Biofilm and colony growth create microenvironments that require coordinated localization and function of cell envelope components. However, the mechanisms by which membrane organization contributes to cell envelope organization during multicellular growth remain poorly understood. Mycobacterium smegmatis contains an inner membrane domain (IMD) at the subpolar regions of the cell that contributes to cell envelope synthesis. Prior research identified PonA2 as important for de novo formation of distinct plasma membrane domains during planktonic growth. PonA2 is a penicillin-binding protein with transglycosylase (TG) and transpeptidase (TP) activities involved in peptidoglycan synthesis. To investigate the role of PonA2 during colony and pellicle biofilm growth, wild-type, ΔponA2, the complement strain (cponA2), and catalytically inactive PonA2 variants (TG-, TP-, and TG-/TP-) were analyzed. Subpolar IMD localization was maintained in wild-type cells during both colony and pellicle biofilm growth, demonstrating that IMD localization is not exclusive to planktonic growth. Loss of ponA2 resulted in smooth colony and early pellicle biofilm architecture and was associated with reduced abundance of trehalose monomycolate, trehalose dimycolate, and glycopeptidolipids. ΔponA2 cells in colony growth exhibited shortened cell morphology and lacked detectable IMD localization, whereas ΔponA2 cells in pellicle biofilm growth retained bacillary morphology and subpolar IMD localization. In contrast, the catalytically inactive PonA2 variants maintained rugose multicellular architecture, bacillary morphology, and subpolar IMD localization similar to that of wild-type. Together, these findings identify a previously unrecognized noncatalytic role for PonA2 in maintaining normal colony and pellicle biofilm development in M. smegmatis.IMPORTANCEPlanktonic, pellicle biofilm, and colony growth expose mycobacteria to distinct environmental conditions that can affect cell-envelope organization and survival. Prior research has shown that mycobacteria form subpolar plasma membrane domains that support polar cell elongation in planktonic growth, but it remains unclear whether this organization is conserved in multicellular biofilm and colony growth, where cells experience nutrient and oxygen gradients and altered cell-to-cell interactions. Our current study analyzed the plasma membrane domain formation across the two growth states and showed that membrane domain localization is conserved, while the mechanisms required to maintain these domains differ depending on growth conditions. These findings suggest that mycobacteria use growth state-specific mechanisms to coordinate membrane organization.

Vibrio cholerae is the causative agent of the human diarrheal disease cholera. Previous studies identified "hyperswarming" (HS) mutant V. cholerae with enhanced motility and decreased virulence factor expression, but the genetic basis of HS was unknown. Through whole-genome sequencing, we have identified point mutations within toxT, which encodes the master virulence regulator, as being responsible for the HS phenotype. These mutations are loss of function, and they eliminate transcription of cholera toxin and toxin-coregulated pilus genes. Using single-cell video tracking, these HS strains were also observed to exhibit enhanced motility/chemotaxis (increased speed and decreased directional change). Two methyl-accepting chemoreceptors (methyl-accepting chemotaxis proteins), AcfB and TcpI, are positively regulated by ToxT, and expression of both AcfB and TcpI within HS mutants restores wild-type motility. Moreover, a V. cholerae strain with a hyperactive ToxT (N106A) exhibits reduced motility (decreased speed and increased directional change), and knocking out acfB and tcpI in this strain restores wild-type motility. AcfB and TcpI localize to the bacterial cell poles, suggesting they incorporate into the chemotaxis arrays to modulate swimming behavior. Our findings demonstrate that the absence of the ToxT-dependent MCPs AcfB and TcpI is responsible for the HS phenotype, demonstrating a direct role of ToxT in the swimming behavior of V. cholerae.IMPORTANCEVibrio cholerae causes the human diarrheal disease cholera, and the master virulence regulator ToxT is responsible for the expression of the most important virulence factors, cholera toxin and the toxin-coregulated pilus, necessary for disease. Motility is also important for the bacteria to cause disease, and in this study, we provide evidence that ToxT also modulates motility, providing a regulatory link between motility and the other virulence factors required for the pathogenic lifestyle of V. cholerae.

