Journal of Bacteriology

Bacteria in natural environments frequently encounter nutrient limitation leading to growth arrest and must balance the potential benefits of continuing to respond to the environment by making new proteins against the costs of depleting limited resources. We previously showed that the RNA polymerase-binding regulator SutA enhances transcription of hundreds of genes during nutrient limitation in Pseudomonas aeruginosa, suggesting that it might be part of a regulatory network facilitating limited new protein synthesis. Here, we sought to expand our understanding of this network by identifying transcriptional regulators influencing sutA expression. Using northern blotting, western blotting, and reporter assays, we found that the sigma factors FliA and RpoS, and the DNA-binding regulator Lrp, impact expression from a proximal sutA promoter during the transition to stationary phase. This constellation of regulators and the dynamics of SutA expression lead us to propose that SutA is part of a regulatory network that facilitates scavenging. Scavenging includes motility toward possible nutrient sources and uptake mechanisms for these nutrients, activities which require an investment of resources but can yield important benefits during starvation. In vitro transcription experiments, proteomic analysis and reporter assays suggest that SutA directly supports new protein synthesis driven by RpoS and indirectly supports flagellar motility, perhaps by helping maintain protein biosynthetic capacity against the metabolic costs of motility. SutA expression is controlled by multiple regulatory inputs, including negative autoregulation, and the protein appears to be short-lived. These properties are consistent with a role in supporting short, controlled bursts of gene expression during nutrient limitation. Author StatementMany bacteria engage in cycles of colonising a nutrient-rich location, using the available nutrients, and then dispersing in search of a new location to colonise. While searching for new nutrients in a low-resource environment, bacteria will be starved and must coordinate resource-intensive processes such as new protein synthesis, motility, and nutrient uptake so that each crucial activity can be accomplished but none use too much of the limited pool of resources. We previously identified a regulator in Pseudomonas aeruginosa called SutA, which facilitates new protein synthesis under starvation conditions. Here, we have identified regulators of SutA expression. We find that the housekeeping sigma factor RpoD drives expression during growth, but at the entry to stationary phase, where SutA has obvious impacts on cellular physiology, the stress sigma factor RpoS, the flagellar sigma factor FliA, and the amino acid sensing transcription factor Lrp are important. Finally, we find that all cells in a nutrient-limited population express some SutA, but appear to do so in infrequent bursts, and that the protein is likely unstable. Together, these findings suggest that SutA contributes to the coordination of resource use while bacteria scavenge for new nutrients, facilitating limited amounts of new protein synthesis.

Biofilm and colony growth creates microenvironments that require coordinated localization and function of cell surface molecules within the membrane. However, the mechanisms by which membrane organization regulates these surface molecules during these biofilm-associated growth are poorly understood, limiting our understanding of how bacteria adapt and survive in multicellular communities. Mycobacterium smegmatis contains an inner membrane domain (IMD) at the subpolar regions of the cell that helps mediate cell envelope synthesis. Prior research has identified ponA2 as critical for de novo formation of distinct plasma membrane domains in planktonic growth. PonA2 is a penicillin-binding protein that catalyzes peptidoglycan synthesis by transglycosylase (TG) and transpeptidase (TP) activities. To investigate the role of PonA2 in membrane domain organization in biofilm and colony growth, wild-type, {Delta}ponA2, the complement strain (cponA2), and catalytic inactive variants of PonA2 (TG-, TP-, and TG-/TP-) were analyzed. The IMD subpolar localization in wild-type was preserved in biofilm and colony growth, indicating that IMD localization is not exclusive to planktonic growth. Both biofilm and colony growth of {Delta}ponA2 showed significant structural deformities compared to wild-type. In contrast, the catalytic inactive mutants produced biofilm and colony structures that resembled wild-type, suggesting that PonA2 has additional noncatalytic functions during multicellular growth. The IMD localization of the catalytic inactive mutants was minimally impacted, suggesting that neither catalytic domain is required for IMD localization in biofilm and colony growth. Together, these findings advance our understanding of the complex mycobacterial membrane biology. ImportancePlanktonic, pellicle biofilm, and colony growth expose mycobacteria to distinct environmental conditions that can affect cell-envelope organization and survival. Prior research has shown that mycobacteria form subpolar plasma membrane domains that support polar cell elongation in planktonic growth, but it remains unclear whether this organization is conserved in multicellular biofilm and colony growth, where cells experience nutrient and oxygen gradients and altered cell-to-cell interactions. Our current study analyzed the plasma membrane domain formation across the two growth states and showed that membrane domain localization is conserved, while the mechanisms required to maintain these domains differ depending on growth conditions. These findings suggest that mycobacteria use growth state specific mechanisms to coordinate membrane organization.

