Bacteria and phages are locked in a co-evolutionary arms race where each entity evolves mechanisms to restrict the proliferation of the other. Phage-encoded defense inhibitors have proven powerful tools to interrogate how defense systems function. A relatively common defense system is BREX (bacteriophage exclusion); however, how BREX functions to restrict phage infection remains poorly understood. A BREX system encoded by the ulfamethoazole and rimethoprim (SXT) integrative and conjugative element, Ind5, was recently identified in , the causative agent of the diarrheal disease cholera. The lytic phage ICP1 (nternational Centre for Diarrhoeal Disease Research, Bangladesh holera hage ) that co-circulates with encodes the BREX-inhibitor OrbA, but how OrbA inhibits BREX is unclear. Here, we determine that OrbA inhibits BREX using a unique mechanism from known BREX inhibitors by directly binding to the BREX component BrxC. BrxC has a functional ATPase domain that, when mutated, not only disrupts BrxC function but also alters how BrxC multimerizes. Furthermore, we find that OrbA binding disrupts BrxC-BrxC interactions. We determine that OrbA cannot bind BrxC encoded by the distantly related BREX system encoded by the aSXT Ban9, and thus fails to inhibit this BREX system that also circulates in epidemic . Lastly, we find that homologs of the Ind5 BrxC are more diverse than the homologs of the Ban9 BrxC. These data provide new insight into the function of the BrxC ATPase and highlight how phage-encoded inhibitors can disrupt phage defense systems using different mechanisms.IMPORTANCEWith renewed interest in phage therapy to combat antibiotic-resistant pathogens, understanding the mechanisms bacteria use to defend themselves against phages and the counter-strategies phages evolve to inhibit defenses is paramount. Bacteriophage exclusion (BREX) is a common defense system with few known inhibitors. Here, we probe how the vibriophage-encoded inhibitor OrbA inhibits the BREX system of , the causative agent of the diarrheal disease cholera. By interrogating OrbA function, we have begun to understand the importance and function of a BREX component. Our results demonstrate the importance of identifying inhibitors against defense systems, as they are powerful tools for dissecting defense activity and can inform strategies to increase the efficacy of some phage therapies.

Leaderless bacteriocins are a unique class of bacteriocins that possess antimicrobial activity after translation and have few cases of documented resistance. Aureocin A53 and lacticin Q are considered two of the most well-studied leaderless bacteriocins. Here, we used genome mining to search for novel aureocin A53-like leaderless bacteriocins in GenBank and MGnify. We identified 757 core peptides across 430 genomes with 75 species found currently without characterized leaderless bacteriocin production. These include putative novel species containing bacteriocin gene clusters (BGCs) from the genera (sp. NBC_00237) and (sp. SL85). To date, all characterized leaderless bacteriocins have been found within the phylum Bacillota, but this study identified 97 core peptides within the phylum Actinomycetota. Members of this phylum are traditionally associated with the production of antibiotics, such is the case with the genus . Actinomycetota is an underexplored phylum in terms of bacteriocin production with no characterized leaderless bacteriocin production to date. The two novel leaderless bacteriocins arcanocin and arachnicin from Actinomycetota members sp. and sp., respectively, were chemically synthesized and antimicrobial activity was verified. These peptides were encoded in human gut (PRJNA485056) and oral (PRJEB43277) microbiomes, respectively. This research highlights the biosynthetic potential of Actinomycetota in terms of leaderless bacteriocin production and describes the first antimicrobial peptides encoded in the genera and .IMPORTANCEBacteriocins are gathering attention as alternatives to current antibiotics given the increasing incidence of antimicrobial resistance. Leaderless bacteriocins are considered a commercially attractive subclass of bacteriocins due to the ability to synthesize active peptide and low levels of documented resistance. Therefore, in this work, we mined publicly available data to determine how widespread and diverse leaderless bacteriocins are within the domain of bacteria. Actinomycetota, known for its antibiotic producers but lacking described and characterized bacteriocins, proved to be a rich source of leaderless bacteriocins-97 in total. Two such peptides, arcanocin and arachnicin, were chemically synthesized and have antimicrobial activity. These bacteriocins may provide a novel source of novel antimicrobials that could aid in the development of future alternative antimicrobials and highlight that the Actinomycetota are an underexplored resource of bacteriocin peptides.

Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact sensing by the flagellum plays a central role in activating colonization programs. adheres to surfaces using a polysaccharide adhesin called the holdfast. In , disruption of the flagellum through interactions with a surface or mutation of flagellar genes increases holdfast production. Our group previously identified several genes involved in flagellar surface sensing. One of these, , codes for a protein with homology to the flagellar C-ring protein FliN. We show here that a fluorescently tagged FssF protein localizes to the flagellated pole of the cell and requires all components of the flagellar C-ring for proper localization, supporting the model that FssF associates with the C-ring. Deleting results in a severe motility defect, which we show is due to a disruption of chemotaxis. Epistasis experiments demonstrate that promotes adhesion through a stator-dependent pathway when late-stage flagellar mutants are disrupted. Separately, we find that disruption of chemotaxis through deletion of or other chemotaxis genes results in a hyperadhesion phenotype. Key genes in the surface sensing network (, , and ) contribute to both ∆dependent and ∆dependent hyperadhesion, but these genes affect adhesion differently in the two hyperadhesive backgrounds. Our results support a model in which the stator subunits of the flagella incorporate both mechanical and chemical signals to regulate adhesion.IMPORTANCEBacterial biofilms pose a threat in clinical and industrial settings. Surface sensing is one of the first steps in biofilm formation. Studying surface sensing can improve our understanding of biofilm formation and develop preventative strategies. In this study, we use the freshwater bacterium to study surface sensing and the regulation of surface attachment. We characterize a previously unstudied gene, , and find that it localizes to the cell pole in the presence of three proteins that make up a component of the flagellum called the C-ring. Additionally, we find that is required for chemotaxis behavior but dispensable for swimming motility. Lastly, our results indicate that deletion of and other genes required for chemotaxis results in a hyperadhesive phenotype. These results support that surface sensing requires chemotaxis for a robust response to a surface.

Species of the genus occupy diverse aquatic environments ranging from brackish water to warm equatorial seas to salty coastal regions. More than 80 species of have been identified, many of them as pathogens of marine organisms, including fish, shellfish, and corals, causing disease and wreaking havoc on aquacultures and coral reefs. Moreover, many species associate with and thrive on chitinous organisms abundant in the ocean. Among the many diverse species, the most well-known and studied is , discovered in the 19th century to cause cholera in humans when ingested. The field blossomed in the late 20th century, with studies broadly examining evolution as a human pathogen, natural competence, biofilm formation, and virulence mechanisms, including toxin biology and virulence gene regulation. This review discusses some of the historic discoveries of biology and ecology as one of the fundamental model systems of bacterial genetics and pathogenesis.

Although the development of disinfection technologies with novel mechanisms has stagnated, we demonstrate the bactericidal effects and mechanisms of high-speed nanodroplet generation technology. The first development of this technology in 2017 gushes out a water droplet of 10 nm in size at 50 m/s; however, the target surface does not become completely wet. Nanodroplets were exposed to biofilm models of , , , and . This phenomenon was verified when the nanodroplets collide with the surface of the bacteria at an impact pressure of ~75 MPa. was exposed to nanodroplets for 30 seconds at 75 MPa, which exploded the bacterial body and completely sterilized. Eighteen MPa damaged the bacterial surface, causing peptidoglycan leakage. was repaired and survives in this state. In contrast, in Gram-negative bacteria, nanodroplets with 18 MPa penetrated some biofilm-forming bacteria but did not hit all of them, and the viable count was not significantly reduced. Although all three bacterial species were completely sterilized at 75 MPa, the disinfectant effect was affected by the biomass of the biofilm formed. In summary, our findings prove that nanodroplets at 18 MPa on the bacterial surface were ineffective in killing bacteria, whereas at 75 MPa, all four bacterial species were completely sterilized. The disinfection mechanism involved a high-velocity collision of nanodroplets with the bacteria, physically destroying them. Our results showed that disinfection using this technology could be an innovative method that is completely different from existing disinfection techniques.

