ABSTRACT
Multidrug efflux systems belonging Resistance-Nodulation-Division (RND) superfamily are ubiquitous in Gram-negative bacteria. RND efflux systems are often associated with multiple antimicrobial resistance but also contribute to the expression of diverse bacterial phenotypes including virulence, as documented in the intestinal pathogen Vibrio cholerae, the causative agent of the severe diarrheal disease cholera. Transcriptomic studies with RND efflux-negative V. cholerae suggested that RND-mediated efflux was required for homeostasis, as loss of RND efflux resulted in the activation of transcriptional regulators, including multiple environmental sensing systems. In this report we investigated six RND efflux responsive regulatory genes for contributions to V. cholerae virulence factor production. Our data showed that V. cholerae gene VC2714, encoding a homologue of Escherichia coli OmpR, was a virulence repressor. The expression of ompR was elevated in an RND-null mutant and ompR deletion partially restored virulence factor production in the RND-negative background. Virulence inhibitory activity in the RND-negative background resulted from OmpR repression of the key ToxR regulon virulence activator aphB, and ompR overexpression in WT cells also repressed virulence through aphB. We further show that ompR expression was not altered by changes in osmolarity, but instead was induced by membrane intercalating agents that are prevalent in the host gastrointestinal tract, and which are substrates of the V. cholerae RND efflux systems. Our collective results indicate that V. cholerae ompR is an aphB repressor and regulates the expression of the ToxR virulence regulon in response to novel environmental cues.
INTRODUCTION
The Gram-negative bacterium Vibrio cholerae is the causative agent of the life-threatening diarrheal disease cholera. V. cholerae is an aquatic organism and infects humans following the consumption of V. cholerae contaminated food or water. After ingestion V. cholerae colonizes the small intestine epithelium to cause disease by a process that is dependent upon virulence factor production. The two most important V. cholerae virulence factors are the toxin coregulated pilus (TCP), which mediates intestinal colonization, and cholera toxin (CT), an enterotoxin that is responsible for the secretory diarrhea that is the hallmark of the disease cholera (1). CT and TCP production are under the control of a hierarchical regulatory system known as the ToxR regulon (2). Activation of the ToxR regulon begins with expression of two cytoplasmic transcriptional regulators, aphA and aphB (3, 4). AphA and AphB function synergistically to activate tcpP expression. TcpP then binds along with ToxR to the toxT promoter to activate toxT expression. ToxT directly activates the expression of the genes that encode for CT and TCP production (2).
The expression of adaptive responses is important for the success of V. cholerae as a pathogen. This includes tight regulation of the ToxR regulon which is known to limit virulence factor production to specific niche within the host. Thus, the ToxR regulon has evolved to respond to specific environmental signals within the host (5). Other genes, which are important for survival and persistence in aquatic ecosystems, must be repressed during host entry for successful colonization (6-8). Late in infection, in preparation for host exit, V. cholerae downregulates virulence genes while activating genes required for dissemination and transmission (9-12). Although the genetic mechanisms involved in ToxR regulon activation have been extensively studied, little is known about how environmental signals influencing ToxR regulon expression in vivo.
V. cholerae is exposed to disparate environments in the aquatic ecosystem and the human gastrointestinal tract. V. cholerae survival and growth in these niches requires rapid adaptation to environmental conditions. V. cholerae enters humans from aquatic ecosystems that are typically aerobic and alkaline. The bacterium must then pass through the gastric acid barrier of the stomach before entering the duodenum and migrating to the epithelial surface where it colonizes the crypts of the small intestine. Successful transition between these dissimilar environments requires that V. cholerae modulate its transcriptional responses so that specific genes are only expressed in appropriate niches. In V. cholerae, like most bacteria, this is achieved by environmentally responsive regulatory systems that monitor the extracellular environment using a range of membrane bound sensors such as ToxR and two-component signal transduction systems (TCS) (13).
TCS are widespread phospho-relay systems that modulate gene expression in response to environmental cues. They consist of a membrane-bound histidine kinase sensor protein coupled with a cytosolic response regulator. In the presence of appropriate stimuli, the sensor auto phosphorylates a conserved histidine residue before transferring the phosphate to a conserved aspartate residue on the response regulator to activate the response regulator. Activated response regulators function to modulate adaptive responses by effecting the expression of target genes. Response regulators are typically transcription factors, but also can function by other mechanisms (14). The adaptive responses mediated by TCS are broad and include virulence, motility, metabolism and stress responses.
