684 is most likely coding for a non-functional gene product due to a large gene deletion (Dötsch et al., 2009; Klockgether et al., 2010). Expression of 15 genes was found to be increased (P ⱕ 0.05) by greater than twofold in the mutant strain, whereas expression of 40 genes was reduced relative to PAO1-reference cultures (Table 1). Among the upregulated genes were mexT, mexS and the mexEF-oprN operon. It has previously been demonstrated that MexT does not only activate the expression of MexS and the Resistance-Nodulation-Division (RND) efflux pump MexEF-OprN, which is responsible for the increased resistance to chloramphenicol, trimethoprim and fluorochinolones, but that MexT is also a global LysR-type transcriptional regulator and induces the expression of various other genes independent of the MexEF-OprN efflux pump (Tian et al., 2009a,b). Most interestingly, all but one (PA0807) of the upregulated genes in PAO1PA3271::Tn5 have been previously shown to be transcriptionally induced by MexT (Tian et al., 2009a). This implicates that overexpression of mexT in the PAO1PA3271::Tn5 mutant largely determines the global transcriptional profile. MexT was furthermore shown to inhibit the expression of a number of virulence traits. This inhibition seems to be at least partially dependent on the induction of MexEF-OprN. Among the genes that were previously shown to be significantly reduced in a mexT overexpressing P. aeruginosa strain were hcnA and pqsA (Tian et al., 2009a). HcnA and © 2011 Blackwell Publishing Ltd, Molecular Microbiology, 83, 536–547
MexT-dependent quinolone signalling in P. aeruginosa 539 A
B
Fig. 3. PA3271 modulates pyocyanin and rhamnolipid production. A. The reduced pyocyanin production of the PA14-PA3271:MrT7 could be complemented by introducing the wild-type gene on the pME6032 vector. Bacteria were grown in BHI broth supplemented with 100 mg ml-1 tetracycline. Pyocyanin was extracted after 12, 24, 36 and 48 h of growth. B. The reduced rhamnolipid production of the PA14-PA3271::MrT7 (carrying the pME6032 vector control) could be complemented by introducing the wild-type gene on the pME6032 vector. The rhlR transposon mutant PA14-rhlR::MrT7 was used as a negative control. Bacteria were grown in LB broth supplemented with 100 mg ml-1 tetracycline. Rhamnolipids were extracted after 16 h of growth. The mean values of three biological replicates are shown, and error bars represent the standard deviation from the mean. Statistical significance is indicated by asterisks (one-tailed t-test, P-value of ⱕ 0.05).
two other MexT-repressed genes (PA1869 and PA4141) also exhibited a significantly reduced expression in PAO1PA3271::Tn5; however, we did not observe a differential in pqsA expression. Nevertheless, in agreement with the reduction of AQ production in PAO1-PA3271::Tn5, several AQ-dependent genes were repressed in the mutant, including the genes rhlA and rhlB, coding for the rhamnosyltransferase subunits A and B, the elastase lasB gene, and genes involved in the biosynthesis of phenazine, as well as the siderophores, pyoverdine and pyochelin.
