Molecular Microbiology

The bacterial peptidoglycan (PG) cell wall, a polymer made of amino-acid-bearing glycan strands, maintains cell shape, provides structural integrity, and protects against osmotic lysis. PG maintenance is an active process that requires regulated PG breakdown to make space for insertion of new PG strands. PG breakdown is accomplished by autolysins, i.e. endogenous enzymes with cell wall cleavage activity. The lytic transglycosylases (LTGs), a class of autolysins, for example, cleave glycan strands during PG remodelling. LTGs are broadly conserved and are highly redundant in bacteria, but their physiological role is poorly-defined. In this study, we interrogated physiological consequences of LTG insufficiency in Vibrio cholerae using TnSeq to gain insights about roles of these enzymes. We identify an uncharacterized transcription factor, Vca0578, which alleviates defects associated with the {Delta}6LTG mutant. We demonstrate that Vca0578 positively regulates the expression of zapC, a typically non-essential Z-ring associated protein. In the absence of zapC, cell division was impaired during perturbations of cell envelope homeostasis caused by absence of LTGs, or by exposure to antibiotics inhibiting cell elongation; either condition rendered zapC conditionally essential. This essentiality could be overcome by increasing FtsZ levels. Lastly, we found that ZapC also contributes to both width and length homeostasis during normal growth. This work thus uncovers a novel transcriptional circuit that contributes to effective cell division in{Delta} 6LTG cells, and suggests an essential role for ZapC in cell division under stress conditions that cause perturbation of cell width homeostasis. AUTHOR SUMMARYBacteria must maintain their outer shell (the cell envelope) in the face of changes in the environment. For this, they use elaborate systems that remodel the cell envelope. How some of these systems work is not well understood. In this study, we describe a new gene circuit that is required to keep cells dividing when the cell envelope is compromised. We found that Vca0578, a putative transcription factor, controls expression of the zapC gene. The protein ZapC then helps bacteria grow and divide when the cell envelope is under stress, for example, in the presence of certain antibiotics. Thus, we have discovered a regulatory circuit that promotes bacterial growth and antibiotic resistance under stress.

The phosphodiesterase (PDE) NbdA (NO-induced biofilm dispersion locus A) consists of a membrane-integrated MHYT domain, a degenerated diguanylate cyclase (DGC) AGDEF domain and an EAL domain. The integral membrane domain MHYT is proposed to sense a so far unknown extracellular signal and transfers the information to the cytosolic enzyme domains to modulate cellular c-di-GMP level. Here, we show that full length NbdA from Pseudomonas aeruginosa PAO1 is an active PDE in vivo. In line with its PDE activity, overexpression leads to slightly reduced global c-di-GMP levels, and reduced twitching motility. Surprisingly, overexpression of truncated cytosolic NbdA variants exhibited increased c-diGMP levels, suggesting previously uncharacterized DGC activity despite lacking a canonical GGDEF motif. While full-length NbdA overexpression resulted in only slight c-di-GMP reduction, cytosolic variants induced a significant increase, indicating a potential for nonenzymatic effects like protein-protein interactions. Further investigation revealed a connection between NbdA and type IV pilus (T4P) function. Overexpression of NbdA conferred resistance to the T4P-dependent phage DMS3vir, suggesting interference with T4P assembly or function. Microscopic analysis demonstrated dynamic localization of NbdA, partially co-localizing with T4P components, supporting a role in T4P regulation. However, no clear link was re-established with flagellar motor switching or chemotaxis signaling. These findings position NbdA in the complex signaling network of c-diGMP and T4P-mediated surface behavior in P. aeruginosa. Future work will focus on elucidating the precise mechanisms of NbdAs PDE activity and its interplay with other DGC/PDE networks. ImportanceIn this work, we show the in vivo activity of the membrane-bound phosphodiesterase NbdA of Pseudomonas aeruginosa, its role in c-di-GMP homeostasis, cellular localization and implications in surface behavior. Using strains overexpressing NbdA and truncated protein variants, we detected a strong defect in growth on solid surfaces and an altered phage susceptibility. Co-localization experiments supported further the hypothesis of interaction with the type IV pilus apparatus. We propose for NbdA to be part of the protein network responsible for c-di-GMP level modulation at the cell pole and thereby regulating the function of type IV pilus apparatus.