The genus Yersinia includes three human pathogens-Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica-where biofilm formation plays distinct roles in transmission and environmental persistence. All three species share the core biofilm machinery-the hmsHFRS operon, encoding enzymes for poly-β-1,6-N-acetylglucosamine (PNAG) exopolysaccharide production, and the c-di-GMP signaling controlling this production-but their regulatory networks and biofilm outputs differ. Transcriptional control of hms involves an integrated network that includes the Rcs phosphorelay (reshaped by rcsA pseudogenization), PhoP/PhoQ, EnvZ/OmpR, BarA/UvrY, Fur/Zur, CRP-cAMP, RovM/RovA, and multiple sigma factors. Post-transcriptionally, the Hfq-sRNA and CsrA-CsrB/CsrC systems modulate gene expression; the latter is directly validated in Y. pestis and Y. pseudotuberculosis, whereas its role in Y. enterocolitica is inferred from genomic conservation. At the translational and enzymatic levels, polyamines sustain Hms protein production, while redox signals govern the HmsC-HmsD disulfide switch, controlling diguanylate cyclase activity. This multi-layer architecture ensures that biofilm output is tuned to species-specific ecological niches. In host adaptation, Y. pestis transitioned from a foodborne enteric pathogen to a flea-borne lifestyle through key genetic events-acquisition of ymt, pseudogenization of rcsA, inactivation of y2909/y3389, and ureD-which are not shared with Y. pseudotuberculosis or Y. enterocolitica, thereby explaining their different biofilm-dependent transmission strategies. Studies in Caenorhabditis elegans have identified host O-glycosylation modifications as receptors for biofilm attachment. This review summarizes progress on the molecular mechanisms, regulatory networks, and host-adaptive functions of Yersinia biofilm formation and proposes future research directions to address knowledge gaps. Future priorities include the identification of c-di-GMP effectors, flea-specific signals, 3D biofilm imaging, and CRISPR-based detection.

Brucella species are intracellular bacterial pathogens that cause brucellosis, one of the most common zoonoses worldwide. This review traces how Brucella, first discovered in the 19th century through investigations of human and bovine disease, became a modern experimental model system. Work on Brucella has illuminated how intracellular bacteria evade and adapt to host defenses and how they remodel host cell biology to establish a replicative niche. It has also yielded insights that extend beyond pathogenesis into bacterial cell envelope architecture and development. Though long regarded as a small, homogeneous group of mammalian pathogens, the genus has expanded in recent years as "non-classical" Brucella have been isolated from unexpected animal hosts, opening new opportunities to understand the fascinating host preferences and evolutionary history of these closely related Alphaproteobacteria. More than a century after their discovery, Brucella species remain versatile models for the study of intracellular infection, bacterial cell biology, and physiology, and the evolution of host association.

