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1 Morehouse School of Medicine, Department of Microbiology, Biochemistry and Immunology, 720 Westview Dr. SW, Atlanta, GA, USA
2 Computer Architecture Department, Facultad de Ciencias Físicas, Universidad Complutense de Madrid, Madrid 28040, Spain
Correspondence
Anisia. J. Silva
asilva-benitez{at}msm.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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crp mutant of El Tor biotype V. cholerae. Here we show that CRP is required for the biosynthesis of cholera autoinducer 1 (CAI-1) and affects the expression of multiple HapR-regulated genes. As expected, the crp mutant produced more cholera toxin and enhanced biofilm. Expression of flagellar genes, reported to be affected in hapR mutants, was diminished in the crp mutant. However, an epistasis analysis indicated that cAMP–CRP affects motility by a mechanism independent of HapR. Inactivation of crp inhibited the expression of multiple genes reported to be strongly induced in vivo and to affect the ability of V. cholerae to colonize the small intestine and cause disease. These genes included ompU, ompT and ompW encoding outer-membrane proteins, the alternative sigma factor 
E required for intestinal colonization, and genes involved in anaerobic energy metabolism. Our results indicate that CRP plays a crucial role in the V. cholerae life cycle by affecting quorum sensing and multiple genes required for survival of V. cholerae in the human host and the environment.
The GEO platform accession number for the TIGR V. cholerae version 2 microarray used in this study is GLP4353. The GEO series record number for the raw data files is GSE7519.
A complete list of differentially expressed genes in the crp mutant is provided as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), enhances enterotoxicity in rabbit ileal loops (Silva et al., 2006) and contributes to reactogenicity in volunteers receiving live genetically attenuated cholera vaccines (Benitez et al., 1999). Expression of HA/protease is regulated by quorum sensing (Jobling & Holmes, 1997; Zhu et al., 2002). Quorum sensing is a process by which bacteria communicate with one another by secreting extracellular signalling molecules termed autoinducers. In V. cholerae, two autoinducer/sensor systems have been identified. System 1 consists of cholera autoinducer 1 (CAI-1), synthesized by the activity of CqsA, and its cognate receptor CqsS (Miller et al., 2002). System 2 consists of an AI-2 molecule, synthesized by the activity of LuxS, and its cognate receptor LuxPQ (Miller et al., 2002). At low cell density the autokinase domains of CqsS and LuxPQ become phosphorylated and phosphorus is transferred to LuxU and then LuxO (Henke & Bassler 2004; Miller et al., 2002). LuxO-P activates expression of four small regulatory RNAs (sRNAs), Qrr1–4, which in conjunction with the RNA-binding protein Hfq destabilize hapR mRNA (Lenz et al., 2004; Bejerano-Sagie & Xavier, 2007). When the amount of CAI-1 and AI-2 produced by growing bacteria reaches a threshold value, CqsS and LuxPQ switch from kinase to phosphatase. The flow of phosphorus is reversed and LuxO becomes dephosphorylated and inactive (Henke & Bassler, 2004; Lenz et al., 2004; Miller et al., 2002). At this stage (high cell density), HapR and HA/protease are expressed (Zhu et al., 2002). A third two-component system controlling quorum sensing is VarS/VarA, which regulates transcription of the sRNAs CsrB, CsrC and CsrD (Lenz et al., 2005; Bejerano-Sagie & Xavier, 2007). The sRNAs CsrBCD control the activity of the global regulator CsrA (Lenz et al., 2005; Bejerano-Sagie & Xavier, 2007), which enhances the activity of LuxO-P at low cell density. Recently the small nucleoid protein Fis has been reported to enhance degradation of hapR mRNA at low cell density (Lenz & Bassler, 2007).
The HapR regulator plays a pivotal role in regulating virulence gene expression and biofilm development (Hammer & Bassler, 2003; Zhu & Mekalanos, 2003). At low cell density the regulator AphA activates expression of TcpP/H (Häse & Mekalanos, 1998; Kovacikova & Skorupski, 2001), which in concert with a second pair of transmembrane regulators (ToxR/S) activates expression of the soluble regulator ToxT (Miller et al., 1989). ToxT acts positively at the ctxA and tcpA promoters to activate production of cholera toxin (CT) and the toxin co-regulated pilus (TCP) (DiRita et al. 1991). At high cell density, HapR represses the transcription of aphA to diminish CT and TCP production (Kovacikova & Skorupski, 2002), represses the exopolysaccharide genes involved in biofilm formation (Yildiz et al., 2004) and activates hapA (Jobling & Holmes, 1997; Zhu et al., 2002).
