Journal of Bacteriology

Carbonic anhydrase (E.C. 4.2.1.1) is an enzyme that catalyzes the reversible hydration of CO2 to carbonic acid (H2CO3), which dissociates to bicarbonate (HCO3-) at intracellular pH. Copious amounts of CO2 are produced by catabolism, although much of this is lost by diffusion from the cell. In Bacillus subtilis, anabolic processes rely largely on bicarbonate as a substrate rather than CO2. While CO2 reacts spontaneously with water to yield bicarbonate, the rate of this reaction is too slow to keep up with cell requirements. B. subtilis encodes three putative {beta}-class carbonic anhydrases, here renamed canA(yvdA), canB(ytiB), and canC(ybcF). The canC gene is encoded in an operon with ndhF-mpsB(ybcC), which encodes a candidate MpsAB-type bicarbonate transporter. Here we demonstrate that a strain lacking canA, canB, canC, and mpsB ({Delta}4) has a severe growth defect at atmospheric CO2. This defect can be overcome by growing cells with supplemental CO2 or by plating at high cell density. We isolated suppressors of {Delta}4 and identified mutations in resD that suppress the requirement for supplemental CO2 to support growth. ResD functions as a global regulator of genes important for both aerobic and anaerobic respiration. We demonstrate that a resD null mutation results in metabolic changes that lead to an increased generation of CO2. We infer that this results in an increase in spontaneous bicarbonate formation that is sufficient to support cell growth. We conclude that the requirement for bicarbonate concentrating mechanisms may be bypassed under conditions that increase endogenous CO2 generation.

Redox reactions mediated by ferredoxin/flavodoxin-NADP+ oxidoreductases (FNRs) and their associated electron-carrier proteins, ferredoxins and flavodoxins, are essential in biology. Although the biochemical activities of these redox proteins are conserved, their precise physiological roles can differ among organisms and cannot be easily inferred. Here we have defined an essential role for Bacillus subtilis YumC, a member of a distinct group of bacterial FNRs that resemble thioredoxin reductase. We have used targeted protein degradation, cytological profiling, metabolomics, and genetic complementation to show that YumC catalyzes the transfer of electrons from NADPH, through ferredoxin (Fer) or through the flavodoxin YkuP, to the isoprenoid biosynthesis pathway, and specifically to the redox enzyme IspG. When YumC was degraded, isoprenoid biosynthesis was compromised, and the level of undecaprenyl phosphate, the isoprenoid lipid carrier for peptidoglycan building block translocation, was diminished. Degradation of YumC or of Fer in a {Delta}ykuP strain led to defective peptidoglycan biosynthesis, activation of the {sigma}M-dependent cell-wall stress response, and lethality. The introduction into B. subtilis of an alternative pathway for isoprenoid biosynthesis that does not require input from electron-carrier proteins could complement the degradation of Fer in a {Delta}ykuP strain, but not the degradation of YumC. This finding indicates that YumC is required for other essential processes that do not necessarily involve Fer and YkuP. This work provides an explanation for why YumC is essential, reveals how reducing power is delivered to isoprenoid biosynthesis in B. subtilis, and illustrates how the varied roles of redox systems among bacteria depend upon metabolic context.