We used transposon sequencing (Tn-seq) to define genetic requirements for intracellular survival of Brucella neotomae, a rodent-associated species. A near-saturating mutant library was subjected to selection during infection of J774A.1 macrophages, identifying 54 genes required for intracellular fitness. These included core components of the VirB type IV secretion system, multiple regulatory factors, an aquaporin gene with a strong fitness defect, and a set of metabolic genes involved in amino acid biosynthesis. Targeted mutagenesis revealed that methionine and histidine biosynthesis are indispensable for intracellular growth, whereas tryptophan biosynthesis was required for full intracellular fitness, with mutants exhibiting significant but incomplete attenuation. Notably, these auxotrophs grew normally in minimal medium under axenic conditions, indicating that their requirement is specific to the intracellular environment. Amino acid supplementation rescued intracellular growth in a concentration- and time-dependent manner, consistent with increased metabolic demand during intracellular replication. Disruption of the aquaporin gene similarly impaired intracellular survival, suggesting a role for water homeostasis during adaptation to the macrophage vacuolar environment. Beyond metabolic and osmotic adaptation, we identify OmpR1 as an upstream regulator of B. neotomae virulence. Biochemical, genetic, and transcriptional analyses establish a hierarchical regulatory cascade in which OmpR1 activates the BvrR/BvrS system, which in turn controls VjbR and downstream VirB expression. Under the conditions examined, OmpR1 is required for activation of this cascade. Consistent with this, OmpR1 loss is not rescued by VjbR and requires BvrR activity for restoration of intracellular growth. Phylogenetic analysis places OmpR1 in a distinct lineage relative to other well-characterized Brucella transcriptional regulators, suggesting that this regulatory pathway has been underappreciated across the genus. Together, these findings reveal that intracellular fitness in Brucella depends on metabolic capacity, osmotic homeostasis, and a hierarchical regulatory cascade centered on OmpR1. Author SummaryBrucella species are bacteria that survive and replicate inside immune cells called macrophages, where they cause persistent infection. To live within these cells, the bacteria must carefully balance their metabolism with the expression of genes required for virulence. We used a genome-wide genetic approach to determine which genes are specifically required for intracellular survival of Brucella neotomae, a rodent-associated species. We found that several amino acid biosynthesis pathways, including those required to produce methionine and histidine, are essential for replication inside macrophages but are not required during growth in laboratory media. This indicates that the intracellular environment imposes nutrient limitations not apparent in culture. We also discovered that a gene encoding an aquaporin, which regulates water movement across the bacterial membrane, is critical for intracellular survival, highlighting the importance of maintaining water balance within the host cell vacuole. In addition, we identify OmpR1 as an upstream regulator that controls a hierarchical virulence cascade required for intracellular growth. Our findings show that successful infection depends on metabolic capacity, virulence regulation and water homeostasis, and provide new insight into how Brucella adapts to its host environment.

Survival of bacteria in their natural habitat requires dynamic responses and adaptation to environmental cues. In Bacillus subtilis, one adaptive strategy is cannibalism, a form of programmed cell death during post-exponential development. Cannibalism enhances multicellular differentiation by prolonging or preventing commitment to endospore formation under starvation conditions. B. subtilis produces three cannibalism toxins: the sporulation delay protein, the sporulation killing factor, and the epipeptide EPE. Production of the latter is encoded in the epeXEPAB operon. Expression of this operon is transcriptionally controlled by the stationary phase regulators Spo0A and AbrB. Here, we demonstrate that EPE production is also post-transcriptionally regulated by two RNA binding proteins, Kre and SpoVG. Deletion of comK, the master regulator of competence development, abolished EPE production. This defect was reversed by additionally deleting kre. The RNA-binding protein, Kre, binds the epeX transcript and acts as a bidirectional ComK repressor, indicating that ComK indirectly regulates EPE biosynthesis via Kre. A second RNA-binding protein, SpoVG, also binds to the epeX mRNA. While Kre acts as a negative regulator, SpoVG was essential for EPE production. These findings reveal a novel regulatory connection between competence and cannibalism, expanding our understanding of how programmed cell death is coordinated in B. subtilis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/716078v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@57e20dorg.highwire.dtl.DTLVardef@1b9f4e5org.highwire.dtl.DTLVardef@17cfbc9org.highwire.dtl.DTLVardef@76824d_HPS_FORMAT_FIGEXP M_FIG C_FIG

