Molecular Microbiology

Flagella are large transenvelope nanomachines but how they transit the peptidoglycan in Gram positive bacteria is poorly understood. A recent model suggested that flagellar basal bodies diffuse in the membrane and become captured at locations in the peptidoglycan with a pore diameter that could accommodate the axle-like flagellar rod. Mutation of penicillin binding protein 1 (PBP1/PonA), a cell wall repair protein thought to decrease peptidoglycan pore frequency and/or size, resulted in a severe growth defect and cell lysis in the ancestral strain of Bacillus subtilis that was dependent on flagellar synthesis. Genetic analysis indicated that toxicity was due to completion of the flagellar hook, which activated the flagellar sigma factor SigD. SigD, in turn, activated a suite of peptidoglycan hydrolases that caused cellular lysis when PBP1 was absent. In addition, mutations that resulted in high levels of the stress response factor Spx could lessen the toxicity, while PBPX, a putative teichoic acid D-alanylase, was required for autolysis. In sum our results indicate that flagellar synthesis, not normally associated with cell viability, causes cell wall stress and under some conditions, cell death. Moreover, our work indicates that cost of envelope integrity by flagellar synthesis may be underappreciated due to strain domestication, and suggests that specialized systems may compensate for the cost of assembly of transenvelope machines in general. SIGNIFICANCEBacteria assemble nanomachines through the cell envelope but how the machines transit the peptidoglycan is poorly understood. Here we find that assembly of trans-envelope flagella results in cell lysis of Bacillus subtilis when the peptidoglycan repair protein PBP1 is absent. Lysis was due to multiple peptidoglycan lyases expressed as a consequence of flagellar assembly, and lytic activity required another PBP homolog, PBPX. Our work indicates that flagella, not normally thought to impact cell viability, can be lethal at the level of cell envelope integrity.

Mycobacterium tuberculosis (Mtb) cultured in minimal medium at acidic pH arrests its growth when provided specific single carbon sources, including glycerol, propionate, and lactate, a phenomenon we refer to as acid growth arrest. To define mechanisms of acid growth arrest on lactate, transposon mutants that suppress growth arrest were selected. Four mutants had insertions in phoT and one had an insertion in pstC2, both components of a phosphate ABC transporter. Mtb grows in minimal media supplemented with lactate at acidic pH when phosphate is depleted, showing that Mtb growth arrest on lactate is dependent on phosphate. The combination of lactate and phosphate at acidic pH causes cytoplasmic acidification below pH 6.7 in wild type Mtb, but a phoT::Tn mutant maintains a cytoplasmic pH of >7.2. Membrane potential in wild type Mtb is slightly decreased by lactate in a dose-dependent manner but is higher in the phoT::Tn mutant. Thus, acidic pH, phosphate, and lactate act together to dissipate proton motive force (PMF), a stress that is associated with acid growth arrest. Transcriptional profiling further supports that lactate causes PMF stress including induction of electron transport chain genes. The phoT::Tn mutant grown in lactate at acidic pH upregulates the senX3/regX3 regulon and using a regX3 mutant, we demonstrate that growth on lactate at low phosphate requires regX3. We propose a model where 1) the combined impact of acidic pH, lactate, and phosphate drives cytoplasmic pH acidification and decreased PMF, thus promoting acid growth arrest, and 2) low phosphate or a mutated phosphate transporter causes upregulation of senX3-regX3, which may induce ESX-5 and PPE/PE-based import mechanisms, thereby altering the mycomembrane or nutrient uptake in a manner that promotes growth on lactate at acidic pH. ImportanceMycobacterium tuberculosis (Mtb) grows well on lactate as a sole carbon source at neutral pH, but not at acidic pH. This study sought to understand why there is a pH-dependent growth restriction on lactate. A genetic selection for mutants that can grow on lactate at acidic pH identified mutants defective in phosphate transport. We found that limiting phosphate through depleting extracellular availability or inactivating a phosphate transporter promotes growth on lactate at acidic pH, and that this growth is dependent on the phosphate responsive two-component regulatory system SenX3-RegX3. Furthermore, we show that lactate, phosphate, and acidic pH combine to cause cytoplasmic pH acidification, a metabolic stress that is associated with acid growth arrest on lactate.