One of the better characterized TCS is the EnvZ-OmpR system that is ubiquitous in Gram-negative bacteria (15). EnvZ is the membrane associated sensor kinase and OmpR the response regulator that functions as a transcription factor. EnvZ-OmpR was first discovered in Escherichia coli and shown to regulate the expression of its two major outer membrane porin proteins (OMP), ompC and ompF, in response to environmental osmolarity (16-18). The function of OmpR as an osmoregulator has been extended to a number of other bacteria genera (19-21). OmpR has also been linked to other phenotypes in Gram-negative bacteria including virulence (19, 20, 22-26) and acidic tolerance (21, 27-31). The V. cholerae OmpR homologue (open reading frame (ORF) VC2714) has been poorly studied, and its role in V. cholerae biology is unknown.
The RND efflux systems are ubiquitous tripartite transporters in Gram-negative bacteria that play critical roles in antimicrobial resistance. Many RND efflux systems exhibit broad substrate specificity and have the capacity to efflux multiple substrates that are both structurally and functionally unrelated (32, 33). The RND systems play critical roles in antimicrobial resistance by exporting toxic compounds from the cytosol and periplasm into the extracellular environment. Although RND efflux pumps have been widely studied for their role in multiple antibiotic resistance, they also impact many other physiological phenotypes in bacteria (34). This was recently documented in V. cholerae where the RND systems were shown to be required for cell homeostasis (33, 35, 36). The absence of RND efflux in V. cholerae resulted in downregulation of the ToxR regulon and altered expression of genes involved in metabolic and environmental adaptation (37, 38), including several TCS. The results of these studies suggested that RND-mediated efflux modulated homeostasis by effluxing cell metabolites which served as concentration-dependent environmental cues to initiate transcriptional responses via periplasmic sensing systems. This observation suggested the possibility that these TCS may have contributed to the virulence attenuation observed in efflux impaired V. cholerae.
In this work we investigated six regulatory genes that were induced in the absence of RND-mediated efflux for their contribution to virulence factor production in V. cholerae. This revealed that VC2714, encoding a homolog of E. coli OmpR, functioned as a virulence repressor in V. cholerae. We documented that VC2714 repressed the expression of the key virulence regulator aphB. We further showed that ompR expression was regulated in response to detergent-like compounds which are prevalent in the host gastrointestinal tract and are substrates of the RND transporters. Our collective results suggest that the V. cholerae EnvZ-OmpR TCS has evolved to regulate virulence in response to novel environmental stimuli.
RESULTS
V. cholerae ompR represses virulence factor production
The loss of RND-mediated efflux resulted in downregulation of the ToxR regulon and diminished CT and TCP production (37), suggesting that there is one or more factors linking efflux to virulence factor production. Transcriptional profiling of an RND negative V. cholerae mutant during growth under AKI conditions (i.e. virulence inducing conditions) showed that the expression of a number of regulatory genes, including several TCSs, were increased in the absence of RND efflux (38). We hypothesized that one or more of these regulatory genes may have contributed to RND efflux-dependent virulence repression. To test this, we expressed six regulators (i.e. VC0486, VC1320-VC1319, VC1081, VC1638, VC1825, VC1320 and VC2714) from the arabinose-regulated promoter in pBAD33 in WT V. cholerae strain JB58 during growth under AKI conditions in the presence of 0.05% arabinose and quantified CT production. VC0486 encodes an uncharacterized DeoR family regulator. VC1320 (carS) and VC1319 (carR) encode the CarRS TCS that is involved in regulating LPS remodeling and vps production (39-41); carR (pTB15)and the carRS (pTB3) were independently expressed in V. cholerae. VC1081 encodes an uncharacterized response regulator. VC1638 was recently shown to regulate the expression of vca0732 in response to polymyxin B (42). VC1825 is an AraC-family regulator that regulates a PTS transporter (43). VC2714 encodes an uncharacterized response regulator. The results showed that only pTB11, expressing VC2714, repressed CT production (Fig. 1A). VC2714 encodes a homolog of the E. coli osmotic stress regulator OmpR, with 92.1% amino acid sequence similarity, and hereafter will be referred to as ompR.
To further verify that V. cholerae ompR was a virulence repressor we repeated the above experiment in WT strain JB58 harboring plasmid pTB11 during growth under AKI conditions in the presence of increasing arabinose concentrations and quantified CT and TcpA production. The results showed an arabinose-dependent inhibition of both CT and TcpA production (Fig. 1B). Based on these results we concluded that ompR functions as a virulence repressor in V. cholerae.