Mycobacterium abscessus is a non-tuberculous Mycobacterium (NTM) that is often associated with respiratory infections in persons with cystic fibrosis. High levels of antibiotic resistance make M. abscessus infections difficult to treat, highlighting the urgent need for alternative therapeutic strategies. Mycobacteriophage therapy-treatment of infections with viruses that infect Mycobacterium hosts-shows considerable promise, but the narrow host specificity of the current mycobacteriophage repertoire limits this approach to personalized therapies. Here, we characterize the anti-M. abscessus activity of mycobacteriophage-encoded Lysin B enzymes (lysterases). Lysterases are serine-esterases that cleave the ester bonds linking mycolic acids to either arabinogalactan or trehalose in the mycobacterial cell envelope to complete the lysis process. In this study, we explored their ability to kill a spectrum of clinically isolated M. abscessus strains when administered exogenously. Six phylogenetically distinct lysterases were purified, characterized on the surrogate M. smegmatis, and their activity against a diverse panel of 24 M. abscessus clinical strains was assessed. The enzymes exhibit a range of activities against these strains, but most have broad-spectrum activity with only minor variations among the M. abscessus isolates. However, the activities can be quite potent, resulting in up to six orders of magnitude of reduction in viability within 4 h with one activity unit of the enzymes, and act synergistically with amikacin. Overall, mycobacteriophage-encoded lysterases show considerable potential for antimycobacterial control, especially for topical applications.IMPORTANCEMycobacterium infections are clinically challenging, and there is a high failure of resolving Mycobacterium abscessus infections using antibiotics. Mycobacteriophages show promise in treating M. abscessus infections, but there is substantial variation among clinical isolates in their phage infection profiles. Mycobacteriophages are unusual in coding for lysis enzymes-lysterases-that target cell wall exposed constituents, and these can have potential antibacterial activity. Exploring the vast sequence diversity of thousands of phage-encoded lysterases identifies a subset that was expressed, purified, and tested for their ability to kill a broad range of M. abscessus clinical strains. Lysterases with the broadest and greatest activity offer substantial potential for treatment of M. abscessus infection, especially where they display synergism with current antibiotics.

The gram-negative cell envelope requires coordinated remodeling of the peptidoglycan layer and outer membrane to preserve envelope integrity during growth and division. The conserved Tol-Pal system contributes to envelope maintenance, although the physiological consequences of its loss vary across environmental contexts and among gram-negative bacteria. Here, we examined the contribution of Tol-Pal across diverse growth environments in Acinetobacter baumannii. Loss of Tol-Pal did not cause a substantial population growth defect, and septal peptidoglycan incorporation remained largely preserved under standard laboratory conditions. In contrast, nutrient-rich media, conditions that alter osmotic balance, and a host-like environment exposed defects in cell division organization and cell morphology. Tol-Pal-deficient cells also exhibited modest but reproducible reductions in outer membrane barrier robustness, and disruption of the Mla phospholipid transport system further exacerbated these defects. Consistent with these phenotypes, loss of Tol-Pal reduced bacterial fitness under environmental stress and host-associated models. Together, these findings indicate that Tol-Pal contributes to outer membrane barrier robustness and that additional consequences of its loss become apparent under conditions that challenge the cell envelope.IMPORTANCEThe physiological consequences of disrupting conserved envelope systems can vary with the conditions in which bacteria grow. In Acinetobacter baumannii, loss of Tol-Pal has little effect on population growth under routine laboratory conditions but modestly compromises outer membrane barrier robustness, whereas specific environmental and host-associated conditions reveal additional defects in cell division organization and bacterial fitness. These findings highlight how environmental context shapes the physiological consequences of Tol-Pal deficiency and provide a framework for understanding why the contribution of this conserved envelope system differs among gram-negative bacteria.

The nucleoside diphosphates linked to moiety-X (Nudix) hydrolases are a superfamily of proteins defined by the characteristic Nudix box sequence, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. The Nudix hydrolase associated with CRISPR-Cas-associated adenosine deaminases (CAAD) from Limisphaera ngatamarikiensis (LngNH) mitigates CAAD-induced toxicity by hydrolyzing ITP to IMP and pyrophosphate. Here, we determined crystal structures of LngNH in the apo form and in complex with ITP using catalytic mutants and validated key interactions by site-directed mutagenesis. The hypoxanthine of ITP is deeply buried in a hydrophobic pocket and engages a distinct hydrogen-bond network, whereas the triphosphate tail is coordinated by conserved residues and divalent metal ions. Together, these findings provide a structural framework for CAAD-linked nucleotide sanitization and suggest that CRISPR-associated Nudix hydrolases function as local self-protection modules that limit toxicity from noncanonical inosine nucleotides.IMPORTANCENucleotide quality-control enzymes protect cells from harmful metabolic by-products. Here, we characterize a CRISPR-associated nucleoside diphosphates linked to moiety-X (Nudix) hydrolase that removes ITP, a toxic noncanonical nucleotide generated during antiviral defense. Biochemical and structural analyses show how this enzyme specifically recognizes and hydrolyzes ITP while avoiding related cellular nucleotides. Our findings suggest that CRISPR-associated nucleotide deaminase systems require "self-protection" mechanisms to prevent damage from their own antiviral activities. These results expand our understanding of how bacteria maintain nucleotide homeostasis during immune responses and reveal an additional regulatory layer within CRISPR-associated defense systems. The structural framework reported here also provides a foundation for future studies of nucleotide sanitization enzymes in microbial physiology and antiviral immunity.

