Journal of Bacteriology

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.

Bacteria use post-transcriptional regulatory mechanisms to rapidly adjust gene expression during environmental change. In the gut-associated genus Bacteroides, these mechanisms remain poorly defined as these organisms lack canonical RNA chaperones like Hfq and CsrA that coordinate post-transcriptional stress responses in many well-studied model bacteria. Most Bacteroides possess conserved RNA recognition motif-1 (RRM-1) domain-containing RNA-binding proteins (more common in eukaryotes than bacteria) that have been proposed to act as global RNA chaperones. Here, we show that these RNA binding proteins (RBPs) are central to cold stress adaptation. Simultaneous deletion of all rbp genes produces a cold-sensitive growth defect across multiple Bacteroides species, while single deletions do not, revealing conserved functional redundancy. RBP transcripts and proteins accumulate rapidly after temperature downshift, and loss of RBPs extensively reprograms the transcriptome. Cold sensitivity of Bacteroides rbp mutants is not caused by defects in ribosome assembly or rRNA maturation. Instead, we find that in Bacteroides thetaiotaomicron, RBPs act together with BT1884, the sole canonical cold shock protein possessed by this organism. The combined loss of RBPs and BT1884 produces a synthetic severe cold sensitivity phenotype, defining two functionally redundant cold stress systems belonging to unrelated protein families. Strains lacking RBPs show reduced survival under simultaneous cold and oxygen stress, the conditions Bacteroides cells are expected to encounter during host-to-host transmission. Together, these findings establish RRM-1 RBPs as non-canonical cold shock proteins that enable cold adaptation and environmental survival in Bacteroides and suggest how these organisms withstand the stresses of transmission between hosts. IMPORTANCEBacteroides species are among the most abundant and stable members of the human gut microbiome, and they are also among the most readily transmitted between people. Reaching a new host requires surviving conditions outside the gut, including cold and oxygen exposure, yet how these bacteria withstand such stress is not well understood. Most bacteria manage stress using a well-defined set of RNA-binding proteins, but Bacteroides lack these canonical factors. We show that Bacteroides instead rely on a different family of RNA-binding proteins, more typical of eukaryotes than bacteria, to survive cold stress, and that these proteins promote survival under the conditions encountered during transmission. This work identifies a molecular system that allows an abundant and ecologically successful gut bacterium to endure the environmental challenges of moving between hosts.

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.

Bacterial peptidoglycan (PG) undergoes a variety of chemical modifications. O-acetylation at the C6 hydroxyl group of N-acetylmuramic acid is a widespread PG-modification found across diverse bacterial phyla. It contributes to virulence in pathogenic bacteria because the O-acetyl group reduces the activity of the PG-degrading host defense enzyme, lysozyme. Beyond its role in host defense evasion, recent studies suggest that PG O-acetylation also regulates the activity of endogenous lytic transglycosylase (LT) autolysins. The ethanologenic alpha-proteobacterium Zymomonas mobilis O-acetylates its PG, which is associated with tolerance to environmental stresses, including salt. To better understand how PG O-acetylation contributes to stress tolerance, we investigated the predicted lytic transglycosylase SleB. Intriguingly, the sleB gene is located adjacent to the pat operon, which encodes the proteins responsible for PG O-acetylation. We showed that loss of SleB caused impaired growth and morphology, and a significant reduction of crosslinks in the PG of Z. mobilis. Furthermore, the sleB mutant was sensitive to environmental stress resembling the sensitivity of the patA mutant. Collectively, our findings unravelled an important role of SleB in PG remodelling and stress resilience.

