Biology subjects

Dalia, A. B.

Publications and source records attributed to Dalia, A. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Nitrogenase regulation in Vibrio natriegens differs from other γ-proteobacteria

Regulation of nitrogenase, which converts nitrogen gas (N2) into ammonium (NH4+), typically involves a conserved set of regulatory proteins across diverse N2-fixing (diazotrophic) bacteria. However, the interactions and relative influence of these regulators can vary between species. Thus, one cannot make assumptions about nitrogenase regulation when working with uncharacterized diazotrophs like Vibrio natriegens, a {gamma}-proteobacterium of growing interest for synthetic biology. Little is known about V. natriegens nitrogenase regulation, which could be used to exploit inexpensive N2 for various applications, including NH4+ production. Here, we characterized the roles of several annotated V. natriegens nitrogenase regulatory proteins in response to NH4+ versus N2. Using functional genomics, targeted mutations, and reporter assays, we identified a typical regulatory hierarchy where the two-component system NtrBC governs a nitrogen-scavenging regulon that includes NifA, the transcriptional activator of nitrogenase genes. Unlike other diazotrophic {gamma}-proteobacteria, NifA was sufficient to activate nitrogenase gene expression, as a mutant lacking NtrBC grew normally with N2 after a lag phase. Thus, NtrBC was dispensable, but still important for timely nitrogenase expression. Furthermore, NtrBC was negatively regulated by the nitrogen-responsive PII proteins GlnB and GlnK; disruption of both PII proteins led to NtrBC-dependent nitrogenase overactivity, marked by NH4+ excretion. The redundant repression of NtrBC by GlnB and GlnK more closely resembles that of non-diazotrophic E. coli than other diazotrophic {gamma}-proteobacteria. Together, our findings provide a framework for V. natriegens nitrogenase regulation that can be leveraged for applications like NH4+ production. HIGHLIGHTSO_LIA genetic examination of Vibrio natriegens nitrogenase regulation is performed C_LIO_LINtrBC is important for early nitrogenase gene expression but is not essential C_LIO_LINifA autoactivation is sufficient for nitrogenase expression C_LIO_LIPII proteins GlnB and GlnK are redundant negative regulators of nitrogenase C_LIO_LIGenetic targets are identified that result in excretion of NH4+ C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/728972v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@aac4adorg.highwire.dtl.DTLVardef@1565c0corg.highwire.dtl.DTLVardef@b545a6org.highwire.dtl.DTLVardef@efb052_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2026

Low affinity DNA-binding promotes cooperative activation of natural transformation in Vibrio cholerae

DNA-binding transcriptional regulators control gene expression in response to environmental cues. A subset of these proteins, called transmembrane transcriptional regulators (TTRs), directly bind DNA to regulate transcription while remaining anchored in the cytoplasmic membrane. Prior work has shown that in the presence of the polysaccharide chitin, two TTRs, TfoS and ChiS, coordinate to induce the expression of TfoR, a small RNA that is critical for natural transformation in Vibrio cholerae. Specifically, it was shown that ChiS recruits the PtfoR locus to the membrane, which allows for the subsequent activation of this promoter by TfoS. However, it was also shown that increasing TfoS protein levels bypasses this coordination, allowing TfoS to activate the promoter independently. It therefore remains unclear what molecular mechanisms drive the requirement for ChiS in native conditions. Here, we show that ChiS binds PtfoR with a higher affinity than TfoS. We hypothesized that the low affinity of TfoS for PtfoR helps reinforce its dependence on ChiS for activation. To test this, we isolated a mutant allele of the TfoS DNA-binding domain that has a higher affinity for PtfoR. We show that this high-affinity TfoS allele promotes ChiS-independent activation of PtfoR and dysregulates chitin-dependent phenotypes in V. cholerae. These results demonstrate that the relative DNA-binding affinity of these TTRs facilitates their coordination, and that this coordination is necessary for optimal V. cholerae fitness on chitin. IMPORTANCEDNA-binding transmembrane transcriptional regulators (TTRs) are critical for some bacterial species to properly sense and respond to their environments. Recent work highlights that pairs of TTRs can coordinate their activities to regulate gene expression, allowing them to sensitively control behaviors like virulence and horizontal gene transfer. However, the mechanisms that enable this coordination remain poorly understood. Here, we show that the relative DNA-binding affinity of paired TTRs is a critical feature that can drive their coordination.