To establish infection, uropathogens must overcome several host defenses including the glycosaminoglycan (GAG) layer coating the apical surface of the bladder urothelium. GAGs are thought to protect against urinary tract infection (UTI) by serving as scaffolding sites for commensals, providing barrier function and preventing uropathogen adherence. However, the ability of uropathogens to degrade and utilize GAGs and the contribution of these activities toward UTI progression is largely unknown. We previously discovered that the uropathogen Proteus mirabilis, a common cause of catheter-associated UTI (CAUTI), degrades the GAG chondroitin sulfate (CS). In this study we sought to define the kinetics and regulation of CS degradation by diverse P. mirabilis strains clinically isolated from both recurrent UTI and CAUTI patients. We found variation in CS degradation kinetics between P. mirabilis strains and media types. However, CS degradation depended on conserved putative chondroitin sulfate ABC endo- and exolyases in all strains. Furthermore, we found that CS degradation in Pm123 was repressed by urea and that this repression was dependent on P. mirabilis urease activity. Complementation of the Pm123 endolyase into urea-insensitive HI4320 resulted in a urea-sensitive CS degradation phenotype suggesting functional differences between the Pm123 and HI4320 endolyases. Sequence alignment and structural modeling analysis identified two unique point mutations within the Pm123 endolyase that may contribute to urea sensitivity. Finally, unlike urea-insensitive P. mirabilis strains, Pm123 demonstrated attenuated swarming and loss of chondroitin endolyase activity had no effect on Pm123 virulence in a mouse CAUTI model. Our results suggest that the kinetics and regulation of CS degradation differ between P. mirabilis strains and in urea-sensitive strains, thus reduces the contribution of CS degradation to urovirulence during murine CAUTI. ImportanceThis work demonstrates that the ability to degrade a common component of bladder mucosal surfaces, chondroitin sulfate, is a phenotype that is shared by multiple strains of the common catheter-associated UTI (CAUTI) pathogen P. mirabilis. We find that this activity is dependent on encoded chondroitin ABC endo- and exolyases, first described in Proteus vulgaris. Additionally, we discovered that for P. mirabilis strain Pm123, degradation of CS is negatively regulated by the presence of urea, a major component of urine. The repression of CS degradation by urea is dependent on the activity of the P. mirabilis urease enzyme, which breaks down urea producing ammonia which raises pH. We found expression of the Pm123 CS endolyase was sufficient to confer a urea-sensitive CS-degradation phenotype and identified two unique mutations within the Pm123 enzyme that may contribute to urea sensitivity. Finally, we find that while CS-degradation plays a role in progression and severity of murine CAUTI model in urea-insensitive P. mirabilis, there was not significant difference in CAUTI outcomes between the urea-sensitive Pm123 wild-type and chondroitinase knockout strains. This study represents a major step forward in understanding the diversity of CS degradation activity and regulation among clinical strains of the critically important CAUTI pathogen P. mirabilis as well as its contribution to urovirulence.

Lig E is a periplasm-targeted ATP-dependent DNA ligase found in many Gram-negative bacteria including Neisseria gonorrhoeae. Although Lig E has been shown to have a role in biofilm formation, many Lig E-possessing bacteria are also naturally competent, suggesting a possible function in transformation with extracellular DNA. Here, we demonstrate that Lig E participates in bacterial competence by increasing transformation with nuclease-damaged extracellular DNA that contains single-stranded or cohesive breaks. We show that increased transformation with this restricted DNA is ATP-dependent, and that the ATP concentration increases in the extracellular milieu during maintenance of N. gonorrhoeae in liquid culture. Impact StatementNatural transformation is an important route of horizontal gene transfer that enables competent bacteria to acquire novel phenotypic traits such as antibiotic resistance or virulence factors. By demonstrating that Lig E increases transformation of N. gonorrhoeae with damaged resistance-encoding DNA, we provide a mechanism which competent bacteria can use to overcome nucleolytic damage sustained by environmental DNA, making this more readily available as a source of novel and potentially pathogen-enhancing genes.