OmpR contributes to virulence repression in RND-efflux deficient V. cholerae
To verify that ompR was upregulated in RND-deficient V. cholerae as previously indicated in a transcriptomics dataset, (38), we introduced the ompR-lacZ transcriptional reporter plasmid pKD9 into JB58 and the isogenic RND efflux-negative strain JB485, and quantified ompR expression in both strains following growth in LB broth, minimal T-media, and under AKI conditions. The results showed significantly increased ompR expression in JB485 relative to WT during growth under AKI conditions, but no significant difference in LB broth or minimal T-medium (Fig. 2A). These findings confirmed the previous study and suggested that the RND-efflux dependent induction of ompR transcription was specific to AKI growth conditions.
We next tested if ompR contributed to the virulence repression observed in the RND-negative strain JB485. To address this, we created ompR deletion strains in WT JB58 and RND-negative strain JB485 and quantified CT and TcpA production in WT, JB485 and their respective isogenic ΔompR mutants. Consistent with previous studies (37), the RND-negative strain produced significantly reduced amounts of CT and TcpA relative to WT (Fig. 2B) and deletion of ompR in WT did not significantly affect CT or TcpA production. By contrast, deletion of ompR in JB485 partially restored CT and TcpA production relative to wild type, but the magnitude of the increase did not reach WT levels (Fig. 2B). Together these data suggested that ompR contributed to virulence attenuation in the RND negative background, but that other factors are also involved in virulence repression.
V. cholerae OmpR represses aphB expression
The above results suggested that OmpR was a virulence repressor, but the mechanism by which it attenuated virulence factor production was unclear. As CT and TCP production are positively regulated by the ToxR regulon, we hypothesized that OmpR repressed components of the ToxR regulon. If this was true, then ompR deletion in JB485 should increase the expression of the affected ToxR regulon genes, relative to the parental strain JB485. We therefore compared ToxR regulon gene expression in JB485 and its isogenic ΔompR mutant during growth under AKI conditions. The results showed that ompR deletion in RND-negative strain JB485 did not significantly affect aphA expression (Fig. 3A) but did result in increased aphB expression and the ToxR regulon genes downstream from aphB (i.e. tcpP, toxT, ctxA and tcpA; (Fig. 3B, C, D, E and F). JB485 and JB485ΔompR had comparable levels of toxR expression, indicating that virulence repression by OmpR was not due to reduced toxR expression (Fig 3G). As aphB is one of the most upstream regulators in the ToxR regulon, these results suggested that OmpR attenuated virulence factor production by repressing aphB in JB485.
To test if OmpR affected ToxR regulon expression in efflux sufficient cells, we repeated the above experiments in WT during growth under AKI conditions. The results showed that ompR deletion in WT did not affect aphA expression but resulted in increased expression of aphB and its downstream target tcpP (Fig. 3I and 3J), but not the other ToxR regulon genes (Fig. 3 panels H, J, K, L, M and N). This is consistent with the observation that deletion of ompR did not affect CT or TcpA production in WT (Fig. 2B). Collectively, these results supported the conclusion that OmpR is an aphB repressor and that ompR regulation of aphB is relevant in WT cells during growth under AKI conditions.
Ectopic ompR expression represses aphB transcription in V. cholerae
To further confirm that OmpR has the ability to repress aphB we tested if ectopic ompR expression altered aphB expression in V. cholerae and the heterologous host E. coli. In the first set of experiments we expressed ompR from pTB11 in WT JB58 bearing lacZ transcription reporters for aphA and aphB during growth under AKI conditions in the presence of varying arabinose concentrations to induce ompR expression. The results showed a small arabinose dose-dependent increase in aphA expression (Fig. 4A); the biological significance of this finding is unclear. By contrast, we observed an arabinose dose-dependent decrease in aphB expression (Fig. 4B), confirming that OmpR is an aphB repressor. Although OmpR may have weak ability to induce aphA expression, its ability to repress aphB appears to be dominant, as the net consequence of ompR regulation of aphA and aphB is repression of tcpP (Fig. 3 panels C and J).
