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Title: The nucleoid occlusion protein SlmA is a direct transcriptional activator of chitobiose utilization in Vibrio cholerae}
Article:
Abstract:
Chitin utilization by the cholera pathogen Vibrio cholerae is required for its persistence and evolution via horizontal gene transfer in the marine environment. Genes involved in the uptake and catabolism of the chitin disaccharide chitobiose are encoded by the chb operon. The orphan sensor kinase ChiS is critical for regulation of this locus, however, the mechanisms downstream of ChiS activation that result in expression of the chb operon are poorly understood. Using an unbiased transposon mutant screen, we uncover that the nucleoid occlusion protein SlmA is a regulator of the chb operon. SlmA has not previously been implicated in gene regulation. Also, SlmA is a member of the TetR family of proteins, which are generally transcriptional repressors. In vitro, we find that SlmA binds directly to the chb operon promoter, and in vivo, we show that this interaction is required for transcriptional activation of this locus and for chitobiose utilization. Using point mutations that disrupt distinct functions of SlmA, we find that DNA-binding, but not nucleoid occlusion, is critical for transcriptional activation. This study identifies a novel role for SlmA as a transcriptional regulator in V. cholerae in addition to its established role as a cell division licensing factor.
Introduction:
V. cholerae, the bacterium responsible for the diarrheal illness cholera, is naturally found in the marine environment. In this niche, this pathogen forms biofilms on the shells of microscopic crustaceans. The shells of these organisms are primarily composed of chitin, an insoluble polymer of β-1, 4 linked N-acetylglucosamine( GlcNAc). Chitin is the second most abundant biopolymer on the planet, and Vibrio species play an important role in recycling chitin in the aquatic environment[1]. The pathway for degradation and utilization of this carbon and nitrogen source is conserved among the Vibrionaceae[2]. In addition to promoting the survival of V. cholerae in the aquatic environment, biofilm formation on chitin is also important for promoting transmission of this pathogen to its human host. Indeed, cholera outbreaks are seasonal in endemic areas and closely associated with blooms in chitinous zooplankton[1]. Furthermore, filtration of water through sari cloth in these areas( which has an effective pore size that will eliminate chitin biofilms but cannot filter planktonic bacteria) reduces the incidence of waterborne transmission[3, 4]. Chitin oligosaccharides also induce natural competence, a mechanism of horizontal gene transfer, in V. cholerae[5, 6]. Thus, Vibrio-chitin interactions are important for the persistence, transmission, and evolution of this important human pathogen in its environmental reservoir. The expression of genes required for chitin degradation, uptake, and utilization are regulated by the orphan sensor kinase ChiS[7, 8]. ChiS activity is normally repressed by chitin binding protein( CBP), the periplasmic substrate binding protein for the chitobiose ABC transporter[7]. This repression is relieved in the presence of chitin oligosaccharides, which bind to CBP. ChiS can also be activated, however, by deletion of CBP[7]. ChiS is not predicted to directly bind DNA. Without a cognate response regulator, the mechanism of gene regulation downstream of ChiS activation is poorly understood. One locus regulated by ChiS is the chb operon, which encodes the genes required for uptake and utilization of the chitin disaccharide chitobiose[8]. Here, we perform an unbiased screen to identify factors downstream of ChiS required for activation of the chb operon and characterize one newly identified transcriptional activator of this locus.
Discussion:
Here, we show that the cell division licensing factor SlmA is required for activation of chitobiose utilization genes in V. cholerae. We show that this regulation is direct because SlmA binds to Pchb, the promoter that regulates these genes. Furthermore, we demonstrate that this direct regulation is required for growth of V. cholerae on chitobiose. While we show that SlmA is a direct activator of Pchb, the exact mechanism of activation is still unclear. We have shown that the location of the SBS within Pchb is important for activation. Additionally, we show that the sequence between the SBS and the-35 sequence is required for activation, consistent with this region harboring a coactivator binding site. Because the SBS is located 167 base pairs away from the TSS, it is unlikely that SlmA directly interacts with RNA Polymerase to mediate activation. We also explored the possibility that SlmA binding may occlude a repressor binding site thereby allowing for activation of Pchb indirectly. We formally tested this hypothesis by performing a Tn mutant screen for repressors in a ΔslmA Δcbp Pchb-lacZ reporter strain, however, this screen did not identify any putative repressors( ~70, 000 mutants visually screened). As a result, we propose a mechanism in which SlmA requires a presently unknown ChiS-dependent coactivator for activation of Pchb( Fig 5). A candidate for this unknown factor was not identified in the Tn-mutant screen for activators that identified SlmA. This screen, however, may not have been saturating or, alternatively, this factor may be essential for viability or masked by genetic redundancy. Because ChiS is an orphan sensor kinase, the most logical coactivator would be a cognate response regulator. However, it has been shown that the conserved histidine and aspartate residues of ChiS that are critical for autophosphorylation and phosphorelay activity, respectively, are not important for activation of Pchb[20]. Thus, while the identity of the regulator that acts downstream of ChiS remains unknown, we believe it is unlikely to be a classical response regulator. It is possible that this coactivator bridges SlmA and RNA polymerase to