Bacteria naturally form densely structured populations wherein both space and nutrients become locally limited. In densely populated Pseudomonas fluorescens Pf0-1 colonies, spatiogenetic patches with drastically reduced cellular density naturally emerge through spontaneous mutations in rsmE. RsmE is a posttranscriptional regulator that binds to specific mRNAs, including those that consequently repress the production of extracellular secretions. We have previously shown that {Delta}rsmE produces an extracellular polysaccharide (EPS) and biosurfactant that collectively function to define the spatiogenetic structure and locally outcompete the WT. Here, we identify additional RsmE-regulated secretions through a combination of RNA-sequencing and LC-MS/MS. In particular, a type VI secretion system (T6SS) was exclusively produced by {Delta}rsmE. Confocal microscopy imaging of WT and genetically modified {Delta}rsmE cocultures showed that the T6SS kills WT cells that invade the low- density spatiogenetic structure. However, its impact on competition with the WT was both quantifiably and spatially limited in the absence of the normally co-produced EPS and biosurfactant, largely due to the consequently altered spatial structure. These results highlight the importance of understanding both the individual and collective spatial functions of extracellular secretions, especially in the ecological context of densely structured microbial populations. IMPORTANCEIn crowded wild type (WT) Pseudomonas fluorescens Pf0-1 colonies, rsmE mutants spontaneously emerge by forming a low cellular density spatial structure that is devoid of WT cells. RsmE functions primarily to repress the production of various extracellular secretions, including an extracellular polysaccharide and biosurfactant, that collectively form the unique spatial structure. Here, we describe an RsmE-regulated type VI secretion system that kills the invading WT cells and protect the low-density structure. Importantly, individual RsmE-regulated secretions carry out unique functions, but their efficacies largely depend on other co-regulated products. Extracellular secretions that are mechanistically similar to those described here are co- produced across diverse biofilm-forming and virulent bacterial species, where they also likely play complex molecular and ecological roles.

Many bacteria engage in quorum sensing (QS), a cell-cell communication system used to coordinate group behaviors. In one type of QS, acyl-homoserine lactone signals generated by LuxI homologs bind to LuxR homolog transcription factors, usually resulting in gene activation. The genome of Pseudomonas aeruginosa encodes three such LuxR homologs: LasR, RhlR, and QscR. Of these, LasR regulates the most genes, including that encoding RhlR. There is strong evidence that, during chronic infections, lasR and other genes encoding LuxR-type regulators are under strong selective pressure for mutations that both inactivate and modulate their function. Thus, we wondered if some mutations in the lasR gene might result in a protein with affinity for promoters usually regulated by the other LuxR homologs; to do so, we investigated the DNA-binding domain (DBD) of LasR through alanine substitution. As expected, we found that most alanine substitutions across the LasR DBD led to loss of function, as did previously identified clinical LasR DBD variants. Additionally, some alanine mutants were indistinguishable from the wild type. We describe a handful of variant LasR polypeptides that unexpectedly exhibit enhanced regulation on a RhlR-regulated gene, which conferred a fitness defect when competed against the wild type. Most other LasR variants had a competitive advantage. Our results suggest a pathway for expansion of the regulon of LuxR-homolog transcription factors, but also that such mutations may be disfavored due to the incurred metabolic burden. ImportanceMany bacteria generate chemical signals to alter gene expression in response to changes in population density, a phenomenon called quorum sensing. One type of quorum sensing relies on acyl-homoserine lactone (AHL) signals. In this type of quorum sensing, first described in the bioluminescent bacterium Vibro fisheri, a LuxI homolog produces the AHL, which binds to a LuxR homolog that typically activates gene expression. The opportunistic pathogen Pseudomonas aeruginosa has two such LuxR homologs, LasR and RhlR, each of which has its own specific regulon. We focused on the transcription factor LasR and investigated structural determinants of its binding to target promoters using an alanine substitution approach. We discovered that some DNA-binding mutations can expand the range of LasR-regulated genes. Our work provides insight into understanding what promoters LuxR homologs bind to and, more generally, how these proteins might evolve over time to change the group of genes that they regulate.

