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Applied and Environmental Microbiology, November 2007, p. 7482-7487, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01564-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Cyclic AMP Receptor Protein Modulates Colonial Morphology in Vibrio cholerae{triangledown}

Weili Liang,1,2 Anisia J. Silva,1 and Jorge A. Benitez1*

Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, 720 Westview Dr. SW, Atlanta, Georgia,1 State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing 102206, People's Republic of China2

Received 10 July 2007/ Accepted 21 September 2007


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ABSTRACT
 
Inactivation of the quorum-sensing regulator HapR causes Vibrio cholerae El Tor biotype strain C7258 to adopt a rugose colonial morphology that correlates with enhanced biofilm formation. V. cholerae mutants lacking the cyclic AMP (cAMP) receptor protein (CRP) produce very little HapR, which results in elevated expression of Vibrio exopolysaccharide (vps) genes and biofilm compared to the wild type. However, {Delta}crp mutants still exhibited smooth colonial morphology and expressed reduced levels of vps genes compared to isogenic hapR mutants. In this study we demonstrate that deletion of crp and cya (adenylate cyclase) converts a rugose {Delta}hapR mutant to a smooth one. The smooth {Delta}hapR {Delta}crp and {Delta}hapR {Delta}cya double mutants could be converted back to rugose by complementation with crp and cya, respectively. CRP was found to enhance the expression of VpsR, a strong activator of vps expression, but to diminish transcription of VpsT. Ectopic expression of VpsR in smooth {Delta}hapR {Delta}crp and {Delta}hapR {Delta}cya double mutants restored rugose colonial morphology. Lowering intracellular cAMP levels in a {Delta}hapR mutant by the addition of glucose diminished VpsR expression and colonial rugosity. On the basis of our results, we propose a model for the regulatory input of CRP on exopolysaccharide biosynthesis.


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INTRODUCTION
 
Cholera is a paradigm waterborne disease caused by Vibrio cholerae serogroups O1 and O139, which continue to cause seasonal outbreaks in heavily populated regions in Asia, Africa, and Latin America. V. cholerae is transmitted through contaminated food and drinking water as well as person to person through the fecal-oral route. The existence of an aquatic reservoir of serogroup O1 and O139 toxigenic strains has not been established. However, their capacity to survive and persist in estuarine and brackish waters is widely accepted (6, 8, 22). V. cholerae has been shown to alternate between a free-swimming planktonic lifestyle and biofilm communities attached to biotic and abiotic surfaces. In the biofilm lifestyle, V. cholerae has been found in association with phytoplankton and zooplankton (11, 12). In addition, large clumps of aggregated partially dormant V. cholerae cells can be detected in surface water as biofilms that resist cultivation in conventional microbiological media (7). These aggregates can be recovered as virulent V. cholerae cells by inoculation into rabbit ileal loops (7).

It has been shown that V. cholerae cells in biofilm communities are more resistant to environmental stresses and protozoan predation (11, 12, 13, 18, 31). The V. cholerae rugose colonial morphology variant initially described by White (27) has been shown to produces more exopolysaccharide and biofilm than the smooth colonial variant (28). Furthermore, the rugose variant has been demonstrated to be more resistant to chlorinated water (20, 21, 28) and osmotic and oxidative stresses than the smooth variant (26, 28).

The rugose colonial morphology has been demonstrated to be a highly multifactorial phenotype subject to complex regulatory mechanisms. The genes responsible for exopolysaccharide biosynthesis (vps) are clustered in two operons in which vpsA and vpsL are the first genes of operons I and II, respectively (28). Regulation of vps genes involves cyclic diguanylate signaling (16, 17, 25), the positive regulators VpsT (3) and VpsR (29), and the negative regulator CytR (10). In some El Tor biotype strains, the quorum-sensing regulator HapR negatively affects biofilm formation and rugosity by repressing the expression of vps genes (9, 30, 31). As a result, hapR mutants of these strains exhibit enhanced biofilm formation and rugose colonial morphology. In addition, a flagellum-dependent vps signaling cascade has been reported in some El Tor biotype strain (14). Finally, a third vps signaling pathway initiated by nutrient starvation has been hypothesized for strain N16961; the absence of hapR and flaA genes in this strain does not produce rugose colonies (14).