Short-chain fatty acids (SCFAs) such as acetate and propionate represent important carbon and energy sources for environmental pseudomonads, requiring coordinated regulation to ensure efficient assimilation while maintaining metabolic homeostasis. In Pseudomonas alloputida KT2440, the two-component system MxtR/ErdR (CrbS/CrbR) is known to activate acetate assimilation through regulation of acsA-I. However, the full extent of the MxtR/ErdR regulon and its physiological role beyond acetate metabolism have remained unclear. We demonstrate that MxtR/ErdR directly regulates the methylcitrate cycle and coordinates both acetate and propionate metabolism. To define the MxtR/ErdR regulatory network, we combined comparative transcriptomics, physiological analyses, promoter-reporter assays, electrophoretic mobility shift assays (EMSAs) and targeted mutagenesis. Comparative transcriptomic analyses of the mxtR-H806N and {Delta}erdR mutants revealed extensive changes in gene expression, including coordinated downregulation of genes involved in propionate metabolism alongside genes associated with central carbon metabolism, transport, chemotaxis, and signal transduction. Consistent with these transcriptional changes, deletion of either mxtR or erdR abolished growth on propionate. Promoter-reporter assays and EMSAs demonstrated direct binding of ErdR to a conserved imperfect inverted repeat upstream of the prp gene cluster and prpE. Mutational analyses confirmed the functional importance of this binding motif for promoter activation. In addition, MxtR/ErdR contributed to pyruvate utilization through regulation of transport-associated genes, whereas deletion of downstream target genes only caused modest phenotypes, indicating that the physiological effects of MxtR/ErdR arise from coordinated regulation of multiple pathways. Our findings substantially expand the MxtR/ErdR regulon and identify this signaling system as a central regulator of SCFA homeostasis in P. alloputida KT2440.

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.

Bacillus subtilis is the best-studied Gram-positive microorganism, but little is known about how the presence and utilization of diverse carbon sources impacts its production of biofilm. Here, we have identified gluconate as a carbon source that induces biofilm wrinkling in B. subtilis colony biofilms. Targeted phenotypic analysis revealed that the genes encoding the canonical structural biofilm components (tasA, epsA-O, and bslA) are required for this response. Import and metabolism of gluconate, however, are not required to induce these phenotypic changes. We show that the effect of gluconate on B. subtilis is linked to iron availability: production of the siderophore bacillibactin is decreased by gluconate and B. subtilis mutants unable to import bacillibactin grow better and wrinkle more in the presence of gluconate. In addition, supplementation of single-carbon media with iron dramatically increased growth and wrinkling of a B. subtilis bacillibactin mutant grown on gluconate but did not impact B. subtilis grown on glucose. We conclude that gluconate acts as a signal to increase biofilm in B. subtilis and increases iron availability in a bacillibactin-independent manner.

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.

The Pseudomonas aeruginosa type III secretion system (T3SS) is an important virulence determinant used to mute and/or impair host immune defenses. Traditional transposon mutagenesis screens have been instrumental in identifying genes required for T3SS gene expression but our understanding of how those genes orchestrate regulatory control remains incomplete. A comprehensive inventory of the genes involved in the control of T3SS gene expression would help define regulatory mechanisms and provide additional targets for anti-virulence therapeutics. Here, we designed a fluorescently tagged transposon mutagenesis screen (FT-Tn-seq) and identified over 70 new genes important for T3SS gene expression. A role for 31 of those genes was validated using a secondary screen. Several candidate genes were selected for further studies. We demonstrate that deletion of shaC, which encodes a sodium-proton antiporter subunit, results in a significant defect in T3SS gene expression. That defect results from a combined effect on exsA transcription and ExsA translation. Deletion of wspF, a cyclic-di-GMP phosphodiesterase, results in a significant reduction of T3SS expression in strain PAK, but not in PA103. The effect on the T3SS is likely due to elevated levels of c-di-GMP in PAK. Finally, we identified a new sRNA (ivy) that participates in the control of the T3SS. Expression of ivy inhibits T3SS gene expression and requires the RNA-chaperone Hfq for inhibitory activity. Findings from this study will open new research avenues to better understand regulation of T3SS gene expression in P. aeruginosa and develop T3SS-specific therapeutic interventions.

