ABSTRACT
Vibrio vulnificus is a potent opportunistic human pathogen that contaminates the human food chain by asymptomatically colonizing seafood. The expression of the 9-gene brp exopolysaccharide locus mediates surface adherence and is controlled by the secondary signaling molecule c-di-GMP and the regulator BrpT. Here, we show that c-di-GMP and BrpT also regulate the expression of an adjacent 5-gene cluster that includes the cabABC operon, brpT, and another VpsT-like transcriptional regulator gene, brpS. The expression of the 14 genes spanning the region increased with elevated intracellular c-di-GMP levels in a BrpT-dependent manner, save for brpS, which was positively regulated by c-di-GMP and repressed by BrpT. BrpS repressed brpA expression and was required for rugose colony development. The mutation of its consensus WFSA c-di-GMP binding motif blocked these activities, suggesting that BrpS function is dependent on binding c-di-GMP. BrpT specifically bound the cabA, brpT, and brpS promoters, and binding sites homologous to the Vibrio cholerae VpsT binding site were identified upstream of brpA and brpT. Transcription was initiated distal to brpA, and a conserved RfaH-recruiting ops element and a potential Rho utilization (rut) terminator site were identified within the 100-bp leader region, suggesting the integration of early termination and operon polarity suppression into the regulation of brp transcription. The GC content and codon usage of the 16-kb brp region was 5.5% lower relative to that of the flanking DNA, suggesting its recent assimilation via horizontal transfer. Thus, architecturally, the brp region can be considered an acquired biofilm and rugosity island that is subject to complex regulation.
IMPORTANCE Biofilm and rugose colony formation are developmental programs that underpin the evolution of Vibrio vulnificus as a potent opportunistic human pathogen and successful environmental organism. A better understanding of the regulatory pathways governing theses phenotypes promotes the development and implementation of strategies to mitigate food chain contamination by this pathogen. c-di-GMP signaling is central to both pathways. We show that the molecule orchestrates the expression of 14 genes clustered in a 16-kb segment of the genome that governs biofilm and rugose colony development. This region exhibits the hallmarks of horizontal transfer, suggesting complex regulatory control of a recently assimilated genetic island governing the colonization response of V. vulnificus.
INTRODUCTION
Bacteria in the environment exist in a free-living planktonic state and in sessile multicellular communities attached to a surface known as biofilms (1). The sessile lifestyle is a widespread survival strategy in the bacterial kingdom. The initial steps of biofilm formation involve surface approach and probing using flagella and pili, respectively (2). Transient surface sensing ultimately leads to the production and secretion of extracellular polysaccharides (EPS) and proteins that facilitate permanent surface colonization. Biofilms are medically, industrially, and environmentally important, as the resident bacteria are more resistant to environmental stressors, such as antibiotic exposure, oxidative agents, ionic stress, and desiccation.
The process of biofilm formation in many bacteria is governed by the second messenger cyclic diguanylate (c-di-GMP) (3). c-di-GMP is synthesized from two GTP molecules by diguanylate cyclase (DGC) enzymes containing GGDEF domains. Second messenger turnover is catalyzed by phosphodiesterase (PDE) enzymes that harbor EAL or HD-GYP domains (4). Typically, DGC and PDE activity is functionally regulated via associated sensor domains that respond to external cues, such as changes in oxygen level, light, nitric oxide, temperature, nutrient limitation, and surface contact (5). c-di-GMP binds to many different types of effector proteins to elicit a change in protein function that leads to the inhibition of flagellar motility, allosteric activation of EPS synthesis, and transcriptional activation or repression of genes that promote or antagonize biofilm development, respectively (6).
The paradigm for c-di-GMP-dependent biofilm formation in Vibrio species is VpsT-mediated regulation of EPS expression in V. cholerae (7). VpsT binds c-di-GMP and activates the transcription of vps genes required for biofilm formation and for rugose colony development, a distinct physiological state characterized by a wrinkly colony morphology, increased biofilm formation, and stress resistance (8–10). A similar scenario has been reported for Vibrio parahaemolyticus, where CpsQ must bind c-di-GMP to activate the expression of the biofilm-promoting EPS encoded by the cps locus (11, 12). CpsQ is homologous to VpsT and bears a canonical VpsT-like c-di-GMP binding motif. An additional VpsT-like regulator, CpsS, represses cps expression (13).