microbiology↗

bioRxiv2026

The stoichiometry of minor-to-major pilins regulates the dynamic activity of the type IVa competence pilus in Vibrio cholerae

Type IVa pili (T4aP) are bacterial surface appendages that perform various functions including twitching motility, reversible surface attachment, microcolony formation, surface sensing, and DNA uptake for natural transformation. Pivotal to each of these functions is the ability of T4aP to be dynamically extended and retracted from the cell surface. However, the factors that regulate this dynamic activity remain poorly understood in most systems. To address this question, we employ the competence T4aP from Vibrio cholerae as a model system. T4aP are composed of major and minor pilin subunits, named based on their relative abundance in the pilus filament. Prior work has established that minor pilins form a complex that initiates T4aP assembly. This allows for the subsequent addition of major pilins to the filament, which promotes T4aP extension. Here, we uncover that the stoichiometry of minor-to-major pilins is a crucial determinant of T4aP dynamic activity. Specifically, we show that either 1) overexpressing minor pilins or 2) underexpressing the major pilin results in a dramatic increase in the frequency of T4aP dynamics. These results indicate that the stoichiometry of minor-to-major pilins, not their absolute abundance, is one mechanism that regulates T4aP dynamic activity. AUTHOR SUMMARYType IVa pili (T4aP) are a broadly conserved family of filamentous bacterial appendages that help bacteria colonize surfaces, move towards or away from stimuli, and gain new traits through a mechanism of horizontal gene transfer called natural transformation. T4aP are primarily composed of protein subunits called major and minor pilins, named based on their relative abundance in the pilus filament. Bacteria can dynamically extend and retract pilus filaments from their surface through polymerization and depolymerization of these pilins. This dynamic activity is critical for the activities that T4aP carry out. However, the factors that regulate this dynamic activity remain incompletely understood. Here, we find that the ratio of minor-to-major pilins is one factor that regulates the frequency of dynamic activity. Minor pilins are a universally conserved feature of T4aP in diderms. So, it is tempting to speculate that the minor-to-major pilin ratio represents a broadly conserved mechanism for controlling dynamic T4aP activity in diverse bacterial species.

microbiology↗

bioRxiv2025

Spatial constraint drives negative frequency dependent selection of phage weaponization

Biofilm growth and phage exposure are common to diverse bacterial species. Studying phage-host interaction and population dynamics in biofilms with cellular resolution remains a significant challenge, especially when attempting to recapitulate the natural environments that microbes occupy. Here we study the population dynamics of phage K139 lysogenized and non-lysogenized Vibrio cholerae when growing in biofilms on the surface of chitin particles in seawater, replicating key features of V. cholerae ecology in the marine environment. We find that lysogenized V. cholerae, via spontaneous lytic induction and phage release, kill and displace non-lysogenized bacteria on chitin surfaces. After lysogens become common, however, they can no longer displace remaining non-lysogenized cells. Using a combination of modeling approaches and microscopy experiments, we show that the lysogens capacity to displace non-lysogens depends on the ability of phages released by spontaneous induction to reach susceptible non-lysogens. Phage access to non-lysogenized bacterial hosts declines once lysogens become common, and this occurs due to the spatial constraints inherent to biofilm growth as well as to superinfection immunity, which neutralizes phage particles adsorbed to lysogens. Once lysogens comprise the majority of the host population, they can be selected against, because they still incur the cost of spontaneous induction without gaining the benefit of killing non-lysogen cells via phage release. The cost of lytic induction, phage-mediated killing of non-lysogens, and constraints on phage mobility within host bacterial biofilms together yield population dynamics that are consistent with negative frequency dependent selection for lysogenized cells under physiologically realistic growth conditions. Significance StatementBacteria often produce and live within biofilm communities in natural environments, where they also encounter many threats including bacteriophages. Here we show how temperate phages can confer a competitive advantage to their hosts via lytic induction and phage release within biofilms of V. cholerae on lab-grown marine snow particles. However, the spatial constraints of biofilm architecture and phage-neutralizing superinfection immunity place limits on the extent to which lysogens can competitively displace non-lysogens via phage release.