We have shown that the cyclic AMP (cAMP) receptor protein (CRP) is required for the expression of cholera autoinducer I (15). Accordingly, deletion of crp in V. cholerae C7258 (El Tor, Ogawa, 1991 Perú isolate) resulted in very low expression of the quorum-sensing regulator HapR (15, 23). The V. cholerae {Delta}crp mutant expressed elevated vpsA, vpsL, and biofilm compared to the wild type (15). However, although the {Delta}crp mutant expressed extremely low levels of hapR mRNA, it produced less vpsA and vpsL mRNA compared to an isogenic rugose {Delta}hapR mutant and displayed smooth colonial morphology. These results suggested that CRP could be required for the expression of other factors required for maximal exopolysaccharide biosynthesis. In this study we address the relationship between CRP, HapR, and colonial morphology. To this end, we constructed isogenic {Delta}crp, {Delta}hapR, and {Delta}hapR {Delta}crp deletion mutants in the genetic background of the smooth strain C7258. The construction of these mutants (Table 1) has been described previously (15). We prepared colonies from LB agar plates for scanning electron microscopy as described earlier (28). As shown in Fig. 1A, the {Delta}crp mutant exhibited smooth colonial morphology, while the {Delta}hapR mutant AJB51 (Fig. 1B) was rugose. Furthermore, deletion of crp from the rugose {Delta}hapR mutant yielded a smooth {Delta}hapR {Delta}crp double mutant (Fig. 1C). This result is consistent with the finding that {Delta}crp and {Delta}hapR {Delta}crp mutants expressed less vpsA and vpsL than the isogenic {Delta}hapR mutant did (15). To confirm that the smooth colony morphology of strain WL51 ({Delta}hapR {Delta}crp) was due to deletion of crp, we restored the active crp allele by transformation with pBADCRP7 (Table 1). The transformant exhibited the rugose colonial morphology of the {Delta}hapR single mutant (Fig. 1D). Since the activity of CRP is determined by intracellular cAMP levels (4), we constructed a {Delta}hapR {Delta}cya double mutant lacking adenylate cyclase. To this end, chromosomal DNA flanking the cya gene was amplified using primer pairs Cya7/Cya902 and Cya1759/Cya2567. These fragments and a Kmr gene from pUC4K (GenBank accession no. X06404) were sequentially cloned in pUC19 to yield p{Delta}Cya-Km (Table 1). The cya deletion/insertion was transferred to pCVD442 (5) and propagated in the permissive Escherichia coli strain SM10{lambda}pir (19). The resulting suicide vector pCVD{Delta}Cya-Km (Table 1) was transferred to strain AJB51 by conjugation, and the {Delta}hapR {Delta}cya mutant WL52 was obtained by sucrose selection as described previously (15). We show that, similar to the {Delta}hapR {Delta}crp mutant, the {Delta}hapR {Delta}cya double mutant exhibited smooth colonial morphology (Fig. 1E). For complementation, the cya open reading frame (ORF) was amplified using primers Cya7 and Cya2567 and cloned in the expression vector pTTQ18 (24) to yield pTT-Cya (Table 1). Transformation of the {Delta}hapR {Delta}cya strain with plasmid pTT-Cya expressing cya from the Tac promoter restored the rugose colonial phenotype even without the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) (Fig. 1F). These results clearly establish a role for the cAMP-CRP complex in regulating V. cholerae colonial variation.