Transcription of hapA occurs in the stationary phase and requires the cAMP receptor protein (CRP) and RpoS (Benitez et al., 2001; Silva & Benitez, 2004). Although the hapA promoter contains a putative cAMP–CRP binding site, we have not observed binding of purified V. cholerae cAMP–CRP to the hapA promoter (Silva & Benitez, 2004). Instead, we observed that a crp : : Km mutant produced lower levels of hapR and rpoS mRNA (Silva & Benitez, 2004). The V. cholerae hapR promoter does not contain a cAMP–CRP binding site, suggesting that CRP does not act directly on hapR.
The CRP global regulator is the determinant of carbon catabolite repression. Carbon catabolite repression can be defined as the inhibition of gene expression and/or protein activity by the presence of a rapidly metabolizable carbon source in the growth medium (Brückner & Titgemeyer, 2002; Kolb et al., 1993; Stülke & Hillen, 1999). This is achieved through activation of adenylate cyclase by a component of the phosphoenolpyruvate-dependent phosphotransferase system (Deutscher et al., 2006). Activation of adenylate cyclase leads to high intracellular levels of cAMP (Ganguly & Greennough, 1975; Kolb et al., 1993) and formation of the cAMP–CRP complex, which binds to responsive promoters to activate or repress transcription (Kolb et al., 1993). The cAMP–CRP complex binds as a dimer to the consensus sequence TGTGA-(N6)-TCACAA, which can be found within, adjacent or upstream of responsive promoters (Brückner & Titgemeyer, 2002; Kolb et al., 1993; Stülke & Hillen, 1999). In Gram-negative bacteria, carbon catabolite repression is a major global regulatory mechanism that influences many signal transduction pathways. CRP has been shown to negatively affect the expression of virulence factors, CT and TCP in classical and El Tor biotype strains grown in LB medium (Skorupski & Taylor, 1997). However, crp mutants are defective in intestinal colonization. This suggests that CRP positively controls additional factors required for V. cholerae survival in the small intestine. Intestinal colonization is a multifactorial process requiring many genes. It has been shown that V. cholerae mutants defective in the alternative sigma factors S (RpoS) and E (RpoE), outer-membrane proteins OmpU and OmpT and motility exhibit diminished colonization of the suckling mouse intestine (Merrell et al., 2000; Kovacikova & Skorupski, 2002a; Provenzano & Klose, 2000; Silva et al., 2006).
To better understand the role of crp in pathogenesis, we performed global gene expression profiling of a crp V. cholerae mutant of the El Tor biotype. Inactivation of crp affected production of CAI-1, multiple HapR-regulated genes, motility, the alternative sigma factor RpoE, outer-membrane proteins and genes involved in anaerobic respiration.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and 2. E. coli strains were grown in LB medium at 37 °C with agitation (250 r.p.m.). V. cholerae strains were grown in LB medium at 37 °C. For HA/protease production, they were grown in Bacto tryptic soy broth (TSB) at 37 °C, and for CT production, in AKI medium at 30 °C (Iwanaga et al., 1986). Plasmid DNA was introduced into V. cholerae by electroporation (Marcus et al., 1990). Culture media were supplemented with ampicillin (Amp, 100 µg ml–1), tetracycline (Tet, 10 µg ml–1), kanamycin (Km, 100 µg ml–1), X-Gal (40 µg ml–1) or polymyxin B (PolB, 100 units ml–1) as required.