In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth when nutrients become available. To identify uncharacterized factors involved in the exit from dormancy, we performed a transposon-sequencing screen taking advantage of the loss of spore heat resistance that accompanies germination. We reasoned that transposon insertions that impair but do not block germination will lose resistance more slowly than wild type after exposure to nutrients and will therefore survive heat treatment. Using this approach, we identified most of the known germination genes and several new ones. We report an initial characterization of 15 of these genes and a more detailed analysis of one (). Spores lacking (renamed ) are impaired in germination in response to both L-alanine and L-asparagine, D-glucose, D-fructose, and K. GerY is a soluble protein synthesized under control in the mother cell. A YFP-GerY fusion localizes around the developing and mature spore in a manner that depends on CotE and SafA, indicating that it is a component of the spore coat. Coat proteins encoded by the operon and are also required for efficient germination, and we show that spores lacking two or all three of these loci have more severe defects in the exit from dormancy. Our data are consistent with a model in which GerY, GerT, and the GerP proteins are required for efficient transit of nutrients through the coat to access the germination receptors, but each acts independently in this process.

is able to survive exposure to high concentrations of transition metals, but is also able to grow under metal starvation conditions. A prerequisite of cellular zinc homeostasis is a flow equilibrium combining zinc uptake and efflux processes. The mutant strain ∆e4 of the parental plasmid-free strain AE104 with a deletion of all four chromosomally encoded genes of previously known efflux systems ZntA, CadA, DmeF, and FieF was still able to efflux zinc in a pulse-chase experiment, indicating the existence of a fifth efflux system. The gene , encoding a protein of the cation diffusion facilitator (CDF) family, is located in proximity to the gene, encoding a P-type ATPase. Deletion of in the ∆e4 mutant resulted in a further decrease in zinc resistance. Pulse-chase experiments with radioactive Zn(II) and stable-isotope-enriched Zn(II) provided evidence that CdfX was responsible for the residual zinc efflux activity of the mutant strain ∆e4. Reporter gene fusions with indicated that the MerR-type regulator ZntR, the main regulator of expression, was responsible for zinc- and cadmium-dependent upregulation of expression, especially in mutant cells lacking one or both of the previously characterized efflux systems, ZntA and CadA. Expression of also proved to be controlled by ZntR itself as well as by zinc and cadmium availability. These data indicate that the region provides with a backup system for the zinc-cadmium-exporting P-type ATPase ZntA, with CdfX exporting zinc and CadA cadmium.IMPORTANCEBacteria have evolved the ability to supply the important trace element zinc to zinc-dependent proteins, despite external zinc concentrations varying over a wide range. Zinc homeostasis can be understood as adaptive layering of homeostatic systems, allowing coverage from extreme starvation to extreme resistance. Central to zinc homeostasis is a flow equilibrium of zinc comprising uptake and efflux reactions, which adjusts the cytoplasmic zinc content. This report describes what happens when an imbalance in zinc and cadmium concentrations impairs the central inner-membrane zinc efflux system for zinc by competitive inhibition for this exporter. The problem is solved by activation of Cd-exporting CadA or Zn-exporting CdfX as additional efflux systems.

NADPH is a redox cofactor that drives the anabolic reactions. Although major NADPH generation reactions have been identified in , some minor reactions have not been identified. In the present study, we explored novel NADPH generation reactions by monitoring the fluorescence dynamics after the addition of carbon sources to starved cells, using a metagenome-derived blue fluorescent protein (mBFP) as an intracellular NADPH reporter. Perturbation analyses were performed on a glucose-6-phosphate isomerase (PGI) deletion strain and its parental strain. Interestingly, mBFP fluorescence increased not only in the parental strain but also in the ΔPGI strain after the addition of xylose. Because the ΔPGI strain cannot metabolize xylose through the oxidative pentose phosphate pathway, this suggests that an unexpected NADPH generation reaction contributes to an increase in fluorescence. To unravel this mystery, we deleted the NADPH generation enzymes including transhydrogenase, isocitrate dehydrogenase, NADP-dependent malic enzyme, glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH) in the ΔPGI strain, and revealed that G6PDH and 6PGDH contribute to an increase in fluorescence under xylose conditions. assays using purified enzymes showed that G6PDH can produce NADPH using erythrose-4-phosphate (E4P) as a substitute for glucose-6-phosphate. Because the (0.65 mM) for E4P was much higher than the reported intracellular E4P concentrations in , little E4P must be metabolized through this bypass in the parental strain. However, the flux would increase when E4P accumulates in the cells owing to genetic modifications. This finding provides a metabolic engineering strategy for generating NADPH to produce useful compounds using xylose as a carbon source.IMPORTANCEBecause NADPH is consumed during the synthesis of various useful compounds, enhancing NADPH regeneration is highly desirable in metabolic engineering. In this study, we explored novel NADPH generation reactions in using a fluorescent NADPH reporter and found that glucose-6-phosphate dehydrogenase can produce NADPH using erythrose-4-phosphate as a substrate under xylose conditions. Xylose is an abundant sugar in nature and is an attractive carbon source for bioproduction. Therefore, this finding contributes to novel pathway engineering strategies using a xylose carbon source in to produce useful compounds that consume NADPH for their synthesis.

