Journal of Bacteriology

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.

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.

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.

Bacteria routinely respond to changing environments, with vastly different with nutrient availability. When nutritional changes are predictable, bacteria can deploy mechanisms to anticipate them. In these cases, an organism's past environment can shape its response to its current environment, which is often referred to as a history-dependent behaviour (HDB). Using nutritional shift assays, we investigated how prior growth phase influences subsequent growth across different carbon sources. Here, we show evidence of a novel HDB regulating galactose metabolism in Escherichia coli, where previous growth to exponential phase leads to a growth advantage in a galactose environment, with no comparable effect observed in all other carbon environments tested. We demonstrate a role of the galactose repressors, GalR and GalS, in regulating this behaviour, as it is lost in strains deficient in either repressor. Further, we show how this HDB is generalizable across a collection of E. coli isolates, but not Salmonella - a closely related species with a similar natural history. Together, these findings build on the existing knowledge of HDBs governing carbohydrate metabolism by demonstrating how growth phase history can influence an organism's response to fluctuating nutritional environments.

Opportunistic pathogens must coordinate virulence-factor production with changing conditions encountered during colonization and infection. Group B Streptococcus (GBS), a common human opportunistic pathogen, uses the CovRS two-component system to regulate virulence, but how the CovS sensor kinase is tuned to environmental conditions remains unclear. Here, we identify CflP, a noncatalytic membrane protein of the CPBP family, as an accessory regulator of CovS. CflP associates with CovS and promotes CovS-dependent phosphorylation of the response regulator CovR, thereby repressing virulence-associated genes, including the {beta}-hemolysin/cytolysin operon. Loss of CflP also enhances cytotoxicity toward human immune cells. CflP acts independently of Abx1, a previously identified homologous CPBP protein that promotes CovS phosphatase activity. CflP abundance is regulated post-transcriptionally by Mg2+ availability, linking an environmental cue to CovRS signaling. Comparative genomic analysis revealed distinct patterns of natural variation in these CovS regulators: cflP is predominantly maintained as an intact gene, although specific amino acid substitutions can compromise CflP abundance, whereas abx1 is frequently disrupted by recurrent frameshifts. Together, these findings reveal an accessory regulatory layer that tunes CovS activity in response to environmental conditions and whose natural variation may contribute to strain-dependent differences in CovRS regulation. More broadly, our findings illustrate how noncatalytic membrane proteins can modulate bacterial signaling to balance virulence-factor production with adaptation to changing host environments.

Bacteria utilize specialized protein secretion systems, particularly the type VI secretion system (T6SS), to dominate interbacterial competitions and kill the prey bacteria in a contact-dependent manner. Here, we report that disruption of the thioesterase gene encoded in the gramibactin (a bacterial siderophore) biosynthetic gene cluster activates the T6SS in the Burkholderia gladioli strain NGJ1, even in the absence of prey. The thioesterase mutant exhibits enhanced antibacterial activity, and ectopic expression of the gene complements the phenotype. Notably, thio mutant as well as thioesterase-complement strains show enhanced sensitivity to oxidative stress. The qRT-PCR analysis showed that the thio mutant had upregulated expression of RpoN1, a sigma factor, which positively regulates T6SS functions. We demonstrate that rpoN1 mutant is hypersensitive to oxidative stress and defective in T6SS activation, whereas overexpression of RpoN1 enhances oxidative stress tolerance in NGJ1 and activates T6SS. We propose that, during interbacterial conflicts encountered during plant colonization, NGJ1 faces an oxidative-stress-enriched environment that modulates thioesterase expression, induces RpoN1, and activates T6SS. Overall, our study exemplifies the broader physiological role of Thioesterase in maintaining intracellular ROS homeostasis in bacteria and stimulating antibacterial activity.