In Gram-negative bacteria, co-translational insertion of membrane proteins into the plasma membrane may be coupled to ongoing transcription, a phenomenon known as transertion. Transertion results in a physical shift of the coding gene from the nucleoid towards the membrane and is one of the determinants of the shape of the nucleoid and placement of cell division in Gram-negative bacteria. In contrast, the existence of functional coupling of transcription and translation in Bacillus subtilis and potentially other Gram-positive bacteria has been questioned, suggesting that transertion may not happen. Here, by imaging vertically oriented B. subtilis cells, we show that the gene of a transmembrane protein changes its localization from inside the nucleoid to the plasma membrane upon induction of its transcription. Localization is restored to the nucleoid when induction of transcription has ceased. The shift of the gene towards the membrane is strictly dependent on transcription, its induction, translation and transmembrane nature of the coded protein. These results suggest that, at least, some principles of cellular regulation based on functional coupling of transcription and translation may be conserved between Gram-positive and Gram-negative bacteria.

The CenIR regulatory system of Clostridioides difficile comprises a predicted transcriptional repressor, CenI, and a predicted membrane metalloprotease, CenR. The physiological role of CenIR and activating signal(s) are not known. CenIR belongs to the BlaIR family of regulators that mediate resistance to {beta}-lactam antibiotics. In canonical BlaIR systems, binding of a {beta}-lactam to the extracellular transpeptidase domain of BlaR triggers proteolysis of BlaI and thus induction of a closely linked {beta}-lactamase gene. However, CenR lacks a {beta}-lactam-binding domain and transposon mutagenesis indicated CenI is essential for viability even when {beta}-lactams are not present. Here we confirmed essentiality of CenIR and determined its regulon contains [~]12 genes, including an exported protein of unknown function (CDR_0474) that is induced about 500-fold and a peptidoglycan hydrolase (Cwp6) that is induced about 7-fold when cells are depleted of CenIR. There are no essential genes or {beta}-lactamases in the regulon. Phenotypic characterization of CenIR-depletion strains revealed slower growth, mild elongation and cell lysis. Deletion of cdr_0474 corrected all three defects, while deletion of cwp6 only rescued the lysis phenotype. It was possible to delete cenIR in either a {Delta}cdr_0474 or {Delta}cwp6 background. We propose that CenIR is essential because its absence leads to lysis due to Cwp6 overproduction. Bioinformatic analyses revealed the predicted extracellular sensing domains in annotated "BlaR" proteins are diverse. Thus, BlaIR systems are not dedicated to defense against {beta}-lactams but probably enable bacteria to adapt to a variety of environmental stimuli. ImportanceMany of the regulatory systems for controlling cell envelope biogenesis and stress responses have yet to be studied. Here we characterize a Clostridioides difficile BlaIR-like regulatory system that we have named CenIR for cell envelope. Unlike canonical BlaIR systems, which bind {beta}-lactams and induce a {beta}-lactamase, CenIR lacks a {beta}-lactam binding domain and is essential for viability even in the absence of antibiotics. We identified the genes in the regulon and found that CenIR is essential because its absence leads to overproduction of the Cwp6 peptidoglycan hydrolase. We also show that most annotated BlaIR-like systems lack a {beta}-lactam-binding domain, from which we infer that these systems have much broader physiological roles than generally appreciated.

Horizontal gene transfer is an important evolutionary process by which DNA is exchanged between cells that are physically co-located but not direct evolutionary descendants. Horizontal transfer of highly divergent DNA is relatively easy to detect and can produce major phenotypic changes, exemplified by acquisition of antibiotic resistance determinants. However, transfer of high-identity DNA, for example between strains of the same species, is likely to be more frequent, harder to detect, and highly impactful in aggregate. In this work, we demonstrate that recombination between soil isolates of the alphaproteobacterium Novosphingobium aromaticivorans can exchange chromosomal DNA, leading to multiple unselected recombination events spanning approximately 10% of the chromosome. Chromosomal recombination was directional, more efficient near an integrative and conjugative element (ICE), and required a relaxase found in the ICE. Recombination could not be observed into strains from closely related Novosphingobium species. In combination, these results suggest that ICE-mediated recombination can efficiently recombine DNA within N. aromaticivorans, increasing the adaptive potential of the species while also enforcing species boundaries through preferential intraspecific recombination. ImportanceHorizontal gene transfer is a key process in bacterial evolution. Mechanisms for transfer of mobile genetic elements are well-characterized, but less is known about how chromosomal DNA is recombined. In this work, we demonstrate that integrative and conjugative elements can efficiently recombine chromosomal DNA between strains of Novosphingobium aromaticivorans but not between different Novosphingobium species. We conclude that ICE-mediated chromosomal recombination can be an important adaptive mechanism within a species, due to its ability to recombine nearby chromosomal alleles, but also serves to delineate species-specific gene pools as a result of its limited phylogenetic range.