The ability of bacteria to interact with and respond to their environment is crucial to their lifestyle and survival. Bacterial cells routinely need to engage with extracellular target molecules, in locations spatially separated from their cell surface. Engagement with distant targets allows bacteria to adhere to abiotic surfaces and host cells, sense harmful or friendly molecules in their vicinity, as well as establish symbiotic interactions with neighboring cells in multicellular communities such as biofilms. Binding to extracellular molecules also facilitates transmission of information back to the originating cell, allowing the cell to respond appropriately to external stimuli, which is critical throughout the bacterial life cycle. This requirement of bacteria to bind to spatially separated targets is fulfilled by a myriad of specialized cell surface molecules, which often have an extended, filamentous arrangement. In this review, we compare and contrast such molecules from diverse bacteria, which fulfil a range of binding functions critical for the cell. Our comparison shows that even though these extended molecules have vastly different sequence, biochemical and functional characteristics, they share common architectural principles that underpin bacterial adhesion in a variety of contexts. In this light, we can consider different bacterial adhesins under one umbrella, specifically from the point of view of a modular molecular machine, with each part fulfilling a distinct architectural role. Such a treatise provides an opportunity to discover fundamental molecular principles governing surface sensing, bacterial adhesion, and biofilm formation.

The transposon Tn is a prevalent composite element often detected in enteric bacteria, including those obtained from clinical samples. The Tn is flanked by two IS elements that work together in mediating transposition. IS-right (ISR) promotes transposition, while IS-left lacks a functional transposase and cannot transpose independently. ISR contains a weak promoter crucial for transposase transcription (pIN), along with two outward-oriented promoters, pOUT and OUTIIp, which may influence the expression of adjacent genes flanking the transposition site. Here, we report the identification of a novel outward-facing promoter, pOUT70, and a functional translation initiation region (TIR) within the last 70 nucleotides of ISR. Furthermore, we show that pOUT70 is negatively regulated by ArgR and positively controlled by IHF, and we demonstrate that pOUT70 enables growth phase-dependent expression of a truncated yet constitutively active version of the histidine kinase BarA. These findings underscore the significance of IS elements in enhancing downstream gene expression, and highlights the role of outward-facing promoters in derepressing virulence factors or acquiring antibiotic resistance.

In mycobacteria, σ is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of σ. The search revealed a number of proteins; prominent among them was MoaB2. The σ-MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors (, closely related σ) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σ was identified to play a role in the σ-MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σ-dependent (but not σ-dependent) transcription and may increase the stability of σ in the cell. We propose that MoaB2, by sequestering σ, has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σ, paving the way for future investigation of this phenomenon.IMPORTANCEMycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σ, the primary sigma factor, and characterize its effects on transcription and σ stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.

Many bacteria produce type IV pili (T4P), surfaced-exposed protein filaments that enable cells to interact with their environment and transition from planktonic to surface-adapted states. T4P are dynamic, undergoing rapid cycles of filament extension and retraction facilitated by a complex protein nanomachine powered by cytoplasmic motor ATPases. Dedicated assembly motors drive the extension of the pilus fiber into the extracellular space, but like any machine, this process is tightly organized. These motors are coordinated by various ligands and binding partners, which control or optimize their functional associations with T4P machinery before cells commit to the crucial first step of building a pilus. This review focuses on the molecular mechanisms that regulate T4P extension motor function. We discuss secondary messenger-dependent transcriptional or post-translational regulation acting both directly on the motor and through protein effectors. We also discuss the recent discoveries of naturally occurring extension inhibitors as well as alternative mechanisms of pilus assembly and motor-dependent signaling pathways. Given that T4P are important virulence factors for many bacterial pathogens, studying these motor regulatory systems will provide new insights into T4P-dependent physiology and efficient strategies to disable them.