Clostridioides difficile is an urgent threat to human health. Current treatments for C. difficile infections (CDIs) are antibiotics and microbiome restoration therapy (MRT) for recurrent cases. However, antibiotics contribute to antibiotic resistance and recurrent CDIs, and the long-term sustainability and accessibility of MRTs remain to be determined. Since a dysbiotic gut microbiome is the primary risk factor for CDI, a better understanding of the interactions between C. difficile, the microbiome, and the host will aid development of treatments with improved precision. Emerging evidence supports that butyrate, a prominent end product of gut microbiome metabolism, is a key determinant of C. difficile pathogenesis. Notably, C. difficile releases more of its toxins (TcdA and TcdB) in butyrate-rich environments. Here, we demonstrate that butyrate-dependent toxin release is not driven by two previously characterized modes of toxin release (e.g., TcdE-dependent secretion or Cwp19-dependent autolysis). Instead, butyrate enhances the expression of a broadly conserved endolysin (EndD), which is responsible for butyrate-dependent toxin release. We additionally demonstrate that endD-dependent toxin release does not universally occur under all growth conditions, that its expression is dependent on the late-stage sporulation sigma factor SigK, and that endD enhances butyrate-dependent sporulation. Overall, our findings provide deeper insight into butyrate-dependent effects on C. difficile pathogenesis and set the stage for future work to better understand the molecular and genetic underpinnings of endD regulation.IMPORTANCEClostridioides difficile is a leading cause of infectious diarrhea. The gut microbiome-derived short-chain fatty acid butyrate triggers C. difficile to release its major virulence factors, toxins TcdA and TcdB. Here, we show that butyrate-dependent toxin release is independent of classical secretion or autolytic pathways and is instead mediated by EndD, an endolysin broadly conserved among C. difficile clinical isolates. We also demonstrate that endD expression is governed by the sporulation factor SigK and enhances sporulation, directly linking transmission and virulence. Uncovering how C. difficile couples metabolic sensing to EndD-mediated toxin release and sporulation advances our understanding of pathogen-microbiome interactions and highlights potential targets for non-antibiotic therapeutics.

The MprBA two-component system (TCS) plays a critical role in Mycobacterium tuberculosis persistence and forms a signaling pathway with the alternative sigma factors SigE and SigB. Here, we demonstrate that the MprBA-SigE-SigB signaling pathway is activated in Mycobacterium smegmatis under multiple stress conditions associated with respiratory inhibition, including genetic inactivation of the aa3 cytochrome c oxidase, treatment with the bcc1 complex inhibitor Q203, hypoxia, high ionic strength, and acidic pH. Strikingly, acidic pH elicited the strongest activation of MprA-dependent sigE expression, suggesting that MprB integrates multiple stress signals beyond respiratory dysfunction. Deletion of mprB resulted in constitutive activation of MprA rather than inactivation, as evidenced by elevated sigE expression, increased MprA phosphorylation, and enhanced rifampicin resistance in an mprB mutant. Using CRISPRi-mediated repression and genetic deletion, we identified the KdpD histidine kinase, a component of the potassium homeostasis KdpDE TCS, as required for phosphorylating and activating MprA in the absence of MprB. These findings reveal unexpected crosstalk between KdpD and MprA. Expression of a truncated MprB lacking the N-terminal sensory domain abolished sigE expression under both neutral and acidic conditions, indicating that this domain is essential for sensing environmental signals and that the truncated protein behaves as a phosphatase-dominant form under the conditions tested. Growth analysis revealed that MprA, SigE, and SigB are required for optimal growth under acidic conditions, demonstrating the importance of the MprBA-SigE-SigB pathway for survival of M. smegmatis under acidic stress.IMPORTANCEThe MprBA TCS contributes to mycobacterial stress adaptation and persistence by regulating SigE- and SigB-mediated stress response pathways. Here, we show that the MprBA TCS is activated under respiration-inhibitory and acidic conditions in Mycobacterium smegmatis, suggesting that this pathway responds to environmental cues relevant to host infection. We also identify an unexpected regulatory relationship between the MprBA and KdpDE systems: in the absence of MprB, MprA can be phosphorylated and activated by the noncognate histidine kinase KdpD. These findings suggest that MprB phosphatase activity is critical for maintaining low basal MprA activity and preventing inappropriate KdpD-mediated activation under non-stress conditions.