In the second set of experiments we expressed V. cholerae ompR from pTB11 in E. coli bearing aphA-lacZ or aphB-lacZ transcriptional reporters to address whether OmpR acted directly at the respective promoters. The E. coli strains were cultured to mid-log phase in the presence of varying arabinose concentrations when aphA-lacZ or aphB-lacZ expression was quantified. The results showed that arabinose addition had little effect on aphA expression (Fig. 4C). By contrast, there was an arabinose dose-dependent decrease in aphB expression (Fig. 4D), consistent with OmpR being an aphB repressor. Further, these results suggested that OmpR may act directly at the aphB promoter; however, we cannot exclude the possibility that OmpR could act through an intermediate that is present in both E. coli and V. cholerae. Collectively, these results supported the conclusion that OmpR negatively regulated the ToxR regulon via directly repressing aphB transcription.
V. cholerae ompR is induced by bile salts and detergents
While the above data showed that OmpR functions as a virulence repressor through repression of aphB, we wished to address the environmental factors that modulate OmpR activity in V. cholerae. OmpR has been extensively studied in the Enterobacteriaceae where it has been shown to function as an osmoregulator that mediates adaptive responses to osmotic stress (18, 22, 44). We therefore tested if V. cholerae ompR functioned as an osmoregulator by quantifying ompR-lacZ expression during growth under AKI conditions in standard AKI broth (86 mM NaCl), AKI broth with NaCl (21.5mM), and AKI with excess NaCl (250 mM). As shown in Fig. 5A, the NaCl concentration did not significantly affect ompR expression, suggesting that ompR was not regulated in response to osmolarity. Consistent with this, growth analysis showed that ompR was dispensable for growth in high osmolarity in broth up to 500 mM NaCl (Fig. 5B). From these results we concluded that V. cholerae OmpR was not regulated in response to medium osmolarity and therefore likely responds to different environmental stimuli than what is observed in the Enterobacteriaceae.
The finding that ompR was induced in the absence of RND-mediated efflux (Fig. 2A) suggested that small molecules that accumulate intracellularly in the absence of RND efflux may play a role in ompR expression. Previous studies showed that a major function of the V. cholerae RND efflux systems was in resistance to hydrophobic and detergent-like molecules including bile salts, fatty acids and detergents (37, 45). We therefore tested if bile salts or detergents affected ompR expression as described above. The results showed that the addition of deoxycholate, bile salts, Oxgall, and SDS to the growth media increased ompR expression (Fig. 5C). We also tested another small molecule, indole. Indole is a V. cholerae metabolite that is an RND-efflux substrate and virulence repressor (45, 46). The data showed that indole did not affect ompR expression, suggesting that altered ompR expression was specific for compounds with detergent-like properties. As detergents are associated with envelope stress due to their membrane intercalating properties, we hypothesized that ompR may be induced in response to envelope stress. To test this, we quantified ompR expression following the induction of membrane stress by ethanol treatment (47). The results of these experiments showed that there was an ethanol dose-dependent increase in ompR expression (Fig 5D). Taken together, these results suggested that V. cholerae ompR is likely regulated in response to membrane perturbations resulting from exposure to membrane intercalating agents.
Conditioned AKI broth nullifies ompR induction in RND-negative V. cholerae strain JB485
Based on the results above we hypothesized that hydrophobic and/or non-polar compounds present in AKI broth were accumulating in the RND efflux-deficient strain JB485 and activating ompR transcription. To test this, we generated conditioned AKI media by passing AKI broth through a Sep Pak C18 Cartridge to deplete non-polar and hydrophobic compounds from the media. We then quantified ompR expression in WT strain JB58 and RND-negative strain JB485 harboring pDK9 (ompR-lacZ) following growth under AKI conditions in AKI broth and in the C18-conditioned AKI broth. The results showed increased ompR expression in JB485 during growth in AKI broth as expected (Fig. 6). Growth of WT JB58 in the conditioned AKI media did not affect ompR expression, when compared to expression in standard AKI media. However, growth of JB485 in the conditioned AKI media alleviated the increase in ompR transcription observed in unconditioned AKI broth. To determine if the hydrophobic compounds from AKI media that were retained on the C18 column were responsible for ompR induction in JB485 we eluted the retained compounds from the C18 cartridges used to extract AKI and LB broth. We then determined if the respective eluates contained ompR-inducing activity by adding it them to LB broth cultures of JB485 and WT and quantifying ompR-lacZ expression. The results showed that the addition of the AKI broth C18 column eluate, but not LB C18 column eluate, activated ompR expression in JB485, while neither eluate had an effect on ompR expression in WT (Fig. 6). Collectively, these data suggested that hydrophobic and/or non-polar compounds present in AKI media were responsible for increased ompR expression in the RND negative strain JB485. The fact that conditioned media did not affect ompR expression in WT indicated that this phenotype was RND-dependent. Significantly, we also observed that the increase in ompR expression in JB485 was not dependent on growth under AKI conditions (i.e. static growth followed by shaken growth), as ompR expression was also enhanced in cultures grown in AKI broth under non-inducing conditions (not shown). This observation, combined with the finding that ompR was not induced in RND-negative JB485 during growth in LB broth or T-media (Fig. 2A), suggested that the ompR-inducing molecules were only present in AKI broth. From these experiments, we concluded that hydrophobic and/or non-polar compounds that are present in AKI media, but not LB media, were responsible for ompR activation in JB485. Further, because this phenotype was RND-efflux dependent, we infer that the inducing compounds are substrates for the V. cholerae RND efflux systems. The nature of these molecules will require further investigation.