Vibrio vulnificus is a potent human pathogen that can cause infection via the ingestion of contaminated seafood or contact with an open wound (14). It has the highest per case treatment cost and death rate for a foodborne illness (15). Oysters are readily colonized by and concentrate V. vulnificus and represent a major entry point of V. vulnificus into the human food chain (16). We previously demonstrated that BrpT is required for biofilm formation and rugose colony development in V. vulnificus (17). BrpT shares homology with VpsT and activates expression of the brpA promoter (PbrpA). The activity of BrpT deviates from the VpsT and CpsQ paradigms in that BrpT does not need to bind c-di-GMP prior to binding to PbrpA (18). Instead, c-di-GMP regulates the expression of brpT. Recently, c-di-GMP was also shown to regulate the expression of the cabABC operon, which encodes a system for the secretion of a calcium binding matrix protein (CabA) that is required for biofilm and rugose colony formation (19).
Here, we explore the c-di-GMP regulon of V. vulnificus and begin to elucidate the complex network of transcriptional regulators governing brp expression and rugose development. A significant portion of the V. vulnificus genome is c-di-GMP regulated, similar to other Vibrios. In contrast to the vpsT regulon, we demonstrate a tight brpT regulon under elevated intracellular c-di-GMP concentrations and show that the expression of the cabABC operon is both c-di-GMP and BrpT dependent. Furthermore, we demonstrate that another VpsT-like transcriptional regulator, BrpS, the gene for which is adjacent to brpT, represses brpA expression and is also required for rugosity. Both of these BrpS functions are dependent on its ability to bind c-di-GMP. Adding to the complexity, a conserved operon polarity suppressor (ops) element, which is bound by the Rho transcription termination antagonist RfaH, and a potential Rho-dependent termination site (rut) were identified in the PbrpA leader region. Lastly, the GC content and codon usage of this region were markedly different from the surrounding region, suggesting its acquisition in a recent gene transfer event. Collectively, the architectural organization of this 16-kb region, the coregulation of its genes by c-di-GMP and BrpT, the possible intervention of RfaH and Rho, and its hallmark signatures of horizontal transfer suggest complex regulatory control of a genetic island governing biofilm and rugose colony formation, phenotypes that underpin the evolution of V. vulnificus as a potent human pathogen and a successful environmental organism.
RESULTS
Impact of elevated c-di-GMP on global gene expression.A comparative transcriptome sequencing (RNA-seq) analysis was used to assess the global impact of elevated intracellular c-di-GMP levels on the V. vulnificus transcriptome. The RNA profile of wild-type control cells with an unaltered intracellular c-di-GMP level was compared to the profile of wild-type cells with elevated intracellular c-di-GMP levels (i.e., expressing DcpA). Relative to control cells, 468 genes (approximately 9% of the genome) were differentially regulated in response to high intracellular c-di-GMP levels (Fig. 1A, top, and B; see also Table S3 in the supplemental material). The expression of 173 genes increased and 295 genes decreased, and the affected pathways impacted carbohydrate and amino acid metabolism, cell surface modification, transport, signal transduction, virulence, motility, and biofilm formation, among others. Elevated intracellular c-di-GMP levels appeared to activate the expression of genes for transport and polysaccharide production while repressing genes involved in signal transduction, energy production, motility, and virulence. In addition, a large number of hypothetical genes were both activated and repressed under these conditions. Thus, c-di-GMP regulated the expression of a significant proportion of the V. vulnificus genome and impacted a variety of cellular pathways.
Impact of elevated c-di-GMP on global and BrpT-dependent gene expression. (A) Volcano plots of differentially expressed gene in wild-type (WT) cells under elevated (E) versus unaltered (U) intracellular c-di-GMP concentrations (top), and in WT and ΔbrpT cells under elevated intracellular c-di-GMP conditions (bottom). Red points indicate genes exhibiting a >2.5-fold change in gene expression with a P value of <0.05. (B) Pie charts displaying the major functional classes of differentially expressed genes under elevated cellular c-di-GMP levels.