Integrative and conjugative elements (ICEs) are widespread in bacteria and typically confer important phenotypes to their hosts. Following excision from the host chromosome, an ICE can transfer to a new cell through the element-encoded conjugation machinery (a type IV secretion system) to create a transconjugant. Tn916, a well-studied ICE that confers tetracycline resistance, is found in several Gram-positive pathogens and has been widely studied in Bacillus subtilis. Unlike most ICEs, Tn916 causes host-cell death following its activation (excision and gene expression). We found that orf7 and orf8 (renamed sig7 and sig8), two regulatory genes in Tn916, encode a two-subunit (split) sigma factor of RNA polymerase, Sig7-8, that activates transcription from Porf7 (now Psig7) and is required for excision of the element. Importantly, production of Sig7-8, either from Psig7 or another strong promoter, caused cell death. Using a Cre recombinase reporter to detect gene expression, we detected little or no transcription from Psig7 in transconjugants. This lack of transcription from Psig7 in transconjugants was essential for fitness of both the element and its host: forcing transcription from Psig7 in transconjugants caused either loss of Tn916 or death of nascent transconjugants. Our findings indicate that, unlike other well-studied ICEs (e.g., ICEBs1) that use repressors to control gene expression and are expressed in nascent transconjugants, most Tn916 genes remain off following transfer to a new host. This regulation prevents Tn916 from killing transconjugants and allows successful horizontal transfer and maintains the potential for future activation in the new host.

Maintaining proper cation homeostasis is essential for the survival of all living organisms. During infection, Salmonella must adapt to host-imposed magnesium (Mg2+) limitation. To compensate, Salmonella induces high-affinity Mg2+ transporters and synthesis of polyamines, organic cations that partially substitute for Mg2+. Once Mg2+ levels are restored and cells enter stationary phase, excess cations must be exported; failure to do so results in loss of viability. The CorC-domain protein PaeA exports excess polyamines, whereas MgpA and CorC reduce intracellular Mg2+ levels. Here, we identify two new CorC-domain proteins, MgpB (formerly YegH) and CorB, as mediators of Mg2+ homeostasis, showing that they also reduce intracellular Mg2+ accumulation and protect cells from endogenous excess-cation stress. Genetic analyses revealed the functional redundancy among these proteins in conferring resistance to exogenous high Mg2+, endogenous stress induced after Mg2+ starvation, and egg whites. Deletion of mgpB and/or corB conferred little to no phenotype. However, these mutations showed synergistic phenotypes in strains lacking MgpA. This synergistic relationship was also evident in mouse competition assays. Lack of MgpA, CorC, MgpB, and CorB leads to an increase in intracellular Mg2+ levels. Moreover, expression of mgpB and corB are increased in high Mg2+, all consistent with their role as putative Mg2+ exporters. Together, these findings identify MgpB and CorB as new mediators of Mg2+ homeostasis and reveal a multilayered system that enables Salmonella to finely balance intracellular cation levels during environmental and host-imposed stress.

Spatial organization during bacterial fission is critical to ensure that division results in identical, viable progeny cells. In Escherichia coli, one important system that accomplishes this is the Min system, comprising MinC, MinD, and MinE proteins. The Min system regulates assembly of the FtsZ-ring to ensure proper placement of the division machinery at midcell. We explored the dynamics of the Min system and demonstrate that Min oscillation and release of MinD from the membrane is modulated by MinC. Deletion of the minC gene from a strain expressing minD fused to the gene encoding green fluorescent protein (Gfp) resulted in faster pole-to-pole oscillation of Gfp-MinD in vivo, leading to a shorter period. This was also observed in a strain expressing the MinC(R133A), a variant mutated at the MinC-MinD interface. In phospholipid binding and release assays in vitro, we observed that the concentration of MinE required to release MinD was lower with MinC, suggesting that MinC activates MinD for MinE-dependent release. Oscillation is produced by a diffusion-capture-release mechanism occurring at the membrane with proteins proceeding toward a pole. To determine if an increase in the diffusive proportion of MinD leads to faster oscillation, we constructed MinD membrane targeting sequence (MTS) variants to reduce the membrane associated pool of MinD. MTS variants induced a range of oscillation defects, with MinD(KR265AA) oscillating faster in vivo. Together our results show that MinC is an effector that modifies Min oscillation by priming MinD for MinE-dependent membrane release and fine-tunes the Min oscillation rate in dividing cells.

