Molecular Microbiology

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.

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.

In most bacteria, peptidoglycan contains mainly 4-3 crosslinks formed by penicillin-binding proteins (PBPs). But in the opportunistic pathogen Clostridioides difficile, 70% of the crosslinks are 3-3 crosslinks formed by L,D-transpeptidases (LDTs), and LDTs are essential for viability. PBPs and LDTs use different acyl donors for crosslinking; PBPs require a pentapeptide, while LDTs require a tetrapeptide. Here, we determined the source of the tetrapeptides in C. difficile and investigated the consequences of reengineering PG crosslinking from predominantly 3-3 to exclusively 4-3. We found that two D-alanyl-D-alanine carboxypeptidases (DD-CPase), DacA and DacC, supply LDTs with tetrapeptides during vegetative growth. Deleting these enzymes was sufficient to bypass the normal requirement for LDTs. The resulting mutant ({Delta}dacAC {Delta}ldt) was remarkably healthy despite the absence of 3-3 crosslinks. Its only major phenotypic defect was a 3- to 4-log decrease in sporulation, which could, however, be overcome by deleting a third DD-CPase, dacB. These findings fill gaps in our understanding of the pathway for LD-transpeptidation in C. difficile and imply that LDTs are not essential components of the elongasome or divisome, both of which function well in the complete absence of LDTs, provided there is sufficient pentapeptide to sustain crosslinking by PBPs. Thus, LDTs are essential for viability because C. difficile has intrinsically high levels of DD-CPase activity. Finally, we propose a model for how PBPs and LDTs work together during PG synthesis. In this model, PBPs construct a sparsely crosslinked PG sacculus that is subsequently strengthened with crosslinks introduced by LDTs.

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.

ABC transporters are key determinants of bacterial adaptation, yet their functional plasticity remains poorly understood. Here, we characterize the type I ABC transporter YhdWXYZ and uncover a striking functional divergence linked to the presence of its substrate-binding protein (SBP). In Escherichia coli K-12, where yhdW is a pseudogene, deletion of yhdWXYZ increases susceptibility to mecillinam and lomefloxacin and leads to intracellular accumulation of lomefloxacin, indicating a role in antibiotic homeostasis. In contrast, in Citrobacter rodentium, which encodes a complete YhdWXYZ system, deletion of the transporter does not affect antibiotic susceptibility. Biochemical analyses demonstrate that the YhdW SBP of C. rodentium binds asparagine with high affinity; however, genetic and physiological assays indicate that YhdWXYZ is not a primary asparagine importer under laboratory conditions, suggesting redundancy with other transport systems. Importantly, in vivo infection experiments reveal that YhdWXYZ contributes to early colonization and persistence in the host, as mutants display reduced bacterial loads and altered intestinal pathology in mice. Together, these findings show that loss of the SBP in E. coli is associated with a functional shift of YhdWXYZ toward antibiotic homeostasis, whereas in C. rodentium, the complete transporter contributes to host adaptation. This work highlights the evolutionary and functional flexibility of ABC transporters in bacterial physiology and pathogenesis.

Mycobacterium tuberculosis (Mtb) encodes two multigene families with 169 members that are exclusive to mycobacteria, the pe and ppe genes. These genes have unusual sequences including low-complexity repeat regions, but their functions--and whether they share a common function--have long been unclear. Recently, several members of the pe/ppe family were shown to transport nutrients across the outer Mtb membrane, a role for which no other proteins have yet been identified. Whether nutrient transport is a family-wide function and the range of nutrients transported by the PE/PPEs remains unclear. Sulfur is an essential nutrient for Mtb physiology and pathogenesis. To test whether PE/PPE transporters contribute to sulfur acquisition, we analyzed the transcriptional response of Mtb to sulfate by RNA sequencing. The pe22/ppe36 genes were induced in sulfate-limiting conditions. Deletion of pe22/ppe36 impaired growth in low-sulfate media and reduced intracellular sulfate levels, effects that were reversed by heterologous expression of the Mycobacterium smegmatis porin MspA. The response of sulfur-responsive genes to sulfur was muted in the pe22/ppe36 deletion strain, and mass spectrometry showed lower sulfolipid and sulfur metabolite levels in the deletion strain. These findings identify PE22/PPE36 as a specific sulfate uptake system and supports the emerging idea of PE/PPE proteins as nutrient uptake systems across the Mtb outer membrane. Significance statementThe mechanism of nutrient transport across the porin-less mycobacterial outer membrane and the function of the large pe/ppe gene family have been longstanding questions in mycobacterial biology. This study shows that the Mycobacterium tuberculosis PE22/PPE36 complex serves as a selective conduit for inorganic sulfate. Deletion of this complex disrupts intracellular sulfur homeostasis and triggers a metabolic seesaw that depletes cell-surface sulfolipids. Our findings expand the PE/PPE transport paradigm and suggest a direct link between nutrient acquisition and TB transmission.