microbiology↗

bioRxiv2025

Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

Diverse bacterial species utilize surface appendages called type IV pili (T4P) to interact with their environment. These structures are dynamically extended and retracted from the cell surface, which is critical for diverse functions. Some T4P systems rely on two distinct motor ATPases, PilT and PilU, whose combined activities are required to power forceful T4P retraction. However, the mechanism by which these motors coordinate to facilitate T4P retraction has remained unclear. Here, we utilize the competence T4P in V. cholerae as a model system to elucidate the molecular basis for PilT-PilU coordination during T4P retraction. Specifically, we modeled the interactions between PilT and PilU using AlphaFold 3 and molecular dynamics (MD) simulations. We then empirically tested these models using a combination of cytological and high-resolution genetic approaches. Our results reveal that interactions between PilT and the PilU C-terminus are critical for these motors to coordinate to drive T4P retraction. Finally, we show that PilT-PilU interactions are broadly conserved in T4P systems from diverse bacterial species, and we experimentally validate that they are required for T4P retraction in Acinetobacter baylyi. Together, this work expands our fundamental understanding of T4P dynamics, and more broadly it provides mechanistic insight into how these ATPases coordinate to assemble some of the strongest biological motors in nature. SIGNIFICANCEDiverse bacterial species use filamentous surface appendages called type IV pili (T4P) to move along surfaces, take up DNA for horizontal gene transfer, and stick to biotic and abiotic surfaces. The forceful retraction of these filaments is often required for these behaviors. In many T4P systems, the combined activity of two distinct motor ATPase proteins is required for forceful retraction; however, a detailed understanding of how these motor proteins interact to promote forceful retraction is currently lacking. Here, we use an integrated approach to uncover the molecular mechanism for motor ATPase coordination. Furthermore, we show that this mechanism is broadly conserved in diverse T4P systems.

microbiology↗

bioRxiv2025

Integrative Structure and Function of the Vibrio cholerae Competence Pilus Machine

Type IV pili are hair-like, surface-exposed polymers involved in many fundamental biological processes. The competence pilus (CP) is responsible for natural transformation, the ability to acquire exogenous DNA. Here we purified the Vibrio cholerae CP and used electron cryo-microscopy (cryo-EM) to obtain a 3.3 [A] map of the fiber by helical reconstruction. We then used electron cryo-tomography (cryo-ET) to reveal the in-situ architecture of the competence pilus machine (CPM). Compared to other type IVa pilus machines, the CPM has unique characteristics including multiple conformational states, a ring-like density formed by PilQs C-terminal domain absent in the previously solved SPA structure, and the presence of a double ATPase ring in the cytoplasm. Using integrative modeling with the cryo-ET map as an envelope, we generated a full-length pseudoatomic model of the CPM. Molecular dynamics (MD) simulations of the entire CPM embedded within realistic bacterial membranes and the peptidoglycan cell wall revealed that the model demonstrates structural integrity under physiological conditions. Steered MD simulations of pilus extension and retraction through PilQ revealed gate-opening mechanisms and demonstrated the channels mechanical resistance and flexibility during translocation.

cell biology↗

bioRxiv2025

Structural modeling reveals the allosteric switch controlling the chitin utilization program of Vibrio cholerae