Cyanobacteria utilize type IV pili for many behavioural responses, such as phototaxis, aggregation, floating, and DNA uptake. Type IV pilus-dependent functions are regulated by the nucleotide second messengers, c-di-GMP and cAMP. In this study, we investigated the role of a recently identified c-di-GMP receptor (CdgR) in cyanobacteria that harbours a ComFB domain. ComFB-domain proteins are widespread in cyanobacteria and are also present in heterotrophic bacteria. We demonstrated that the CdgR homolog from the cyanobacterium Synechocystis sp. PCC 6803, a model organism for studying type IV pilus-dependent functions, specifically binds to c-di-GMP. Genetic and phenotypic analyses revealed that Synechocystis CdgR is involved in phototactic motility and natural competence. Inactivation of cdgR resulted in altered expression of specific sets of minor pilins, which are essential for motility or natural competence. We identified interactions between CdgR and the CRP-family transcription factors, SyCRP1 and SyCRP2. Disruption of these CdgR-SyCRP1 and CdgR/SyCRP2 complexes is initiated by elevated c-di-GMP levels. Moreover, the assembly and stability of these complexes are influenced by other cyclic nucleotides, such as cAMP and c-di-AMP. These observed interactions imply a complex regulatory mechanism by which CdgR influences gene expression in response to cyclic nucleotide messenger signalling, particularly c-di-GMP. The present findings highlight the importance of CdgR in c-di-GMP signalling and its role in regulating type IV pilus-dependent functions in Synechocystis. The modulation of the expression of specific minor pilin genes by CdgR, through interactions with the transcription factors SyCRP1 and SyCRP2, contributes to the establishment of multiple type IV pilus functions and adaptive behaviours of cyanobacteria.

Staphylococcus aureus is one of the most frequently co-isolated pathogens in polymicrobial infections, where interspecies interactions contribute to enhanced virulence, persistence, and antimicrobial tolerance. Nutrient availability plays a central role in these interactions as microorganisms compete for resources required to sustain essential cellular processes. For instance, branched-chain amino acids (BCAAs) are critical for protein synthesis, and valine synthesis pathway precursors are essential for energy production. In S. aureus, BCAAs are also the precursors for branched-chain fatty acids (BCFAs), the dominant fatty acids in the S. aureus membrane. We previously identified a second pathway that uses branched-chain carboxylic acids (BCCAs) and the high-affinity acyl-CoA synthetase MbcS to catalyze the synthesis of BCFA precursors. However, the physiological role of this pathway and the conditions triggering its activation remain unclear. Here, we show that mbcS is restricted to S. aureus and closely related human-associated staphylococci. Phylogenetic analyses suggest that MbcS arose from a refunctionalization event and represents a non-orthologous replacement for the phosphotransbutyrylase (Ptb) and butyrate kinase (Buk) enzymes. Consistent with this model, Ptb and Buk from Staphylococcus pseudintermedius catalyze the formation of branched-chain acyl-CoAs from BCCAs, but only at high substrate concentrations. We further show that mbcS expression is upregulated in a codY mutant, implicating this pathway in BCAA-limited conditions. In support, we show that mbcS is required for optimal fitness during intra-species competition. Together, our findings support a model in which the MbcS-dependent pathway enables S. aureus to scavenge BCFA precursors under nutrient-limited conditions, providing a competitive advantage in polymicrobial environments. ImportanceStaphylococcus aureus is a major contributor to polymicrobial infections, where competition for nutrients can influence bacterial physiology and survival. A deeper understanding of how S. aureus adapts to nutrient limitation is therefore essential to explain its success as a human pathogen. In S. aureus, the acyl-CoA synthetase MbcS supports BCFA synthesis from BCAA-derived carboxylic acids and aldehydes, which are released into the environment as by-products of bacterial metabolism. Herein, we provide evidence that S. aureus acquired the acyl-CoA synthetase MbcS as an adaptive trait. This metabolic innovation allows this bacterium to maintain membrane homeostasis under nutrient limitation and compete against neighboring bacteria. Our findings highlight an adaptive strategy that may contribute to the persistence of S. aureus in polymicrobial infections.