Furthermore, AQ-dependent phenotypes such as the production of pyocyanin and rhamnolipid were reduced in PAO1-PA3271::Tn5 (Fig. S1). It was recently demonstrated that MexEF-OprN is involved in the secretion of HHQ (Lamarche and Deziel, 2011). If this pump is upregulated in the PAO1PA3271::Tn5 strain one would expect to identify lower levels of AQs and as a secondary effect a reduced transcription of the pqsA–E biosynthetic operon, since AQs bind to the transcriptional regulator PqsR and enhance the transcription of pqsA–E. Since under our experimental conditions this secondary effect on the transcription of the pqsA–E biosynthetic operon could not be detected, we monitored the expression of the pqsA–E genes in P. aeruginosa cultured under different conditions over time. Therefore, we introduced the PpqsA::gfp(ASV) reporter plasmid pAC37 (obtained from Tim TolkerNielsen; Yang et al., 2007) into PAO1-PA3271::Tn5 and the PAO1-reference strain. As depicted in Fig. S2, the GFP expression controlled by the pqsA promoter was significantly weaker and delayed in PAO1-PA3271::Tn5. Furthermore, we grew the bacteria within biofilms in flow chambers for 48 h and monitored the expression of the pqsABCDE operon by analysing microcolonies using confocal laser scanning microscopy. As shown in Fig. S3, the PAO1-reference exhibited strong GFP expression after 24 h and a significantly weaker expression level after 48 h. In contrast, cells of PAO1-PA3271::Tn5 showed on both days a significantly reduced GFP expression in all analysed microcolonies. Overexpression of the MexEF-OprN pump in the PA3271::Tn mutants In order to verify the transcriptome results, we monitored the expression of the MexEF-OprN pump by introducing the plasmid pUCPmexE-DsRed harbouring the mexE promoter DsRed fusion into the P. aeruginosa PAO1 wild type and the respective PA3271::Tn strain and quantified the red fluorescence. PAO1-PA3271::Tn5 clearly exhibited an enhanced activity of the mexE promoter (262.8 RFU ⫾ 6) as compared to the PAO1 wild type (87.5 RFU ⫾ 1.7) after 36 h of incubation (t-test, P-value ⱕ 0.01). Furthermore, in accordance with the overexpression of the MexEF-OprN pump, the PA3271::Tn mutant exhibited an increased resistance against ciprofloxacin. By the use of E-tests the minimal inhibitory concentration (MIC-values) of ciprofloxacin was 0.5 mg ml-1 for PAO1PA3271::Tn5 and 0.38 mg ml-1 for PA14-PA3271::MrT7 as compared to 0.094 mg ml-1 for both PAO1 and PA14 wildtype strains. Complementation of PA14-PA3271::MrTn7 with the PA3271 gene in trans fully restored the susceptibility profile of the PA14 wild type.
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Table 1. P. aeruginosa genes differentially regulated in the mutant PAO1-PA3271::Tn5 in comparison to PAO1-reference, determined using a microarray.
Genea Upregulated PA0807 PA1970 PA2491 PA2492 PA2493 PA2494 PA2495 PA2759 PA2811 PA2812 PA2813 PA3229 PA4356 PA4623 PA4881 Downregulated PA0122 PA0140 PA1094 PA1300 PA1473 PA1869 PA2033 PA2067 PA2068 PA2069 PA2193 PA2300 PA2426 PA3237 PA3371 PA3475 PA3478 PA3479 PA3724 PA3811 PA3812 PA3814 PA3815 PA4141 PA4156 PA4206 PA4207 PA4211 PA4221 PA4225 PA4228 PA4338 PA4469 PA4470 PA4471 PA4625 PA4709 PA4738 PA4898 PA5220
Change (fold)b
Designation
Description
ampDh3
ampDh3 Hypothetical protein Probable oxidoreductase Transcriptional regulator MexT Efflux membrane fusion protein MexE Efflux transporter MexF Multidrug efflux OM protein OprN Hypothetical protein Probable permease of ABC-transporter Probable ATP-component of ABC transporter
mexT mexE mexF oprN yadH yadG yliJ xenB
ahpF fliD
hcnA chiC pvdS
pheC rhlB rhlA lasB hscB iscA iscS
mexH mexI phzB1 fptA pchF pchD
fumC1
phuS opdK