Signal transduction by histidine kinases (HKs) is nearly ubiquitous in bacterial species. HKs can either sense ligands directly or indirectly via a cognate solute binding protein (SBP). The molecular basis for SBP-dependent signal reception, however, remains poorly understood in most cases. CBP and ChiS are the SBP-HK pair that activate the chitin utilization program of Vibrio cholerae. Here, we elucidate the molecular basis for allosteric regulation of CBP-ChiS by generating structural models of this complex in the unliganded and liganded states, which we support with extensive genetic, biochemical, and cell biological analysis. Our results reveal that ligand-binding induces a large conformational interface switch that is distinct from previously described SBP-HKs. Structural modeling suggests that similar interface switches may also regulate other uncharacterized SBP-HKs. Together, these results extend our understanding of signal transduction in bacterial species and highlight a new approach for uncovering the molecular basis of allostery in protein complexes. SIGNIFICANCE STATEMENTAll living things use protein receptors to sense and respond to environmental changes via a process termed signal transduction. However, how these proteins sense environmental stimuli remains poorly understood in many cases. In this study, we study allosteric activation of the chitin sensor, ChiS, by chitin-binding protein, CBP, in Vibrio cholerae as a model system. Using a combination of structural modeling, genetics, and biochemistry we uncovered the molecular basis underlying CBP-ChiS allosteric regulation, which we find is distinct from previously described systems. This work expands our understanding of bacterial signal transduction and highlights an approach for uncovering new modes of allosteric regulation.

microbiology↗

bioRxiv2025

The molecular basis for DNA-binding by competence T4P is distinct in Gram-positive and Gram-negative species

Competence type IV pili (T4P) are bacterial surface appendages that facilitate DNA uptake during horizontal gene transfer by natural transformation. These dynamic structures actively extend from the cell surface, bind to DNA in the environment, and then retract to import bound DNA into the cell. Competence T4P are found in diverse Gram-negative (diderm) and Gram-positive (monoderm) bacterial species. While the mechanism of DNA-binding by diderm competence T4P has been the recent focus of intensive study, relatively little is known about DNA-binding by monoderm competence T4P. Here, we use Streptococcus pneumoniae as a model system to address this question. Competence T4P likely bind to DNA via a tip-associated complex of proteins called minor pilins, and recent work highlights a high degree of structural conservation between the minor pilin tip complexes of monoderm and diderm competence T4P. In diderms, positively charged residues in one minor pilin, FimT, are critical for DNA-binding. We show that while these residues are conserved in ComGD, the FimT homolog of monoderms, they only play a minor role in DNA uptake for natural transformation. Instead, we find that two-positively charged residues in the neighboring minor pilin, ComGF (the PilW homolog of monoderms), play the dominant role in DNA uptake for natural transformation. Furthermore, we find that these residues are conserved in other monoderms, but not diderms. Together, these results suggest that the molecular basis for DNA-binding has either diverged or evolved independently in monoderm and diderm competence T4P. AUTHOR SUMMARYDiverse bacteria use extracellular structures called competence type IV pili (T4P) to take up DNA from their environment. The uptake of DNA by T4P is the first step of natural transformation, a mode of horizontal gene transfer that contributes to the spread of antibiotic resistance and virulence traits in diverse clinically relevant Gram-negative (diderm) and Gram-positive (monoderm) bacterial species. While the mechanism of DNA binding by competence T4P in diderms has been an area of recent study, relatively little is known about how monoderm competence T4P bind DNA. Here, we explore how monoderm competence T4P bind DNA using Streptococcus pneumoniae as a model system. Our results indicate that while monoderm T4P and diderm T4P likely have conserved structural features, the DNA-binding mechanism of each system is distinct.

microbiology↗

bioRxiv2024

DprA recruits ComM to facilitate recombination during natural transformation in Gram-negative bacteria

