Biology subjects

Klancher, C. A.

Publications and source records attributed to Klancher, C. A..

6 recordsLinked to original sources

bioRxiv2024

Two transmembrane transcriptional regulators coordinate to activate chitin-induced natural transformation in Vibrio cholerae

Transcriptional regulators are a broad class of proteins that alter gene expression in response to environmental stimuli. Transmembrane transcriptional regulators (TTRs) are a subset of transcriptional regulators in bacteria that can directly regulate gene expression while remaining anchored in the membrane. Whether this constraint impacts the ability of TTRs to bind their DNA targets remains unclear. Vibrio cholerae uses two TTRs, ChiS and TfoS, to activate horizontal gene transfer by natural transformation in response to chitin by inducing the tfoR promoter (PtfoR). While TfoS was previously shown to bind and regulate PtfoR directly, the role of ChiS in PtfoR activation remains unclear. Here, we show that ChiS directly binds PtfoR upstream of TfoS, and that ChiS directly interacts with TfoS. By independently disrupting ChiS-PtfoR and ChiS-TfoS interactions, we show that ChiS-PtfoR interactions play the dominant role in PtfoR activation. Correspondingly, we show that in the absence of ChiS, recruitment of the PtfoR locus to the membrane is sufficient for PtfoR activation when TfoS is expressed at native levels. Finally, we show that the overexpression of TfoS can bypass the need for ChiS for PtfoR activation. All together, these data suggest a model whereby ChiS both (1) recruits the PtfoR DNA locus to the membrane for TfoS and (2) directly interacts with TfoS, thereby recruiting it to the membrane-proximal promoter. This work furthers our understanding of the molecular mechanisms that drive chitin-induced responses in V. cholerae and more broadly highlights how the membrane-embedded localization of TTRs can impact their activity. AUTHOR SUMMARYLiving organisms inhabit diverse environments where they encounter a wide range of stressors. To survive, they must rapidly sense and respond to their surroundings. One universally conserved mechanism to respond to stimuli is via the action of DNA-binding transcriptional regulators. In bacterial species, these regulators are canonically cytoplasmic proteins that freely diffuse within the cytoplasm. In contrast, an emerging class of transmembrane transcriptional regulators (TTRs) directly regulate gene expression from the cell membrane. Prior work shows that two TTRs, TfoS and ChiS, cooperate to activate horizontal gene transfer by natural transformation in response to chitin in the facultative pathogen Vibrio cholerae. However, how these TTRs coordinate to activate this response has remained unclear. Here, we show that ChiS likely promotes TfoS-dependent activation of natural transformation by (1) relocalizing its target promoter to the membrane and (2) recruiting TfoS to the membrane proximal promoter through a direct interaction. Together, these results inform our understanding of both the V. cholerae chitin response and how TTR function can be impacted by their membrane localization.

microbiology↗

bioRxiv2022

The molecular mechanism for carbon catabolite repression of the chitin response in Vibrio cholerae

Vibrio cholerae is a facultative pathogen that primarily occupies marine environments. In this niche, V. cholerae commonly interacts with the chitinous shells of crustacean zooplankton. As a chitinolytic microbe, V. cholerae degrades insoluble chitin into soluble oligosaccharides. Chitin oligosaccharides serve as both a nutrient source and an environmental cue that induces a strong transcriptional response in V. cholerae. Namely, these oligosaccharides induce the chitin sensor, ChiS, to activate the genes required for chitin utilization and horizontal gene transfer by natural transformation. Thus, interactions with chitin impact the survival of V. cholerae in marine environments. Chitin is a complex carbon source for V. cholerae to degrade and consume, and the presence of more energetically favorable carbon sources can inhibit chitin utilization. This phenomenon, known as carbon catabolite repression (CCR), is mediated by the glucose-specific Enzyme IIA (EIIAGlc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). In the presence of glucose, EIIAGlc becomes dephosphorylated, which inhibits ChiS transcriptional activity by an unknown mechanism. Here, we show that dephosphorylated EIIAGlc interacts with ChiS. We also isolate ChiS suppressor mutants that evade EIIAGlc-dependent repression and demonstrate that these alleles no longer interact with EIIAGlc. These findings suggest that EIIAGlc must interact with ChiS to exert its repressive effect. Importantly, the ChiS suppressor mutations we isolated also relieve repression of chitin utilization and natural transformation by EIIAGlc, suggesting that CCR of these behaviors is primarily regulated through ChiS. Together, our results reveal how nutrient conditions impact the fitness of an important human pathogen in its environmental reservoir. AUTHOR SUMMARYVibrio cholerae is a facultative human pathogen that most commonly inhabits aquatic environments and can cause the disease cholera when ingested in the form of contaminated food or drinking water. The survival of this pathogen in the marine environment is dependent upon its ability to colonize chitin, an insoluble polysaccharide that is the main constituent of the shells of crustacean zooplankton. V. cholerae degrades chitin into soluble oligomers, which can be taken up by this microbe and used as a nutrient. Importantly, chitin oligomers also induce the activity of a chitin-sensing protein, ChiS, to activate the expression of genes required for both (1) eating chitin and (2) undergoing horizontal gene transfer by natural transformation. Previous work demonstrated that more energetically favorable carbon sources, like glucose, can inhibit chitin-induced behaviors. However, the mechanism underlying this response remained unclear. Here, we show how a dephosphorylated intermediate in the glucose uptake pathway - EIIAGlc - interacts with ChiS to repress its activity. Furthermore, we demonstrate that this repression of ChiS by EIIAGlc is responsible for inhibiting both growth on chitin and natural transformation. Together, our results reveal how nutrient conditions impact the fitness of a major human pathogen in its environmental reservoir.