Probable glutathione S-transferase Hypothetical protein Xenobiotic reductase Hypothetical protein Hypothetical protein Alkyl hydroperoxide reductase subunit F Flagellar capping protein FliD Probable sigma -70 factor Hypothetical protein Probable acyl carrier protein Hypothetical protein Probable hydrolase Probable MFS transporter Probable carbamoyl transferase Hydrogen cyanide synthase HcnA Chitinase Sigma factor PvdS Hypothetical protein Hypothetical protein Cyclohexadienyl dehydratase precursor Rhamnosyltransferase chain B Rhamnosyltransferase chain A Elastase LasB Heat shock protein HscB Probable iron-binding protein IscA L-cysteine desulphurase Hypothetical protein Hypothetical protein Probable TonB-dependent receptor Efflux membrane fusion protein Efflux transporter Probable phenazine biosynthesis protein Fe(III)-pyochelin outer membrane receptor Pyochelin synthetase Pyochelin biosynthesis protein PchD Hypothetical protein Hypothetical protein Fumarate hydratase Hypothetical protein Hypothetical protein Probable hemin degrading factor Hypothetical protein Histidine porin OpdK Hypothetical protein
Signal intensity [mut]c
Signal intensity [ref]d
2.2 3.8 22.9 4.3 13.9 20.7 6.9 8.7 2.1 2.8 3.3 25.0 3.1 7.2 22.7
3882.79 317.57 1522.98 361.23 1798.97 1123.06 894.01 536.54 171.15 583.82 1114.23 3453.24 406.30 1460.08 3209.76
1741.78 83.51 66.50 84.88 129.09 54.33 129.91 61.69 81.67 210.07 337.13 137.96 129.46 203.50 144.16
-4.5 -5.1 -2.5 -2.9 -4.0 -4.0 -3.4 -2.8 -3.3 -6.9 -3.8 -11.1 -4.4 -22.6 -2.7 -2.5 -7.0 -8.9 -6.2 -2.1 -2.3 -3.1 -3.9 -7.4 -12.1 -5.3 -5.2 -4.4 -10.0 -3.9 -6.5 -3.3 -14.8 -18.1 -23.3 -2.7 -2.3 -2.7 -2.3 -5.2
574.31 297.74 708.43 89.02 107.48 96.32 84.45 107.65 116.25 100.42 394.27 120.26 161.12 58.97 487.62 110.09 121.24 284.21 999.14 161.06 1278.04 1159.29 765.09 998.94 65.65 281.61 83.25 444.11 168.79 104.90 145.38 406.31 141.03 97.29 115.39 155.77 192.79 625.03 201.88 130.08
2583.10 1504.28 1771.60 253.64 432.03 384.02 285.96 301.78 378.71 695.58 1498.26 1336.10 701.16 1332.02 1299.93 277.93 847.39 2539.81 6168.82 341.07 2932.78 3565.58 2955.76 7348.10 793.12 1478.04 431.73 1936.00 1679.22 409.97 484.84 129.47 2080.48 1759.12 2688.95 414.62 446.35 1688.60 456.96 680.49
a. According to the P. aeruginosa genome website (http://www.pseudomonas.com/). b. Regulation of genes differentially expressed in the PAO1-PA3271::Tn5 mutant relative to the PAO1-reference. A positive number indicates transcript upregulation in the PAO1-PA3271::Tn5 mutant. c. Signal intensity PAO1-PA3271::Tn5. d. Signal intensity PAO1-reference.
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Fig. 4. MexT-mediated effects on pyocyanin and rhamnolipid production. Overexpression of mexT in the PA14 wild type (PA14 pME::mexT) mimics the PA14-PA3271::MrT7 mutant phenotype in respect to pyocyanin and rhamnolipid production. For pyocyanin extraction bacteria were grown 12 h in BHI, and rhamnolipids were extracted after 16 h of growth in LB broth. Both media were supplemented with 1 mM IPTG and 100 mg ml-1 tetracycline.
MexT overexpression mimics the PA14-PA3271::MrTn7 phenotype We furthermore analysed whether in PA14-PA3271::MrT7 de-repression of MexT was responsible for the observed phenotype. We therefore overexpressed mexT in the PA14 wild-type strain and monitored rhamnolipid and pyocyanin production. As depicted in Fig. 4, the mexT overexpressing P. aeruginosa PA14 strain exhibited – similar to the respective PA14-PA3271::MrTn7 – a reduced pyocyanin and rhamnolipid production as compared to the wild type. Structural homology of the PA3271 sensor kinase to E. coli ArcB The PA3271 gene encodes for an orphan putative hybrid sensor kinase belonging to the group of ITR-type of
histidine kinases carrying Input, Transmitter and Receiver domains according to the classification by Rodrigue et al. (2000). The protein encoded by PA3271 shares structural homology to ArcB in E. coli and to the membraneassociated unorthodox histidine kinase BvgS which is involved in virulence factor expression in Bordetella pertussis. Figure 5 depicts the structural homology of E. coli ArcB and B. pertussis BvgS in comparison to the 1159-amino-acid-long PA3271 protein. The N-terminal transmembrane domain of PA3271 is predicted [SMART database (http://smart.embl-heidelberg.de)] to have 12 membrane-spanning a-helices followed by a Per-ArntSim (PAS) motif, a histidine kinase A phosphoacceptor domain (HisKA), a histidine kinase-like ATPase domain (HATPase_c) and a cheY-homologous receiver (REC) domain at its C-terminus. Despite the overall similarity of the structural domain organization, the identity/similarity at the amino acid level is low.