ATP, most widely known as the primary energy source for numerous cellular processes, also exhibits the characteristics of a biological hydrotrope. The viable but nonculturable (VBNC) and persister states are two prevalent dormant phenotypes employed by bacteria to survive challenging environments, both of which are associated with low metabolic activity. Here, we investigate the intracellular ATP concentration of individual VBNC and persister cells using a sensitive ATP biosensor QUEEN-7μ and reveal that both types of cells possess a lower intracellular ATP concentration than culturable and sensitive cells, although there is a certain overlap in the intracellular ATP concentrations between antibiotic-sensitive cells and persisters. Moreover, we successfully separated VBNC cells from culturable cells using fluorescence-activated cell sorting based on the intracellular ATP concentration threshold of 12.5 µM. Using an enriched VBNC cell population, we confirm that the precipitation of proteins involved in key biological processes promotes VBNC cell formation. Notably, using green light-illuminated proteorhodopsin (PR), we demonstrate that VBNC cells can be effectively resuscitated by elevating their intracellular ATP concentration. These findings highlight the crucial role of intracellular ATP concentration in the regulation of bacterial cell fate and provide new insights into the formation of VBNC and persister cells.IMPORTANCEThe viable but nonculturable (VBNC) and persister states are two dormant phenotypes employed by bacteria to counter stressful conditions and play a crucial role in chronic and recurrent bacterial infections. However, the lack of precise detection methods poses significant threats to public health. Our study reveals lower intracellular ATP concentrations in these states and establishes an ATP threshold for distinguishing VBNC from culturable cells. Remarkably, we revive VBNC cells by elevating their intracellular ATP levels. This echoes recent eukaryotic studies where modulating metabolism impacts outcomes like osteoarthritis treatment and lifespan extension in . Our findings underscore the crucial role of intracellular ATP levels in governing bacterial fate, emphasizing ATP manipulation as a potential strategy to steer bacterial behavior.

Cobamides (Cbas) are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life as co-catalyst of diverse reactions. There are several structural features that distinguish Cbas from one another. The most relevant of those features discussed in this review is the lower ligand, which is the nucleobase of a ribotide located in the lower face of the cyclic tetrapyrrole ring. The above-mentioned ribotide is known as the nucleotide loop, which is attached to the ring by a short linker. In Cbas, the nucleobase of the ribotide can be benzimidazole or derivatives of it, purine or derivatives of it, or phenolic compounds. Given the importance of Cbas in prokaryotic metabolism, it is not surprising that prokaryotes have evolved enzymes that cleave part or the entire nucleotide loop. This function is advantageous when Cbas contain nucleobases that somehow interfere with the function of Cba-dependent enzymes in the organism. After cleavage, Cbas are rebuilt via the nucleotide loop assembly (NLA) pathway, which includes enzymes that activate the nucleobase and the ring intermediate, followed by condensation of activated intermediates and a final dephosphorylation reaction. This exchange of nucleobases is known as Cba remodeling. The NLA pathway is used to salvage Cba precursors from the environment.

We have previously shown that the nucleoid-associated protein, HU, uses its DNA-binding surfaces to bind to bacterial outer-membrane lipopolysaccharide (LPS), causing HU to act as a glue aiding the adherence of DNA to bacteria, e.g., in biofilms. We have also previously shown that HU and DNA coacervate into a state of liquid-liquid phase separation (LLPS), within bacterial cells and also . Here, we show that HU and free LPS (which is ordinarily shed by bacteria) also condense into a state of phase separation. Coacervates of HU, DNA, and free LPS are less liquid-like than coacervates of HU and DNA. cells bearing LPS on their surfaces are shown to adhere to (as well as advance into) coacervates of HU and DNA. HU appears to play a role, therefore, in maintaining both intracellular and extracellular states of phase separation with DNA that are compatible with LPS and LPS-bearing with LPS determining the liquidity of the biofilm-simulating coacervate.