DISCUSSION
V. cholerae is an inhabitant of the aquatic ecosystem which can colonizes the human gastrointestinal tract to cause disease. The ability of V. cholerae to replicate in these two disparate ecosystems is dependent upon its ability to rapidly adapt to the changing environments it encounters. For example, upon host entry, V. cholerae must adjust to dramatic changes in temperature, pH, salinity, oxygen tension, and the presence of antimicrobial compounds. At the same time, colonization of the intestinal tract requires the expression of niche-specific genes (e.g. virulence factors). Prior to exiting the host, V. cholerae must also regulate the expression of genes that are important for transmission and dissemination (9-12). How all of these responses are integrated in response to the dynamic environment in the host is poorly understood. What is clear is that periplasmic sensing systems play a critical role in the process. This includes ToxR which regulates host entry, the Cad system that contributes to acid tolerance, the CarRS TCS which mediates antimicrobial peptide resistance, OscR which regulates response to osmolality, and stress response systems like the Cpx system that alleviate stress due to the presence of antimicrobial compounds in this host. (1, 39, 48-50).
In this study we interrogated the function of six regulatory genes on virulence factor production in V. cholerae. All of the tested regulatory genes were identified as being upregulated in an RND-efflux negative V. cholerae mutant (38). As the RND-mediated efflux is required for virulence factor production, these induced regulatory genes represented potential efflux-dependent virulence repressors. We found that ompR contributed to virulence attenuation in the RND-null strain by repressing aphB expression. AphB is a key regulator in the ToxR virulence regulon (3). Previous studies have shown that AphB activity is modulated by low oxygen and acidic pH, but it was unknown whether expression of aphB was itself regulated (51). To our knowledge OmpR is the first regulator shown to modulate aphB expression in V. cholerae. We further demonstrated that ompR was activated in response to membrane intercalating compounds that are abundant in the host, suggesting that this regulatory circuit may be relevant in vivo.
Although the function of OmpR has been widely explored in the Enterobacteriaceae, the function of the V. cholerae OmpR homolog has not been investigated previously. OmpR is known as an osmoregulator in the Enterobacteriaceae that is induced at high salt concentrations to alleviate osmotic stress (16, 52). Herein we report that V. cholerae ompR was not induced in response to osmolality and that ompR was dispensable for growth at high salt concentrations. These findings were consistent with two previous studies on V. cholerae responses to osmolarity (49, 53), neither of which identified ompR as one of the genes to respond to increased osmolarity. In the latter study, OscR was identified as an osmoregulator which regulated motility and biofilm formation (49). We did not observe any effect of ompR on either of these two phenotypes (not shown), suggesting that OscR and OmpR function independently. Taken together these results suggested that V. cholerae OmpR has evolved to respond to different environmental stimuli and fulfil new functions.
Bacterial regulatory networks evolve in response to evolutionary pressures placed on individual species as they inhabit in specific niches (54, 55). TCS have been suggested to evolve under such selective pressures to respond to novel stimuli and regulate diverse target genes to meet the needs of specific bacterial species (56). OmpR-EnvZ is an example of this. While EnvZ-OmpR is ubiquitous in the Gammaproteobacteria, its function appears to have evolved divergently in several bacterial species (20, 23, 24, 31, 57). Our results suggest that this divergent evolution has also occurred in V. cholerae. We speculate that the lifestyle of V. cholerae, which involves growth in murine environments and the human host gastrointestinal tract, has selected for OmpR to respond to novel stimuli, and to fulfil a novel physiological role in V. cholerae. Sequence comparison of the V. cholerae the ompR locus to the E. coli ompR locus supports this hypothesis. While V. cholerae OmpR is 83% identical in amino acid sequence to its E. coli homolog, the V. cholerae EnvZ sensor kinase was only 47% identical to its E. coli counterpart.