c-di-GMP and BrpT regulate the expression of a 5-gene cluster adjacent to the brp locus by specifically binding to embedded promoters.We previously demonstrated that the expression of the brp locus and brpT was c-di-GMP-dependent (17, 18). Comparative RNA-seq analyses suggested that the expression of a genomic region extending beyond the brp locus was also regulated by c-di-GMP (Fig. 2 and Table S3). This region included brpT and all 9 of the brp locus genes (brpABCDFHIJK). Notably, the oppositely orientated 3-gene cabABC operon situated between brpT and the brp locus, in addition to a vpsT-like transcriptional regulator (aot11_00090, designated brpS) adjacent to brpT, also exhibited increased expression under high c-di-GMP conditions. To confirm this, the expression of PcabA and PbrpS reporter constructs was monitored in wild-type cells under unaltered and elevated intracellular c-di-GMP conditions. The expression of both promoters increased at least 4-fold when c-di-GMP levels were elevated (Fig. 3A). The proximity of cabABC and brpS to the brp locus and their responsiveness to c-di-GMP suggested that they might also be regulated by BrpT. To investigate this, we monitored expression of the PcabAgfp and PbrpSgfp reporter constructs in ΔbrpT cells under unaltered or elevated intracellular c-di-GMP concentrations. PcabA expression was not detectable in the wild-type or ΔbrpT background unless intracellular c-di-GMP levels were elevated, whereupon its expression increased 35-fold in wild-type cells relative to ΔbrpT cells (Fig. 3A). Conversely, PbrpS expression was 2-fold higher in the ΔbrpT background relative to that in the wild type under unaltered and elevated intracellular c-di-GMP conditions, suggesting that BrpT repressed PbrpS.
Organization of the c-di-GMP regulated brp polysaccharide region. The traces show the reads per kilobase per million mapped sequence reads (RPKM) to the brp locus for the wild-type strain under unaltered (U) and elevated (E) intracellular c-di-GMP conditions (i.e., expressing DcpA). The expression profiles for genes (green arrows) in the forward and reverse orientations are denoted by the red and blue traces, respectively. Bent arrows are promoters upstream of brpA, brpH, cabA, brpT, and brpS (aot11_00090).
Regulation of the cabA and brpS promoters by c-di-GMP and BrpT. (A) Wild-type (wt) and ΔbrpT V. vulnificus strains expressing dcpA (DcpA) from an arabinose-inducible promoter were grown in LB under inducing conditions. Vector control strains (v) carried the empty expression vector. The levels of PcabAgfp and PbrpSgfp expression relative to the OD600 after 12 h of growth (peak expression) are shown. Plots show the means, and error bars represent the standard deviations. Data are from three technical replicates of three biological replicates for each sample. Statistically significant differences (P < 0.03) among the respective PcabAgfp and PbrpSgfp samples were determined by one-way ANOVA followed by pairwise comparisons with a Bonferroni adjustment and are indicated by different symbols above each bar. (B) BrpT was purified and incubated with labeled DNA fragments corresponding to PcabA and PbrpS. The iamA pilin promoter (PiamA) was used as a negative control for specific binding.
To determine if BrpT was autoregulatory, PbrpT expression was monitored in wild-type and ΔbrpT cells under unaltered and elevated cellular c-di-GMP conditions. A modest but reproducible decrease in PbrpT expression was detected in ΔbrpT cells relative to that in wild-type cells (see Fig. S3A). When monitored in Escherichia coli to minimize potential c-di-GMP regulatory effects, PbrpT exhibited a 3.6-fold increase in expression relative to vector control cells following plasmid-borne expression of brpT from an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter (Fig. S3B). BrpT was also able to specifically bind PbrpT but not the V. vulnificus PiamA pilus promoter (20, 21) (Fig. S3C), further suggesting that BrpT could indeed regulate its own expression. A similar modest but reproducible increase in PbrpS expression was detected in ΔbrpS cells relative to that in the wild type under elevated cellular c-di-GMP conditions, hinting that BrpS, along with BrpT, might repress PbrpS expression (Fig. S3A). However, attempts to investigate this further in E. coli were not possible, as PbrpS expression remained low irrespective of the cellular c-di-GMP levels, and we have not yet identified the c-di-GMP responsive regulator that activates PbrpS expression.
The BrpT-dependent gene expression profile was determined by comparing the transcriptomes of wild-type and ΔbrpT strains under elevated c-di-GMP levels. A total of 18 genes were differentially regulated (Fig. 1A, bottom, and Table S4). The brpABCDFHIJK and cabABC loci were among the 16 genes that exhibited decreased expression in the ΔbrpT background. This suggested that the expression of all of the genes in the region was BrpT-dependent. We previously demonstrated that BrpT could specifically bind to promoters upstream of brpA and brpH (18). To ascertain if BrpT also directly regulated the expression of the cabABC operon and brpS, we purified BrpT and tested its ability to bind DNA fragments that corresponded to the cabA and brpS promoter regions. BrpT bound specifically to both promoters (Fig. 3B) but not to PiamA. Together, these results suggested that the expression of a genomic region spanning the 14 genes from brpA to brpS was regulated by c-di-GMP and dependent upon BrpT. Moreover, elevated c-di-GMP levels trigger the expression of all of the genes across this region, while BrpT appeared to positively regulate the expression of PbrpA, PbrpH, PcabA, and PbrpT but negatively regulate PbrpS expression.