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  		TABLE 1. Strains, plasmids, and primers used in this study


Figure 1
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  		FIG. 1. Scanning electron microscopy of {Delta}crp, {Delta}cya, and {Delta}hapR colonies. (A) Strain WL7258 ({Delta}crp); (B) strain AJB51 ({Delta}hapR); (C) strain WL51 ({Delta}hapR {Delta}crp); (D) WL51 transformed with pBADCRP7; (E) WL52 ({Delta}hapR {Delta}cya); (F) WL52 ({Delta}hapR {Delta}cya) transformed with pTT-Cya. Strains were streaked onto LB agar, and colonies were allowed to develop at 30°C for 24 h. Expression of crp was induced by the addition of 0.02% L-arabinose. Scanning electron microscopy was performed with a magnification of x50.

We first considered the possibility of cAMP-CRP directly interacting with the vpsA and vpsL promoters. However, analysis of the DNA sequence located upstream of vpsA and vpsL using the Virtual Footprint software program (http://www.prodoric.de/vfp) did not reveal any cAMP-CRP binding site. Therefore, we hypothesized that cAMP-CRP modulation of vpsA and vpsL is due to cAMP-CRP modulation of HapR and other factors required for maximal expression of vps genes. To investigate the mechanisms by which cAMP-CRP affects colonial morphology, we measured the expression of several regulators of vps gene expression in {Delta}hapR and {Delta}crp single and double mutants using quantitative real-time reverse transcription-PCR (qRT-PCR). Because it is well established that hapR is expressed and functions in cells at a high cell density (9, 31), V. cholerae strains were grown in LB at 37°C, and cells were collected at an optical density at 600 nm (OD600) of 1.5. Total RNA was isolated using the RNeasy kit (Qiagen Laboratories) combined with DNase treatment on column and qRT-PCR conducted using the iScript two-step RT-PCR kit with SYBR green (Bio-Rad Laboratories). Relative expression values (R) were calculated using the equation R = 2–({Delta}CT target – {Delta}CT reference) where CT is the fractional threshold cycle and recA mRNA was used as internal reference. Gene-specific primers for cytR, vpsT, and vpsR used in this analysis are listed in Table 1. The {Delta}crp mutant expressed reduced levels of CytR, a repressor of biofilm formation (10) (Fig. 2A). Analysis of the DNA sequence located upstream of the cytR ORF using the Virtual Footprint software program revealed the presence of two sites closely matching the TGTGAN6TCANA cAMP-CRP consensus binding sites. This result suggests that cAMP-CRP could act directly at the cytR promoter to activate its transcription. The cytR promoter contains a putative binding site for HapR, and it has been suggested that HapR activates cytR (30). However, cytR mRNA levels were unaffected by the deletion of hapR under our experimental conditions (Fig. 2A). This discrepancy could be due to the use of different strains and to the fact that in our study, RNA was extracted from cells grown to a higher cell density (OD600 of 1.5). In the preceding study, cells were harvested at a different physiological stage (OD600 of 0.3 to 0.4) (30). This difference would be expected to impact the expression and activity of HapR and CRP, respectively.


Figure 2
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  		FIG. 2. Expression of regulators of rugose colonial morphology in V. cholerae {Delta}hapR and {Delta}crp mutants. Strains C7258 (wild type [WT]), AJB51 ({Delta}hapR), WL7258 ({Delta}crp), and WL51 ({Delta}hapR {Delta}crp) were grown in LB medium to an OD600 of 1.5. Abundance of mRNAs encoding cytR (A), vpsT (B), and vpsR (C) was determined by qRT-PCR. Results were normalized to recA mRNA expression. Error bars indicate the standard deviations of at least three independent cultures.

The {Delta}crp mutant expressed elevated vpsT (Fig. 2B), a positive regulator of rugose colonial morphology (3). However, no cAMP-CRP binding sites were found upstream of vpsT, suggesting that CRP modulates expression of this gene indirectly. Deletion of hapR resulted in elevated vpsT mRNA, suggesting that HapR represses vpsT as reported earlier (30). It is noteworthy that the deletion of crp had little effect on vpsT expression in the {Delta}hapR background, suggesting that the CRP modulation of VpsT could be partly due to CRP modulation of HapR.