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.

The horizontally acquired mgtC gene from Salmonella enterica confers this bacterium the abilities to survive episodes of magnesium (Mg2+) starvation, and to replicate in mammalian macrophages. The former property allows bacteria to persist in the environment through periods of Mg2+ depletion, whereas the latter allows S. enterica to overcome self-limiting intestinal colonization and cause an invasive systemic infection in susceptible mammalian hosts. Even though the biochemical function of MgtC is not completely understood, this protein is thought to function primarily by preventing the production of toxic levels of Mg2+-chelating adenosine triphosphate (ATP). In the current work, we investigated the physiological roles of mgtC homologs from an array of bacterial species, by probing the processes controlled by this gene during replication in low Mg2+ medium and in macrophages. We determined that MgtC homologs that do not participate in Pi homeostasis during Mg2+ starvation and do not promote intramacrophage replication in their resident species can partake in these processes when expressed in S. enterica. This indicates that the function of this protein is context dependent. Accordingly, we show that the physiological processes affected by S. enterica MgtC vary, depending on whether the bacteria replicate in low Mg2+ medium or inside macrophages. While these results suggest that MgtC is a regulator, they also demonstrate that horizontally acquired genes can assume different roles, depending on the genome and the biochemical context into which they are inserted. ImportanceThe mgtC gene encodes an inner membrane protein that has been horizontally acquired by multiple bacterial species, including several mammalian pathogens. In Salmonella enterica, MgtC promotes replication in mammalian macrophages and allows this bacterium to survive cytoplasmic magnesium (Mg2+) starvation. These phenotypes are thought to result from MgtCs inhibition of Pi metabolism and ATP production, which prevents the accumulation of toxic levels of Mg2+-chelating ATP and disrupts other physiological processes that are strictly dependent on Mg2+, such as ribosome assembly and translation. In the current study, we show that processes that are controlled by MgtC vary with the genetic and biochemical contexts in which this protein is expressed. While establishing a broader role for MgtC as a regulator, our findings illustrate how horizontally acquired regulatory genes can potentiate regulatory interactions, facilitating the evolution of new traits.

Small RNAs (sRNAs) rarely cause strong growth phenotypes upon overexpression, complicating efforts to link regulatory interactions to physiological outcomes. Here, we report that high levels of the Escherichia coli sRNA OmrA, but not its sibling OmrB, severely inhibit growth in glucose minimal medium. Genetic, biochemical, and physiological analyses indicate that OmrA-dependent toxicity results from reduced flux through the tricarboxylic acid (TCA) cycle. A UV-based suppressor screen identified mutations in the gene encoding Hfq, the RNA-chaperone that aids sRNA-mRNA interactions. Secondly, three independent mutations clustered in the ribosome-binding site of ppc, encoding phosphoenolpyruvate carboxylase, a key anaplerotic enzyme. OmrA directly inhibits Ppc translation via Hfq-dependent base-pairing in the ppc 5' UTR, including the mutated nucleotides obtained in the genetic screen. OmrA is significantly more effective than OmrB in ppc repression in vivo and in vitro, consistent with sequence divergence in their central regions. Supplementation with glutamate, glutamine, or downstream TCA cycle metabolites fully restores growth, linking reduced Ppc levels to metabolic limitation. These results identify ppc as a physiologically relevant OmrA target and suggest how RNA toxicity can uncover central metabolic nodes used by sRNAs to modulate bacterial physiology.