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.

Daughter cell separation in Escherichia coli is driven primarily by two classes of peptidoglycan (PG) hydrolases that work in tandem: N-acetylmuramoyl-L-alanine amidases that strip stem peptides from the PG glycan backbone and lytic transglycosylases (LTs) that break down the PG glycan backbone. Although the relevant amidases have been known for years, which of E. colis eight LTs contribute to this process is less clear. Because the amidases process PG first, the relevant LTs must utilize peptide-free or "denuded" glycan substrates (dnGs). MltA is one of the few E. coli LTs that can break down peptide-free PG glycans in vitro, but its precise physiological roles are not known. Here we show MltA localizes to the division site in constricting E. coli cells and cells lacking MltA accumulated dnGs in septal PG. We found that MltA binds to the anhydroMurNAc ends of glycan chains, which raises the possibility that these structures are enriched in septal PG. Nevertheless, as reported previously, deletion of mltA does not impair daughter cell separation sufficiently to cause a chaining phenotype. Overall, our findings demonstrate that MltA is a physiologically relevant peptidoglycan hydrolase for cell division in E. coli. IMPORTANCEHow bacteria coordinate synthesis and cleavage of septal peptidoglycan remains poorly understood, in part because some of the relevant enzymes have yet to be identified. Here we show that the E. coli lytic transglycosylase MltA is involved in cleaving septal peptidoglycan. Besides elucidating a physiological role for MltA, our work brings the field a step closer to identifying all of the proteins involved in cell division in an important model organism.

The envelope of Gram-negative bacteria like Escherichia coli is multilayered with two membranes sandwiching a peptidoglycan cell wall. The inner membrane is a typical phospholipid bilayer whereas the outer membrane is asymmetric with phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. We recently discovered that inactivation of the conserved peptidoglycan synthesis machinery responsible for cell elongation causes defects in both peptidoglycan and LPS synthesis in E. coli. This finding suggests that the isolation of suppressors that rescue the growth phenotype caused by an impaired cell elongation system is an attractive means of identifying factors involved in coordinating the biogenesis of different envelope layers. Here, we report the results of a global, transposon sequencing-based screen for such suppressors. The inactivation of a number of factors including the phospholipid synthesis enzyme PlsX was found to partially suppress the growth defects of a cell elongation mutant. Deletion of plsX also conferred increased resistance to CHIR-090, an inhibitor of the committed step of LPS synthesis catalyzed by LpxC, suggesting that loss of PlsX function stimulates LPS synthesis. Evidence is presented that increased CHIR-090 resistance is not mediated by changes in the activity of the proteolytic system (YejM-LapB-FtsH) controlling LpxC turnover. Rather, our results are consistent with a model in which the phospholipid precursor acyl-phosphate produced by PlsX serves as an inhibitor of LpxC to lower the rate of LPS synthesis when phospholipid synthesis capacity is reduced. IMPORTANCEOver the last several decades, most proteins essential for Gram-negative cell surface assembly have been characterized. However, relatively little is known about how the synthesis of different envelope layers is coordinated to promote uniform surface growth. Here, we report the results of a transposon sequencing-based genetic screen for mutants that suppress defects in the conserved peptidoglycan synthesis machinery responsible for cell elongation. Inactivation of the plsX gene encoding a phospholipid synthesis enzyme was found to both suppress the growth defect of a cell elongation mutant and to confer elevated resistance to an inhibitor of lipopolysaccharide synthesis. Our results suggest the attractive possibility that the product of PlsX, acyl-phosphate, may play a regulatory role in coordinating the phospholipid and lipopolysaccharide synthesis pathways.