During phage infection, phages reprogram their host to secure energy, building blocks for their genome, and proteins, as well as the transcription and translation machinery required to replicate their genome and produce phage particles. This also includes reprogramming the host transcriptome to modify host gene expression and align it with the needs of the phage during infection. In this study, we present a putative early-expressed protein, Gp204 of Mosigvirus AV110, renamed Host transcription modulator (Htm), that has a broad impact on the extended-spectrum beta-lactamase (ESBL)- associated E. coli transcriptome. Overexpression of Htm decreased the growth rate of ESBL-E. coli and caused DNA condensation. Htm is structurally similar to bacterial enhancer-binding proteins, binds ssDNA, and alters the expression levels of over 700 E. coli genes when expressed. The majority of these genes were upregulated and are involved in multiple pathways, such as galactose metabolism and pentose and glucuronate interconversions, which may promote the synthesis of purines, pyrimidines, and histidine, all of which are required for the replication, transcription, and translation of the phage genome. Furthermore, energy production is likely to be upregulated by increased availability of acetyl-CoA, resulting from, among other factors, fatty acid degradation. Htm also downregulated the expression of the structural genes involved in flagellar assembly and multiple two-component systems. In conclusion, Htm could promote phage progeny production by increasing the availability of nucleotides, amino acids, and energy, while inhibiting non-essential processes, such as flagella formation.

During infection, Klebsiella pneumoniae must withstand host-derived oxidative stress not only by detoxifying reactive oxygen species (ROS) but also by repairing oxidative damage to cellular physiology. How these detoxification and stress-adaptive responses are coordinated remains unclear. Here, we identify a plasmid-encoded tacAT locus as an OxyR-responsive regulatory module that supports peroxide tolerance and in vivo fitness in K. pneumoniae. Guided by the conservation of the OxyR DNA-binding domain and its consensus binding motif, we surveyed promoter-proximal regions of the K. pneumoniae CRK3022 chromosome and plasmids for candidate OxyR-responsive loci. This analysis identified tacAT, a putative type II toxin-antitoxin (TA) module, as the only plasmid-associated candidate. OxyR directly bound the tacAT promoter and contributed to tacAT induction during hydrogen peroxide (H?O?) stress. The TacAT complex exhibited DNA-binding and transcriptional regulatory activity, promoting the expression of genes linked to protein quality control and membrane/envelope homeostasis, including clpB, htpG, cadC, bhsA, and marA. Deletion of tacAT compromised survival under lethal peroxide challenge and attenuated bacterial dissemination, tissue pathology, and inflammatory responses in a murine bacteremia model. These findings define an OxyR-TacAT regulatory branch that connects peroxide sensing with stress-adaptive gene expression, revealing how a TA-associated module can be integrated into stress-adaptive programs during host-associated oxidative stress.

Pre-prolipoprotein phosphatidylglycerol diacylglyceryl transferase (Lgt) catalyzes the first step in the lipoprotein modification pathway and is essential for growth and viability of Helicobacter pylori. We explored the fate of H. pylori cells under Lgt depletion conditions to obtain insight into Lgt function on morphology and physiology. Downregulation of lgt expression led to growth arrest and a heterogenous population of small rod-shaped cells and small round cells with spiky protrusions, a loose outer membrane and disintegrated flagella. H. pylori overcomes a defect in lipoprotein modification through alteration of its gene expression profile resulting in so-called revertants. Outer membrane protein synthesis, carbon metabolism and nutrient uptake was reduced, and protein translation and flagellar biogenesis increased. Lgt specific regulation was observed in phospholipid biosynthesis resulting in an initial increase in cardiolipin followed by an increase in lysophospholipids at the time of adaptation. Suppressors of {Delta}lgt that were selected on solid media under restrictive conditions either lacked chromosomal mutations or had mutations in the cagUT-tetR regulon controlling the expression of lgt. A suppressor without mutations had an altered expression profile that led to downregulation of a DNA methyltransferase and upregulation of HP_0014, a motility associated glycosyltransferase family protein. Transmission electron microscopy and single cell video microscopy showed that a subpopulation of cells is motile in the revertant while the suppressor regained full motility with higher average speed compared to the wild type strain. Overall, our data show that lipoprotein modification by Lgt is important for membrane and flagellar integrity and that the heterogenous character of H. pylori cultures allows individual cells to overcome this cell envelope stress and survive. Our findings are important for antibacterial development strategies targeting essential integral membrane enzymes involved in lipoprotein modification and stimulate exploration of synergetic activity between known antibiotics and novel antibacterial agents targeting novel cellular pathways.