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 Escherichia coli protein YbeX/CorC is encoded in the same operon as the ribosome biogenesis factor YbeY, and its deletion leads to accumulation of 17S pre-rRNA and degradation intermediates of 16S rRNA under magnesium limitation. To further investigate the ybeX deletion phenotype, we used rRNA fluorescence in situ hybridization coupled with flow cytometry (rRNA-FISH-flow) to quantify 16S rRNA, 23S rRNA, and 17S pre-rRNA levels at single-cell resolution in{Delta} ybeX and{Delta} ybeY strains.{Delta} ybeX cells grown under limiting Mg2+ develop striking cell-to-cell heterogeneity in 17S pre-rRNA content during the transition to stationary phase, with up to 25-fold differences between individual cells. Upon regrowth from the stationary phase,{Delta} ybeX cultures display a bimodal distribution of 17S pre-rRNA, revealing two distinct subpopulations -- one retaining high levels of unprocessed pre-rRNA and the other with low levels -- whose relative proportions shift over time, until visible growth resumes. The stoichiometry between mature 16S and 23S rRNAs remains tight in both strains, indicating that the heterogeneity is specific to pre-rRNA processing, rather than a general disruption of ribosome homeostasis. The{Delta} ybeY mutant accumulates 17S pre-rRNA more uniformly across cells and primarily during exponential growth in rich medium, consistent with its direct role in 16S rRNA maturation. These single-cell data suggest that YbeX and YbeY affect ribosomal RNA metabolism through distinct mechanisms and that the extended lag phase of{Delta} ybeX is caused by a heterogeneous clearing of pre-ribosomal intermediates in individual cells.

Rhizobia are an agronomically valuable group of bacteria capable of entering into endosymbiotic relationships with leguminous plants, during which they fix atmospheric nitrogen using energy derived from the metabolism of plant-provided dicarboxylic acids. It is generally assumed that the gluconeogenic catabolism of dicarboxylic acids proceeds via the Embden-Meyerhoff-Parnas pathway in rhizobia. However, rhizobia are classically thought to lack the phosphofructokinase enzyme required for conversion of fructose-1,6-bisphosphate to fructore-6-phosphate as part of this pathway. Here, we demonstrate that a model rhizobium, Sinorhizobium meliloti, encodes a phosphofructokinase, completing the Embden-Meyerhoff-Parnas pathway of this organism. Biochemical characterization of the S. meliloti phosphofructokinase demonstrates that it can catalyze the reversible phosphorylation of fructose-6-phosphate under in vitro conditions in a pyrophosphate-dependent, rather than ATP-dependent, manner. We further show that S. meliloti also encodes a distinct fructose-1,6-bisphosphatase that can phenotypically complement the loss of the phosphofructokinase enzyme. Loss of both enzymes results in a block of the gluconeogenic pathway in S. meliloti and results in S. meliloti being unable to fix nitrogen in symbiosis with alfalfa (Medicago sativa). Phylogenetic analyses and complementation studies demonstrate that PPi-dependent phosphofructokinases are broadly distributed across the phylum Pseudomonadota (syn. Proteobacteria), including most rhizobial species of the class Alphaproteobacteria, suggesting both that PPi-dependent phosphofructokinases are likely more broadly distributed than is generally recognized, and that the catabolism of dicarboxylic acids in most rhizobia proceeds via a PPi-dependent phosphofructokinase. SIGNIFIGANCECentral carbon metabolism is an important biochemical network that bridges the gap between substrate catabolism and biosynthetic reactions in all living organisms. However, much of what we know about metabolism comes from the study of a few model organisms such as the bacterium Escherichia coli. Here, we identified the enzyme catalyzing a key step of central carbon metabolism in rhizobia (nitrogen-fixing bacterial symbionts of legumes), which until now had remained undetected. We show that this enzyme is dependent on pyrophosphate, which is different than the situation in E. coli, helping to explain why previous studies failed to identify this enzyme in rhizobia and highlighting the limitations associated with generalizing our understanding of metabolism from a limited subset of organisms.