Listeria monocytogenes can grow as a saprophyte on decaying plant material, but can also switch to a pathogenic lifestyle. This switch is mediated by the virulence regulator PrfA, which activates the expression of most virulence genes. PrfA activity is tightly regulated by several mechanisms to ensure that virulence genes are only expressed within the host. One of these regulatory mechanisms is the sugar-dependent repression. In the presence of readily metabolizable sugars, which are imported via phosphotransferase systems (PTS) such as cellobiose, PrfA is repressed; however, the precise mechanism is still unknown. Using a sugar screen, trehalose was identified as the first PTS-dependent sugar that supports growth of L. monocytogenes, but does not seem to impact PrfA activity. We demonstrated that the PTS permease TreB is the sole trehalose importer. After import, trehalose-6-phosphate is cleaved by the phosphotrehalase TreA; however, loss of TreA does not fully abolish growth on trehalose suggesting that L. monocytogenes encodes an additional phosphotrehalase. 13C-Labeling experiments revealed that trehalose metabolism is repressed in the presence of glucose, while it can be metabolized in the presence of glycerol. Additionally, these experiments provided evidence that trehalose and cellobiose are metabolized via identical pathways, including glycolysis and the incomplete TCA cycle, although trehalose has a slower uptake and/or metabolization rate. We therefore hypothesize that sugar-dependent PrfA repression correlates with sugar transport and/or consumption rates, potentially due to varying availability of phosphoenolpyruvate (PEP), which serves as both a metabolic intermediate and phosphate donor for PTS-dependent transport.

Cyclic di-AMP (c-di-AMP) is an essential second messenger in Listeria monocytogenes, but its accumulation is detrimental as it disrupts cell wall homeostasis and attenuates virulence. The mechanisms underlying this toxicity remain poorly understood. To understand the molecular basis of this toxicity, we performed a forward genetic screen to identify suppressor mutations that restore {beta}-lactam resistance in a {Delta}pdeA {Delta}pgpH ({Delta}PDE) mutant, which accumulates high c-di-AMP and is susceptible to cell wall-targeting {beta}-lactam antibiotics. We found that the majority of suppressor mutants carried mutations in the mreB gene, which encodes the bacterial actin-like cytoskeletal protein, MreB, that directs lateral peptidoglycan synthesis during cell elongation. These mutations restored {beta}-lactam resistance and ex vivo virulence while still retaining high intracellular c-di-AMP levels. Microscopy analyses indicate that these suppressor mutations reduce MreB activity, as evidenced by cell widening, and that they phenocopy sublethal treatment with the MreB inhibitor A22. Consistently, A22 treatment also rescued {beta}-lactam sensitivity in the {Delta}PDE mutant, supporting a functional link between MreB activity and c-di-AMP toxicity. Mechanistically, c-di-AMP accumulation impaired cell division/septation and reduced peptidoglycan synthesis under cell wall stress, whereas MreB mutations restored both transglycosylation and transpeptidation activities and promoted cell division. These effects were independent of potassium homeostasis, suggesting a distinct pathway linking c-di-AMP to cell wall regulation in L. monocytogenes. Together, our findings demonstrate that dysregulated MreB activity contributes to cell wall defects at elevated c-di-AMP levels and highlight the importance of coordinating cytoskeletal dynamics with cell division to maintain cell envelope integrity.

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.