It is common for bacteriophages to encode proteins that are strongly inhibitory to growth of the bacterial host, and about 10% of mycobacteriophage-encoded proteins have this property. Adephagia is a Cluster K1 mycobacteriophage and prior cloning and expression of 66 Adephagia non-structural genes identified 14 that are toxic when expressed in Mycobacterium smegmatis. One of the expressed proteins, the 70-residue gp48, is highly toxic when expressed in both M. smegmatis and Mycobacterium abscessus and acts by binding to and inactivating the function of Msmeg_1828, a mannose-1-phosphate guanylyltransferase (Mpg). Mpg is an essential enzyme for biosynthesis of GDP-mannose, a precursor of several key cell wall constituents including lipoarabinomannan and phosphatidylinositol mannosides. The crystal structure of Adephagia gp48 shows that the N-terminal 43 residues form two alpha helices that strongly promote dimer formation; the C-terminal 27 residues are disordered but are predicted to bind Fe-S clusters via cysteine and histidine residues. Adephagia gp48 inhibits Mpg guanylyltransferase activity in vitro and is predicted to interrupt Mpg dimer formation. The C-terminal metal binding activity of gp48 is not required for toxicity, and non-toxic mutants have substitutions in the N-terminal alpha helices that are involved in dimerization. The selective advantage of Mpg inactivation for the phage is unclear, but it may protect from competing phages that require GDP-mannose derived molecules for efficient infection.

In the environment, bacteria mainly exist in multicellular, surface-adhered communities called biofilms. One key feature of the biofilms is a self-produced extracellular matrix that embeds cells within a structured community and contributes to their adhesion, organization, and retention. The matrix of most characterized biofilms contains three primary components: polysaccharides, protein, and extracellular DNA (eDNA). Upon biofilm induction, Bacillus subtilis produces and secretes a polysaccharide known as exopolysaccharide (EPS). The 15 genes of the epsA-O operon encode proteins that coordinate the assembly, export, and polymerization of EPS oligosaccharide (glycan) subunits. While much progress has been made to elucidate the chemical structure of EPS, further work is needed to determine the contributions of some eps genes to EPS biosynthesis experimentally. Here, we generated a collection of marker-less, in-frame deletions targeting each eps gene in the epsA-O operon in B. subtilis strain NCIB 3610 using the Cre system. Biofilm phenotypes of resulting mutants were used to assess each gene's importance to biofilm development. Impact The epsA-O single-gene deletion collection was designed to enable systematic assessment of the contribution of each eps gene to EPS production and biofilm-associated phenotypes. When combined with current knowledge of EPS structure, this collection will help clarify the roles of individual genes in EPS biosynthesis. This approach can also be easily adapted for the systematic analyses of other large, complex operons in diverse bacterial species.

Pseudomonas aeruginosa is a metabolically versatile opportunistic pathogen capable of adapting to diverse and challenging environments, including the oxygen- and nutrient-limited conditions encountered during chronic infections. Its ability to utilize a broad range of carbon sources and to switch from aerobic to anaerobic respiration contributes to its persistence within the host and its resilience to therapeutic interventions. In particular, denitrification enables P. aeruginosa to grow under oxygen-limited conditions by using nitrate and nitrite as alternative electron acceptors, a process particularly relevant in biofilms and chronic infections such as cystic fibrosis. Ubiquinone (UQ) is essential for respiratory metabolism in P. aeruginosa, which possesses two distinct UQ biosynthetic pathways that ensure UQ production across a broad range of oxygen concentrations. Recent studies have identified the O2-independent UQ biosynthetic pathway as a key determinant of chronic infection. Because this pathway is restricted to a limited group of bacteria, we hypothesized that its selective inhibition could represent a strategy to specifically target P. aeruginosa under anaerobic conditions. To test this hypothesis, we screened analogues of 4-hydroxybenzoic acid (4-HB), the earliest precursor of the UQ biosynthetic pathway, and identified 4-aminobenzoic acid, also known as para-aminobenzoic acid (pABA), as a potential inhibitor. pABA primarily affects the anaerobic metabolism of P. aeruginosa by acting as a competing substrate for UQ biosynthetic enzymes, thereby interfering with UQ biosynthesis. This inhibitory effect positions pABA as a valuable tool for investigating, and potentially targeting, anaerobic metabolism in P. aeruginosa.