The {Delta}crp mutants expressed little VpsR (Fig. 2C), which has been reported to be a strong activator of vpsA and vpsL (29). No potential cAMP-CRP binding sites could be detected upstream of the vpsR coding sequence. It has been recently reported that VpsR is a stronger activator of vps expression than VpsT (2). Thus, the lower expression of VpsR in the {Delta}hapR {Delta}crp mutant could explain why inactivation of CRP in the {Delta}hapR background tends to diminish expression of vpsA and vpsL and convert the rugose {Delta}hapR mutant to the smooth form. This interpretation is in agreement with the finding that {Delta}hapR {Delta}vpsR double mutants display smooth colonial morphology (2). We have observed that the deletion of crp in the {Delta}hapR background has a more pronounced effect on vpsL, which contains a putative VpsR binding motif (30). It is noteworthy that the deletion of crp diminished VpsR expression in both the wild-type and {Delta}hapR backgrounds, but a negative effect on vpsA and vpsL expression was observed only in the {Delta}hapR background. We propose that the down-regulation of HapR and CytR and overexpression of VpsT in the {Delta}crp mutant partly compensates for reduced expression of VpsR resulting in a discrete increase in vps expression that is not big enough to switch the colonial morphology to rugose. We did not observe repression of vpsR by HapR in our study (Fig. 2C). In fact, the {Delta}hapR mutant expressed slightly less VpsR (Fig. 2C). These results contrast with a previous report suggesting that HapR represses VpsR in a different strain (2, 30). Strain differences and the cell density of cultures could be responsible for these discrepancies. Occurrence of strain variation is further suggested by the fact that no repression of VpsR by HapR was observed in mutants derived from strain C6706 of the El Tor biotype (9).

Since deletion of crp resulted in significantly lower vpsR expression in the {Delta}hapR background (Fig. 2C), we considered the possibility of low vpsR expression being responsible for the smooth colonial morphology of {Delta}hapR {Delta}crp and {Delta}hapR {Delta}cya mutants. To test this hypothesis, we used primers VpsR78 and VpsR1431 to clone vpsR in plasmid pTTQ18 (24). The resulting plasmid, pTTVpsR (Table 1), expresses VpsR under the control of the inducible Tac promoter. Expression of VpsR from a heterologous promoter restored colonial rugosity to {Delta}hapR {Delta}crp and {Delta}hapR {Delta}cya double mutants (Fig. 3C and F).


Figure 3
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  		FIG. 3. Spot colonial morphology of {Delta}crp and {Delta}cya mutants expressing VpsR. Overnight cultures of strain WL51 ({Delta}hapR {Delta}crp) and WL52 ({Delta}hapR {Delta}cya) were diluted 1:200, and 2 µl of each dilution was spotted onto LB agar containing 100 µg/ml ampicillin as required. (A) Strain WL51 with empty vector pTTQ18; (B) WL51 with pTTVpsR; (C) WL51 with pTTVpsR induced with 10 mM IPTG; (D) strain WL52 with empty vector pTTQ18; (E) WL52 with pTTVpsR; (F) WL52 with pTTVpsR induced with 10 mM IPTG. Plates were incubated for 24 h at 30°C, and colonies were photographed with a Canon EOS camera with a 60-mm macro lens.

CRP plays a key role in carbon catabolite repression, a process by which the presence of a favorable carbon source (i.e., glucose, fructose, or sucrose) in the medium inhibits gene expression and/or activity of enzymes involved in the catabolism of other carbon sources (4). We have observed that the addition of 5% glucose to LB agar but not glycerol inhibited expression of rugose colonial morphology in the {Delta}hapR mutant AJB51 (data not shown). We hypothesized that lowering intracellular cAMP levels by the addition of glucose should abolish expression of rugose morphology by reducing VpsR expression. To test this hypothesis, we extracted RNA from cells grown with and without 5% glucose and performed qRT-PCR. As shown in Fig. 4, glucose significantly diminished expression of vpsR. We used primer pairs VpsA434/VpsA676 and VpsL607/VpsL775 and qRT-PCR to determine the expression of the vpsA and vpsL genes, which are located downstream of vpsR. As expected, glucose repression of VpsR was reflected in significantly reduced expression of vpsA and vpsL (Fig. 4). These results further confirm a role for cAMP and CRP in the modulation of V. cholerae colonial morphology phase. The inhibition of the rugose colonial morphology by sugars has been previously reported (1). Our results suggest that carbon catabolite repression participates among other factors in the regulation of exopolysaccharide biosynthesis.