Salmonella enterica serovars Typhimurium (STM) and Paratyphi A (SPA) cause clinically distinct diseases, yet the molecular bases for their different lifestyles remain incompletely understood. Genome degradation, a hallmark of typhoidal Salmonella, results in extensive pseudogenization across multiple functional pathways. Here, we investigate how such gene inactivation rewires cyclic-di-GMP (c-di-GMP) signaling and flagellar motility regulation in SPA vs. STM. We show that YhjH, a conserved phosphodiesterase (PDE), is required for motility in STM but not in SPA, despite retaining PDE activity in both serovars. We demonstrate that this functional divergence is caused by pseudogenization of ycgR in SPA, which truncates the flagellar brake protein YcgR to a nonfunctional peptide, severing the link between c-di-GMP levels and flagellar motor control. Site directed mutagenesis in the YhjH active site and ectopic expression of intact YcgR from STM that restored YhjH-dependent motility regulation in SPA, confirmed this molecular mechanism. Additionally, using a bacterial two-hybrid (BACTH) genetic screen, we identified a serovar-specific interaction between YhjH and the general stress protein YciG in SPA, but not in STM. Computational RNA folding analysis predicted substantial differences in mRNA secondary structure and stability between the SPA and STM yhjH alleles, suggesting a potential role for synonymous mutations in this serovar-specific interaction. Together, these findings reveal how genome degradation can rewire regulatory networks, uncovering a fundamental difference in motility control between typhoidal and non-typhoidal Salmonella and suggest that these differences allow SPA motility under conditions that suppress motility in STM. IMPORTANCESalmonella enterica serovars Typhimurium (STM) and Paratyphi A (SPA) cause fundamentally different diseases in humans, yet the molecular basis for their distinct lifestyles and pathogenicity remains poorly understood. Genome degradation is a hallmark of typhoidal Salmonella, but its functional consequences for regulatory networks are largely unexplored. Here, we demonstrate that pseudogenization of ycgR in SPA dismantles c-di-GMP-mediated flagellar motor control, liberating SPA from an inhibitory brake that suppresses motility in STM. Additionally, we uncover a serovar-specific interaction between the phosphodiesterase YhjH and the general stress protein YciG in SPA, demonstrating that genome degradation can drive regulatory network rewiring beyond simple gene loss. These differences in motility regulation may facilitate the systemic pathogenesis and unique lifestyle of SPA.

Chromosome segregation is an essential process in monoploid bacteria, in which faithful inheritance of the genetic material is required for viability of daughter cell. In several alpha-proteobacteria, the polar organizing protein PopZ plays a crucial role in chromosome segregation by anchoring the chromosome partitioning machinery at a cell pole. While this process has been studied in monoploid species, it remains unclear whether this type of segregation mechanisms play a significant role in bacterial cells that carry multiple copies of chromosome. The alpha-proteobacterium Zymomonas mobilis exhibits ethanologenic physiology, and thus, is a promising chassis for biofuel production. Recent studies showed that Z. mobilis may serve as a non-model system for studying bacterial cell biology, owing to its relatively simple physiology and reduced genome size. Z. mobilis has previously been shown to be polyploid, and possesses a homolog of PopZ, a key regulator of chromosome organisation and cell polarity in alpha-proteobacteria. This raises the question of whether PopZ is involved in the organization of multiple chromosome copies. Here, we investigated the function of PopZ in Z. mobilis. Unlike in previously studied alpha-proteobacterial species, PopZ is dispensable for growth and cell morphology in Z. mobilis. However, the loss of PopZ altered the polar accumulation of chromosome segregation protein ParB, indicating an involvement in chromosome organization in Z. mobilis. These findings indicate that Z. mobilis might possess a chromosome segregation machinery that is dispensable under favourable growth conditions but might become important under certain conditions that reduce chomorome copy numbers.

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.

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.

Bacteria often evolve resistance to phage infection by altering the cell-surface structures required for viral adsorption. However, the role extracellular metabolites play in influencing phage susceptibility and the evolution of phage resistance remains unclear. Here, we evaluated whether sustained exposure to putrescine, a polyamine released during phage-mediated cell lysis, alters susceptibility of the pathogen Pseudomonas aeruginosa to the type IV pili-dependent phage DMS3vir. Using adaptive laboratory evolution over [~]66 generations, we evolved P. aeruginosa with or without putrescine and with or without DMS3vir. As expected, direct phage exposure rapidly led to complete phage resistance. Interestingly, populations exposed to putrescine also developed phage resistance by the end of the experiment, despite having never encountered the phage. Whole-population genome sequencing revealed parallel mutations in genes associated with type IV pili and the global transcriptional regulator mexT. Using transposon insertion mutants in the type IV ATPases pilT and pilB, we confirmed that disruption of these genes leads to DMS3vir phage resistance. We also used a type IV pilus biogenesis factor fimV transposon mutant, which showed a putrescine-dependent reduction in phage susceptibility. These findings show that sustained exposure to a host-derived metabolite can drive the evolution of phage resistance through modification of key phage-adsorption sites and regulatory genes. Our work identifies elevated polyamine exposure as a selective pressure that promotes type IV pili-mediated phage resistance, even in the absence of phage exposure. IMPORTANCEPseudomonas aeruginosa is a major cause of hospital-acquired infections and a key priority for phage-based therapies. Previous work has shown that the polyamine putrescine is released into the extracellular environment during cell lysis. These signals can then transiently reduce susceptibility to bacteriophage infection and alert neighboring cells to danger. Our research demonstrates that long-term exposure to putrescine can drive heritable phage resistance without prior exposure to phage. We show that resistance is linked to mutations in genes involved in type IV pili assembly. This work further demonstrates the critical role that polyamines can play in promoting phage resistance in bacterial communities.