Bone infections, which are predominantly caused by Staphylococcus (S.) aureus, can be difficult to treat and have high rates of chronicity and reoccurrence. We previously identified that osteoclasts, the cells that break down bone matrix, may contribute to disease progression by allowing S. aureus to replicate intracellularly. There we identified that this bacteriums ability to grow intracellularly is tied to the maturation of osteoclasts. In this study we addressed whether osteoclast differentiation supports intracellular growth by changing the host cells response to infection or by altering the host cell environment to better support S. aureus. Using dual species RNA-sequencing we analyzed host and bacterial transcripts of infected osteoclast and precursor bone marrow macrophage (BMM) cultures. Host transcript analysis suggests that infected osteoclasts are slow to upregulate bacterial response genes compared to BMMs. We also identify that the S. aureus transcriptional response is primarily determined by the host cell type, and that bacteria in osteoclasts upregulate carbon metabolism genes compared to those inside BMMs. By utilizing intracellular survival assays on S. aureus mutants deficient in carbon metabolism and related pathways we determine that S. aureus require glycolysis, acetyl-CoA synthesis, and aspartate biosynthesis for proliferation inside osteoclasts, although bacteria can survive without them. With differentiation, osteoclasts increase glutamine uptake, and this metabolite is required for S. aureus intracellular growth. Taken together, these findings suggest that osteoclasts support S. aureus intracellular growth by providing nutrients required to replicate in the context of a blunted antimicrobial response. IMPORTANCEInfectious osteomyelitis, bone infection, is frequently caused by the bacterium Staphylococcus aureus. Intracellular infection of cells that build bone, osteoblasts, and cells that resorb bone, osteoclasts, have been implicated in disease progression by providing a niche for immune evasion. While S. aureus in osteoblasts are largely quiescent, bacteria in osteoclasts proliferate and therefore may be a source of reemergent infection. Factors that promote this growth in osteoclasts are poorly characterized. In this study we find that osteoclasts have a diminished transcriptional response to infection and show that S. aureus acquire glucose and glutamine, which have high flux in osteoclasts, to support intracellular growth. We further observe that S. aureus in osteoclasts require aspartate synthesis to grow intracellularly. This work highlights the importance of host cellular metabolism for the intracellular fate of S. aureus as an added factor beyond the direct antimicrobial response.

Mycobacteria form rough and smooth colonies. The Mycobacterium marinum strain 1218S is a smooth colony forming variant isolated from the 1218R strain, which forms rough colonies and is more virulent than 1218S in infecting fish. Genes for the type VII secretion ESX-1 system, which includes mycobacterial virulence genes, have been partially duplicated in M. marinum and is refered to as ESX-6. We recently reported that several ESX-1 genes are missing in the 1218S strain. On the basis of the complete genomes of these two and three other M. marinum strains we provide insight into strain differences and similarities focusing on 1218R and 1218S, and ESX genes, selected virulence genes, and LOS genes, which are involved in lipooligosaccharide synthesis and smooth colony formation. We provide RNA-Seq data for 1218R and 1218S and two other well-characterized M. marinum strains suggesting that loss of ESX-1 genes in 1218S results in increased transcript levels of ESX-6 genes. Furthermore, while there is no difference in gene synteny and sequence of LOS genes comparing 1218R and 1218S, with the exception of duplication of lsr2, a regulator of LOS genes, in 1218S. Our RNA-Seq data show increased transcript levels of LOS genes in stationary 1218S cells relative to 1218R indicating that transcription and/or RNA degradation of LOS genes influence smooth and rough colony formation. We finally provide data suggesting that Ms1 RNA affect the transcription of LOS genes (and ESX-1 genes), and that loss of ESX-1 genes influence biofilm formation.

The Mycobacterium genus includes more than 190 species that occupies diverse ecological niches. Some are nonpathogenic and environmental, whereas others cause severe diseases both in humans and animals, e.g. tuberculosis (TB) and leprosy. SelenoCysteine (SeC) is present in all three domains of life. Here we report the presence of the SeC-machinery (selA, selB, selC and selD) genes and selenoprotein (fdhA) genes in roughly 40% of 244 mycobacterial genomes. Their presence is distributed evenly among slow and rapid growing mycobacteria and our data indicate that they were acquired through horizontal gene transfer. Some mycobacteria however lost these genes during the evolution of the genus. We provide RNA-Seq data showing transcript levels of the SeC-machinery genes and fdhA in different mycobacteria grown under different conditions. Finally, we suggest that selC (the tRNASeC gene), positioned immediately upstream of selA-selB, is involved in the regulation of the expression of the SeC-machinery genes selA-selB. Together our data expand our understanding of selenocysteine metabolism and its evolution within the Mycobacterium genus.