Natural transformation (NT) represents one of the major modes of horizontal gene transfer in bacterial species. During NT, cells can take up free DNA from the environment and integrate it into their genome by homologous recombination. While NT has been studied for >90 years, the molecular details underlying this recombination remain poorly understood. Recent work has demonstrated that ComM is an NT-specific hexameric helicase that promotes recombinational branch migration in Gram-negative bacteria. How ComM is loaded onto the post-synaptic recombination intermediate during NT, however, remains unclear. Another NT-specific recombination mediator protein that is ubiquitously conserved in both Gram-positive and Gram-negative bacteria is DprA. Here, we uncover that DprA homologs in Gram-negative species contain a C-terminal winged helix domain that is predicted to interact with ComM by AlphaFold. Using Helicobacter pylori and Vibrio cholerae as model systems, we demonstrate that ComM directly interacts with the DprA winged-helix domain, and that this interaction is critical for DprA to recruit ComM to the recombination site to promote branch migration during NT. These results advance our molecular understanding of recombination during this conserved mode of horizontal gene transfer. Furthermore, they demonstrate how structural modeling can help uncover unexpected interactions between well-studied proteins to provide deep mechanistic insight into the molecular coordination required for their activity. SIGNIFICANCE STATEMENTBacteria can acquire novel traits like antibiotic resistance and virulence through horizontal gene transfer by natural transformation. During this process, cells take up free DNA from the environment and integrate it into their genome by homologous recombination. Many of the molecular details underlying this process, however, remain incompletely understood. In this study, we identify a new protein-protein interaction between ComM and DprA, two factors that promote homologous recombination during natural transformation in Gram-negative species. Through a combination of bioinformatics, structural modeling, cell biological assays, and complementary genetic approaches, we demonstrate that this interaction is required for DprA to recruit ComM to the site of homologous recombination.

microbiology↗

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↗

bioRxiv2024

SbcB facilitates natural transformation in Vibrio cholerae in an exonuclease-independent manner

Natural transformation (NT) is a conserved mechanism of horizontal gene transfer in bacterial species. During this process, DNA is taken up into the cytoplasm where it can be integrated into the host genome by homologous recombination. We have previously shown that some cytoplasmic exonucleases inhibit NT by degrading ingested DNA prior to its successful recombination. However, one exonuclease, SbcB, counterintuitively promotes NT in Vibrio cholerae. Here, through a systematic analysis of the distinct steps of NT, we show that SbcB acts downstream of DNA uptake into the cytoplasm, but upstream of recombinational branch migration. Through mutational analysis, we show that SbcB promotes NT in a manner that does not rely on its exonuclease activity. Finally, we provide genetic evidence that SbcB directly interacts with the primary bacterial recombinase, RecA. Together, these data advance our molecular understanding of horizontal gene transfer in V. cholerae, and reveal that SbcB promotes homologous recombination during NT in a manner that does not rely on its canonical exonuclease activity. IMPORTANCEHorizontal gene transfer by natural transformation contributes to the spread of antibiotic resistance and virulence factors in bacterial species. Here, we study how one protein, SbcB, helps facilitate this process in the facultative bacterial pathogen Vibrio cholerae. SbcB is a well-known for its exonuclease activity (i.e., the ability to degrade the ends of linear DNA). Through this study we uncover that while SbcB is important for natural transformation, it does not facilitate this process using its exonuclease activity. Thus, this work helps further our understanding of the molecular events required for this conserved evolutionary process, and uncovers a function for SbcB beyond its canonical exonuclease activity.

microbiology↗

bioRxiv2024

Bacterial cross-feeding can promote gene retention by lowering gene expression costs