Type IVa pili (T4aP) are widespread and enable bacteria to translocate across surfaces. T4aP engage in cycles of extension, surface adhesion, and retraction, thereby pulling cells forward. Accordingly, the number and localization of T4aP are critical to efficient translocation. Here, we address how T4aP formation is regulated in , which translocates with a well-defined leading and lagging cell pole using T4aP at the leading pole. This localization is orchestrated by the small GTPase MglA and its downstream effector SgmX that both localize at the leading pole and recruit the PilB extension ATPase to the T4aP machinery at this pole. Here, we identify the previously uncharacterized protein SopA and show that it interacts directly with SgmX, localizes at the leading pole, stimulates polar localization of PilB, and is important for T4aP formation. We corroborate that MglA also recruits FrzS to the leading pole, and FrzS stimulates SgmX recruitment. In addition, FrzS and SgmX separately recruit SopA. Precise quantification of T4aP-formation and T4aP-dependent motility in various mutants supports a model whereby the main pathway for stimulating T4aP formation is the MglA/SgmX pathway. FrzS stimulates this pathway by recruiting SgmX and SopA. SopA stimulates the MglA/SgmX pathway by stimulating the function of SgmX, likely by promoting the SgmX-dependent recruitment of PilB to the T4aP machinery. The architecture of the MglA/SgmX/FrzS/SopA protein interaction network for orchestrating T4aP formation allows for combinatorial regulation of T4aP levels at the leading cell pole resulting in discrete levels of T4aP-dependent motility.

Plant pathogenic bacteria encounter a drastic increase in apoplastic pH during the early stages of plant immunity. The effects of alkalization on pathogen-host interactions have not been comprehensively characterized. Here, we used a global transcriptomic approach to assess the impact of environmental alkalization on pv. DC3000 . In addition to the Type 3 Secretion System, we found expression of genes encoding other virulence factors such as iron uptake and coronatine biosynthesis to be strongly affected by environmental alkalization. We also found that the activity of AlgU, an important regulator of virulence gene expression, was induced at pH 5.5 and suppressed at pH 7.8, which are pH levels that this pathogen would likely experience before and during pattern-triggered immunity, respectively. This pH-dependent control requires the presence of periplasmic proteases, AlgW and MucP, that function as part of the environmental sensing system that activates AlgU in specific conditions. This is the first example of pH-dependency of AlgU activity, suggesting a regulatory pathway model where pH affects the proteolysis-dependent activation of AlgU. These results contribute to deeper understanding of the role apoplastic pH has on host-pathogen interactions.IMPORTANCEPlant pathogenic bacteria, like , encounter many environmental changes including oxidative stress and alkalization during plant immunity, but the ecological effects of the individual responses are not well understood. In this study, we found that transcription of many previously characterized virulence factors in pv. DC3000 is downregulated by the level of environmental alkalization these bacteria encounter during the early stages of plant immune activation. We also report for the first time the sigma factor AlgU is post-translationally activated by low environmental pH through its natural activation pathway, which partially accounts for the expression Type 3 Secretion System virulence genes at acidic pH. The results of this study demonstrate the importance of extracellular pH on global regulation of virulence-related gene transcription in plant pathogenic bacteria.

is a Gram-positive, opportunistic human pathogen that is a leading cause of skin and soft tissue infections and invasive disease worldwide. Virulence in this bacterium is tightly controlled by a network of regulatory factors. One such factor is the global regulatory protein CodY. CodY links branched-chain amino acid sufficiency to the production of surface-associated and secreted factors that facilitate immune evasion and subversion. Our previous work revealed that CodY regulates virulence factor gene expression indirectly in part by controlling the activity of the SaeRS two-component system (TCS). While this is correlated with an increase in membrane anteiso-15:0 and -17:0 branched-chain fatty acids (BCFAs) derived from isoleucine, the true mechanism of control has remained elusive. Herein, we report that CodY-dependent regulation of SaeS sensor kinase activity requires BCFA synthesis. During periods of nutrient sufficiency, BCFA synthesis and Sae TCS activity are kept relatively low by CodY-dependent repression of the operon and the isoleucine-specific permease gene . In a null mutant, which simulates extreme nutrient limitation, de-repression of and directs the synthesis of enzymes in redundant and import pathways to upregulate production of BCFA precursors. Overexpression of independent of CodY, is sufficient to increase membrane anteiso BCFAs, Sae-dependent promoter activity, and SaeR ~ levels. Our results further clarify the molecular mechanisms by which CodY controls virulence in .IMPORTANCEExpression of bacterial virulence genes often correlates with the exhaustion of nutrients, but how the signaling of nutrient availability and the resulting physiological responses are coordinated is unclear. In CodY controls the activity of two major regulators of virulence-the Agr and Sae two-component systems (TCSs)-by unknown mechanisms. This work identifies a mechanism by which CodY controls the activity of the sensor kinase SaeS by modulating the levels of anteiso branched-chain amino acids that are incorporated into the membrane. Understanding the mechanism adds to our understanding of how bacterial physiology and metabolism are linked to virulence and underscores the role virulence in maintaining homeostasis. Understanding the mechanism also opens potential avenues for targeted therapeutic strategies against infections.