Figure 4
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  		FIG. 4. Repression of VpsR by glucose. Strain WL51 ({Delta}hapR) was grown in LB (control) and LB supplemented with 5% glucose. Expression of vpsR, vpsA, and vpsL was determined by qRT-PCR. Error bars indicate the standard deviations of at least three independent cultures.

Our results add new information to the highly complex regulatory circuitry controlling exopolysaccharide biosynthesis and colonial rugosity in V. cholerae. In Fig. 5, we propose a model for the regulatory input of cAMP-CRP in exopolysaccharide biosynthesis and rugose colonial morphology. Deletion of crp results in diminished expression of the positive regulator VpsR, which is required for expression of rugose colonial morphology (Fig. 2C). However, this event is partially compensated for by reduced expression of the negative regulators HapR (15) and CytR (Fig. 2A) and overexpression of VpsT (Fig. 2B). As a result, {Delta}crp mutants express elevated vpsA and vpsL than the wild type does (15), but this increase is not big enough to induce rugose colonial morphology. The reduced motility of {Delta}crp mutants could be a second mechanism compensating for the reduced expression of VpsR. We have shown that the {Delta}crp mutant WL7258 is less motile than the wild type and expresses reduced FlaA (15). Inactivation of flaA has been reported to enhance the expression of vps genes (14). According to our model, maximal expression of vps genes resulting in rugose colonial morphology occurs when the vps repressor HapR is eliminated by mutation, but cAMP-CRP is available to induce the positive regulator VpsR. Further studies are required to understand how cAMP-CRP regulates VpsR. We considered the possibility that cAMP-CRP modulates the expression of GGDEF and EAL domain proteins that regulate VpsR by controlling intracellular levels of cyclic diguanylate (16, 17). However, we have not found any effect of deleting crp on the transcription of genes cdgA, mbaA, and cgdC encoding GGDEF/EAL domain proteins (data not shown). These results do not rule out the possibility of cAMP-CRP influencing cyclic diguanylate levels by affecting the activity of the above proteins or modulating other GGDEF/EAL domain proteins. VpsR has been reported to be a {sigma}54-dependent activator (29). Interestingly, gene expression profiling of strain WL7258 revealed down-regulation of multiple {sigma}54-dependent genes affecting motility and C4-dicarboxylate transport (15).


Figure 5
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  		FIG. 5. Model for the regulatory input of cya and crp in exopolysaccharide biosynthesis and rugose colonial morphology.

Our results clearly establish cAMP-CRP as a regulator of exopolysaccharide biosynthesis and rugose colonial morphology. The recognition of the role of cAMP-CRP in this process will facilitate further studies to determine how environmental cues influencing intracellular cAMP levels (i.e., carbon starvation) affect the ability of V. cholerae to adopt the rugose colonial morphology variant and resist environmental stresses.


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ACKNOWLEDGMENTS
 
This work was supported by PHS grants RO1AI063187 and 2S06GM008248-20 from the National Institutes of Health to J.A.B. and A.J.S., respectively.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, 720 Westview Dr. SW, Atlanta, GA 30310-1495. Phone: (404) 756-6661. Fax: (404) 752-1179. E-mail: jbenitez{at}msm.edu Back

{triangledown} Published ahead of print on 5 October 2007. Back


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Applied and Environmental Microbiology, November 2007, p. 7482-7487, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01564-07
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