Cross-feeding is prevalent in microbial communities. Through time, cross-feeding is thought to enrich for loss-of-function mutations, thereby creating or reinforcing dependencies between community members. However, few studies have compared how cross-feeding affects the evolutionary trajectory of partners compared to monoculture conditions. Here we compared mutations that were differentially enriched in bacterial monocultures versus cocultures pairing phototrophic Rhodopseudomonas palustris with fermentative Escherichia coli in an obligate cross-feeding relationship based on the exchange of nitrogen and carbon for 650-800 generations. Opposite trends for the number of differentially enriched mutations were observed for each species; R. palustris accumulated more unique mutations in monoculture whereas E. coli accumulated more unique mutations in coculture. Contrary to expectations, the emergence of additional dependencies was more apparent for both species in monoculture, even though additional layers of cross-feeding involving iron and adenine were present in coculture. We reasoned that iron and adenine cross-feeding occurred at levels sufficient to repress gene expression in the recipient, thereby promoting gene retention by lowering gene cost. Thus, the influence of cross-feeding on evolutionary trajectories can vary with organisms and conditions, and there are situations where cross-feeding can limit, rather than promote, emergent dependencies. IMPORTANCEBacteria commonly engage in cross-feeding where nutrients are transferred between neighbors. Cross-feeding is thought to alleviate energy expenditures for genes whose role can be met by cross-fed nutrients, leading to eventual gene loss. However, few examples have been documented, especially in comparison to monocultures where bacteria are grown without a cross-feeding partner. Here we grew cocultures pairing phototrophic Rhodopseudomonas palustris with fermentative Escherichia coli alongside corresponding monocultures for 650-800 generations. While coculture conditions required obligate exchange of nitrogen and carbon, additional cross-feeding of adenine and iron likely occurred. Contrary to expectations, iron and uncharacterized dependencies emerged in monocultures but not in cocultures. Low expression for iron scavenging and adenine synthesis genes in cocultures suggested that cross-feeding repressed gene expression, thereby lowering gene cost. Thus, whereas there are likely cases where cross-feeding makes costly genes dispensable, there are also cases where cross-feeding lowers gene cost, thereby promoting gene retention.

microbiology↗

bioRxiv2023

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae

The vertebrate hosts immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass-spectrometry-based profiling, metabolomics, expression assays and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular reactive sulfur species (RSS), specifically sulfane sulfur. We first present a comprehensive sequence similarity network analysis of the arsenic repressor (ArsR) superfamily of transcriptional regulators where RSS and reactive oxygen species (ROS) sensors segregate into distinct clusters. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity and remaining DNA-bound following treatment with various ROS in vitro, including H2O2. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

biochemistry↗

bioRxiv2023

A simple mechanism for integration of quorum sensing and cAMP signalling in V. cholerae

Many bacteria use quorum sensing to control changes in lifestyle. The process is regulated by microbially derived "autoinducer" signalling molecules, that accumulate in the local environment. Individual cells sense autoinducer abundance, to infer population density, and alter their behaviour accordingly. In Vibrio cholerae, quorum sensing signals are transduced by phosphorelay to the transcription factor LuxO. Unphosphorylated LuxO permits expression of HapR, which alters global gene expression patterns. In this work, we have mapped the genome-wide distribution of LuxO and HapR in V. cholerae. Whilst LuxO has a small regulon, HapR targets 32 loci. Many HapR targets coincide with sites for the cAMP receptor protein (CRP) that regulates the transcriptional response to carbon starvation. This overlap, also evident in other Vibrio species, results from similarities in the DNA sequence bound by each factor. At shared sites, HapR and CRP simultaneously contact the double helix and binding is stabilised by direct interaction of the two factors. Importantly, this involves a CRP surface that usually contacts RNA polymerase to stimulate transcription. As a result, HapR can block transcription activation by CRP. Thus, by interacting at shared sites, HapR and CRP integrate information from quorum sensing and cAMP signalling to control gene expression. This likely allows V. cholerae to regulate subsets of genes during the transition between aquatic environments and the human host.

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↗

bioRxiv2022

The canonical PilT retraction ATPase promotes both extension and retraction of the MSHA type IVa pilus in Vibrio cholerae