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.

Sulfolobus acidocaldarius is a thermoacidophilic archaeon used as a model system for studying fundamental cellular processes and for emerging biotechnological applications. However, the limited availability of selectable markers restricts advanced genetic manipulation in this organism. Here, we report the development of a tryptophan auxotrophy-based selection system in S. acidocaldarius. A {Delta}trpBA mutant was constructed in the {Delta}pyrE background strain using a classical pop-in/pop-out recombination strategy. The resulting mutant exhibited little growth defects in rich medium, likely due to exogenous tryptophan supplied by complex nutrients, but failed to grow in a newly developed defined Brock-based amino acid dropout medium lacking tryptophan. Exploiting both uracil and tryptophan auxotrophies, we achieved dual-plasmid co-transformation and co-expression of the surface layer proteins and a dominant-negative mutant of the AAA-ATPase Vps4, revealing that the accumulation of surface layer lattice forming protein SlaA at the midzone of division-arrested cells together with its membrane anchor SlaB. Together, these results provide evidence for spatial regulation of S-layer assembly during archaeal cytokinesis while expanding the genetic toolkit available for S. acidocaldarius. ImportanceSulfolobus acidocaldarius is a key archaeal model organism for studying cellular processes shared with more complex life and is increasingly used for biotechnological applications. Here, we establish tryptophan auxotrophy as a new selectable marker in S. acidocaldarius, expanding the range of genetic selection systems available in this organism. By developing a defined Brock-based dropout medium, we enable stringent amino acid auxotrophy selection and precise control over nutrient composition. This system can be combined with existing uracil-based selection to support dual auxotrophy workflows, enabling co-transformation, simultaneous expression of multiple proteins, and more sophisticated genetic manipulation strategies. Using both markers, we show that S-layer proteins are localised to the division bridge in cytokinesis-arrested cells. This exemplifies ways in which the expanding molecular genetic tool kit available for Sulfolobus acidocaldarius is furthering our understanding of archaeal cell biology.

Contact with solid surfaces activates signaling pathways that promote biofilm formation in many bacteria. The alphaproteobacterium Caulobacter crescentus uses its flagellum to sense surfaces and responds by synthesizing an adhesive called the holdfast. The C. crescentus surface sensing pathway can be activated by mutating genes required for the assembly of the flagellum or genes required for chemotaxis. However, flagellar assembly and chemotaxis mutations activate distinct surface sensing pathways that differ in the activation of the diguanylate cyclase PleD. Here, we used a genome-wide screen to identify cmrA (CCNA_02061) as a crucial determinant of hyperadhesion in the chemotaxis mutant {Delta}cheYII. Genetic analysis showed that cmrA is important for activation of PleD in a context-specific manner. It is dispensable in wild-type and late-stage flagellar ({Delta}flgH) mutant backgrounds but promotes adhesion in early-stage flagellar assembly ({Delta}fliF), chemotaxis ({Delta}cheYII) and stator ({Delta}motB) mutant backgrounds. Fluorescently tagged CmrA displays a mostly cytoplasmic localization in genetic backgrounds where cmrA is dispensable for adhesion but localizes to the cell pole in backgrounds where it regulates adhesion. Structural modeling indicates that CmrA is a degenerate, catalytically inactive GGDEF/EAL domain containing protein, but cmrA alleles with mutated conserved c-di-GMP coordinating residues are unable to support hyperadhesion. Our results indicate that altering the directional switching of MotAB stators recruits CmrA to the cell pole where it activates PleD to drive surface adaptation. Ultimately, this work underscores the complexity of flagellar surface sensing by highlighting how the many rotational states of the motor stimulate distinct but overlapping c-di-GMP signaling pathways.