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In all cases, mutants were obtained by cloning a chromosomal DNA fragment containing a deletion of the target gene in pCVD442. The resulting vector was transferred by conjugation to the receptor strain and sucrose selection was applied to isolate segregants retaining the mutant allele. Briefly, to construct crp mutants WL7258 and WL51 (Table 1
), chromosomal DNA flanking the crp ORF was amplified using primer pairs CRP362/CRP1221 and CRP1784/CRP2595, respectively. The amplified DNAs were sequentially cloned in pUC19 to generate plasmids pCRP1, pCRP2 and pCRP (Table 1). The chromosomal fragment containing the crp deletion was transferred from pCRP to pCVD442 (Donnenberg & Kaper, 1991) to generate pCVDCRP (Table 1). The suicide vector pCVDCRP was constructed in E. coli SM10
pir and mobilized to strains C7258 and AJB51 (Table 1) by conjugation. Exconjugants were selected in LB medium containing Amp and PolB and streaked on LB agar containing 15 % (w/v) sucrose. Sucrose-resistant colonies were tested for Amp sensitivity and deletion of crp was confirmed by DNA sequencing using primers CRP873 and CRP2105. A similar strategy was used to construct the hapR mutant AJB51, rpoS mutant AJB50 and cqsA mutant AJB61 (Table 1). Suicide vectors pCVDhapR, pCVDrpoS2 and pCVDcqsA (Table 1) were transferred by conjugation to C7258; the corresponding mutants were obtained by sucrose selection and confirmed by DNA sequencing. Finally, to construct the luxS insertion mutant M36, luxS was amplified using primers LuxSF1 and LuxSR1301. The amplicon was cloned in pUC18 and luxS was subsequently inactivated by insertion of a Km cassette from pUC4K (GenBank accession no. X06404). The luxS : : Km allele was transferred to pCVD442 and the resulting plasmid introduced by conjugation into C7258. The luxS : : Km mutant M36 was obtained by sucrose selection and confirmed by DNA sequencing.
Differential gene expression analysis.
The V. cholerae version 2 microarray (GEO platform accession number GLP4353) consisting of 3811 70-mer oligonucleotides representing ORFs from V. cholerae strain N16961 was provided by The Institute for Genome Research Pathogen Functional Genomics Resource Center (http://pfgrc.tigr.org/). Wild-type strain C7258 and its crp mutant were grown in LB at 37 °C with agitation (250 r.p.m.) to OD600 1.5. Cells were collected by centrifugation at 4 °C and immediately subjected to RNA extraction. Total RNA was extracted using the Trizol Plus RNA purification system (Invitrogen) followed by the RNeasy MinElute Cleanup (Qiagen). The purity and integrity of RNA samples was verified by UV spectrophotometry and denaturing agarose gel electrophoresis. Using 15 µg total RNA per sample, cDNA was synthesized in the presence of 3-aminoallyl-2'-deoxyuridine 5'-triphosphate by reverse transcription with Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Cy-5 fluorescent dye (Amersham Biosciences) was coupled to the reference (wild-type) cDNA; Cy-3 was coupled to the test (mutant) cDNA. Labelled cDNAs were purified using the MiniElute PCR purification kit (Qiagen) and the cDNA probes mixed and applied to the array surface for hybridization overnight at 42 °C for 16 h. The slides were scanned using the Agilent G2565AA Microarray Scanner System and images processed using the Agilent Technologies Scanner Control Software 6.13 and Agilent Feature Extraction Software 8.5.1.1 (Agilent Technologies). For differential expression, we used the false discovery rate (FDR) to decide the cut-off values for significant genes. To calculate FDR, we used the rank product (RP) test statistic (Breitling et al., 2004), which is a non-parametric method to detect differentially regulated genes in replicated experiments. An average fold change (AFC) greater than 1.5 and a FDR threshold lower than 0.05 was considered to define significantly differentially expressed genes. Results reported in this study represent the average of four experiments. Selected genes found to be differentially expressed in the microarray study were confirmed by quantitative real-time RT-PCR (qRT-PCR).
Reverse transcription PCR.