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.

The cell wall of the Gram-positive bacterium Enterococcus faecalis is decorated with the enterococcal polysaccharide antigen (EPA), consisting of a core rhamnan backbone linked covalently with a strain-variable teichoic acid-like (TA) polymer. Current models propose that the TA decoration is a repeating polymer composed of two alternating subunits, designated TAI and TAII, which are attached to the rhamnan core via a mild-acid labile phosphodiester bond from the initiating TAI subunit. In this study, we characterize the EpaU autolysin encoded within the EPA biosynthetic gene cluster. We demonstrate that the cell wall-binding domain of EpaU associates with the intact TA domains of EPA synthesized with the aid of the glycosyltransferases EpaR and EpaX. We further show that EpaU is a potent autolysin that binds generally over the E. faecalis cell surface, suggesting that it functions as a remodeling peptidoglycan hydrolase. The absence of EpaU leads to increased ampicillin resistance and elevated intracellular levels of the second messenger c-di-AMP. These data suggest that E. faecalis possesses a mechanism that senses the integrity of the peptidoglycan meshwork and employs c-di-AMP to regulate cell turgor, potentially altering the antibiotic resistance. ImportanceEnterococcus faecalis is an important opportunistic pathogen that can cause severe nosocomial infections. Knowledge of how bacteria remodel the cell wall is key to understanding many important cellular processes, such as antibiotic resistance, cell division, biofilm formation, and stress resistance. In this study, we shed new light on the structural details of the main cell wall polysaccharide, Enterococcal Polysaccharide Antigen (EPA), and its interaction with EpaU, an autolysin that cleaves peptidoglycan during cell wall remodeling. We also report a link between EpaU and the regulation of turgor pressure via cyclic dinucleotide signaling. This work contributes to a more complete picture of E. faecalis cell wall and may provide insight into the development of antimicrobial agents based on autolysins.

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.

1.In 2024, the World Health Organisation (WHO) classified Klebsiella pneumoniae as a maximum priority pathogen for the development of new alternatives to antibiotics. In this context, understanding the regulation of key virulence mechanisms is essential. Here, we investigated the role of the orphan quorum-sensing receptor SdiA in modulating virulence-associated processes during macrophage infection. Deletion of sdiA ({Delta}sdiA) significantly increased susceptibility to phagocytosis, as demonstrated using an amoeba predation model in which mutant strains formed larger clearance zones compared to wild-type bacteria. This phenotype was also observed in murine macrophages, where {Delta}sdiA strains exhibited increased adhesion (1.5 to 2.5-fold) and phagocytic uptake. Reduced uronic acid levels were also quantified in mutant strains, indirectly indicating a diminished capsule production, likely contributing to this enhanced phagocytosis. Despite enhanced uptake, {Delta}sdiA strains showed increased intracellular survival and replication rates within macrophages, leading to reduced host cell viability. This effect occurred despite loss of interbacterial killing capacity against E. coli, suggesting that enhanced intracellular fitness is not driven by classical antibacterial offensive mechanisms. Notably, mutant-infected macrophages displayed increased generation of reactive oxygen species (ROS), NF-{kappa}B expression, and pro-inflammatory cytokines (mCXCL10 and mTNF) production, indicating that macrophage defence mechanisms are not impaired during mutant infection. Overall, bacterial survival of {Delta}sdiA could result from overwhelming, rather than actively suppressing, host defences. Together, these findings identify SdiA as a negative regulator of phagocytosis and intracellular survival in K. pneumoniae and highlight a context-dependent role in virulence. This work provides new insights into the regulatory networks governing host-pathogen interactions and bacterial adaptation to the intracellular environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/725935v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d45bfdorg.highwire.dtl.DTLVardef@e3547forg.highwire.dtl.DTLVardef@c078f9org.highwire.dtl.DTLVardef@46408a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Loss of sdiA strongly affects phagocytosis, as mutant strains showed increasing adhesion (1.5 to 2.5-fold) and phagocytic uptake. Diminished capsule production could be contributing to this enhanced phagocytosis, as reduced uronic acid levels were also quantified in mutant strains. Despite being internalized at higher rates, mutants exhibited enhanced intracellular survival and replication, reducing macrophage viability. This fitness advantage occurred independently of classical offensive mechanisms, as evidenced by a lost ability to kill E. coli. Notably, mutant-infected macrophages mounted a stronger immune response, marked by elevated ROS, NF-{kappa}B expression, and pro-inflammatory cytokines production (mCXCL10 and mTNF). Together, these findings suggest that strains survive by overwhelming, rather than suppressing, host immune defences. Created with Biorender (https://www.biorender.com/). C_FIG HighlightsO_LISdiA deletion in K. pneumoniae increases susceptibility to phagocytosis. C_LIO_LIThe mutant strains exhibit reduced uronic acid levels, indicative of capsule production. C_LIO_LISdiA mutants show enhanced intracellular survival and higher macrophage death. C_LIO_LIMutant infected macrophages have higher NF-{kappa}B, TNF, and CXCL10 responses. C_LIO_LISdiA-deficient strains lose predatory capacity against E. coli. C_LI