Staphylococcus aureus encounters diverse environmental conditions during colonization and infection, including fluctuations in nutrient availability, oxidative stress, and oxygen limitation. Adaptation to these environments requires regulatory systems that coordinate stress responses with metabolic remodeling. The extracytoplasmic function sigma factor SigS contributes to stress adaptation and virulence in S. aureus and directly activates expression of the sroAB operon, which encodes the small proteins SroA and SroB. While previous work demonstrated that SroA participates in feedback regulation of sigS expression, the broader physiological role of SroA has remained unclear. To define the regulatory functions of SroA, we performed RNA sequencing following inducible overexpression of sroA in S. aureus. Transcriptome analysis revealed extensive remodeling of gene expression, with approximately 200 transcripts significantly altered. Transcriptome analysis revealed coordinated repression of metabolic pathways (including nitrate respiration and nucleotide biosynthesis) alongside activation of stress-response and nutrient acquisition genes. Northern blot and quantitative RT-PCR analysis confirmed repression of narG and narJ transcripts following SroA overexpression. Consistent with these transcriptional changes, nitrate reduction assays demonstrated that SroA overexpression reduces nitrate respiration activity. In addition to repression of nitrate respiration genes, SroA overexpression broadly suppressed genes involved in de novo purine and pyrimidine biosynthesis. In contrast, transcripts associated with stress responses and nutrient acquisition, including the SOS-associated gene sosA and the phosphate transport gene pstS, were upregulated. Together, these findings identify SroA as a regulator that links stress-responsive signaling to metabolic remodeling in S. aureus, particularly through modulation of nitrate respiration pathways. ImportanceStaphylococcus aureus must rapidly adapt its metabolism to survive the diverse environments encountered during colonization and infection, including conditions where oxygen availability is limited. In this study, we identify a previously uncharacterized role for the small protein SroA in regulating metabolic adaptation in S. aureus. Transcriptome analysis revealed that SroA strongly represses genes involved in nitrate respiration, a pathway that enables bacteria to maintain energy production when oxygen is scarce. Consistent with these transcriptional changes, SroA overexpression reduced nitrate respiration activity. These findings reveal a regulatory link between stress-responsive signaling pathways and respiratory metabolism, expanding our understanding of how S. aureus adapts to oxygen-limited environments encountered during infection.

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.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that coordinates lifestyle transitions, virulence, and developmental processes. In Clostridioides difficile, elevated c-di-GMP levels inhibit sporulation, but the underlying mechanism remained unclear. Here, we identified a conserved family of small, membrane-associated proteins encoded by c-di-GMP riboswitch-regulated genes. Transcriptomic analyses revealed that c-di-GMP represses these genes, and reporter assays demonstrated riboswitch-dependent transcriptional regulation via premature termination mechanism. Overexpression of a single member, CD1980.2, was sufficient to trigger the transcriptional activation of sporulation genes, including sigma factors and their regulons, and to increase spore formation. Conversely, sporulation efficiency decreased proportionally with the number of deleted small protein genes, and the strain lacking all seven genes displayed a severe sporulation defect, underscoring their cumulative and functionally redundant roles. Elevating c-di-GMP levels in the deletion mutant did not further reduce sporulation, supporting a model in which c-di-GMP inhibits spore formation by repressing the expression of this small-protein family. Our work establishes these small proteins, encoded by c-di-GMP riboswitch-regulated genes, as important mediators of developmental output in C. difficile. Their redundancy, conservation, and integration into riboswitch regulatory pathways highlight their central role in spore formation, a process essential for pathogen persistence and transmission. These findings expand the repertoire of components regulated by second-messenger signaling in bacterial physiology.

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.

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.