Diverse bacterial species use type IVa pili (T4aP) to interact with their environments. The dynamic extension and retraction of T4aP is critical for their function, but the mechanisms that regulate this dynamic activity remain poorly understood. T4aP are typically extended via the activity of a dedicated extension motor ATPase and retracted via the action of an antagonistic retraction motor ATPase called PilT. These motors are generally functionally independent, and loss of PilT commonly results in T4aP hyperpiliation due to undeterred pilus extension. However, for the mannose-sensitive hemagglutinin (MSHA) T4aP of Vibrio cholerae, the loss of PilT results in a loss of surface piliation, which is unexpected based on our current understanding of T4aP dynamics. Here, we employ a combination of genetic and cell biological approaches to dissect the underlying mechanism. Our results demonstrate that PilT is necessary for MSHA pilus extension in addition to its well-established role in promoting MSHA pilus retraction. Through a suppressor screen, we also provide genetic evidence that the MshA major pilin impacts pilus extension. Together, these findings contribute to our understanding of the factors that regulate pilus extension and describe a previously uncharacterized function for the PilT motor ATPase. AUTHOR SUMMARYMany bacteria use filamentous appendages called type IVa pili to interact with their environment. These fibers dynamically extend and retract through the activity of ATPase motor proteins. In most pilus systems, deletion of the retraction motor results in uninterrupted pilus extension, leading to hyperpiliation. However, in the MSHA pilus system of V. cholerae, deletion of the retraction motor, pilT, results in a decrease in the number of surface pili. Here, we show that PilT is unexpectedly required for MSHA pilus extension in addition to its defined role in promoting pilus retraction. These results extend our understanding of the complex mechanisms underlying the dynamic activity of these broadly conserved filamentous appendages.

microbiology↗

bioRxiv2022

Transertion is used for localized expression and assembly of Vibrio parahaemolyticus T3SS2

Using environmental cues, bacteria commit to the assembly of transmembrane complexes such as the type III secretion system 2 (T3SS2), a membrane-bound, syringe-like secretory apparatus used during infection to inject host cells with virulence factors. Here we report Vibrio parahaemolyticus uses transertion, localized transcription, translation, and membrane insertion, to assemble its T3SS2. Upon binding bile acids, the membrane bound receptor and transcription factor VtrA/VtrC captures the T3SS2 pathogenicity island at the inner membrane. Activated VtrA/VtrC induces production of VtrB, the membrane bound master T3SS2 transcriptional regulator. VtrB then induces the membrane-proximal T3SS2 genes to undergo transertion for assembly of the membrane inserted secretion machinery. Transertion is a process that can be used for the efficient assembly of membrane-bound molecular complexes in response to extracellular signals. One-Sentence SummaryLocalized transcription, translation, and membrane insertion of multi-protein complexes in bacteria in response to host cues.

microbiology↗

bioRxiv2021

Retraction ATPase motors are promiscuous among type IVa pilus systems

Bacterial surface appendages called type IVa pili (T4aP) promote diverse activities including DNA uptake, twitching motility, and virulence. These activities rely on the ability of T4aP to dynamically extend and retract from the cell surface. Dynamic extension relies on a motor ATPase commonly called PilB. Most T4aP also rely on specific motor ATPases, commonly called PilT and PilU, to dynamically and forcefully retract. Here, we systematically assess whether motor ATPases from three orthologous T4aP can functionally complement Vibrio cholerae mutants that lack their endogenous motors. We found that the PilT and PilU retraction ATPases from the three T4aP systems tested are promiscuous and promote retraction of the V. cholerae competence T4aP despite a high degree of sequence divergence. In contrast, the orthologous extension ATPases from the same T4aP systems were not able to mediate extension of the V. cholerae competence T4aP despite exhibiting a similar degree of sequence divergence. Also, we show that one of the PilT orthologs characterized does not support PilU-dependent retraction and provide some data to indicate that the C-terminus of PilT is important for PilU-dependent retraction. Together, our data suggest that retraction ATPases may have maintained a high degree of promiscuity for promoting retraction of T4aP, while extension ATPases may have evolved to become specific for their cognate systems. IMPORTANCEOne way that bacteria interact with their environments is via hair-like appendages called type IVa pili (T4aP). These appendages dynamically extend and retract from the cell surface via the action of distinct ATPase motors. T4aP are present in diverse bacterial species. Here, we demonstrate that retraction motors from three T4aP are promiscuous, and capable of promoting retraction of a heterologous T4aP system. By contrast, the extension ATPase motors from these same T4aP systems are specific and cannot promote extension of a heterologous T4aP. Thus, these results suggest that T4aP extension may be more tightly regulated compared to T4aP retraction.

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