Bacterial pathogens must evolve regulatory mechanisms that balance the necessity of virulence genes with the costs associated with their expression. Salmonella Pathogenicity Island-2 (SPI-2) is a genomic locus that encodes a Type 3 Secretion System (T3SS) and secreted effectors necessary for intracellular survival and replication. Recent work has shown that SPI-2 is expressed heterogeneously, suggesting uniform or aberrant expression of the system carries fitness costs. Here, we report a negative correlation between microcolony growth and SPI-2 expression, suggesting a growth cost to expression of SPI-2. To quantify and mechanistically evaluate this cost, we employ knockout and synthetic expression of the SPI-2 master regulator ssrB to eliminate or force the expression of SPI-2 genes. We find that ssrB expression causes deficits throughout the growth curve and puts cells at a competitive disadvantage in acidic, nutrient limited media. We further show that these deficits are environment specific, and not observed until stationary phase in neutral, rich media. We also observe unexpected cell morphology effects of ssrB expression, suggesting its effects on the cell extend beyond its regulation of SPI-2 genes. Using known ssrB mutations we find that DNA-binding activity, but not phosphorylation, of SsrB is necessary for growth costs. Lastly, we show that this growth cost is incurred even in the absence of the SPI-2 genetic locus, indicating that it is inherent to expression of ssrB, rather than its primary downstream virulence gene targets. These results demonstrate that SPI-2 expression is coupled to context specific cell growth and fitness deficits induced by its master regulator, offering a potential benefit to heterogeneous expression. SIGNIFICANCEVirulence factors are often heterogeneously expressed in bacteria, offsetting the resource and energetic burdens they impose. Characterizing such burdens reveals the evolutionary pressures that act on virulence genes and uncovers the logic behind regulatory mechanisms that drive heterogeneity in their expression. This study evaluates the growth and fitness costs associated with expression of Salmonella Pathogenicity Island-2 (SPI-2), which encodes a Type 3 Secretion System and secreted effectors required for intracellular survival and replication. The expression of the SPI-2 master regulator ssrB is shown to impose environment-specific growth and fitness deficits. These deficits persist in the absence of T3SS genes, suggesting that costs originate from SsrB acting on other target genes. These results provide critical context to recent observations of heterogeneous SPI-2 expression, elucidating the fitness cost this heterogeneity has evolved to offset.