To establish infection and cause disease, the intracellular pathogen Shigella must successfully navigate a series of host defenses and distinct microenvironments within the human body. One way Shigella navigates these enviroments is by using the secondary messenger c-di-GMP, which regulates many different bacterial behaviours. C-di-GMP is synthesized by diguanylate cyclases (DGCs) and broken down by c-di-GMP specific phosphodiesterases (PDEs). In this study, we investigated how Shigellas c-di-GMP specific PDEs impact c-di-GMP turn-over and subsequently biofilm and virulence phenotypes. We knocked out each of Shigellas six c-di-GMP specific PDEs to determine how these PDEs impact biofilm, virulence and c-di-GMP levels within the bacterial cell. We found that these PDEs negatively regulate c-di-GMP levels while modulating Shigellas virulence and biofilm behaviour. We also noted that altering expression of these Shigella PDEs changes bacterial cell size. Transcriptome analysis revealed that a Shigella {Delta}pdeB strain showed reduced expression of many genes, including the virulence genes ipgD and ipgE, as well as genes associated with lipid metabolism. We confirmed that a Shigella {Delta}pdeB strain had altered levels of stearic acid, and expression of pdeB alters Shigella antibiotic susceptibility. This study highlights the complexities of c-di-GMP signaling in regulating numerous Shigella pathways.

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.

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.

Pseudomonas aeruginosa is an opportunistic human pathogen that is hospital-endemic, forming biofilms on medical equipment and causing thousands of hospital-acquired infections each year. The success of P. aeruginosa as an opportunistic pathogen is linked to its phenotypic and genotypic adaptability. Relatedly, P. aeruginosa forms a small colony subpopulation in response to stresses like oxygen limitation and antibiotic exposure. Additionally, P. aeruginosa coordinates population-level decisions using a mechanism of cell-cell communication called quorum sensing. P. aeruginosa uses these signaling pathways to control virulence factor production and biofilm formation in the host. We show that certain quorum-sensing mutations promote phenotypic variation; specifically, deletion of lasR and autoinducer modulating mutations in rhlI enhanced small colony formation in a time course-dependent manner. Using transcriptome analyses of isogenic small and large colony variants, we show that small colony formation is driven in part by the PhoPQ two-component signal transduction system in quorum-sensing mutant backgrounds. Specifically, our data show that unphosphorylated PhoP represses rhlR gene expression, and that subsequent de-repression of quorum sensing contributes to the production of virulence factors and the small colony phenotype. In total, these findings provide insight on how mutations evolved by clinical strains might serve as a bet-hedging strategy to promote the formation of a small colony phenotype and alter quorum-sensing signaling within a subpopulation of a community.

Staphylococcus aureus relies on coordinated regulatory networks to adapt to environmental stress and host-associated conditions. The alternative sigma factor SigB plays a central role in this process and is controlled by the anti-anti-sigma factor RsbV, which functions as a key regulatory node in the pathway. While numerous small regulatory RNAs (sRNAs) have been identified in S. aureus, relatively few have been directly linked to the SigB stress response network. Here, we investigated the role of the small RNA Teg16 in post-transcriptional regulation of the SigB stress response pathway. Computational prediction identified a region of complementarity between Teg16 and the translational initiation region of rsbV. To test a potential regulatory effect based on this prediction, teg16 was overexpressed, and rsbV transcript levels were measured by quantitative RT-PCR. Teg16 overexpression resulted in reduced rsbV transcript levels and decreased expression of SigB-dependent genes, including asp23 and the carotenoid (crt) biosynthesis operon responsible for staphyloxanthin pigment production. In addition, strains carrying the teg16 expression construct exhibited altered hemolytic activity under the conditions tested, suggesting effects on virulence-associated phenotypes. We further examined whether Teg16 influences the global regulator CodY and observed reduced codY transcript levels at early time points following teg16 overexpression. Together, these results extend a previously identified regulatory relationship between Teg16 and CodY and raise the possibility of a feedback relationship linking post-transcriptional regulation to metabolic control. These findings identify Teg16 as a previously uncharacterized regulator that connects small RNA-mediated control to the SigB stress response network in S. aureus. ImportanceThe alternative sigma factor SigB is a central regulator of stress adaptation in Staphylococcus aureus and influences both metabolism and virulence-associated phenotypes. While numerous small regulatory RNAs (sRNAs) have been identified in this organism, few have been functionally linked to control of the SigB pathway. Here, we identify the small RNA Teg16 as a regulator of rsbV, a key modulator of SigB activity. Teg16-dependent repression of rsbV is associated with reduced expression of SigB-dependent genes and measurable changes in phenotype, including decreased pigment production and altered hemolytic activity. In addition, our findings extend a previously identified relationship between Teg16 and the global regulator CodY, suggesting integration of post-transcriptional regulation with metabolic control. These results establish Teg16 as a previously uncharacterized component of the SigB regulatory network and provide new insight into how small RNAs contribute to stress adaptation in S. aureus.