The PA3271 sensor kinase is responsive to the oxidation status of a quinone electron carrier In the E. coli ArcAB system the histidine kinase ArcB activity is reduced in the presence of the oxidized forms of quinones (Malpica et al., 2004). In addition to ArcB, the intracellular PAS domain of the membrane-associated unorthodox histidine kinase BvgS in B. pertussis was shown to be involved in sensing the intracellular oxidation state of the cells via the respiratory chain electron carrier ubiquinone (Bock and Gross, 2002). Due to the structural similarities, we wondered whether the P. aeruginosa PA3271 sensor kinase may also be redox-responsive. We cloned the cytosolic portion of the PA3271 gene comprising PAS, HisKA, HATPase-c and receiver domain (amino acids 624– 1159) into the glutathione (GST) expression vector pGEX-6-P1 resulting in the hybrid plasmid pGEX-6P1::3271c. The truncated sensor kinase fusion protein
Fig. 5. Domain organization of P. aeruginosa PA3271 compared to that of E. coli ArcB and B. pertussis BvgS. The numbers in shaded areas between the domains of PA3271 and BvgS and of PA3271 and ArcB display levels of identity/similarity between the domains. Black vertical bars show the positions of transmembrane domains. PBPb, bacterial periplasmic substrate-binding proteins; PAS, energy sensing domain; HisKa, histide kinase dimerization domain; HATPase_c, histidine kinase ATPase domain; REC, receiver domain; Htp, histidine phosphotransfer domain. © 2011 Blackwell Publishing Ltd, Molecular Microbiology, 83, 536–547
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Discussion
Fig. 6. Ubiquinone-0 (Q0) inhibits autophosphorylation of the PA3271 sensor kinase’s cytosolic domain. On the top panel (A) a representative autoradiograph of the PA3271 sensor kinase after 3 min of reaction in the presence of oxidized Q0 (0.25 mM), Q0 (0.25 mM) with 1 mM dithionite (DTT), DTT alone (1 mM), s-methyl methanethiosulphonate (MMTS) alone and MMTS with DTT is shown. The lower panel (B) shows the corresponding Bis-Tris gel, which illustrates PA3271 sensor kinase protein quantity for each reaction condition. This figure is representative of four independent experiments. Similar results are obtained after 7 min of reaction time.
lacking the N-terminal transmembrane domain was then used in autophosphorylation assays and incubated in the presence of soluble analogues of ubiquinone-0 (Q0) and [gg-32P] adenosine triphosphate (ATP). As demonstrated in Fig. 6, autophosphorylation was clearly inhibited by ubiquinone. However, as opposed to the E. coli ArcB the thiol-reducing agent dithionite (DTT) did not enhance the in vitro phosphorylation of the PA3271 sensor kinase but instead drastically reduced autophosphorylation even in the absence of an oxidizing agent. This indicates that DTT per se affects the phosphorylation kinetics of the PA3271 sensor kinase most likely due to a negative effect on protein conformation. Similarly, the addition of s-methyl methanethiosulphonate (MMTS), which reacts specifically with the free sulphhydryl groups on cysteine side chains to form Cys-S-CH3, inhibited autophosphorylation of the PA3271 sensor kinase. The addition of up to 500 mM PQS did not have an effect on autophosphorylation of the sensor kinase (data not shown). It has been suggested that in E. coli ArcB two cystein residues (Cys-180 and Cys-241) are involved in the mechanisms of ArcB regulation since they are required for the action of the inhibiting agents (Alvarez and Georgellis, 2010). However, in contrast with most other ArcB homologues that possess two conserved cysteine residues, PA3271 of P. aeruginosa possesses eight cysteine residues that are located at positions 50, 124, 259, 697, 990, 1043, 1063, 1074 of the protein. Thus, although our results clearly indicate that the physiological redox state is signalled to the PA3271 sensor kinase by the oxidized form of quinone electron carriers, the molecular event of redox signalling remains to be shown.