Vibrio cholerae, the facultative pathogen underlying cholera, naturally inhabits warm aquatic estuaries. Environmental persistence is enhanced by the ability of V. cholerae to colonize host reservoirs and form multicellular biofilms, causing seasonally endemic outbreaks in many tropical regions. Most toxigenic strains utilize the type IVa mannose-sensitive hemagglutinin (MSHA) pilus for host reservoir colonization and biofilm formation. Temperature and salinity can alter V. cholerae biofilm formation, yet their impact on MSHA production specifically remains largely unknown. Here, we utilized transcriptional reporters of predicted msh promoters (msh-P1/msh-P2/msh-P3) and functional assays, to determine temperature and salinity impacts on msh expression and pilus biogenesis. Under standard laboratory conditions (30{degrees}C, 1% NaCl) only msh-P1/P2 are active and inversely-coordinated with one another. Both msh-P1/P2 activity were elevated by high temperature (37{degrees}C) and low salinity (0.25%/0.5% NaCl), and reduced by low temperature (20{degrees}C/25{degrees}C) and high salinity (2%/3% NaCl). Temperature-mediated alterations in promoter activity were not immediately reflected in changes to cell-surface MSHA levels, whereas high salinity led to decreased MSHA production. Combining high temperature (37{degrees}C) and high salinity (2%/3% NaCl), attenuated the salinity-mediated reduction of msh-P1/P2 activity. Biofilm biomass levels were only substantially heightened at 25{degrees}C and 20{degrees}C, likely a result of no temperature-dependent changes in cell-surface MSHA, and additional temperature-controlled biofilm regulation previously described. We also found msh-P1/P2 promoter activity and MSHA production varies widely across toxigenic O1 and O139 serogroups despite complete sequence homology. These results shed new light on how key signals regulate MSHA pilus production to support V. cholerae persistence in aquatic environments.

A subgroup of membrane-anchored bacterial Alkaline Phosphatase superfamily members transfers phospholipid headgroups from phospholipids to diverse acceptor molecules. As part of type II and hybrid type I/II cellulose biosynthesis gene clusters BcsG transfers phosphorylethanolamine pEtN from phospholipid to the emerging 1,4 {beta}-D-glucan chain synthesized by the BcsABC cellulose biosynthesis nanomachine. BcsG homologs throughout the phylogenetic tree possess a uniquely structured tripartite linker that connects the fifth transmembrane domain with the periplasmic catalytic domain. A N-terminal unstructured sequence of highly variable length and biased amino acid composition, the N-linker, is followed by an -helix and an unstructured sequence of high similarity and constant length leading into the first {beta}-strand of the catalytic domain. With BcsG from Salmonella typhimurium synthesizing pEtN cellulose as a model, linkers were shown to be promiscuous and linker length positively correlated with transfer efficiency of the pEtN headgroup and protein stability. As the donor substrate, the phosphatidylethanolamine content of the membranes affects pEtN transfer efficiency. With Alkaline Phosphatase superfamily members to be promiscuous enzymes the catalytic domain of OpgE transfers pEtN to osmoregulated periplasmic glucan. BcsG-OpgE hybrid proteins cause colony morphology alterations thus potentially transferring pEtN to the BcsA synthesized glucan chain. With BcsG with shortest linkers not associated with a cellulose biosynthesis gene cluster, multiple hypotheses can be proposed that select for different length of the N-linker in BcsG homologs located within a cellulose biosynthesis gene cluster in other Pseudomonadati.