Recently, our group showed that the dental pathogen Streptococcus mutans is inherently more tolerant to high zinc stress than other streptococci, a phenotype associated with the presence of a P-type ATPase exporter named ZccE, virtually unique to S. mutans. In addition to zccE, a previous transcriptome analysis revealed that S. mutans upregulates genes involved in glutathione uptake during initial exposure to zinc stress. Glutathione, a major supplier of organic sulfur that also plays key roles in antioxidant defense and xenobiotic detoxification, forms coordination complexes with a variety of metals, including zinc, thereby functioning as a buffer that protects cells from metal intoxication. To investigate the contribution of glutathione zinc tolerance in S. mutans, the gshT gene, which encodes the substrate-binding subunit of a glutathione transporter, was deleted in both the parent and {Delta}zccE strains and the ability of these mutants to overcome zinc stress through intracellular glutathione accumulation determined. Targeted metabolomics revealed that S. mutans accumulates glutathione in a GshT-dependent manner following zinc stress, a response that was strikingly amplified in the {Delta}zccE strain. Although glutathione supplementation had a minimal and non-significant impact on growth of either parent or mutant strains in sub-inhibitory zinc concentrations, the {Delta}gshT strain exhibited increased zinc sensitivity in a plate-based assay. However, the {Delta}zccE{Delta}gshT mutant displayed enhanced zinc tolerance compared to the {Delta}zccE single mutant. While glutathione alone did not alter zinc levels in the UA159 or {Delta}zccE strains, the combination of zinc and glutathione nearly doubled intracellular zinc levels in {Delta}zccE compared to cells grown in zinc only. We conclude that while glutathione may play a minor role in S. mutans zinc tolerance, uncontrolled glutathione uptake observed in {Delta}zccE facilitates zinc entry, as glutathione:Zn2+ complexes inadvertently promote zinc intoxication via a Trojan horse mechanism.

Bacteria frequently face challenges adapting to changing environmental conditions to survive and thrive, such as shifting resource utilization. Methylotrophs capable of growth on reduced single-carbon compounds are prevalent in the phyllosphere (aerial plant surfaces), where they face continual and predictable shifts in the availability of different plant-produced carbon sources. We examined the ability of the methylotroph Methylobacterium extorquens PA1 to adapt to repeated shifts between two different carbon and energy sources: the one-carbon compound methanol and the multi-carbon organic acid succinate, both present in the phyllosphere. Evolved lineages of wild-type cells all increased their capacity for rapid transition between the carbon sources through high frequencies of loss-of-function mutations affecting a previously uncharacterized gene, named cstR for carbon source transition regulator, which encodes an orphan single-domain response receiver. Characterization showed that mutant strains were more competitive bidirectionally in the succinate-methanol transition. Though evolved populations of the {Delta}efgA and {Delta}ttmR strains, which are defective in the succinate-to-methanol transition, experienced similar phenotypic improvements in carbon-source transitions, we did not observe cstR mutations rise to prominence as extensively or frequently in these lineages. Transcriptomic work revealed loss-of-function to cstR impacted expression of genes involved in motility/chemotaxis, energy metabolism, and stress response, among others, suggesting that it coordinates responses to metabolic cues that are prevalent in certain carbon source and growth phase transitions. Loss of cstR function did not compromise exogenous formaldehyde tolerance in the {Delta}efgA and {Delta}ttmR mutants, breaking a previously described tradeoff between these two phenotypes. However, this loss did lead to defects under exposure to certain stressors, including heat, desiccation, oxidative stress agents, and particularly pH stress. Altered levels of NAD+/NADH across conditions, improved growth under acidic pH, and diminished ATP and increased mortality under heightened pH together support a model where CstR is responsible for coordinating cell signaling to manage the balance between growth and maintaining stress resilience.