In P. aeruginosa PAO1 there are 50 (putative) sensor kinases. Seventeen of those belong to the small group of so-called unorthodox sensor proteins because they harbour an intermediate receiver at the C-terminus in addition to the classical transmitter domain. Whereas most of the genes encoding for the proposed sensor kinases are organized within one operon with their cognate response regulators, 14 sensor kinases, all of them unorthodox sensor kinases, are encoded by orphan genes. Functional descriptions of such unorthodox or hybrid kinases and their respective response regulators have been performed in a few bacteria, for example, LuxN/LuxO in Vibrio harveyi (Freeman and Bassler, 1999), ArcB/ArcA in E. coli, V. cholerae and H. influenzae (Iuchi and Lin, 1992), BvgS/BvgA in B. pertussis (Beier and Gross, 2006) and VirA/VirG in Agrobacterium spp. (Winans et al., 1986; Leroux et al., 1987). These histidine kinases regulate the expression of many genes which are involved in bacterial virulence in response to diverse environmental conditions (Bekker et al., 2006). For the ArcB and BvgS sensor kinases it was shown that the oxidized form of quinones specifically interferes with the kinase activity and turns off the enzymatic activity, whereas the reduced form remains without effect (Bock and Gross, 2002; Malpica et al., 2004; 2006). Both sensor kinases, ArcB and BvgS, harbour a PAS domain in the cytoplasmic portion between the transmembrane and the transmitter domain. PAS domains are signalling domains that function as input modules able to perceive oxygen, redox potential, light and other stimuli (Möglich et al., 2009). In this study, we describe the identification of an unorthodox hybrid sensor kinase in P. aeruginosa (MxtR) that harbours a cytoplasmatic PAS domain and that shares structural and functional characteristics with the ArcB/ BvgS unorthodox sensor kinases. We provide evidence that autophosphorylation of this sensor kinase is inhibited by ubiquinone in vitro and that this sensor kinase is involved in the repression of the MexT regulon. Previous studies have shown that the Arc regulons in different bacterial pathogens can be strikingly various. For example, whereas ArcA is affecting the activity of the TCA cycle under anaerobic conditions in E. coli, its role in mediating metabolism in Shewanella oneidensis is rather minor (Gao et al., 2008), although E. coli ArcB was shown to be able to functionally replace ArcS in S. oneidensis (Lassak et al., 2010). Furthermore, although the E. coli ArcA regulon generally mediates regulation of operons involved in respiratory metabolism, it also affects chromosomal replication at oriC (Lee et al., 2001), flagella synthesis (Kato et al., 2007), cell division and stress-induced survival (Nishijyo et al., 2001). Moreover, the expression of the general stress sigma factor RpoS was found to be © 2011 Blackwell Publishing Ltd, Molecular Microbiology, 83, 536–547
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directly repressed by ArcA (Mika and Hengge, 2005) and the ArcAB system is also important for bacterial resistance to reactive oxygen species (ROS) under aerobic conditions (Loui et al., 2009). The lifestyles and natural environments of E. coli and P. aeruginosa are substantially different and it has been suggested that regulatory systems adopted by individual species are adjusted accordingly (Perez and Groisman, 2009). In P. aeruginosa efficient adaptation to a microaerophilic environment seems to be crucial for survival and persistence, e.g. during the chronic infection of the CF lung, and P. aeruginosa has been shown to respond to various stimuli likely to be encountered within the human host (Zaborina et al., 2007; Zaborin et al., 2009). The cognate response regulator of the PA3271 sensor kinase MxtR in P. aeruginosa remains unknown; however, our transcriptional studies implicate that a main target of this regulator is MexT, a LysR-type transcriptional regulator. We found not only many MexT-regulon genes that were upregulated in the mutant, but also a reduced expression of the AQs and the AQ-dependent virulence factors pyocyanin and rhamnolipids. The latter phenotype seems to be at least in part due to a MexT-dependent upregulation of the MexEF-OprN efflux pump, which has been shown to be involved in the export of HHQ (Köhler et al., 2001; Martinez et al., 2009; Lamarche and Deziel, 2011). Taken together, our studies have identified a novel sensor kinase of a two-component system in P. aeruginosa which is involved in the regulation of interbacterial signalling in response to environmental conditions. Future studies will focus on the identification of the cognate response regulator and the elucidation of the involvement of the MexT-LysR type transcriptional regulator in the transduction of the bacterial response.