Distal initiation of brp transcription.Under elevated intracellular c-di-GMP conditions, RNA reads mapping to PbrpA indicated that transcription was initiated at least 100 bp upstream of brpA (Fig. 4A), suggesting that the binding site for BrpT was likely even further upstream. A PbrpAgfp promoter deletion series was constructed to map the potential BrpT binding site(s). Transcriptional fusions bearing sequential deletions from 325 bp to 150 bp upstream of the brpA start ATG (Fig. 4B) were transformed into E. coli that carried a compatible brpT expression plasmid. The 325-bp promoter fragment supported a high level of BrpT-dependent gfp expression relative to that of the vector control. A 10-fold drop in expression was observed when the promoter was whittled to 200 bp, and no expression could be detected from a 150-bp promoter fragment. An alignment of the V. cholerae vspL (PvpsL) and PbrpA promoters revealed a putative BrpT binding site that showed homology to the VpsT binding site in PvpsL (Fig. 4C). This site is 272 bp upstream of the brpA start ATG and correlates with the expression data. Some divergence was apparent in the BrpT binding site, the majority of which was localized to the left end, causing degradation of its palindromic nature relative to the VpsT binding site. A homologous site was also revealed when the vpsT (PvpsT) and PbrpT promoters were aligned (see Fig. S1).
Distal initiation of brp transcription. (A) The gray trace shows the reads per kilobase per million mapped sequence reads (RPKM) to the brpA promoter region in wild-type cells under elevated intracellular c-di-GMP conditions. TSS, the major transcription start site. Numbering is relative to the brpA start codon. (B) PbrpAgfp deletion constructs (top) and a plot (bottom) showing the level of PbrpA expression in E. coli relative to the OD600 for each construct. v, vector control cells; BrpT, following brpT induction. Shown are the means, and error bars represent the standard deviations. Statistically significant differences in expression (*, P < 0.001) between the PbrAgfp promoter deletions (200 and 150 bp) and the full-length construct (325 bp) were determined using the Student t test (two-tailed distribution with two-sample equal variance). (C) Alignment of the VpsT and BrpT binding sites in the vpsL and brpA promoters.
BrpS is required for rugose colony development.The physiological role of BrpS was unknown, but its proximity to brpT and dependence on c-di-GMP for, for expression led us to surmise that it might be related to biofilm formation. A phylogenetic tree of known and putative transcriptional regulators of the VpsT family from V. cholerae, V. parahaemolyticus, and V. vulnificus suggested that BrpS was more closely related to the activator VpsT than was BrpT, suggesting that it too might be an activator (18). However, the V. parahaemolyticus repressor CpsS also branched within the clade, confounding a functional interpretation for BrpS. To ascertain if BrpS function was related to brp expression, we monitored PbrpAgfp expression in wild-type and ΔbrpS backgrounds under unaltered (vector) and elevated (DcpA) intracellular c-di-GMP concentrations. The expression of dcpA in wild-type bacteria resulted in an 8.4-fold increase in gfp expression (Fig. 5A; see also Fig. S4). PbrpA expression in the ΔbrpS strain was 2-fold higher than in wild-type cells and increased 12-fold when c-di-GMP levels were elevated. This increase could be countered by an ectopically integrated copy of brpS expressed from its native promoter; however, the mutation of its predicted c-di-GMP binding site from WFSR to WFSA (BrpSR134A) negated this effect. This suggested that brpS function is likely dependent on c-di-GMP binding. To ascertain if the increase in PbrpA expression in a brpS mutant amounted to a phenotypic change, rugose colony development was assessed under unaltered and elevated intracellular c-di-GMP levels. Colony morphologies were very similar for all of the strains under unaltered intracellular c-di-GMP concentrations. Under elevated intracellular c-di-GMP levels, rugosity developed in the wild type and the complemented (ΔbrpS-C) strain but failed to do so in the null or BrpSR134A complemented strains. However, the opacity of the ΔbrpS and BrpSR134A complemented strains clearly increased, in agreement with elevated PbrpA expression. The need for a functional BrpS in rugose colony development was cemented by the failure of the ΔbrpS strain to become rugose even when PbrpA expression was maximized by overexpressing BrpT while simultaneously elevating the intracellular c-di-GMP level (Fig. 5B; Fig. S4). In contrast, elevating the c-di-GMP level alone induced rugosity in wild-type cells, and the phenotype was even more pronounced following simultaneous overexpression of BrpT. These results suggested that deletion of brpS led to a derepression of brpA expression but that BrpS was also required for the development of the rugose phenotype. A possible target promoter was PcabA. Indeed, PcabAgfp expression in wild-type cells was twice that observed in ΔbrpS cells, and the defect could be complemented by ectopic expression of brpS (see Fig. S5). This suggested that BrpS, along with BrpT, activated PcabA.