OmpR functioned as a virulence repressor and its expression was activated in response to compounds that are prevalent in the host gastrointestinal tract (e.g. bile salts and detergents). This likely explains the upregulation of ompR in the RND-negative background, as cells lacking RND-mediated efflux are hypersensitive to membrane intercalating compounds due to its diminished ability to actively efflux these compounds from within the cell (37, 45, 58) We speculate that virulence repression in the RND-null mutant resulted from the intracellular accumulation of non-polar and hydrophobic molecules that are present in AKI media (e.g. fatty acids and detergent-like molecules). This hypothesis is supported by the finding that WT and RND-negative V. cholerae strains have comparable ompR expression when cultured in C18 cartridge conditioned AKI media. We are currently investigating the exact compound(s) in AKI media responsible for RND-dependent ompR induction. These molecules are likely the substrates of the RND transporters and thus accumulated in the RND-negative mutant, resulting in ompR induction and subsequent virulence repression. Bile salts and detergent-like molecules (e.g. fatty acids) are also found at high concentrations in the lumen of the small intestine, suggesting the possibility that OmpR could contribute to spatial and temporal virulence regulation observed in vivo (59). This tight regulation of virulence factor production is paramount to the pathogenic success of V. cholerae. Thus, it is interesting to speculate that V. cholerae OmpR is one of multiple factors that converge on the ToxR regulon to ensure that it is only expressed in the appropriate in vivo niche. It is interesting to note that bile salts and fatty acids have pleiotropic effects on the ToxR regulon. Fatty acids have been shown to negatively affect ToxT activity (60). Bile acids, fatty acids and other detergent-like compounds also signal through ToxR to repress aphA (38, 61). Thus, there seems to be a coordinated response to these environmental cues that impacts virulence factor production in V. cholerae.
The induction of V. cholerae ompR in response to non-specific membrane intercalating agents suggests that OmpR could also function as part of a generalized membrane stress response. Consistent with this, there is evidence that OmpR in the Enterobacteriaceae may be a component of other stress response systems (62, 63). A conserved response to membrane stress in bacteria includes suppressing membrane protein production as a mechanism to alleviate envelope stress. Thus, OmpR-dependent virulence repression in V. cholerae could conceivably contribute to a membrane stress response because the ToxR regulon controls the expression of many membrane-bound and secreted proteins, including the two major outer membrane porins OmpU and OmpT (64). However, analysis of WT and ΔompR whole cell lysates by SDS-PAGE staining did not reveal any effect of ompR on production of OmpU and OmpT (not shown); which is consistent with the finding that ompR did not affect toxR expression, or protein production (Fig. 3 and not shown). This contrasts what is observed in other bacterial species where OmpR regulates the expression of outer membrane porins (16-18, 21, 65).
MATERIALS AND METHODS
Bacterial strains and culture conditions
The bacterial strains and plasmids used in this study are listed in Table 1. E. coli strains EC100Dpir+ and SM10λpir were used for cloning and plasmid conjugation, respectively. V. cholerae stain JB58 was used as wild-type (WT) in all experiments. Bacterial strains were routinely grown at 37°C in lysogeny broth (55) or on LB agar. AKI growth conditions were used to induce V. cholerae virulence gene expression as previously described (66). Modified T medium was prepared as previously described (67). Antibiotics were used at the following concentrations: streptomycin (56), 100 µg/mL; carbenicillin (Cb), 100 µg/mL; chloramphenicol (Cm), 20 μg/ml for E. coli and 2 μg/ml for V. cholerae. C18 conditioned AKI media was prepared as follows. Sepa-Pak C18 cartridges (Waters) were preconditioned with 10 mL of 100% methanol followed by 10 mL of sterile ddH2O before 50 mL of AKI broth was passed through the cartridge and the flow through collected and used as conditioned AKI broth. Molecules that were retained on the C18 columns following the passage of LB or AKI broth media were eluted from the column with 10 ml of 100% methanol. The eluates were concentrated by evaporation. The resulting residue was resuspended in a volume of LB broth that was identical to the volume of the extracted AKI broth and filter sterilized prior to use.