Experimental procedures Bacterial strains and culture conditions The bacterial strains and plasmids used in this study are described in Table S1. Transposon mutants of the clinical small colony variant strain SCV 20265 were generated by the use of EZ::TN™ Transposon mutagenesis tool (Epicentre Biotechnologies, USA). P. aeruginosa and E. coli strains were routinely cultured at 37°C in Brain Heart Infusion medium (BHI, supplied by Becton, Dickinson and Company), and Luria broth (LB) medium. Tetracyclin and ampicillin were used at a concentration of 100 mg ml-1, carbenicillin at a concentration of 400 mg ml-1 and gentamicin at a concentration of 15 mg ml-1.
agar plate (Oxoid) by the use of a cotton swab, and 2 ml of a P. aeruginosa SCV 20265 stationary phase culture methanol extract (containing high concentrations of AQs) was spotted onto the centre of the agar plate. More than 5000 mutants (on approximately 2500 agar plates) were monitored for the appearance of an iridescent metallic sheen on the surface of the lawns following an incubation at 37°C for 14–16 h. Transposon insertions in mutants affected in the production of the metallic sheen were determined by the amplification of the transposon flanking regions using a linker as described (Kwon and Ricke, 2000).
Resistance testing To determine the MIC-values of the bacterial strains against ciprofloxacin, three colonies from an overnight culture grown on LB-Agar were resuspended in 10 ml PBS and poured over a Mueller Hinton agar plate. After decanting, E-test stripes purchased from bioMérieux were placed on top of the agar and incubated at 37°C overnight before analysis by optical inspection.
Extraction and quantification of extracellular P. aeruginosa AQ metabolites 2-Alkyl-4(1H)-quinolone metabolites were extracted at given time points from P. aeruginosa BHI broth cultures (50 ml of culture medium in 250 ml flasks, shaken at 180 r.p.m.). Therefore, bacterial cultures were extracted with two volumes of dichloromethane by vigorous shaking (Bredenbruch et al., 2005). After centrifugation at 3500 g for 10 min, the lower organic layer was separated from the liquid supernatant and subsequently dried by evaporation. 2-Alkylquinoline derivatives were analysed by GC-MS after trimethylsilylation [50% pyridine, 50% BSTFA (bistrimethylsilyltrifluoroacetamide) containing 1% TMC (trimethylchlorosilane)] (70°C, 1 h) with a Thermo-Finnigan GCQ ion trap mass spectrometer (Finnigan MAT, San Jose, CA) running in the positive-ion electron impact (EI) mode equipped with a 30 m DB5 capillary column as described by Bredenbruch et al. (2005). Quantification was performed by electronic integration of the most abundant fragment ion traces at m/z 231 (HHQ) and 304 (PQS) and correction of the integrals by the relative intensities of the respective fragment ions.
Construction of pME6032::PA3271 A 3778 bp fragment consisting of the PA3271 coding sequence and its 300 bp upstream region was amplified with HotStar HiFidelity Polymerase (Qiagen) from PA14 chromosomal DNA using the forward primer PA3271PLongEcoRI and reverse PA3271LRevSacI. The purified PCR fragment was digested with EcoRI/SacI restriction endonucleases and cloned into the similarly digested broad-host range vector pME6032.
Screen for AQ non-responsive mutants For the screen of the responsiveness of SCV 20265 transposon mutants towards the exogenous addition of AQs, the mutants were streaked onto half of a Columbia sheep blood
Pyocyanin measurement For pyocyanin assays, P. aeruginosa strains were grown with aeration in BHI broth (PA14) and DeMoss (PAO1) medium at
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37°C and pyocyanin levels were determined in culture supernatants as a modification of the method previously described by Xu et al. (2005). Samples of grown cultures were centrifuged to remove bacterial cells, and 500 ml aliquots of culture supernatants were extracted with 500 ml of chloroform. After vigorous shaking for 1 min and centrifugation (3 min, 16 000 g) the organic phase was extracted with 250 ml of 0.2 N HCl. The absorbance of this solution at 520 nm was measured using a NanoDrop spectrophotometer.