Enterococci are Gram-positive bacteria that colonize the gastrointestinal tract. Clinically relevant enterococci are intrinsically resistant to antibiotics in the cephalosporin family, and prior therapy with cephalosporins is a major risk factor for the acquisition of an enterococcal infection. One important determinant of intrinsic cephalosporin resistance in enterococci is the two-component signal transduction system CroS/R. The CroS sensor kinase senses cephalosporin-induced cell wall stress to become activated and phosphorylates its cognate response regulator CroR, thereby enhancing CroR-dependent gene expression to drive cephalosporin resistance. CroS possesses a short (~30 amino acids) extracellular segment between its two transmembrane domains near the N-terminus, but whether this extracellular segment is important for sensing cephalosporin stress, or possesses any other function, has remained unknown. Here, we explored the role of the CroS extracellular segment through mutagenesis and functional studies. We found that mutations in the CroS extracellular segment biased CroS to adopt a more active state during ceftriaxone stress, which led to an increase in CroR-dependent gene expression and hyper-resistance to ceftriaxone. Importantly, these mutants still responded to ceftriaxone-mediated stress by enhancing CroS activity, indicating that the extracellular segment of CroS does not directly bind a regulatory ligand. Overall, our results suggest that although the extracellular segment of CroS does not directly bind a regulatory ligand, it can modulate the magnitude of CroS signaling for phosphorylation of CroR to regulate cephalosporin resistance through the resulting changes in CroR-dependent gene expression.

is a common opportunistic pathogen and a model organism for studying bacterial sociality. A social behavior of that is critical for its success as a pathogen is its ability to form protective biofilms. Many of 's social phenotypes are regulated by quorum sensing-a type of cell-cell communication that allows bacteria to respond to population density. Although biofilm formation is known to be affected by quorum sensing, evidence for direct regulation of biofilm production by quorum regulators has remained elusive. In this work, we show that production of the major biofilm matrix polysaccharide Psl in PAO1 is regulated by the quorum regulators LasR and RhlR in stationary-phase cultures. Secretion of Psl into the culture medium requires LasR, RhlR, and the quorum signal molecules -3-oxo-dodecanoyl-homoserine lactone and -butanoyl homoserine lactone. Psl production in strains unable to synthesize the homoserine lactone signals can be restored by exogenous introduction of the signal molecules. We found that LasR and RhlR perform different roles in the regulation of Psl production: LasR acts at the promoter of the operon and activates transcription of the Psl biosynthetic genes, while RhlR activates translation of the transcripts. This work contributes to our understanding of the overlapping but distinct functions of the Las and Rhl quorum-sensing systems and implicates both in the direct regulation of biofilm matrix production.IMPORTANCE biofilms are responsible for many treatment-resistant infections in humans. Many cooperative behaviors in are controlled by quorum sensing, but evidence for a direct role of quorum sensing in the regulation of biofilm matrix production has been scant. In this work, we show that the Las and Rhl quorum-sensing systems have distinct roles in regulating production of the matrix polysaccharide Psl and that this regulation happens at the level of transcription (Las) and translation (Rhl) of the operon. These findings deepen our understanding of overlapping functions of Las and Rhl quorum sensing and the complex regulation of biofilm development in .

Contact-dependent inhibition (CDI) is a mechanism of interbacterial competition in Gram-negative bacteria. Bacteria that contain CDI systems produce a large, filamentous protein, CdiA, on their cell surfaces. CdiA contains a C-terminal toxin domain that is transported across the outer membranes (OMs) of neighboring bacteria. Once inside a target bacterium, the toxin is released from the CdiA protein via a proteolytic mechanism that has not been well characterized. We have developed an assay to monitor this toxin release process and have identified several conserved amino acids that play critical roles in the autocatalytic mechanism. Our results indicate that a hydrophobic, membrane-like environment is required for CdiA to fold, and the proteolysis occurs through an asparagine cyclization mechanism. Our assay thus provides a starting point for analyzing the conformational state of the CdiA protein when it is inserted into a target cell's OM and engaged in transporting the toxin across that membrane.