Plasmid and mutant construction
Oligonucleotides used in this study are listed in Table 1. Chromosomal DNA from WT strain JB58 was used as the template for cloning experiments. The ompR-lacZ reporter plasmid pDK9 was generated as follows. The ompR promoter region was amplified by PCR using the P-VC2714-F-XhoI and P-VC2714-R-XbaI oligonucleotide primers. The resulting amplicon was digested with XhoI and XbaI restriction endonucleases and ligated into similarly digested pTL61T vector to generate the plasmid pDK9. The ompR expression vector pTB11 was created by amplifying ompR using the VC2714-F-SacI and VC2714-R-SmaI oligonucleotide primers. The resulting 766 bp fragment was digested with SacI and SmaI restriction endonucleases and ligated into similarly digested pBAD33 to generate pTB11. The other expression plasmids (pTB3, pTB5, pTB7, pTB9 and pTB15) were made in a similar manner. The primers used for the construction of these latter plasmids is available upon request. The ompR (VC2714) deletion construct was constructed as follows. Primers pairs ompR-F1/ompR-R2 and ompR-F2/ompR-R1 were used in separate PCR reactions with N16961 genomic DNA. The two resulting amplicons (∼1.5-kb each) were collected and used as the template for the second-round PCR amplification with the flanking ompR-F1 and ompR-R1 PCR primers. The resulting ∼3-kb amplicon was then digested with the SpeI and SmaI restriction endonucleases before being ligated into similarly digested pWM91 vector to generate pWM91-ΔompR. pWM91-ΔompR was then used to delete ompR through allelic exchange as previously described (37). All plasmids were validated via DNA sequencing.
Transcriptional reporter assays
V. cholerae and E. coli strains containing the indicated lacZ reporters were cultured under AKI conditions, in LB broth, or in modified T medium. At the indicated times aliquots were collected in triplicate and β-galactosidase activity was quantified as previously described (68). The experiment quantifying ompR expression during growth under varying NaCl concentrations was performed as follows. WT strains harboring pDK9 were cultured under virulence-factor inducing conditions in AKI media containing the indicated NaCl concentrations for 5h. Culture aliquots were then collected in triplicate and β-galactosidase production was assessed. The experiments quantifying gene expression responses to bile salts, deoxycholate, SDS, Oxgall, indole and ethanol were performed as follows. The indicated strains were grown in LB broth at 37°C with shaking, or under AKI conditions for 4h when the indicated compounds were added to the cultures. Thereafter the cultures were then incubated with shaking for an additional hour before culture aliquots were collected in triplicate and β-galactosidase production was assessed. All of the transcriptional reporter experiments were performed independently at least three times.
Determination of CT and TcpA production
CT production was determined by GM1 enzyme-linked immunosorbent CT assays as previously described using purified CT (Sigma) as a standard (37). The production of TcpA was determined by Western immunoblotting as previously described (10).
Growth curve experiments
Growth curves were generated in microtiter plates. Overnight cultures of WT and ΔompR strains grown in LB broth were washed in PBS then diluted 1:10,000 in fresh LB broth containing 0.5M NaCl. 200 microliters of the diluted cultures were then aliquoted in triplicate wells of a 96-well microtiter plate. The microtiter plates were then incubated at 37°C with constant shaking, and the OD at 600 nm (OD600) was measured every 30 min using a Biotek Synergy microplate reader.
Quantitative real time PCR
V. cholerae strains were grown under AKI conditions for 3.5 h when total RNA was isolated from the cultures using Trizol (Invitrogen) per the manufacturer’s directions. cDNA was generated from the purified RNA using the Maxima First Strand cDNA Synthesis Kit (Thermo). The expression level of specific genes was quantified by amplifying 25 ng of cDNA with 0.3 μM primers using the SYBR green PCR mix (Thermo) on a StepOnePlus real-time PCR System (Applied Biosystems). The relative expression level of genes in the mutant and WT cultures was calculated using the 2−ΔΔCT method. The presented results are the means ± standard deviation from three biological replicates, with each biological replicate being generated from three technical replicates. DNA gyrase (gyrA) was used as the internal control.
ACKNOWLEDGEMENTS
This work was supported by the National Institutes of Health (NIH) under Award Numbers R01AI132460 and R21AI141934. DEK was supported in part by training grant AI049820. The content is solely the responsibility of the authors.