BrpS is required for rugose colony development. PbrpA expression profiles in the wild-type (wt) and mutant brpS (ΔbrpS) or brpT (ΔbrpT) strains under unaltered (−) or elevated (+) intracellular c-di-GMP levels by expressing DcpA from an arabinose-inducible promoter. The indicated regulators, BrpS or BrpSR134A (A) and BrpT (B), were expressed from an IPTG-inducible promoter. Vector control strains (v) carried the respective empty expression vector. Shown are the means, and error bars represent the standard deviations. Statistically significant differences among the samples (P < 0.05, as determined by one-way ANOVA followed by pairwise comparisons with a Bonferroni adjustment) are indicated by different symbols above each bar. The images below each graph show the opacity and rugose colony development by the same strains.
DISCUSSION
Motility, virulence, polysaccharide production, and cell cycle progression are among the many physiological processes known to be regulated by the secondary messenger c-di-GMP (22). Signal transduction is elicited by physical associations between c-di-GMP and a growing number of effector proteins as well as at least two identified riboswitches (5, 6, 23–26). The final output is a change in activity at the transcriptional (e.g., the V. cholerae regulator, VpsT) or protein (e.g., the E. coli flagellar brake, YcgR) level that ultimately alters cell physiology. Here, we show that approximately 9% of the V. vulnificus genome is differentially expressed in response to elevated intracellular c-di-GMP. This was on par with the overall degree of change observed among other Vibrio species (27–30). The biofilm-associated EPS-encoding brp locus and its activator (brpT) were among the genes in the c-di-GMP regulon. We also identified two additional components of the c-di-GMP/BrpT biofilm regulatory network: brpS, which codes for a BrpT homolog, and the cabABC operon, which is required for the secretion of the calcium binding matrix protein, CabA. Furthermore, we assign a role for BrpS in regulating brp expression and rugose colony development.
VpsT controls the expression of ∼7% of the V. cholerae genome (27). In stark contrast, the V. vulnificus homolog, BrpT, appears to regulate only approximately 16 genes under elevated intracellular c-di-GMP conditions. Thus, VpsT seemingly behaves as a central c-di-GMP sentinel capable of influencing multiple networks, whereas the activity of BrpT is restricted to impact a small subset of biofilm-related pathways. This may be due to differences in the interaction of the regulators with c-di-GMP. The activity of VpsT is directly mitigated by binding to the signaling molecule, enabling a rapid response to fluctuations in the intracellular c-di-GMP concentration (31). In contrast, BrpT does not directly bind to and sense c-di-GMP at physiologically relevant concentrations (18). This likely dampens its responsiveness to changes in intracellular c-di-GMP concentrations, thereby diminishing its effectiveness as a global regulator.