Photosynthetic hydrogen (photoH2) production by the cyanobacterium Synechocystis sp. PCC 6803 is an attractive means for storing solar energy. However, photoH2 yields remain limited by competing electron flux pathways. Recent in vitro characterization suggests that photoH2 production requires electrons from both carbohydrate oxidation and photosynthesis. Engineered fusions between photosystem I (PSI) and hydrogenase (PSI-H2ase) aim to divert electrons toward H2 production and rely exclusively on photosynthesis. Thus, photoH2 production differs fundamentally between wildtype (WT) and PSI-H2ase fusion mutants. Here, we show that photoH2 production in WT is enhanced by supplemented glucose, consistent with the recently reported confurcating nature of HoxEFUYH H2ases. PhotoH2 production was further studied in the new psaE-hoxUYH mutant by simultaneously monitoring electron flux through PSI alongside with turnover rates of O2, CO2 and H2. PsaE-hoxUYH achieved the highest photoH2 yield and longest production period among the currently available PSI-H2ase mutants in Synechocystis, prolonged by removing O2. Upon illumination, psaE-hoxUYH exhibited high initial photoH2 production rates, which decreased in parallel with CO2 fixation and ceased immediately in the presence of O2. In absence of O2, photoH2 production still declined slowly. Therefore, in addition to CO2 fixation and O2, other yet unknown factors might limit photoH2 production under these conditions. Moreover, we traced a previously observed high H2 production phase of unclear origin in psaD-hoxYH cultures to contaminating [FeFe]-H2ases from Clostridium intestinale rather than genuine photoH2 production by the mutant. Together, these findings indicate a complex metabolic interplay tuning photoH2 production in Synechocystis WT and PSI-H2ase fusion mutants.

Enterococcus faecalis is an opportunistic pathogen and facultative anaerobe that primarily relies on fermentative metabolism to colonize a wide range of aerobic and anaerobic environments. In the presence of exogenous heme, E. faecalis can assemble a minimal electron transport chain consisting of membrane-associated primary dehydrogenases, demethylmenaquinone, and the terminal cytochrome bd oxidase (CydAB). This respiratory chain is thought to generate a proton motive force to drive ATP synthesis via the F-type ATP synthase, thereby improving energy conservation under aerobic conditions. However, a cytosolic NADH oxidase (Nox) also consumes NADH and oxygen, potentially competing with the electron transport chain for reducing equivalents and terminal electron acceptors; but the relative physiological contributions of these two oxygen-reducing pathways remain poorly understood. To define the roles of CydAB and Nox under normoxic and hypoxic conditions, we constructed {Delta}cydAB and {Delta}nox mutants. Real-time, in situ measurements revealed {Delta}cydAB had no significant effect on oxygen utilization while in the {Delta}nox it was significantly reduced; revealing Nox as the major consumer of oxygen. Semi-untargeted metabolomic analysis further revealed oxidase-specific alterations in central metabolism with the {Delta}nox causing pronounced shifts in the ATP and NADH ratios; highlighting Nox as a key determinant of intracellular redox and energy homeostasis. Finally, single-cell fluorescence microscopy showed that membrane potential, a component of proton motive force, was substantially diminished only in the absence of both CydAB and Nox, or the F-type ATP synthase. These findings indicate that the F-type ATP synthase is a major generator of proton motive force, even upon aerobic growth, and demonstrate a complementary role for the electron transport chain and Nox in the bioenergetics of E. faecalis.

Burkholderia cenocepacia is an opportunistic pathogen associated with increased disease severity and mortality in cystic fibrosis (CF) patients. We have previously shown that elevated iron and acidic pH in the CF nutritional environment increases B. cenocepacia growth rate and decreases its susceptibility to some of the antimicrobials used clinically to treat CF infections. Here, we aimed to characterize B. cenocepacia physiology and its molecular response under acidic pH and increased zinc and iron concentrations using a modified synthetic CF sputum media (SCFM-FeZn). By investigating B. cenocepacia internal pH homeostasis, we found that it maintains a neutral internal pH when exposed to mildly acidic media at pH 5.5. We also assessed the effect of B. cenocepacia growth on the pH of SCFM-FeZn. When cultured at pH 6.8, B. cenocepacia maintained a media pH of [~]6.5. In contrast, when the culture pH value was initially 5.5, it increased to 6.5 during growth. Using comparative transcriptomics and metabolomics analysis, we identified 990 differentially expressed genes, and 23 differentially abundant metabolites in supernatants at acidic compared to neutral pH. Some of these genes and metabolites were involved in aromatic amino acid metabolism including the upregulated trpE gene, encoding a tryptophan biosynthetic enzyme. A tryptophan auxotrophic trpE deletion strain grew slower in SCFM-FeZn. Overall, this work identifies mechanisms involved in B. cenocepacia adaptation to acidic pH under conditions to model the CF nutritional environment. Some of these mechanisms are also associated with pathogenicity and virulence. ImportancePathogenic bacteria can be exposed to acidic pH inside and outside the host. Their ability to adapt to pH fluctuations contributes to success in host colonization. B. cenocepacia can grow at acidic pH ([~]3.5) and has been recovered from intracellular acidic compartments of amoebas and macrophages. Adaptation to acidic pH depends on molecular mechanisms that maintain a near optimal pH inside the cell for the function of vital processes. A few mechanisms that contribute to its adaptation to acidic pH have been described, but not in conditions reflecting the CF nutritional environment. Here, we identified multiple differentially-regulated systems that are associated with bacterial susceptibility to antimicrobials and pathogenesis. This research provides a better understanding of the role of acidic pH on B. cenocepacia physiology in the CF nutritional context and highlights possible systems that should be further characterized.