Rho is a conserved, ATP-dependent RNA translocase that terminates transcription at hundreds of sites across bacterial genomes. Although the molecular mechanism of Rho-dependent termination is well characterized, its spatial interplay with RNA polymerase (RNAP) within bacterial cells remains elusive. To address this question, we constructed intragenic, in-frame fusions inserting mCherry or sfGFP 48 amino acid residues downstream of the N-terminus of Rho in Salmonella. Strikingly, mCherry--but not sfGFP--renders the first 48 residues of Rho dispensable. Rho{Delta}48::mCherry is viable in single copy and exhibits wild-type termination activity in vitro, whereas the full-length Rho::sfGFP fusion, although viable, slows growth and shows strongly reduced activity. Structured illumination microscopy (SIM) revealed that, despite these functional differences, both constructs exhibit similar localisation patterns relative to fluorescently tagged RNAP in single cells. During exponential growth, both Rho and RNAP form discrete clusters, but with markedly distinct spatial organisations: RNAP clusters associate with the nucleoid, whereas Rho is distributed throughout the cell body. This spatial partitioning persists in stationary phase, where RNAP becomes diffusely associated with a compacted nucleoid while Rho accumulates at the cell periphery. The widespread distribution of Rho at cytoplasmic locations is unexpected and suggests participation in cellular functions beyond its canonical role in transcription termination.

Adaptation of Escherichia coli to osmotic upshift requires rapid accumulation of intracellular solutes to restore turgor and maintain cellular homeostasis. While compatible solutes are well-established contributors to this process, they do not fully account for the early events following osmotic stress. Here, we demonstrate that inorganic phosphate and phosphorylated metabolites play a major and previously underappreciated role in osmoadaptation. Following osmotic upshift under conditions where accumulation of compatible solutes is restricted, E. coli exhibits a substantial increase in intracellular phosphate after a short lag. This increase accounts for a significant fraction of the charge balance required during rapid uptake of K+ and NH4+, the latter supporting glutamate synthesis as a principal counterion. Concomitantly, nucleotide pools display complex, multiphasic dynamics, including a transient decrease in adenylate energy charge whose duration correlates with stress magnitude. In addition, levels of pyrophosphate and key glycolytic intermediates, including dihydroxyacetone phosphate and 1,3-bisphosphoglycerate, increase markedly, indicating redistribution of phosphate into central metabolic pathways. These findings support a model in which phosphate uptake and metabolic redistribution contribute both to intracellular charge balance and to dynamic metabolic reorganisation during osmotic stress. By linking ion transport with central metabolism, this work expands current models of bacterial osmoadaptation and identifies phosphate flux as a key component of the early stress response. IMPORTANCEBacterial survival in fluctuating environments depends on rapid adaptation to osmotic stress. While compatible solutes are central to this process, their contribution does not fully account for early events in Escherichia coli following osmotic upshift. This work demonstrates that inorganic phosphate uptake and redistribution into nucleotide and glycolytic pools contribute substantially to balance the large positive charge entering the cell as it takes up K+ and NH4+ during osmotic upshift. These findings expand current models of bacterial osmoregulation by identifying phosphate flux as a central integrator of ion homeostasis and metabolic adaptation.

GlnH is an amino acid binding protein that senses aspartate to regulate metabolism via the PknG pathway in diverse Actinobacteria. Information about ligand occupancy of periplasmic GlnH is conveyed to PknG via an uncharacterised transmembrane protein GlnX. This pathway is important in the virulence of Mycobacterium tuberculosis, and in regulating valuable industrial fermentations by Corynebacterium glutamicum. GlnH has a "Venus flytrap"-like structure, comprising two lobes that surround the ligand aspartate. However, the conformational changes that allow GlnH to initiate this signalling pathway are unknown. To address this question, we produced GlnH from pathogens M. tuberculosis and Mycobacterium marinum and non-pathogens Mycobacterium smegmatis and C. glutamicum and used X-ray crystallography and cryo-EM to determine their structures. The results show that amino acid specificity is conserved in all homologues. However, GlnH from Mycobacteria was monomeric and bound aspartate with nanomolar affinity, whereas GlnH from C. glutamicum bound aspartate with micromolar affinity and dimerised upon binding. Whilst GlnH of the non-pathogens was stable at neutral pH, GlnH from the pathogens was most stable at acidic pH, reflecting the environment of host phagosomes. Structures were determined for all homologues, but only M. smegmatis GlnH crystallised in both unbound (Apo) and Asp-bound forms. GlnH has an open structure with a cleft between the lobes to permit access to aspartate. The Asp-bound structure is more compact with the lobes locked together, completely enclosing the ligand. AlphaFold was used to design mutations to disrupt the predicted GlnH-GlnX interface, and these variants failed to complement the metabolic defect of glnX knockout in M. smegmatis, supporting the predicted complex and suggesting how the GlnH conformational change is transmitted GlnX to initiate signalling.