Listeria monocytogenes relies on a tightly controlled set of surface-associated and secreted proteins to mediate host interaction and infection. The correct localization and exposure of these proteins at the bacterial surface are critical for virulence, yet the role of cell wall components in organizing this process remains incompletely understood. In particular, wall teichoic acid (WTA) glycosylation has been implicated in anchoring and function of selected surface proteins, but its global impact on protein distribution across the bacterial cell envelope is unclear. Here, we performed a comprehensive proteomic analysis to investigate how WTA glycosylation influences protein distribution in L. monocytogenes. Using isogenic mutants lacking rhamnose ({Delta}rmlT) or GlcNAc ({Delta}lmo1079) WTA glycosylation, we compared the exoproteome, the surface-accessible proteome and the surface-exposed proteome. Loss of WTA glycosylation did not result in a global disruption of the surface proteome but instead induced a redistribution of proteins across extracellular and surface-associated fractions. This effect was dependent on protein anchoring mechanisms, with limited changes observed for LPXTG-anchored proteins, moderate effects on non-covalently associated proteins, and a marked enrichment of lipoproteins in the surface-exposed proteome, particularly in the {Delta}lmo1079 mutant. In parallel, virulence-associated proteins displayed altered accessibility and exposure, with a progressive shift towards increased surface localization and a combination of shared and mutant-specific responses. This global effect was supported by functional annotation, which revealed that the affected proteins were associated with similar biological processes across fractions, highlighting a broad rather than pathway-specific impact of WTA glycosylation loss Together, these findings indicate that WTA glycosylation plays a key role in organizing the bacterial surface by modulating protein retention, exposure and release. Rather than affecting specific proteins, WTA glycosylation broadly shapes the spatial distribution of proteins across the cell envelope, with potential consequences for host- pathogen interactions.

Regulation of RNA pools allows for adaptation to changing environments and stress, which is especially important in pathogenic bacteria such as Mycobacterium tuberculosis. RNA degradation is a significant contributor to RNA abundance, and Ribonuclease (RNase) E has a rate-limiting role in degradation of a majority of mycobacterial transcripts. However, many open questions remain about the RNA substrate requirements and specificities for efficient cleavage by mycobacterial RNase E. Here, using both Mycolicibacterium smegmatis and M. tuberculosis RNase E, we demonstrate that this enzyme is only active on substrates with a minimum length of approximately 27 nt. Furthermore, we show that mycobacterial RNase E prefers substrates with 5 monophosphates rather than 5 triphosphates, and that the positions of cleavage events within substrates are dictated by both sequence and distance from the RNA ends. Our results also suggest that RNase E may be affected by product inhibition. Finally, we show that M. smegmatis RNase E behaves similarly to M. tuberculosis RNase E, validating the use of this model organism for RNA degradation studies.

The release of extracellular vesicles (EV) is a universally conserved process. Bacterial EVs package diverse cargo, including proteins and nucleic acids, and influence bacterial adaptation and survival as well as host-pathogen interactions. Currently, our understanding of the mechanisms underlying global principles in Gram-positive EV biogenesis and release is limited, partly due to labor-intensive vesicle isolation and assessment methods. Here, we describe a moderately high-throughput approach to analyze the Nebraska Transposon Mutant Library to identify genetic determinants of EV production in S. aureus. We show that the agr quorum sensing system dictates EV production in response to nutrient availability, likely through communication with the adaptive stress response. This study demonstrates the contribution of nutritional stress to vesiculogenesis and supports a conserved communication strategy that allows metabolic state to influence EV production.

RNA serves the essential role of priming DNA replication for all organisms. In bacteria, it is estimated that more than 20,000 ribonucleotides accumulate on the lagging strand per round of replication. Despite the importance of RNA primers in initiating DNA synthesis, unresolved primers result in strand breaks and induction of the DNA damage response, leading to genome instability. In bacteria, the prevailing model suggests that DNA polymerase I (Pol I) replaces RNA primers with DNA during lagging strand replication. However, in Bacillus subtilis, we show that {Delta}polA cells have a near wild-type phenotype, demonstrating that cells lacking Pol I still perform efficient Okazaki fragment repair. To determine how cells compensate for the loss of Pol I, we tested the ability of two major replicative polymerases, DnaE and PolC, to participate in lagging strand replication in vitro. We found that DnaE replicates an Okazaki fragment as efficiently as Pol I using strand displacement synthesis. In contrast, PolC is unable to catalyze strand displacement synthesis, but can facilitate repair when combined with a nuclease or a gapped substrate. Together, our work shows that B. subtilis can use several different mechanisms to replicate the lagging strand, ensuring fidelity within the replication cycle.