Francisella tularensis is a highly virulent, gram-negative bacterial pathogen that causes the zoonotic disease tularemia. F. tularensis infects a variety of host cells and replicates intracellularly while evading and interfering with host immune responses. The molecular mechanisms that facilitate the intracellular replication and virulence of F. tularensis are poorly understood. The Francisella genome contains a set of pil genes that code for the assembly of surface fibers termed type IV pili (T4P). T4P are major bacterial virulence determinants, but the function of the pil system during F. tularensis infection and intracellular growth is unclear. T4P are closely related to the type II secretion pathway, and the pil system of a related Francisella species, F. novicida, was shown to function in protein secretion as well as pilus assembly. To identify proteins secreted by F. tularensis, we analyzed the F. tularensis Live Vaccine Strain (LVS) using bio-orthogonal noncanonical amino acid tagging (BONCAT). Using BONCAT in conjunction with proteomics, we identified candidate proteins secreted by the wild-type LVS, as well as candidate proteins whose extracellular abundance decreased in the absence of the PilF ATPase or the PilE4 pilus subunit. Using epitope tagging of selected candidates, we validated T4P-mediated secretion of the ChiA and ChiD chitinases and the KatG catalase by the LVS. These results further our understanding of the pil system and protein secretion pathways in F. tularensis.IMPORTANCEFrancisella tularensis is a highly virulent gram-negative bacterial pathogen and the causative agent of tularemia. F. tularensis lacks secretion systems utilized by other intracellular bacterial pathogens but contains pil genes that encode for type IV pili (T4P) and may also function in protein secretion. T4P are observed on the surface of all Francisella spp., but pil-mediated protein secretion has only been reported for F. novicida, which is not normally pathogenic in humans. In this study, we used bio-orthogonal noncanonical amino acid tagging to identify proteins secreted by F. tularensis, for which there is limited information. We demonstrate that the F. tularensis pil system is capable of protein secretion and validate T4P-mediated secretion of the ChiA and ChiD chitinases and the KatG catalase. These results will facilitate investigation of Francisella virulence mechanisms and may provide targets for therapeutic intervention.

Spores of Bacillus species are metabolically dormant and resistant to many agents but can come back to life in germination triggered by a variety of small molecules; these spore properties vary depending on the conditions and media for spore formation. In the current work, we have compared the properties of Bacillus subtilis spores made in a rich medium and an extremely poor medium. Notably, spores made in the poor liquid medium had lowered resistance to wet heat and somewhat lowered resistance to hydrogen peroxide and formaldehyde. Spores made in the poor liquid medium also have an inner membrane (IM) that is more permeable and fluid compared to the IM in spores made in a rich medium. The poor liquid medium spores also germinated slower than the rich medium spores with several germinant receptor (GR) germinants but faster with the non-GR-dependent germinant dodecylamine. While the reasons for the latter differences are likely the more fluid and permeable IM in the poor medium spores, there were minimal, if any, differences between rich and poor medium spores in their levels of core water or CaDPA, two compounds that alone have major effects on spore resistance properties. These findings provide new information on the resistance and germination properties of spores of B. subtilis.IMPORTANCESpores of various Bacillus species can cause adverse effects such as food spoilage and some human diseases due to their resistance properties and ability to "come back to life" in spore germination. The reason for these effects has been commonly studied using spores prepared in relatively rich media, and there is a large body of literature on this. However, there have been minimal, if any, studies using spores prepared in an extremely poor medium, as was done in the current work. We believe that the findings in this work provide new insight into mechanisms of spore resistance and their germination.