Despite growing interest in the MreBCD morphogenetic complex as a potential antimicrobial target, its function in Pseudomonas aeruginosa remains poorly understood. While previous studies using the MreB inhibitor A22 have established its role in cell shape maintenance and pilus regulation, the impact of mreB deletion has not been comprehensively investigated. Using genetic and microscopy-based approaches, we show that deletion of mreB is viable in P. aeruginosa, but results in spherical cells that lose all forms of motility despite retaining flagella. Importantly, we uncover a previously overlooked polar effect of the in-frame mreB deletion on the downstream mreCD genes and show, using CRISPRi-mediated silencing, that mreCD expression is essential for viability.{Delta} mreB mutants also display increased sensitivity to {beta}-lactam antibiotics and enhanced initial surface attachment, yet form more compact biofilms with reduced dispersal. In mixed-culture biofilms, spherical{Delta} mreB cells are outcompeted by rod-shaped wild-type cells and remain confined to the biofilm base. The identification of natural P. aeruginosa isolates carrying truncated mreB alleles further indicates that loss of MreB function can be tolerated in natural populations. Together, our findings reveal important contributions of the MreBCD system to viability, morphogenesis, motility and biofilm development in P. aeruginosa, providing new insights into bacterial adaptation and informing the development of targeted antimicrobial strategies.

Genome-wide viability catalogs produced by transposon sequencing (Tn-seq) and CRISPR interference (CRISPRi) have successfully mapped the essential genome of Streptococcus mutans. However, particularly for genes annotated as "hypothetical" or uncharacterized, translating these findings into mechanistic biological functions remains a significant bottleneck. In this study, we developed an integrated functional genomics pipeline combining predictive bioinformatics, tunable CRISPRi transcriptional silencing, transmission electron microscopy, transcriptomics, and genetic suppressor screens to characterize nine legacy hypothetical essential genes in S. mutans. Comparative transcriptomics and proteomics revealed a conserved baseline stress signature across diverse essential pathways, marked by the coordinated downregulation of the citZ-citB-idh metabolic locus and insoluble matrix synthesis enzymes (gtfBC), paired with the robust activation of the integrative and conjugative element TnSmu1. Against this backdrop of systemic stress, we successfully resolved the function of SMU_393, defining it as a functional equivalent of the pneumococcal regulator of chromosome segregation, RocS. Depletion of SMU_393 resulted in abnormal cell widening, hypersensitivity to DNA damage, and a significant subpopulation of anucleate cells. Remarkably, these phenotypes were bypassed by a spontaneous surface-exposed missense mutation (dnaAQ197E) within the AAA+ ATPase domain of the replication initiator. Together, this work uncovers an important cell cycle regulator and provides a framework for exploring uncharacterized essential genes of the oral microbiome. ImportanceAlthough genome sequencing has identified thousands of genes required for bacterial survival, the precise biological roles for many of them remain completely unknown. This study implements an integrated functional genomics pipeline to resolve the molecular functions of legacy uncharacterized essential genes in the oral pathogen Streptococcus mutans. We discovered a critical molecular checkpoint that acts as a physical anchor, linking the bacterial chromosome to the cell envelope to ensure that chromosome replication is synchronized with cell division. Remarkably, a single mutation in the replication machinery can fully bypass the loss of this anchor, maintaining proper genetic inheritance even during severe cellular stress. Ultimately, this study provides a pipeline for uncovering highly specific physiological vulnerabilities that can be exploited for targeted therapeutics against oral pathogens.

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.