Gram-negative bacteria must coordinate remodeling of the peptidoglycan cell wall with invagination of the outer membrane to preserve envelope integrity during growth and division. The conserved Tol-Pal system has been implicated in coordinating these processes, yet its physiological contribution to envelope organization remains unclear and may depend on environmental context. Here, we examined the role of Tol-Pal in coordinating envelope remodeling in Acinetobacter baumannii across distinct growth environments. Loss of Tol-Pal did not cause a major population growth defect, and septal peptidoglycan incorporation remained largely preserved under standard laboratory growth conditions. In contrast, under specific environmental conditions--including nutrient-rich media, altered osmotic conditions, and host-like environments--Tol-Pal deficiency disrupted the spatial organization of cell division and cell morphology. Tol-Pal mutants also exhibited modest but reproducible reductions in outer membrane barrier robustness and decreased fitness in environmental and host-associated contexts. Together, these findings demonstrate that Tol-Pal is not an essential component of the core division machinery but instead contributes to the coordinated organization of the Gram-negative envelope under conditions that impose additional physiological demands. More broadly, our results highlight how environmental context can reveal conditional roles for conserved envelope systems that are not apparent during standard laboratory growth. ImportanceThe Gram-negative envelope is a complex, multilayered structure that must remain intact as cells grow and divide across diverse and often challenging environments. Coordination between peptidoglycan remodeling and outer membrane invagination is therefore critical for maintaining envelope organization and cellular fitness. Here, we show that the conserved Tol-Pal system in Acinetobacter baumannii contributes to the spatial organization of cell division and outer membrane robustness under specific environmental conditions. Although Tol-Pal deficiency permits sustained population growth under standard laboratory conditions, its absence disrupts envelope organization and compromises bacterial fitness in environmental and host-associated contexts. These findings demonstrate how environmental conditions can expose conditional roles for conserved envelope systems and highlight the importance of physiological context in shaping bacterial cell envelope organization.

H-NS is an abundant nucleoid-associated protein found in Enterobacterales species. Some conjugative plasmids encode H-NS homologues, which are thought to facilitate plasmid maintenance and reduce the fitness costs associated with plasmid carriage. Here, we characterize HppXCROD2, an H-NS homologue encoded by the IncX4 plasmid pCROD2 of Citrobacter rodentium. Our data indicate that HppXCROD2 has a strong preference for binding pCROD2 over the chromosome or other plasmids. By contrast, chromosomally encoded H-NS displays no preference for plasmid sequence. When expressed from a heterologous plasmid in Escherichia coli, HppXCROD2 showed similar DNA-sequence preference to chromosomally encoded H-NS. Moreover, HppXCROD2 binding to a sequence from pCROD2 was much lower when that sequence was cloned in a laboratory plasmid. Thus, HppXCROD2 preferentially binds DNA in the context of the plasmid where it is encoded, a phenomenon we term "cognate plasmid specificity". We propose that cognate plasmid specificity occurs through recognition of plasmid-specific DNA topology generated by plasmid-encoded topoisomerases. Cognate plasmid specificity may insulate regulation of plasmid genes from the effects of host DNA, while minimizing disruption of host chromosome regulation due to plasmid carriage. IMPORTANCEMany bacteria carry conjugative plasmids, mobile DNA molecules that spread traits such as antibiotic resistance. Some conjugative plasmids encode proteins related to the bacterial DNA-binding protein H-NS. We show that an H-NS-like protein from the IncX4 plasmid pCROD2 binds almost exclusively to the plasmid from which it originates, while largely ignoring the host chromosome. Our findings reveal a previously unrecognized mechanism that allows plasmids to regulate their own genes with high specificity while minimizing interference with host gene expression.

