Biology subjects

Dalia, T. N.

Publications and source records attributed to Dalia, T. N..

At least 19 recordsLinked to original sources

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↗

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↗

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 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↗

bioRxiv2021

Oxygen depletion and nitric oxide stimulate type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. Dynamic extension and retraction of pili is often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To study this question, we use the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we find that V. cholerae cells retract MSHA pili and detach from a surface in microaerobic conditions. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP (c-di-GMP) under microaerobic conditions to induce MSHA pilus retraction. CdpA contains a putative NO-sensing NosP domain, and we demonstrate that nitric oxide (NO) is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that microaerobic conditions result in endogenous production of NO (or an NO-like molecule) in V. cholerae. Together, these results describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. More broadly, these results show how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

microbiology↗

bioRxiv2021

Fresh extension of Vibrio cholerae competence type IV pili predisposes them for motor-independent retraction

Bacteria utilize dynamic appendages called type IV pili (T4P) to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can either occur with the aid of an ATPase motor, or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remains poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken, but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization. SIGNIFICANCEExtracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions, however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus of Vibrio cholerae to study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the external pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.

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

Novel mechanisms of type IV pilus regulation in Acinetobacter baylyi

Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility1. Dynamic extension and retraction of T4P is essential for their function, yet little is known about the mechanisms controlling these dynamics or the extent to which their regulation is conserved across bacterial species. Here, we develop Acinetobacter baylyi as a new model to study T4P by employing a recently developed pilus labeling method2,3. Our findings overturn the current dogma that T4P extension occurs through the action of a single, highly conserved motor, PilB, by showing that T4P synthesis in A. baylyi is dependent on an additional, phylogenetically distinct motor, TfpB. Furthermore, we uncover an inhibitor of T4P extension that specifically binds to and inhibits PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to inhibit T4P synthesis.

microbiology↗

bioRxiv2020

Prophage-dependent neighbor predation fosters horizontal gene transfer by natural transformation

Natural transformation is a broadly conserved mechanism of horizontal gene transfer (HGT) in bacteria (1) that can shape their evolution through the acquisition of genes that promote virulence, antibiotic resistance, and other traits (2). Recent work has established that neighbor predation via Type VI secretion systems (3), bacteriocins (4) and virulent phages (5), play an important role in promoting HGT. Here, we demonstrate that in chitin estuary microcosms, Vibrio cholerae K139 lysogens exhibit prophage-dependent neighbor predation of non-lysogens to enhance HGT. Through predation of non-lysogens, K139 lysogens also have a fitness advantage in these microcosm conditions. The ecological strategy revealed by our work provides a better understanding of the evolutionary mechanisms used by bacteria to adapt in their natural setting and contributes to our understanding of the selective pressures that may drive prophage maintenance in bacterial genomes. IMPORTANCEProphages are nearly ubiquitous in bacterial species. These integrated phage elements have previously been implicated in horizontal gene transfer (HGT) largely through their ability to carry out transduction (generalized or specialized). Here, we show that prophage-encoded viral particles promote neighbor predation leading to enhanced HGT by natural transformation in the water-borne pathogen Vibrio cholerae. Our findings contribute to a comprehensive understanding of the dynamic forces involved in prophage maintenance which ultimately drive the evolution of naturally competent bacteria in their natural environment.

microbiology↗

bioRxiv2020

A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae

The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements Ptac and PBAD to versions that have an additional theophylline-responsive riboswitch (Ptac-riboswitch and PBAD-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with Ptac > PBAD > PBAD-riboswitch > Ptac-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for Ptac > Ptac-riboswitch > PBAD; while the newly developed PBAD-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible PBAD-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of Vibrio cholerae and could be adapted for use in other species.

microbiology↗

bioRxiv2020

CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, the Type IV competence pilus is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. A lack of structural information has hindered our understanding of this process, however. Here, we solved the first ever high-resolution structure of a Type IV competence pilus secretin. A functional tagged allele of VcPilQ purified from native V. cholerae cells was used to determine the cryoEM structure of the PilQ secretin in amphipol to [~]2.7 [A]. This structure highlights for the first time key differences in the architecture of the Type IV competence pilus secretin from the Type II and Type III Secretin System secretins. Based on our cryoEM structure, we designed a series of mutants to interrogate the mechanism of PilQ. These experiments provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation. We prove that it is possible to reduce pilus biogenesis and natural transformation by sealing the gate, suggesting VcPilQ as a new drug target.

biophysics↗

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↗

bioRxiv2019

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear because pilU mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus of Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in {Delta}pilT strains. Thus, these data suggest that PilU is a bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a {Delta}pilU mutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additional in vitro and in vivo data show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.\n\nAuthor SummaryAlmost all bacterial species use thin surface appendages called pili to interact with their environments. These structures are critical for the virulence of many pathogens and represent one major way that bacteria share DNA with one another, which contributes to the spread of antibiotic resistance. To carry out their function, pili dynamically extend and retract from the bacterial surface. Here, we show that retraction of pili in some systems is determined by the combined activity of two motor ATPase proteins.

microbiology↗