Rhamnolipid measurement Rhamnolipid production was monitored by indirect quantification of hexose sugars using the orcinol method as described by Wilhelm et al. (2007). Strains were grown in 5 ml LB broth supplemented with tetracycline for pME6032 plasmid maintenance. A P. aeruginosa PA14 rhlR transposon mutant (PA14-rhlR::MrTn7, ID-19120; Liberati et al., 2006) was used as a rhamnolipid negative control.
PqsA promoter activity measured by GFP fluorescence PqsABCD operon expression was monitored by introducing the PpqsA – gfp(ASV) transcriptional fusion [pAC37, kindly provided by Tim Tolker-Nielsen (Yang et al., 2007)] into the PAO1-PA3271::Tn5 mutant and the respective PAO1reference. Strains were grown in 96-well plates in LB broth at 37°C. Plasmid-derived, pqsA promoter-induced GFP expression was measured using Fusion universal microplate analyser (PerkinElmer).
Biofilm analysis Pseudomonas aeruginosa PAO1 strains were grown in FAB medium (Heydorn et al., 2000) in continuous-culture flow cells (channel dimension, 1 by 4 by 40 mm) at 30°C. Channels were inoculated with 0.2 ml of diluted, early-stationaryphase cultures containing approximately 5 ¥ 107 cells ml-1 and incubated without flow for 1.5 h. The experiment was started with a mean flow of 0.2 mm s-1. Biofilm cells expressing the pqsA promoter-controlled GFP(ASV) from plasmid pAC37 were visualized using a confocal laser scanning microscope (FluoView1000; Olympus) 20 ¥/0.75 N.A. objective (dry) with adequate filter settings. Laser intensity and detector settings were not changed throughout the experiment to allow comparison of fluorescence intensities. Zoomed images of the microcolonies were generated with the software Imaris (same magnification for all samples).
Construction of pGEX-6-P1(3271c) In order to amplify the gene sequence coding for the intracellular part of the PA3271 sensor kinase, the primers GSTPA3271FW and GSTPA3271RV were used in a PCR reaction with P. aeruginosa genomic DNA as template (Table S2). The PCR product was isolated, hydrolysed with EcoRI and BamHI and subsequentially cloned into similarly digested pGEX-6-P1 (promega) resulting in the plasmid pGEX-6-P1::3271c.
Construction of the mexE promoter DsRed fusion pUCPmexE – DsRed For the construction of a mexE promoter DsRed fusion, a 265 bp region upstream of the mexE gene was amplified from P. aeruginosa PAO1 chromosomal DNA using the primers PmexEFW and PmexERV (Table S2). The PCR product was transcriptionally fused to DsRed from pMW212 and subsequently introduced into pUCP20. The resulting plasmid pUCPmexE – DsRed was verified by sequence analysis and introduced into P. aeruginosa PAO1-PA3271::Tn5 and wildtype strain by electroporation.
Construction of pME::mexT The mexT gene was amplified from P. aeruginosa PA14 chromosomal DNA using the primers mexTFW and mexTRV (Table S2). The PCR product was digested with KpnI/HindIII and cloned into the similarly digested pUCP20, resulting in the plasmid pUCP20::mexT, which was verified by sequence analysis. Following, the fragment was subcloned into pBluescript KS+ by SacI/HindIII restriction, to allow subsequent introduction of the mexT gene into pME6032 by SacI/XhoI restriction, resulting in the plasmid pME::mexT. The pME6032 and pME::mexT plasmids were introduced into P. aeruginosa wild-type strains by electroporation. Protein expression was induced by adding 1 mM IPTG (isopropyl-mD-thiogalactopyranoside) to the bacterial culture.