The type VII secretion system (T7SS) is found in many Gram-positive bacteria and secretes toxins with antibacterial activity. Most characterised substrates have an N-terminal LXG domain that interacts with other helical partner proteins to form a composite T7SS targeting signal. Here we describe only the second substrate family to have a reverse domain arrangement. We show that TslM has a C-terminal LXG-like domain and an N-terminal lipase domain that has phospholipase activity. Secretion of TslM requires a single helical partner protein that binds to the TslM C-terminus, and its toxic activity is neutralised by a distinct family of membrane proteins. Genome analysis reveals that Staphylococcus aureus strains have the capacity to encode up to seven paralogous copies of this toxin family. Taken together our findings show that lipases are an important component of the staphylococcal T7SS toxin arsenal, and that toxins with a reverse domain arrangement are more widespread than previously appreciated.

Many protozoan parasites have complex life cycles entailing migration through different organs in their hosts, but the cues guiding them remain poorly understood. Using a semi-solid plate motility assay, we show that early procyclic forms of Trypanosoma brucei, the first stage to develop in the tsetse fly midgut, perceive several metabolites - including glucose, glycerol and proline - as chemoattractants, while the glycolytic end-product succinate acts as a repellent. During adaptation in the fly, T. brucei switches from glucose/glycerol to proline as its primary energy source. We show that the parasite's chemotactic response towards proline requires adenylate cyclase ACP5 and the cyclic AMP response protein CARP3, two components of signalling pathway involved in pH sensing. These results further support a role for T. brucei's expanded repertoire of receptor adenylate cyclases as environmental sensors that guide navigation through the host.

Bacteria routinely exhibit unexpected phenotypic and molecular changes in response to spaceflight and spaceflight-analogue conditions, yet the mechanisms by which they sense and respond to these low fluid shear environments are not fully elucidated. We previously demonstrated that spaceflight and low shear modeled microgravity (LSMMG) altered motility and chemotaxis gene expression in Salmonella enterica serovar Typhimurium (S. Typhimurium), raising the possibility that flagella mediate responses of the pathogen to these environments. Herein, we investigated whether LSMMG culture alters S. Typhimurium motility and examined the role of flagella in regulating pathogenesis-associated stress and infection phenotypes. LSMMG enhanced the swimming motility of wild-type S. Typhimurium relative to 1xg controls; a trend which persisted even in the absence of the global stress response regulators Hfq and RpoS. This finding was unexpected, as {Delta}hfq mutants are typically defective for motility under conventional culture conditions. Motility was also observed in the flagella-deficient {Delta}flhDC mutant following LSMMG and 1xg culture, although the relative motility pattern differed relative to wild-type. Collectively, these results indicate that flagella contribute to LSMMG-enhanced motility, but are not strictly required under these conditions. Conditioned supernatant exchange demonstrated that LSMMG-induced motility changes are cell-intrinsic rather than mediated by extracellular factors. While flagella were dispensable for many pathogenesis-related phenotypes tested, their deletion selectively altered the magnitude of LSMMG-associated thermal stress and intracellular survival in human intestinal epithelial cells. Together, these findings demonstrate that motility and pathogenesis-related responses in S. Typhimurium are governed by multiple regulatory pathways that differentially respond to LSMMG and 1xg conditions. IMPORTANCESpaceflight and spaceflight-analogue conditions alter bacterial physiology in unexpected ways that are important for pathogenesis, yet the mechanisms by which bacteria sense and respond to low fluid shear environments remain incompletely understood. This study shows that low shear modeled microgravity (LSMMG) enhances Salmonella Typhimurium motility and produces unexpected motility phenotypes in mutants lacking Hfq or the flagellar master regulator FlhDC. These findings indicate that flagellar biosynthesis contributes to LSMMG-enhanced motility but is not strictly required for motility under these conditions. We also suggest that flagella influence the magnitude of selected stress and infection phenotypes rather than serving as an absolute requirement for LSMMG responsiveness. Together, these results highlight the complexity of bacterial mechanotransduction under simulated microgravity conditions and advances our understanding of how a foodborne pathogen adapts to physiological low fluid shear environments encountered both in space and during terrestrial infection of the intestinal tract.