Both BrpS and BrpT are required to maintain proper expression of the brp locus; BrpT activates brpA expression while BrpS represses it. Moreover, the expression of brpT and brpS is c-di-GMP dependent. BrpS, like BrpT, belongs to the VpsT-like family of transcriptional regulators and contains a canonical WFSR c-di-GMP binding site. A brpS null mutant was unable to repress PbrpA expression or support rugose colony development, and a BrpS mutant (BrpSR134A) bearing a WFSA c-di-GMP binding motif, where the 4th-position R residue that is absolutely required for c-di-GMP binding (11, 31) was mutated to an A residue, failed to complement the phenotypes of the ΔbrpS strain. This suggests that the repressor function of BrpS is dependent on c-di-GMP binding. A similar circuit exists in V. parahaemolyticus. CpsQ activates cps expression. The V. parahaemolyticus BrpS homolog, CpsS, negatively regulates cps expression and CpsQ suppresses cpsS (11). However, in stark contrast to BrpS, a transposon insertion in CpsS was sufficient to induce elevated PcpsA expression and rugose colony formation (13). BrpS is also required for rugose colony development. This presents a conundrum, whereby brp expression is required for rugosity, as is a functional BrpS; however, the latter represses the expression of the former. This quandary is solved, in part, by the repression of brpS by BrpT. The requirement of BrpS for rugose colony development suggests that, in addition to repressing brpA, BrpS also directs the expression of an additional biofilm matrix component that is required for rugosity. A candidate is the previously described cabABC operon, which is part of the c-di-GMP and BrpT regulons and contributes to calcium-mediated biofilm and rugose colony formation (19). cabABC expression in a brpS mutant was half that of wild-type cells under elevated cellular c-di-GMP concentrations, and this likely had a negative impact on rugose development in this background. A similar circuit exists in V. parahaemolyticus, where the homologous mfpABC locus that participates in biofilm formation and rugosity is regulated by the BrpT homolog, CpsQ (11). Alternatively, BrpS may also regulate the expression of an additional unidentified component(s) of the biofilm matrix that contributes to rugosity.
BrpT is able to directly bind PbrpA, and a dramatic drop in PbrpA expression was observed following the truncation of the promoter from 325 bp to 200 bp upstream of the start codon. This correlated with the position of a 23-bp site showing homology to the VpsT binding site in PvpsL. This BrpT site was 272 bp upstream of brpA. Distal regulation of the V. cholerae vps locus has also been reported and involves HN-S as well as several additional transcription factors. We imagine that regulation at PbrpA may be equally complex and involve a mechanism invoking DNA bending for the initiation of transcription from PbrpA. Moreover, a conserved ops element was identified in the 5′ untranslated region of PbrpA (see Fig. S2 in the supplemental material). The ops element is an 8-bp motif and a conserved 3′ thymine residue (5′-GGCGGTAGYVT-3′) that is bound by RfaH, a regulator that is known to activate the transcription of genes whose products assemble and export surface and extracellular macromolecules (32, 33). RfaH has also been shown to specifically and efficiently recruit ribosomes to mRNA templates harboring ops elements (34). RfaH is recruited to transcribing RNA polymerase (RNAP) complexes in promoter proximal regions upon exposure of a functional ops element in the nontemplate strand, where it interacts with the element and RNAP. RfaH remains associated with the elongation complex, suppressing Rho-mediated termination at downstream transcription pause sites until its dissociation at an intrinsic terminator (32, 35, 36). Rho terminates transcription by binding to C-rich Rho utilization (rut) sites in the 5′ unstructured region of nascent RNA transcripts (37). This stimulates its RNA-dependent helicase activity and 5′ to 3′ translocation along the transcript toward the paused RNAP. Contact with RNAP leads to the ATP-dependent dissociation of the paused transcription elongation complex, resulting in early termination and the repression of gene expression. Notably, a putative C-rich rut site was situated between the brp transcription start site (TSS) and the ops element (Fig. S2). Neither an ops element nor a potential rut site was identified in the vps locus of V. cholerae. Collectively, this combination of factor binding sites suggests that the regulation of brp expression is equally as complex as, but significantly different from, the V. cholerae paradigm. The evolutionary implications of the regulatory differences in EPS expression between V. cholerae and V. vulnificus are significant. Such differences highlight the intricacy of c-di-GMP signaling in Vibrios and may explain how closely related species using similar biofilm regulatory networks can still evolve to occupy distinct niches in shared environments. We are working to determine if the putative rut site is indeed functional and to decipher the contribution of these systems to the regulation of brp expression and biofilm formation.