Prevotella melaninogenica is a Gram-negative bacterium that has been implicated in numerous disorders such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis, and despite its abundance in the human microbiome, there is still limited knowledge on the fundamental physiology describing Prevotella melaninogenica. To properly advance Prevotella biology, it is critical to report and document such characterizations of Prevotella; therefore, the purpose of this study is to examine and define the phenotypic and growth characteristics of P. melaninogenica ATCC 25845. In this study, we found that Prevotella melaninogenica ATCC 25845 is a Gram-negative bacterium that is predominantly short and medium rod-shaped, with occasional filamentous rods. SEM imaging revealed that this filamentation may serve as division centers, points from which multiple daughter cells form. We also describe the antibiotic profile, which showed susceptibility to four classes and resistance to six classes of antibiotics. Carbon source growth kinetic characterization revealed a preference for carbohydrate sources.

Thermophilic bacterial lipases must combine structural stability with the conformational flexibility required for interfacial activation, yet the local organization of their metal-binding sites remains incompletely understood. Here, we determined two X-ray crystal structures of the family I.5 lipase from Geobacillus kaustophilus (GkLip). The 2.47-[A] orthorhombic model contains a Ca2+ ion at the conserved metal-binding site, whereas the 2.30-[A] anisotropic triclinic model contains four non-equivalent protein chains in which no Ca2+ ion was modeled. GkLip retains the canonical /{beta}-hydrolase fold, the Ser114-Asp318-His359 catalytic triad, the Phe17/Gln115 oxyanion-hole region, an extended lid, and a conserved Zn2+-binding site. In the orthorhombic structure, Gly287, Glu361, Asp366, and Pro367 contribute to the Ca2+ coordination environment. Pro367 adopts a C{gamma}-endo pucker in the Ca2+-containing structure; three Ca2+-free chains also adopt the endo pucker, whereas one adopts a C{gamma}-exo pucker. The presence of both Pro367 pucker states in the Ca2+-free structure is consistent with calcium preferentially stabilizing a pre-existing, coordination-compatible conformation. This local organization is further supported by an Arg35-centered network of direct and water-mediated contacts. Comparison with the 94%-identical open G. thermocatenulatus lipase reveals substantial lid rearrangement despite strong conservation of the protein core. Together, these structures highlight localized conformational variability at the calcium-binding region within an otherwise highly conserved thermophilic lipase scaffold.

Metformin is the first-line therapy for type 2 diabetes, yet the mechanisms underlying its glucose-lowering effects remain incompletely understood. Increasing evidence implicates gut microbiota as an important mediator of metformin action, but the bacterial targets of the drug remain poorly defined. Here, we identify bacterial NADH oxidoreductase (NDH-1), the homolog of mitochondrial complex I, as a key target of metformin. Metformin inhibited bacterial growth at millimolar concentrations and, at subinhibitory concentrations, markedly impaired carbohydrate fermentation in Escherichia coli and other NDH-1-containing bacteria. Supplementation with nicotinic acid and Mg2+; restored fermentation, whereas pyruvate bypassed the metabolic defect, implicating impaired NAD+; regeneration and disruption of metabolism upstream of pyruvate formation. Genetic analysis further established NDH-1 dependence: metformin failed to inhibit fermentation in an E. coli delta-nuoF mutant lacking functional NDH-1 but retained activity in an NDH-2-deficient delta-ndh strain. Molecular docking predicted metformin binding within the NDH-1 quinone-binding region, proximal to conserved residues involved in quinone reduction. Consistent with this mechanism, Staphylococcus aureus, which lacks NDH-1, was not affected in carbohydrate fermentation, while several gut-associated probiotic and short-chain fatty acid-producing bacteria, including Lactobacillus, Bifidobacterium, and Akkermansia muciniphila, lacked detectable NDH-1 homologs, suggesting intrinsic resistance to this metabolic effect of metformin. These findings identify bacterial NDH-1 as a metabolic target of metformin and provide a mechanistic framework for its effects on the gut microbiota. Selective inhibition of NDH-1-dependent bacteria could alter microbial substrate utilization and ecological interactions in the intestine, potentially contributing to the microbiome-mediated component of metformin's antidiabetic effects.