mediate transcriptional activation( as depicted in Fig 5). Alternatively, it is possible that the coactivator loops, bends, or structurally alters DNA in the chb promoter to enhance SlmA-dependent recruitment of RNA polymerase. Identifying and characterizing a putative coactivator for Pchb that acts downstream of ChiS will be the focus of future work. Another possible mechanism of activation is that SlmA directly interacts with RNA polymerase through its ability to polymerize and spread on DNA[14]. We have two results, however, that diminish this as a likely model for activation. First, we show that moving the SBS as little as 3 bp in either direction within Pchb abolishes activation( S7 Fig). If DNA spreading were critical we would predict that this perturbation should have a limited effect on transcriptional activation. Further, we have shown that overexpression of SlmA can actually decrease Pchb transcription, which again would not be predicted by a DNA spreading model( S10 Fig). Alternatively, it is possible that increased SlmA spreading( when SlmA is overexpressed) may prevent RNA polymerase from binding, resulting in decreased expression of Pchb. We have shown that there are two transcriptional start sites for the chb locus. The short transcript is strongly induced in the presence of chitin oligosaccharides, while the long transcript is basally produced in the absence of chitin and is only modestly induced in its presence( 2–4 fold). This indicates that there may be an additional level of regulation involving the 5’ untranslated region( UTR) of the chb transcript. A putative hairpin in Pchb also seemed to have some inhibitor effect on Pchb expression( S5 Fig). It is possible that the long transcript and hairpin play an important role in the basal expression of the chb operon, which is required for a rapid response to chitin oligosaccharides. TetR proteins are generally transcriptional repressors, however, there are examples where these proteins can mediate transcriptional activation. One well-studied example is the LuxR protein in Vibrio harveyi( also known as HapR in V. cholerae and SmcR in Vibrio vulnificus)[21, 22]. SlmA is primarily characterized as a TetR family cell wall licensing factor and here we describe its additional role as a transcriptional activator. It has been hypothesized that transcriptional regulatory proteins arose from nucleoid-associated proteins[23]. Their role in binding DNA to properly structure the nucleoid may have evolved to aid in transcriptional regulation by contributing to the rearrangement of DNA structure at promoter regions[24]. One such example is integration host factor( IHF), which binds DNA and results in dramatic DNA bending( bend angle of ~120°), which can promote activation of regulated genes. SlmA binding results in subtle bending of DNA( ~18°)[14]. Thus, SlmA may have evolved from a protein that structures the nucleoid into a cell division licensing factor as well as a transcriptional regulator. Additionally, since SlmA carries out both nucleoid occlusion and transcriptional activation, it is possible that these two activities affect one another. As a result, co-option of SlmA for regulation of Pchb may be a mechanism to integrate the cell division status of the cell with activation of chitin utilization. To our knowledge, this is the first example of gene regulation by a nucleoid occlusion protein. A recent in vitro whole genome binding analysis identified 79 putative SBSs in V. cholerae[17]. We determined that ~25%( 20/79) of these putative SBSs are in intergenic sites( including the one in Pchb), while only ~12% of the genome constitutes intergenic sequence. By contrast, in E. coli, ~8. 3%( 2/24) of SBSs are in intergenic sites[12] and ~11% of the genome is intergenic. Also, analyzing binding sites for Noc, the nucleoid occlusion protein of Bacillus subtilis, ~12%( 9/74) of binding sites are intergenic[25] and ~11% of the genome represents intergenic sequence. By contrast, for binding sites for the terminus macrodomain proteins MatP( known as matS sites) ~24%( 6/25) are in intergenic regions[26]. Thus, it is possible that enriched binding of nucleoid-associated proteins at intergenic regions( as is the case for matS sites in E. coli and SBSs in V. cholerae) is because these sites are more flexible in regards to accommodating mutations compared to coding regions of the genome. Alternatively, SBSs may be enriched in intergenic sites in V. cholerae possibly to regulate the expression of additional genes. This will be the focus of future work. Preliminary RNA-seq analysis of a slmA mutant grown in rich medium, however, did not uncover any additional loci regulated by SlmA in V. cholerae. An effect of SlmA on gene regulation at Pchb, however, was only uncovered by deletion of cbp or growth on chitin( both of which induce this locus). Thus, it may be that additional inducing cues are required to uncover a role for SlmA-dependent regulation at additional genetic loci. Nucleoid occlusion factors are present across diverse bacterial genera and it is tempting to speculate that these proteins may also participate in regulating gene expression in addition to their established roles in cell division licensing.
The cholera pathogen Vibrio cholerae is a natural resident of the aquatic environment and causes disease when ingested in the form of contaminated food or drinking water . In the aquatic environment , the shells of marine zooplankton , which are primarily composed of chitin , serve as an important food source for this pathogen . The genes required for the utilization of chitin are tightly regulated in V . cholerae , however , the exact mechanism underlying this regulation is currently unclear . Here , we uncover that a protein involved in regulating cell division is also important for regulating the genes involved in chitin utilization . This is a newly identified property for this cell division protein and the significance of a common regulator for these two disparate activities remains to be understood .