Structural maintenance of chromosomes (SMC) complexes play conserved roles in chromosome organization, segregation, and repair in all domains of life. In Bacillus subtilis, SMC is required for the segregation of newly replicated origins. To investigate whether other proteins function with SMC in this process, we performed a synthetic lethal screen with an smc hypomorphic allele (smc*) that is mildly defective in chromosome segregation. In addition to recovering previously reported genetic interactions of smc with parB and spoIIIE, our screen identified minJ and divIVA as essential in the smc* background. We show that the synthetic lethality between smc* and ∆minJ or ∆divIVA arises from defects in segregating the replication terminus. Importantly, deletion of minD, which suppresses the cell division defects of ∆minJ and ∆divIVA, restored terminus segregation and viability in the smc* background. These findings support a model in which proper septum formation promotes chromosome terminus resolution and segregation by enabling SpoIIIE-mediated DNA clearance from the division septum during cytokinesis. These findings highlight the interdependence between chromosome segregation and cell division.IMPORTANCEThe SMC complex plays a central role in chromosome organization and segregation in Bacillus subtilis, but the cellular functions that become important when SMC activity is reduced are not well understood. Using a hypomorphic smc allele, we discovered that mutations affecting cell division become essential when chromosome organization and segregation are impaired. Our findings support a model in which efficient septum formation enables proper localization of the SpoIIIE DNA translocase, which in turn resolves and segregates the chromosome terminus region. These results highlight the critical role of cell division in supporting chromosome segregation.

Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems form the adaptive immunity of prokaryotes, conferring sequence-specific protection against genetic parasites. Here, we functionally characterized the type I-F CRISPR-Cas system of Pseudomonas aeruginosa ATCC 10145 (PA10145), which led us to discover the existence of an isolated CRISPR array unique to this system. Its core CRISPR-Cas system is composed of a cas operon flanked by two divergently organized arrays: CRISPR2 and CRISPR1. Meanwhile, the isolated CRISPR array, CRISPR3, was found ~1.3 million bp away from cas. The cas and three CRISPR arrays together function toward adaptive immunity, eliminating plasmids engineered with protospacer targets. If plasmids possessed an intact protospacer adjacent motif (PAM), hyperactive adaptation was stimulated in all CRISPR arrays of PA10145, whereas minimal to no adaptation was observed when the PAM was mutated. Spacer acquisition via interference-driven adaptation proceeds through strand-biased priming in PA10145. Interestingly, the isolated CRISPR3 and cas-adjacent CRISPR2 have nearly identical leader sequences, with only 3 bp mismatches. Of the 1,196 P. aeruginosa genomes analyzed, all 281 isolated arrays only occur as type I-F with similarly matching leaders to CRISPR2. Integrative conjugative elements and prophages only associate with CRISPR2 and CRISPR3, suggesting that isolated arrays might have originated from recombination events involving CRISPR2 as facilitated by these highly transmissible mobile genetic elements. Tracing evolutionary trajectories of the isolated CRISPR3 relative to cas-adjacent arrays revealed that CRISPR3 is horizontally transferred across P. aeruginosa genomes. Taken together, these results implicate the role of horizontally acquired isolated arrays in CRISPR-mediated pan-immunity as gateways to mobilize genetic memories.