We propose the following model for the regulation of brp expression (Fig. 6). Potential Rho-dependent termination may suppress the expression of the brp region by preventing the extension of transcripts initiated under low intracellular c-di-GMP concentrations beyond the RfaH-recruiting ops element. This is relieved when environmental signals alter DGC and/or PDE activity such that intracellular c-di-GMP levels increase. This results in increased brpR expression by an unknown mechanism (an effector [E] protein may bind the signaling molecule and activate brpR expression or brpR repression may be relieved). BrpR may directly bind c-di-GMP, analogous to VpsR, or it may activate brpT expression independent of c-di-GMP binding, similar to BrpT. BrpT oligomers activate brp and cab and further activate its own expression. RfaH can then act to suppress premature transcript termination at downstream pause sites, coactivating brp expression alongside BrpT. BrpT also represses brpS expression, the product of which negatively regulates brpA expression in a c-di-GMP-dependent manner. The net result is an increase in brp expression. The expression of brpS increases as c-di-GMP levels increase, eventually leading to decreased brp expression and boosted cabABC expression, thereby fine tuning the output to balance polysaccharide and matrix protein production during biofilm and rugose development.
Proposed model for BrpT and c-di-GMP-dependent regulation of brp expression. Potential Rho-dependent termination may suppress the expression of the brp region by preventing the extension of transcripts initiated under low intracellular c-di-GMP concentrations beyond the RfaH-recruiting ops element. This is relieved when environmental signals alter DGC and/or PDE activity such that intracellular c-di-GMP levels increase. This results in increased brpR expression by an unknown mechanism (an effector protein [E] may bind the signaling molecule and activate brpR expression or brpR repression may be relieved). BrpR may directly bind c-di-GMP, analogous to VpsR, or it may activate brpT expression without the need to bind c-di-GMP, similar to BrpT. BrpT oligomers can then activate brp and cab and further activate its own expression (brpT). RfaH can then act to suppress premature transcript termination at downstream pause sites, coactivating brp expression alongside BrpT. BrpT also represses brpS expression, the product of which represses brpA expression in a c-di-GMP-dependent manner while coactivating cabA expression. Green and red arrows denote activation and repression by the indicated regulator, respectively. Solid and dashed lines signify direct and unknown interactions, respectively.
Interestingly, Rho has been demonstrated to terminate the transcription of poorly translated RNAs, such as horizontally transferred foreign genes (38), and RfaH has been shown to activate the expression of horizontally acquired operons that encode cell wall components, exopolysaccharides, and pili (39). Neither an ops element nor a potential rut site was identified in the vps locus. The GC content of the vps locus differed by only 1.2% from the genome average, arguing against its recent acquisition by horizontal transfer (Fig. 7). Conversely, both a potential rut site and an ops element were present in PbrpA. The GC content of the 16-kb region spanning the brp region was 4.9% lower than the genome average, and its codon usage pattern was also significantly different, supporting its recent assimilation by horizontal gene transfer (40). Thus, architecturally, the brp region can be considered an acquired biofilm and rugosity island that is subject to complex regulation. The conservation of the brp region in all V. vulnificus strains sequenced to date suggests that it has played an important role in the environmental success of the species.
GC content of the brp, vps, and cps loci and flanking regions. (Top) Radar plots of codon usage based on the coding sequences of 12 conserved core genes (C) and the coding sequences of the brp (B) locus of V. vulnificus. The red and blue flares indicate the relative frequencies of synonymous codons (the scale is in the middle). Codons are grouped by the last nucleotide of the triplet (A, T, C, and G). Preferred codon triplets are shown in the outer ring. (Bottom) Schematic representation of the brp and vps loci and flanking chromosomal DNA. The cab and rbm loci of V. vulnificus and V. cholerae, respectively, are indicated by white lettering. The GC content and length of each segment is indicated.
MATERIALS AND METHODS
Media and strains.BD Difco LB and LB agar were used for these experiments. Antibiotics and additives were used at the following concentrations: chloramphenicol (Cm), 25 μg/ml; gentamicin (Gm), 10 (E. coli) or 35 (V. vulnificus) μg/ml; isopropyl-β-d-thiogalactoside (IPTG), 100 μM; X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside), 20 μg/ml; l-arabinose, 0.2%. The strains and plasmids are listed in Table S1 in the supplemental material.
Colony morphology assays.Colony morphology assays were conducted as previously described. Briefly, overnight cultures of indicated strains were grown at 30°C with the appropriate antibiotics and arabinose inducer, where indicated. Cultures were then diluted 1:100 into fresh LB medium and grown to an optical density at 600 nm (OD600) of 0.5. Cultures (2 μl) were then spotted onto LB agar containing the appropriate antibiotics and/or inducer and incubated for 48 h at 30°C. Images were captured using a Leica MS5 dissecting scope equipped with a Leica DC300F camera.