microbiology↗

bioRxiv2020

The ChiS family DNA-binding domain contains a cryptic helix-turn-helix variant

Sequence specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We have recently shown that ChiS contains a cryptic DBD in its C-terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 [A]. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight "turn" of the HTH is replaced with an insertion containing a {beta}-sheet, a variant which we term the "helix-sheet-helix". Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse Proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs. ImportanceRegulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae the transcription factor ChiS directly regulates gene expression through a cryptic DNA binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA binding domain (DBD) and find that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

microbiology↗

bioRxiv2020

Species-specific quorum sensing represses the chitobiose utilization locus in Vibrio cholerae

The marine facultative pathogen Vibrio cholerae forms complex multicellular communities on the chitinous shells of crustacean zooplankton in its aquatic reservoir. V. cholerae-chitin interactions are critical for the growth, evolution, and waterborne transmission of cholera. This is due, in part, to chitin-induced changes in gene expression in this pathogen. Here, we sought to identify factors that influence chitin-induced expression of one locus, the chitobiose utilization operon (chb), which is required for the uptake and catabolism of the chitin disaccharide. Through a series of genetic screens, we identified that the master regulator of quorum sensing, HapR, is a direct repressor of the chb operon. We also found that the levels of HapR in V. cholerae are regulated by the ClpAP protease. Furthermore, we show that the canonical quorum sensing cascade in V. cholerae regulates chb expression in a HapR-dependent manner. Through this analysis we found that signaling via the species-specific autoinducer CAI-1, but not the inter-species autoinducer AI-2, influences chb expression. This phenomenon of species-specific regulation may enhance the fitness of this pathogen in its environmental niche. ImportanceIn nature, bacteria live in multicellular and multispecies communities. Microbial species can sense the density and composition of their community through chemical cues using a process called quorum sensing (QS). The marine pathogen Vibrio cholerae is found in communities on the chitinous shells of crustaceans in its aquatic reservoir. V. cholerae interactions with chitin are critical for the survival, evolution, and waterborne transmission of this pathogen. Here, we show that V. cholerae uses QS to regulate the expression of one locus required for V. cholerae-chitin interactions.

microbiology↗

bioRxiv2020

ChiS is a noncanonical DNA-binding hybrid sensor kinase that directly regulates the chitin utilization program in Vibrio cholerae

Two-component signal transduction systems (TCSs) represent a major mechanism that bacteria use to sense and respond to their environment. Prototypical TCSs are composed of a membrane-embedded histidine kinase (HK), which senses an environmental stimulus and subsequently phosphorylates a cognate partner protein called a response regulator (RR) that regulates gene expression in a phosphorylation-dependent manner. Vibrio cholerae uses the hybrid HK ChiS to activate the expression of the chitin utilization program, which is critical for the survival of this facultative pathogen in its aquatic reservoir. A cognate RR for ChiS has not been identified and the mechanism of ChiS-dependent signal transduction remains unclear. Here, we show that ChiS is a noncanonical membrane-embedded one-component system that can both sense chitin and directly regulate gene expression via a cryptic DNA binding domain. Unlike prototypical TCSs, we find that ChiS DNA binding is diminished, rather than stimulated, by phosphorylation. Finally, we provide evidence that ChiS likely activates gene expression by directly recruiting RNA polymerase. Together, this work addresses the mechanism of action for a major transcription factor in V. cholerae and highlights the versatility of signal transduction systems in bacterial species. Significance StatementFrom bacteria to humans, the ability to properly respond to environmental cues is critical for survival. The cholera pathogen Vibrio cholerae uses one protein, ChiS, to sense chitin in its environmental reservoir to regulate the expression of genes that are critical for the survival and evolution of this pathogen in this niche. Here, we study how the chitin sensor ChiS works, and discover that it regulates gene expression in an unexpected and unorthodox manner. Thus, this study uncovers how the major regulator ChiS works in this important human pathogen and highlights the versatile mechanisms that living systems use to respond to their environment.

microbiology↗

bioRxiv2017

The Nucleoid Occlusion Protein SlmA Is A Direct Transcriptional Activator Of Chitobiose Utilization In Vibrio cholerae

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, surprisingly, 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.\n\nAUTHOR SUMMARYThe 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.

microbiology↗