The Min system disassembles FtsZ-rings after septation in Bacillus subtilis and is localized to the nascent division plane and cell poles by the protein MinJ. The N-terminal region of MinJ contains transmembrane segments while the C-terminal region of MinJ contains a PDZ domain but its topology and functional domains are poorly understood. Here we empirically test MinJ topology based on a variety of transmembrane prediction models and find that the data is most consistent with Alphafold3, which predicts a 9-pass transmembrane protein with an external N-terminus and internal C-terminus. Deletion analysis indicates that all regions of the protein tested are required for function but deletion of the PDZ domain alone preserves polar localization and interaction with both MinD and DivIVA. Moreover, Alphafold predicts that transmembrane segments 6 and 7 comprise staves of an unusual transmembrane {beta}-sheet and deletion of the putative {beta}-sheet in the absence of MinD results in a minicell frequency that exceeds mutation of MinD alone. Bioinformatic analysis indicates that MinJ is highly conserved within Firmicutes and is co-conserved with MinD and DivIVA with which it interacts. Our data clarify the structure of MinJ and support models in which MinJ has functions in addition to restricting the activity of the Min system.

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.

Ribonucleases (RNases) play a key role in modulating gene expression at the post-transcriptional level, enabling bacteria to rapidly adapt to their environment. The B. subtilis genome encodes more than 20 RNases and understanding the role of each of these enzymes is crucial for a comprehensive knowledge of bacterial post-transcriptional adaptation mechanisms. Among these, RNase J1 and J2, belonging to the {beta}-lactamase enzyme family, form a heterodimer. While the primary function of RNase J1 as a 5-exoribonuclease in B. subtilis has been known for some time, the role of RNase J2, encoded by the rnjB gene, has remained unclear. Indeed, it has been considered a minor degradation factor due its very weak 5-exoribonuclease in vitro, the lack of a growth phenotype and the limited number of mRNAs with altered equilibrium levels in rnjB mutants. In this study, we demonstrate that the absence of RNase J2 influences pellicle and macro-colony biofilm formation, as well as swarming in B. subtilis. We show that RNase J2 plays a far more global role in governing mRNA half-lives than originally thought, by stimulating RNase J1-mediated degradation of a subset of RNase J1s mRNA substrates. An inter-subunit salt-bridge between RNase J1 and J2 is crucial for RNase J2 action. Altogether, our results suggest that RNase J2 acts primarily as a specificity co-factor for RNase J1 rather than as a ribonuclease per se. SignificanceThis study elucidates the role of RNase J2 in RNA degradation in B. subtilis. Through a combination of phenotypic analyses and Rif-seq data, we demonstrate that RNase J2 plays a far more important role than previously appreciated. We provide evidence that physical interactions between RNase J1 and J2 (via salt-bridge) are critical for the degradation of RNase J2 targets, suggesting that RNase J2 acts more as a co-factor of RNase J1 than as a standalone RNase.

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.

Bile salts constitute a major antimicrobial barrier encountered by Salmonella Typhimurium during infections. Here, we identified the interplay of two global regulators, Fnr (fumarate and nitrate reductase) and ArcA (aerobic respiration control protein A), in determining adaptive responses to bile. We utilized WT, {Delta}fnr, {Delta}arcA and {Delta}arcA{Delta}fnr strains and compared the expression and functional responses of S. Typhimurium to bile. The highlights of this study are: First, the {Delta}arcA and {Delta}arcA{Delta}fnr strain form smaller colonies on LB agar plates and the {Delta}arcA{Delta}fnr strain displays lower motility. Second, qRT-PCR expression analysis demonstrates that fnr is induced early with bile, followed by arcA. Also, arcA transcripts are lower in the {Delta}fnr strain and fnr expression is also partially lower in the {Delta}arcA strain. Third, the {Delta}arcA and {Delta}fnr strains display partial sensitivity to bile, whereas the {Delta}arcA{Delta}fnr strain exhibits hypersensitivity to bile. Upon bile exposure, the {Delta}arcA{Delta}fnr strain displays elevated transcripts of the major antioxidant genes (sodA and katG) and outer membrane protein (ompC), higher induction of reactive oxygen species (ROS), and greater membrane damage. Fourth, intracellular nitrite is induced earlier than ROS with bile. Fifth, arcA and fnr protects S. Typhimurium from bile induced stress by activating the nitrate metabolism pathway, which lowers ROS. Functionally, pretreatment of the deletion strains with sodium nitrate reduces ROS, improves membrane integrity and survival with bile. Overall, these findings demonstrate that arcA and fnr function cooperatively to utilize alternate electron acceptors, reduce dependence on aerobic respiration and lower ROS to improve survival of S. Typhimurium during bile stress.

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