V. cholerae strains were grown in LB medium to OD600 1.5 and total RNA extracted as described in the preceding section. To validate microarray data, RNA samples were analysed by qRT-PCR using the iScript two-step RT-PCR kit with SYBR Green (Bio-Rad). Relative expression values (R) were calculated using the equation 
, where CT is the fractional threshold cycle. The recA mRNA was used as reference. The following primer combinations were used: CqsA1424 and CqsA1816 for cqsA mRNA; HapR589 and HapR1046 for hapR mRNA; Fis56 and Fis190 for fis mRNA; FlaA40 and FlaA291 for flaA mRNA; FlaC405 and FlaC592 for flaC mRNA; RecA578 and RecA863 for recA mRNA; RpoE111 and RpoE345 for rpoE mRNA; and VpsL607 and VpsL775 for vpsL mRNA. A control mixture lacking reverse transcriptase was performed for each reaction to exclude chromosomal DNA contamination.
Assay of CAI-1 activity.
Cell-free supernatants of V. cholerae cultures were used as the source of autoinducer molecules. To this end, single colonies were inoculated into 5 ml LB and incubated overnight at 37 °C with shaking. The cultures were then diluted 1 : 100 in fresh LB and grown to the same optical density (OD600 1.5). Cultures were centrifuged at 17 400 g for 10 min and the clear supernatants filtered through a 0.22 µm syringe filter. Cell-free culture supernatants were tested for the presence of CAI-1 activity by inducing light production in the V. cholerae reporter strain MM920 (Table 1). This reporter strain does not produce CAI-1 and does not respond to AI-2. Briefly, reporter strain MM920 was grown overnight with shaking at 30 °C, diluted 1 : 10 in fresh medium, and 70 µl aliquots were transferred to an opaque-wall 96-well microtitre plate. Cell-free culture fluids were added to a final concentration of 30 % (v/v). The plates were incubated at 30 °C with agitation, and light production was measured at 30 min intervals in a GeniosPlus plate reader. The data are reported as peak fold light induction compared to a sterile medium control.
Enzymic and immunosorbent assays.
The amount of HA/protease secreted to the medium was measured using an azocasein assay (Benitez et al., 2001). One azocasein unit is the amount of enzyme that produces an increase of 0.01 optical density units h–1 in this assay. CT was determined by GM1 ELISA using a standard curve of pure CT (Sigma) as described previously (Silva et al., 1998).
Biofilm formation.
Biofilm formation was measured by the crystal violet staining method and results normalized for growth and expressed as the A570/OD600 ratio (Zhu & Mekalanos, 2003). Strains were grown in 5 ml LB medium for 16 h, diluted 1 : 50 in fresh medium and transferred to 96-well flat-bottom microtitre plates. The plates were incubated for 24 h at 30 °C for biofilm development.
Motility assay.
Swarm agar plates consisted of LB medium containing 0.3 % agar. They were inoculated by stabbing with overnight LB cultures grown from single colonies. Plates were incubated for 24 h at 30 °C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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crp mutantcrp mutant WL7258 using the TIGR version 2 V. cholerae microarray. A total of 176 genes, including crp itself (VC2614) were found to be differentially expressed in the crp mutant (AFC>1.5, FDR<0.05), representing 4.4 % of V. cholerae N16961 predicted genes. As expected, apart from conserved hypothetical proteins, the majority of differentially expressed genes fell in the role categories energy metabolism, transport and binding proteins, and cellular processes (Fig. 1). Furthermore, 79 % of the differentially expressed genes were downregulated, suggesting that CRP most frequently acts as a positive regulator in V. cholerae under the conditions used. In Table 3 we provide a short list of differentially expressed genes emphasizing those regulated by quorum sensing and/or involved in cholera pathogenesis. A complete list of differentially expressed genes is provided as supplementary data with the online version of this paper. The raw data files have been deposited in Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) and assigned series record number GSE7519.
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crp mutant (Table 3). In order to validate the microarray results, we performed qRT-PCR. The crp mutant expressed very little cqsA mRNA compared to its wild-type precursor in this assay (Fig. 2a). As expected, cell-free supernatants of the crp mutant contained negligible amounts of CAI-1 activity (Fig. 2b). To confirm that CRP is required for CAI-1 production, we performed a complementation test by introducing plasmid pBADCRP7 (Table 1), which expresses crp from the arabinose (araBAD) promoter. We have shown that this plasmid encodes a biologically active CRP protein that exhibits cAMP-dependent binding to a bona fide cAMP–CRP binding site (Silva & Benitez, 2004). In Fig. 2(b) we show that introduction of this plasmid in the crp mutant fully restored production of CAI-1. We conclude that CRP is required to generate the CAI-1 quorum-sensing signal. We performed an electrophoresis mobility-shift experiment as described previously (Silva & Benitez, 2004) to determine if purified V. cholerae CRP binds the cqsA promoter. However, no cAMP-dependent binding could be demonstrated under the conditions used (data not shown).