Mutant construction and ectopic complementation.The ΔbrpT and ΔbrpS strains were generated by amplifying PCR fragments corresponding to 3 kb upstream and downstream of the target gene (see Table S2 for primer sequences). The fragments were fused and cloned into the pRE112 plasmid by Gibson assembly. Plasmids were maintained in E. coli S17.1λπ for conjugation to V. vulnificus. Integration was selected on LB Cm plates and gridded onto LB 20% sucrose plates for sacB counterselection. Mutants were confirmed to have the expected deletion in brpT or brpS by PCR. The brpS gene, including the promoter, was amplified and cloned into pTX1KebgAKm by Gibson assembly. Plasmids were conjugated from E. coli S17.1λπ to V. vulnificus, and chromosomal cointegrates were selected on LB kanamycin plates.
Reporter constructs.PCR products for transcriptional reporters were amplified from V. vulnificus genomic DNA and fused to gfp for insertion into pSW23T-lacZ by Gibson assembly. Plasmids were conjugated to V. vulnificus for chromosomal integration into lacZ. Cointegrates were selected on LB chloramphenicol plates and confirmed by PCR.
Electrophoretic mobility shift assay.Electrophoretic mobility shift assays (EMSAs) were performed using purified BrpT (18) and infrared 700 (IR700)-labeled probes corresponding to the indicated promoter fragments that were amplified from V. vulnificus genomic DNA. Reaction mixtures contained 5 μM BrpT, 0.2 mg/ml bovine serum albumin (BSA), 0.1 μg/ml poly(dI-dC) in binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM KCl, 75 mM NaCl, 10 mM MgCl2, 5% glycerol, 1 mM dithiothreitol [DTT], and 0.1 mM EDTA). After a 45-min incubation at room temperature in the dark, the reaction mixtures were loaded onto a 5% Tris-borate-EDTA (TBE)-polyacrylamide gel with 10% glycerol and run in 0.5× TBE. The gels were prerun for 1 h at 50 V before loading, and samples were resolved at 50 V for 2 h. Gels were visualized on an Odyssey FC imaging system (LI-COR).
Expression analysis.Strains were grown overnight, diluted in fresh medium containing antibiotic and inducer where indicated, and inoculated in triplicates into 96-well plates. OD600 and florescence measurements (excitation, 485 nm; emission, 528 nm) were taken on a Biotek Synergy HT1 microplate reader. Data from three technical replicates of three biological replicates were collected for all experiments. Plots show the means and error bars represent the standard deviations.
RNA isolation and RNA-seq analysis.Cultures were grown overnight at 30°C with appropriate antibiotics. Overnight cultures were diluted 100-fold into fresh medium with Gm to maintain pSU38GT or pSU38GT-DcpA. When cultures reached an OD600 of 0.5, they were diluted to an OD600 of 0.025 in 5 ml of medium that included 0.2% arabinose and grown for 5 h at 30°C. One OD600 unit was pelleted by centrifugation, and RNA was extracted by using the BioBasic Total RNA miniprep kit. PCR amplification of a 150-bp gyrA fragment using the purified RNA as the template was conducted to verify that it was DNA free. RNA libraries were constructed from 3 biological replicates of each sample and sequenced at the Indiana University Center for Genomics and Bioinformatics. Reads were mapped to the V. vulnificus ATCC 27562 reference genome (41) and quantified using RSEM v1.3.0 (42). Differential expression analysis was performed using EBseq (43).
Statistical analyses.Statistical significance was determined by the Student t test (two-tailed distribution with two-sample equal variance) when directly comparing two conditions or a one-way analysis of variance (ANOVA) followed by pairwise comparisons with a Bonferroni adjustment when comparing data with multiple samples.
ACKNOWLEDGMENTS
We thank Dan Kearns, Ankur Dalia, and members of their labs for insightful discussions.
Funding for this work was provided by Indiana University Bloomington (D.R.-M.).
FOOTNOTES
- Received 30 March 2018.
- Accepted 8 May 2018.
- Accepted manuscript posted online 14 May 2018.
- Address correspondence to Dean A. Rowe-Magnus, drowemag{at}indiana.edu.
Citation Chodur DM, Rowe-Magnus DA. 2018. Complex control of a genomic island governing biofilm and rugose colony development in Vibrio vulnificus. J Bacteriol 200:e00190-18. https://doi.org/10.1128/JB.00190-18.
Supplemental material for this article may be found at https://doi.org/10.1128/JB.00190-18.
REFERENCES
- Copyright © 2018 American Society for Microbiology.