Protein expression and purification Cells of E. coli (pGEX-6-P1::3271c) were grown in LB broth at 20°C under shaking conditions to an optical density of OD600 0.6. Protein expression was induced by adding IPTG to a final concentration of 0.1 mM. After an overnight incubation, bacteria were harvested by centrifugation at 6000 g for 10 min, and bacterial pellets were resuspended in cold lysis buffer (PBS pH 7.4, 1 mM dithioerythritol, complete Mini EDTA-free protease inhibitor cocktail, Roche). One millilitre aliquots of bacterial suspension were pipetted into vials containing 50 ml glass beads (0.1 mm, BioSpec). The vials were submitted to ribolysis using a Hybaid ribolyser (Hybaid) and quickly placed on ice. Vials were subsequently centrifuged for 10 min at 20 000 g at 4°C and the supernatant obtained was loaded onto a column containing PBS equilibrated Glutathione Sepharose High Performance beads (GE Healthcare). After 1 h of incubation at 4°C, the beads were washed with 600 ml PBS and equilibrated in reaction buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2). Due to protein instability, elution from the beads was not performed and fresh GST – 3271c fusion protein, bound to the beads, was directly used for the phosphorylation assays.
Autophosphorylation assay The autophosphorylation assays were carried out at room temperature in a reaction buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2). Prior to the addition of 0.2 mM (2 mCi) of gamma-P32 ATP (specific activity 6000 Ci mmol-1), aliquots of beads containing 50 pmol of proteins were preincubated at 37°C for 30 min with or without 1 mM s-methyl © 2011 Blackwell Publishing Ltd, Molecular Microbiology, 83, 536–547
MexT-dependent quinolone signalling in P. aeruginosa 545
methanethiosulphonate (MMTS), followed by 10 min incubation with either 0.25 mM ubiquinone 0 (Q0), Q0 with 2 mM dithionite (DTT), or 2 mM DTT alone. The phosphorylation reaction was initiated by the addition of gamma-P32 ATP and terminated after 3 min by addition of 10 ml 4 ¥ LDS sample buffer (Invitrogen). Samples were heated at 70°C for 10 min and subsequently submitted to SDS/PAGE electrophoresis, using NuPAGE Novex 10% Bis-Tris gels (Invitrogen). After electrophoresis, the gels were stained with Instant Blue (expedeon), washed with PBS and exposed to Fugifilm M membrane over 5 days. Protein autophosphorylation was determined qualitatively using a PhosphorImager.
Microarray analyses Pseudomonas aeruginosa PAO1-reference and PAO1PA3271::Tn5 cultures were grown at 37°C for 14 h in LB medium (late exponential growth phase). Three independent cultures were pooled, the RNA was immediately stabilized with RNAprotect Bacteria Reagent (Qiagen) and total RNA was isolated using RNeasy Mini Columns (Qiagen). Contaminating genomic DNA was removed by treatment with a DNA-free kit (Ambion, Austin, TX). RNA quality was assessed spectrophotometrically and by agarose gel electrophoresis. Two Affymetrix GeneChips were hybridized for each strain. RNA isolation, cDNA generation, fragmentation, biotinylation, GeneChip hybridization and analysis were performed according to the Affymetrix protocol. Probeset summarization and data normalization was done using RMA (Irizarry et al., 2003). Data were joined with the latest annotation from the website of the P. aeruginosa PAO1 sequence and the community annotation project provided at http://www.pseudomonas.com. Datasets were combined into two groups separating transcriptome data according to their genotype. Averaged signal intensities were used to calculate transcriptional differences between both groups. A Student’s t-test was applied to filter out differentially regulated genes showing similar expression patterns in both replicates. Changes of ⱖ twofold P-value of ⱕ 0.05 were used as the cut-offs for reporting expression changes.
Acknowledgements We thank Tim Tolker-Nielsen (Technical University Copenhagen, Denmark) for providing the PpqsA::gfp reporter plasmid, Andreas Dötsch, Robert Geffers and Vanessa Jensen for helpful discussions and Petra Hagendorf for excellent technical assistance. M.M. was supported by the International Research Training Group 1273 funded by the German Research Foundation (DFG). C.Z. was supported by a Marie Curie Early Stage Research Training Fellowship of the European Community’s Sixth Framework Program under contract number MEST-CT-2004-504990. Financial support from the Mukoviszidose e.V., the BMBF and the HelmholtzGemeinschaft is gratefully acknowledged.
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