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

The mycobacterial cell envelope consists of multiple covalently linked layers that must be synthesized in a coordinated manner to maintain cell wall integrity. Despite the importance of this coordination, its molecular mechanisms remain poorly understood. PgfA (polar growth factor A) interacts with trehalose monomycolate lipids (TMMs) (1) and the TMM transporter MmpL3 (1, 2). PgfA promotes TMM transport in the periplasm and functions as an upstream regulator of polar growth. How TMM transport is linked to the expansion of the entire multi-layered cell wall is unclear. Here, we provide evidence that PgfA regulates peptidoglycan metabolism. We show that PgfA localization correlates with peptidoglycan metabolism and that PgfA can function as both an activator and inhibitor of peptidoglycan metabolism. We further explore the role of TMMs in polar growth and find evidence that periplasmic TMMs are a signaling molecule that may regulate polar peptidoglycan metabolism. Finally, we find an epistatic connection between PgfA overexpression and altered TMM levels that suggests that PgfA and TMMs work in the same pathway to regulate peptidoglycan metabolism. Our data are consistent with a model in which TMM-free PgfA inhibits peptidoglycan metabolism, while TMM-bound PgfA promotes polar peptidoglycan metabolism. This work identifies PgfA as a key protein that coordinates synthesis of the peptidoglycan and mycolic acid envelope layers. ImportanceThe mycobacterial cell envelope consists of multiple covalently linked layers whose synthesis must be coordinated to maintain cell integrity. Despite decades of research on individual envelope components, the molecular mechanisms coordinating synthesis of different layers remain poorly understood. Here, we identify PgfA as a key regulatory protein that coordinates peptidoglycan and mycolate synthesis in mycobacteria. PgfA has both inhibitory and stimulatory effects on peptidoglycan metabolism, depending on the context. Our findings suggest PgfA may act as a regulator that senses mycolate precursor availability and prevents envelope imbalance when these precursors are limiting. This work provides new insight into how mycobacteria coordinate the synthesis of their complex cell envelope, with implications for better understanding mycobacterial physiology and developing antimycobacterial therapeutics.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection worldwide and a leading cause of inflammatory reproductive tract disease and infertility in women. Much of the tissue damage associated with genital chlamydial infection arises from host inflammatory responses rather than direct bacterial cytotoxicity. Epithelial cells lining the female reproductive tract represent the primary host cells infected during chlamydial infection and play key roles in initiating innate immune responses. Among the cytokines produced by infected epithelial cells, type-I interferons have emerged as important regulators of host defense and inflammatory signaling; however, the specific contribution of interferon-{beta} (IFN-{beta}) to epithelial transcriptional responses during chlamydial infection remains incompletely defined. In the present study, we investigated the role of IFN-{beta} in coordinating epithelial immune signaling networks during infection with Chlamydia muridarum. Using wild-type murine oviduct epithelial cells (OE-WT) and IFN-{beta}-deficient epithelial cells (OE-IFN{beta}-KO), we performed pathway-focused RT{superscript 2} Profiler PCR array analyses examining transcriptional responses across four biological pathways: (1) innate and adaptive immune responses, (2) type-I interferon signaling, (3) inflammatory and autoimmune responses, and (4) fibrosis-associated pathways. Infection of OE-WT cells resulted in coordinated induction of cytokines, chemokines, and interferon-stimulated genes associated with antimicrobial defense and immune cell recruitment. In contrast, IFN-{beta} deficiency resulted in widespread dysregulation of these transcriptional programs, including reduced induction of interferon-responsive chemokines such as CCL5 and CXCL10, altered inflammatory cytokine expression, and transcriptional signatures consistent with enhanced tissue remodeling responses. Notably, IFN-{beta} deficiency resulted in increased TNF expression accompanied by reduced IL-6 induction, suggesting disruption of balanced inflammatory signaling networks. Pathway analyses further revealed dysregulated expression of fibrosis-associated genes including Serpine1, Ctgf, and Eng in IFN-{beta}-deficient epithelial cells, indicating potential mechanisms linking interferon signaling to tissue remodeling during infection. Collectively, these findings identify IFN-{beta} as a central regulator of epithelial immune networks during chlamydial infection and suggest that disruption of IFN-{beta} signaling may promote inflammatory and fibrotic pathology within the female reproductive tract. Author SummarySexually transmitted infections caused by Chlamydia trachomatis are a major cause of infertility worldwide. Although antibiotic treatment can eliminate the bacteria, damage to the reproductive tract often results from the bodys own immune response to infection. The epithelial cells lining the reproductive tract are the first cells infected and play an important role in initiating immune responses. In this study, we investigated how a specific immune signaling molecule, interferon-{beta} (IFN-{beta}), regulates the gene expression programs activated in epithelial cells during chlamydial infection. Using pathway-focused gene expression arrays, we found that IFN-{beta} coordinates multiple immune pathways, including interferon signaling, inflammatory cytokine networks, and genes associated with tissue remodeling. When IFN-{beta} was absent, many of these pathways became dysregulated, resulting in altered inflammatory signaling and gene expression patterns linked to fibrosis. These findings suggest that IFN-{beta} functions as a key regulator that helps balance protective immune responses with inflammatory processes that can damage reproductive tissues during infection.