The endonuclear symbiont Holospora obtusa invades the macronucleus of Paramecium caudatum using an infectious-form invasion-tip containing a major 89-kDa periplasmic protein implicated in escape from the host digestive vacuole and macronuclear invasion. Sequence-based analyses placed this protein within a Holospora-specific family and predicted an N-terminal membrane-anchoring module and a long alpha-helical C-terminal region. AlphaFold2 modeling followed by molecular dynamics indicated that the C-terminal region forms an antiparallel, spectrin-repeat-like helical solenoid containing a localized three-helix coiled-coil. The protein exhibits a large pH-dependent charge difference, becoming 55 units more positive at pH 4 than at pH 7. Independent simulations at these pH values differed mainly in the relative orientation of the N-terminal core and C-terminal rod, described by DynDom as an approximately 35 degrees rigid-body rotation about a hinge centered on Gly291. However, none of the nearby glutamates (Glu273, Glu275, and Glu302) formed a pH-dependent salt bridge, and no specific protonation-dependent interaction responsible for the conformational difference was identified. We nonetheless propose, as a testable hypothesis, that acidification of the digestive vacuole may promote this reorientation, thereby repositioning the N-terminal membrane-anchoring module at the bacterial surface as an early step toward escape from the vacuole.

Ethanolamine utilization is an important metabolic capability of enteric bacteria and has been associated with bacterial fitness and adaptation within diverse host environment. In Salmonella enterica serovar Typhimurium, ethanolamine is metabolized within the bacterial microcompartment (Eut MCP), which is composed of five shell protein subunits. Deciphering how self-assembled shell proteins spatially organize the pathway, retain metabolic intermediates and create proper environment for catalysis is crucial for understanding role of Eut MCP in cellular metabolism and pathogenesis. Through genetic mutant construction, growth and metabolic assays, microscopy, and metabolite quantification, we demonstrate that each shell protein fulfills a distinct role to the functioning of the Eut MCP. Our findings provide mechanistic insights into the structural organization and functional dynamics of the bacterial microcompartment organelle.

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.

Jeongeupia sacculi (HS-3) is a novel {beta}-proteobacterium with a unique multicellular life cycle. The genetic tractability and mechanisms underlying differentiation are unknown. Here, we adapt a method of homologous recombination for genetic modification in HS-3 and generate a disruption mutant of rpoS, which encodes the alternative sigma factor{sigma} S commonly associated with general stress response or major behavioral switches. We then characterize phenotypes associated with rpoS disruption. Under low-nutrient conditions, rpoS disruption reduced Congo red binding, consistent with rpoS acting as a regulator of low-nutrient response by upregulating production of biofilm-associated materials. Surprisingly, rpoS disruption did not significantly alter survival in response to other stressors in general stress response assays tested. Our work establishes a method for genetic manipulation in HS-3 and enables future investigation into the life cycle of HS-3, including whether rpoS has a role in differentiation triggered by low-nutrient response.

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.

Elucidating how microbes associate with their hosts and respond to different environments is fundamental to our understanding of symbiosis. Here, we investigate the molecular and genetic mechanisms enabling the honey bee symbiont Bombella apis to colonize and persist in harsh honey bee microenvironments, like royal jelly and the larval gut. By using evolved B. apis strains, we demonstrate that the broadly conserved two-component system (TCS) ChvGI is integral for the bacteriums ability to withstand royal jelly and the larval gut. Furthermore, we show that in B. apis ChvGI also regulates critical bacterial behaviors such as motility, flagellar production, and biofilm formation. In other Alphaproteobacteria, ChvGI is known as a master regulator that mediates the bacterial responses to stressors and is also found to be involved in symbiosis, often through the production of EPS and biofilm formation. Our work underscores the broader significance of ChvGI as an important TCS in Alphaproteobacteria that regulates bacterial behavior in response to different types of stressors and uncovers a genetic mechanism that allows an important honey bee symbiont to survive in diverse host environments.