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). We confirmed by qRT-PCR that the crp strain WL7258 expressed significantly less hapR mRNA (0.03±0.007) compared to its wild-type precursor C7258 (2.8±0.4). To determine the contribution of cqsA to HA/protease production, we compared isogenic luxS : : Km, cqsA, crp and hapR mutants for protease secretion using the azocasein assay (Fig. 2c). There were no significant differences in the levels of HA/protease between the luxS : : Km mutant M36 and wild-type (Fig. 2c). In contrast, inactivation of cqsA and crp significantly reduced HA/protease production. This result suggests that CRP regulates HA/protease production by controlling CAI-1 expression. In addition, our data suggest that, under these experimental conditions, the AI-2 signal is not essential for expression of HA/protease.
Since HA/protease is repressed by glucose (Silva & Benitez, 2004), we investigated whether CAI-1 expression is repressed by glucose, leading to diminished hapR mRNA. To this end, we cultured strain C7258 in HA/protease production medium. At OD600 1, cultures were divided into halves; one half was used as a control and the second half transferred to a new flask containing 0.4 % glucose. As expected, addition of glucose repressed production of HA/protease (data not shown). In Fig. 3 we show that glucose strongly repressed production of CAI-1, leading to lower hapR mRNA levels. No repression of CAI-1 production was observed after adding glucose to the spent supernatant. This result further confirms that CAI-1 production is subject to carbon catabolite repression. Lack of CAI-1 expression in the crp mutant or its repression by exogenous glucose leads to diminished HapR levels that cannot sustain expression of HA/protease.
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crp mutant (Table 3). For instance, hypothetical proteins VCA0880, VCA0881, VCA0882 and acetyl-CoA acetyltransferase (VCA0690) were found to be downregulated in the crp mutant (Table 3) and activated by HapR (Zhu & Mekalanos, 2003).
Gene VC0291, annotated as a NifR3/Smm1 family protein, is a tRNA-dihydrouridine synthase of unknown function in V. cholerae. This protein was found to be upregulated in the crp mutant (Table 3). Polar Tn5 insertions in this locus reduce the expression of the small nucleoid protein Fis (Lenz & Bassler, 2007). We did not observe differential expression of fis in our gene-profiling experiment. Because expression of Fis declines at high cell density (Lenz & Bassler, 2007), we used the more sensitive qRT-PCR assay to investigate the expression of this gene in the mutant. This assay confirmed that fis is enhanced in the crp mutant (1.0±0.3) compared to its wild-type precursor (0.14±0.04). In a separate study we observed that the rpoS mutant AJB50 grown to stationary phase expressed less hapR mRNA (1.9±0.2) than the wild-type (3.9±0.2). In V. cholerae, CRP activates transcription of rpoS in the stationary phase (Silva & Benitez, 2004). Taken together, our results show that CRP can modulate expression of HapR by multiple mechanisms which include regulation of CAI-1, Fis and RpoS.
CRP mutants make more CT and enhanced biofilm
The HapR regulator has been shown to negatively influence CT expression by repressing AphA and TcpPH (Kovacikova & Skorupski 2001, 2002). Expression of aphA (VC2647) and tcpH (VC0827) was strongly upregulated in the crp mutant (Table 3). We examined whether reduced expression of hapR in the crp mutant enhanced CT production in AKI medium. As expected, crp, hapR and hapRcrp mutants made significantly more CT than wild-type (Fig. 4a). Furthermore, production of CT by the hapRcrp double mutant was similar to that by the hapR and crp single mutants, suggesting that cAMP–CRP affects CT production by influencing expression of HapR. The diminished expression of hapR in crp mutants resulted in significantly enhanced biofilm production in LB medium compared to wild-type (Fig. 4b). The crp, hapR and hapRcrp mutants made similar biofilms, suggesting that cAMP–CRP affects this phenotype by acting through HapR. Consistent with the biofilm results we found that the crp mutant expressed elevated levels of the HapR-repressed vpsL exopolysaccharide biosynthesis gene (Fig. 4c). It is of note that the crp mutant expressed lower vpsL levels than the hapR mutant (Fig. 4c).