Bacterial membranes comprise diverse lipids whose proportions vary according to environmental conditions. How cells direct lipid flux toward specific products remains unclear. We address this question in the human pathogen Staphylococcus aureus, where multiple lipid products compete for a common precursor, the major phospholipid, phosphatidylglycerol (PG). One product, lipoteichoic acid (LTA), is essential for cell division, envelope homeostasis, and virulence. Lipids and metabolites were quantified to identify factors that prioritize LTA synthesis over the other PG-derived products. We identify upstream fatty acid synthesis (FASII) pathway as a key control point for LTA production. Inhibition of FASII by antibiotics or gene inactivation causes LTA depletion. FASII inhibition similarly affects LTA in Streptococcus agalactiae, suggesting conservation of this LTA control strategy. Changes in membrane fatty acids do not account for LTA depletion. Instead, we show that FASII inhibition causes a drop in intracellular glycerophosphate (GroP), a precursor for both PG and LTA. Under these conditions of GroP limitation, PG flux favors production of a non-GroP lipid, cardiolipin. Moreover, combined inhibition of FASII and WTA blocks S. aureus growth, confirming the lethality of depleting LTA and WTA simultaneously. This study resolves how S. aureus manages phospholipid flux, by prioritizing the synthesis of GroP-rich LTA or of non-GroP-containing lipids according to FASII-controlled GroP availability.

Bacteria have evolved sophisticated mechanisms to sense and adapt to environmental shifts, particularly when transitioning into the stable, warm temperatures of a host organism. Escherichia coli, a primary inhabitant of the warm-blooded host gut, must rapidly initiate growth upon entry to ensure reproductive success. In this study, we demonstrate that the specific growth rate at the onset of the logarithmic phase (defined as at ODratio=0.2) is specifically stimulated across a medium temperature range (30{degrees}C-42{degrees}C), peaking near the physiological temperature of 37{degrees}C. This adaptive response is strain-specific and depends on both the histone-like nucleoid-associated protein (NAP) H-NS and the presence of the Rac prophage. Using high-frequency automated growth monitoring and statistical modeling, we redefine H-NS not merely as a gene silencer but as a critical "nucleoid structural organizer". Our results indicate that H-NS undergoes a conformational switch at approximately 37{degrees}C, transitioning into a parallel form that provides the necessary physical scaffold for nucleoid reorganization. This reorganization is essential for coordinating transcription and replication during the rapid onset of growth. Crucially, we resolve the "silencing paradox": while H-NS silencing is traditionally thought to be weakened at 37{degrees}C, hns-deficient mutants grow significantly slower because they lack this essential structural scaffold. We conclude that the H-NS-mediated physical organization of the genome is more critical for host adaptation than the mere de-repression of the genomic reservoir, enabling E. coli to effectively transition into a high-growth state for successful host colonization.