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). Two genes, VC1034 and VCA0053, annotated as encoding uridine phosphorylase (Udp) and purine nucleoside phosphorylase, respectively, were downregulated in the crp mutant (Table 3). We examined the expression of udp in wild-type, hapR, crp and hapRcrp mutants by qRT-PCR (Fig. 4d). This experiment confirmed that cAMP–CRP positively regulates udp expression while HapR acts as a repressor (Fig. 4d).
Effect of CRP on motility
Expression of motility has been shown to be required for intestinal colonization (Silva et al., 2006). Several motility and chemotaxis genes – VC2187 (flaC), VC2188 (flaA), VC2143 (flaD), VC0216, VC2190 (flgL), VC2006 (cheV), VC2142 (flaB) and VC2125 (fliN) – were significantly downregulated in the crp mutant (Table 3). Interestingly, flaACD and flgL have also been found to be downregulated in a hapR mutant (Yildiz et al., 2004). To determine the contribution of cAMP–CRP and HapR to the regulation of flagellar genes and motility we confirmed the downregulation of flaA and flaC by qRT-PCR and compared expression of flaA and flaC in wild-type, hapR, crp and hapRcrp backgrounds. As shown in Fig. 5(a), although the hapR mutant expressed lower levels of flaA and flaC, the crp mutation had a more severe effect (Fig. 5a). The crp mutation was epistatic to the hapR defect (Fig. 5a). In a swarm agar test the crp mutant appeared more affected for motility compared to the hapR mutant, and the double mutant exhibited a cumulative effect (Fig. 5b).
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crp mutant. For instance, expression of genes encoding three OMPs, OmpT (VC1854) OmpW (VCA0867) and OmpU (VC0633), was significantly diminished in the mutant (Table 3). Since OmpU has been implicated in resistance to bile, organic acids and antimicrobial peptides (Mathur & Waldor, 2004; Merrell et al., 2001; Provenzano & Klose, 2000; Provenzano et al., 2001; Wibbenmeyer et al., 2002), we confirmed that our crp mutant is more sensitive than the wild-type to growth inhibition by 0.1 % (w/v) sodium deoxycholate in LB medium (Fig. 6a). Expression of rpoE encoding the alternative sigma factor E (VC2467) was also inhibited in the crp mutant. We used qRT-PCR to validate that wild-type strain C7258 produced significantly more rpoE mRNA (0.7±0.2) compared to its isogenic crp mutant (0.12±0.03). Growth of V. cholerae rpoE mutants is inhibited by 3 % (v/v) ethanol (Kovacikova & Skorupski, 2002a). Consistent with the lower expression of rpoE, the crp mutant was found to be more sensitive to 3 % (v/v) ethanol in LB medium (Fig. 6b). Other genes potentially implicated in intestinal colonization and found to be downregulated in crp mutant were VC1779 and VCA0205 encoding C4-dicarboxylate transport proteins, the arginine ABC transporter gene VCA0760, the universal stress protein gene VC0076, and fumarate reductase subunit genes VC2656, VC2657, VC2658 and VC2659 (frdABCD) (Table 3). These genes have been found to be strongly induced in the rabbit ileal loop model (Xu et al., 2003).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The opposing effects of CRP on CT production (Skorupski & Taylor, 1997) and HA/protease expression (Silva & Benitez, 2004), suggest that CRP plays a key role in shifting the metabolism of V. cholerae from a ‘virulence mode’ characterized by production of CT to an ‘exit mode’ typified by production of the HA/protease ‘detachase’ activity (Finkelstein et al., 1992).
Gene expression profiling of a crp mutant revealed significant overlaps with the HapR-controlled regulatory network. Most importantly, production of CAI-1 synthesized by the activity of CqsA showed a nearly absolute dependence on CRP (Fig. 2a, b). The mechanism by which cAMP–CRP regulates cqsA is unknown. However, the absence of putative cAMP–CRP binding sites in the cqsA promoter suggests that cAMP–CRP could act through other regulators. Lack of CAI-1 expression in the crp mutant resulted in downregulation of hapR and differential expression of multiple HapR-dependent genes such as hapA. Interestingly, inactivation of luxS had no effect on HA/protease expression under the conditions used. This result is in agreement with the phenotype of luxS mutants constructed by Zhu & Mekalanos (2003). Taken together, the results indicate that although CAI-1 and AI-2 act in parallel to activate HapR, their contribution might differ significantly under specific conditions. A possible explanation for these observations is that under conditions commonly used to grow V. cholerae in the laboratory the bacterium synthesizes significantly larger amounts of CAI-1 than AI-2. AI-2 is regarded as an inter-species communication molecule and it is possible that small amounts of V. cholerae AI-2 could have a more pronounced effect on other bacteria than on itself. It is also possible that AI-2 could have a major effect on HapR expression under other conditions.
In other Gram-negative bacteria CRP has been reported to influence the expression of quorum-sensing-regulated genes by different mechanisms. For instance, in Vibrio harveyi and V. vulnificus CRP and LuxR concertedly activate quorum-sensing-regulated target genes (Chatterjee et al., 2002; Jeong et al., 2003). However, in Vibrio fischeri and Pseudomonas aeruginosa, CRP acts as an upstream regulator by directly activating transcription of LasR or LuxR (Albus et al., 1997; Dunlap & Greenberg, 1988). In E. coli, CRP has been reported to affect quorum sensing by regulating AI-2 biosynthesis and uptake (Wang et al., 2005).
Based on the finding that cqsA expression is significantly inhibited in crp mutants, we hypothesized that CRP could affect other hapR phenotypes in addition to HA/protease. As shown in Fig. 4(a), crp, hapR and hapRcrp mutants produced elevated levels of CT compared to wild-type. We note here that CRP has been reported to inhibit transcription of tcpH by directly binding to the tcpPH promoter (Kovacikova & Skorupski, 2001). However, our epistasis analysis revealed that the hapRcrp mutant did not make significantly more CT than the hapR mutant. This result suggests that, under our experimental conditions, direct repression of tcpPH by CRP does not play a major role in downregulating the TcpP/H-ToxT-CT signalling pathway.
Both crp, hapR and hapRcrp mutants made enhanced biofilm in LB medium (Fig. 4b). Our double mutant analysis suggested that CRP and HapR could act along the same pathway to affect biofilm formation. Consistent with the biofilm data, the crp mutant expressed elevated levels of the exopolysaccharide biosynthesis gene vpsL. VpsL has been shown to be repressed by HapR and contains a putative HapR binding site (Yildiz et al., 2004). However, crp and hapRcrp mutants made less vpsL mRNA than hapR, suggesting that CRP could affect other factors required for maximal exopolysaccharide expression. Similar results were obtained for the exopolysaccharide biosynthesis gene vpsA (data not shown). We are currently investigating the interactions between cAMP–CRP, HapR and other regulators of exopolysaccharide biosynthesis.
Expression of udp (VC1034) has been related to biofilm formation (Haugo & Watnick, 2002). We found expression of udp to be strongly inhibited in the crp mutant, suggesting that this gene is activated by cAMP–CRP (Fig. 4d). In addition, udp was found to be repressed by HapR (Fig. 4d). These results are consistent with the presence of putative HapR and cAMP–CRP binding sites in the udp promoter (Yildiz et al., 2004; Zolotukhina et al., 2003). The udp gene is also repressed by CytR, a repressor of biofilm formation (Haugo & Watnick, 2002). A cytR superbiofilm mutant expressed elevated udp (Haugo & Watnick, 2002). However, our crp mutant made more biofilm in spite of expressing very low levels of udp (Fig. 4b, d). This result suggests that derepression of udp in cytR mutants is not related to their superbiofilm phenotype.
Several flagellar genes reported to be downregulated in hapR mutants (Yildiz et al., 2004) were also downregulated in the crp mutant, rendering this strain less motile (Fig. 5b). However, double mutant analysis suggested that CRP has its own effect on motility (Fig. 5a). FlaA is the only flagellin demonstrated to be essential for motility in V. cholerae (Klose & Mekalanos, 1998). There are no CRP boxes located upstream of the flaA ORF. Expression of flaA and motility is dependent on the alternative sigma factor 54 (Prouty et al., 2001). Studies on the E. coli 54-dependent glnAp2 promoter have suggested that the cAMP–CRP complex could directly interact with the 54 holoenzyme to affect gene expression without binding to a CRP box (Tian et al., 2001).
It is well established that crp mutants, in spite of expressing elevated CT and TCP, are defective in intestinal colonization (Skorupski & Taylor, 1997). We have found that CRP positively controls multiple genes required for infant mouse colonization. These genes included flagellar genes and several OMPs that have been suggested to play a role in resistance to bile and antimicrobial peptides (Mathur & Waldor, 2004; Merrell et al., 2001; Provenzano & Klose, 2000; Provenzano et al., 2001; Wibbenmeyer et al., 2002). OmpT and OmpW have been reported to be strongly induced in rabbit ileal loops (Xu et al., 2003). Downregulation of ompT in the crp mutant is consistent with the report of Li et al. (2002) showing that CRP directly activates the ompT promoter. As expected, the crp mutants displayed increased sensitivity to sodium deoxycholate. The crp mutant expressed reduced levels of rpoE, encoding the alternative sigma factor E. Conserved functions of E in Gram-negative bacteria include the synthesis, assembly and homeostasis of lipopolysaccharide and outer-membrane porins (Rhodius et al., 2006). E has been shown to contribute to virulence in several pathogenic bacteria, including Salmonella, Haemophilus and V. cholerae (Kazmierczak et al., 2005; Kovacikova & Skorupski 2002a). Furthermore, expression of fumarate reductase subunits frdABCD was significantly diminished in the crp mutant. The above genes have been found to be strongly induced in rabbit ileal loops (Xu et al., 2003). It has been suggested that frdABCD could be required to derive energy from anaerobic respiration using alternative electron acceptors such as fumarate during growth in vivo (Xu et al., 2003). We suggest that downregulation of these genes in the crp mutant could also affect intestinal colonization. In a previous study we showed that CRP positively enhances expression of the general stress regulator RpoS (Silva & Benitez, 2004) reported to be required for intestinal colonization (Merrell et al., 2000). Thus, the reduced expression of multiple factors required for intestinal colonization such as motility, OMPs, RpoE, genes required for anaerobic energy metabolism and RpoS explains the observed failure of crp mutants to colonize the suckling mouse intestine (Skorupski & Taylor, 1997).
In Fig. 7 we propose an integrative model showing multiple interactions by which CRP affects the V. cholerae life cycle. Carbon source and autoinducers act together as external signals to modulate quorum sensing. CRP serves to couple nutritional and cell density sensory information by controlling the biosynthesis of CAI-1, which contributes to activation of HapR. CRP could also influence expression of HapR through its effect on Fis and RpoS. However, these effects most likely influence expression of HapR at different stages of the V. cholerae growth curve. For instance, expression of fis is higher at low cell density (Lenz & Bassler, 2007), RpoS is expressed in the stationary phase (Silva & Benitez, 2004) and CAI-1 accumulates in the medium as the population increases. Induction of HapR by CRP negatively affects production of CT, TCP and biofilm formation. Cells within biofilm communities have been reported to be more resistant to environmental stresses and protozoan grazing (Joelsson et al., 2006; Matz et al., 2005). CRP can contribute to environmental stress resistance by positively influencing RpoS expression. Finally, CRP contributes to intestinal colonization by enhancing motility, RpoE, RpoS and resistance to bile. In summary, CRP plays a key role in the V. cholerae life cycle by integrating multiple signal transduction pathways in response to environmental changes.
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ACKNOWLEDGEMENTS |
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Edited by: W. Quax
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REFERENCES |
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Received 2 February 2007;
revised 29 May 2007;
accepted 5 June 2007.
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=0.01).
, positive effect; 
, inhibitory effect. In this model cAMP, by binding to CRP and activating cqsA expression, is proposed to link carbon catabolite repression and quorum sensing.