During mixed-acid fermentation, Escherichia coli metabolizes glucose to a mixture of acetate, ethanol, formate, lactate, and succinate, together with equimolar amounts of H2 and CO2. These gases are generated via the disproportionation of formate by formate hydrogenlyase (FHL-1). This reaction provides the cell with its only source of internally generated CO2 for carboxylation reactions. Formate is produced by pyruvate formate-lyase (PflB), and its balanced level within the cell is maintained by the activities of PflB, FHL-1, and the bidirectional formate channel, FocA. A formate imbalance results when strains synthesize a FocAN209 variant that continuously and efficiently exports formic acid. During fermentative growth, a FocAN209-producing strain prematurely enters stationary phase and fails to synthesize FHL-1 due to intracellular formate insufficiency. Here, we show that intracellular pH (pHi) of the mutant was increased relative to the parental strain, correlating with reduced ATP levels. Anaerobic growth could be partially restored by cultivation with bicarbonate; however, pHi was lowered only marginally and ATP levels remained low. Determination of the fermentation-product profile revealed that the continuous loss of formate from the cell could not be compensated by bicarbonate supplementation. A severe limitation in both lactate and succinate production was also only partially rescued by bicarbonate. Increased ethanol production indicated that acetyl-CoA was sacrificed in the mutant to re-oxidize reduced pyridine nucleotides. These findings demonstrate that an imbalance in intracellular formate not only negatively impacts carboxylative metabolism but also remodels the spectrum of fermentation products to the detriment of the cell's energy metabolism and pH homeostasis.IMPORTANCEFormate is central to the fermentative metabolism of Escherichia coli. Balanced formate levels are required for DNA synthesis and for CO2 generation by formate hydrogenlyase (FHL-1). An E. coli mutant synthesizing an FocA variant that irreversibly exports formate has a severe growth defect because it cannot make FHL-1, which severely restricts CO2 and negatively impacts pH homeostasis by lowering ATP levels. We show, however, that impaired growth of the mutant can be only partially restored by supplying bicarbonate. The underlying cellular lack of formate cannot be fully compensated by bicarbonate because efficient pyruvate formate-lyase-dependent pyruvate cleavage remodels fermentation away from acetate and toward ethanol production, which ensures redox balance, but generates less ATP by substrate-level phosphorylation.
Mycobacterium tuberculosis (Mtb) causes over 1 million deaths from tuberculosis (TB) every year and remains a major burden on human health. Reducing the deadly impact of TB requires a better understanding of the strategies used by M. tuberculosis to adapt its metabolism, survive, and persist in the human host. Previous enzymological studies reported that the Mtb rv3400 gene encodes a β-phosphoglucomutase; however, its role in M. tuberculosis metabolism had not been investigated. Here, we show that deletion of rv3400 causes a 30-fold increase in β-D-glucose-1-phosphate, confirming its primary function as a β-phosphoglucomutase. Deletion of rv3400 also causes a growth defect when trehalose is the sole carbon source. Targeted metabolomics revealed that metabolites associated with redox homeostasis, including ergothioneine, mycothiol, and mycothione, are decreased in the Mtb Δrv3400 strain compared with the wild type, indicating altered tolerance to redox stress. Consistent with this, the Δrv3400 strain showed increased susceptibility to oxidative stress induced by H2O2 and cumene hydroperoxide, conditions that Mtb encounters during infection. This work advances our understanding of trehalose metabolism in Mtb and suggests that Rv3400 may represent a target for the development of new antimicrobial therapies. IMPORTANCE:Trehalose metabolism plays a cornerstone role in Mycobacterium tuberculosis physiology and virulence. A better understanding of the metabolism of this essential disaccharide is therefore required to develop new strategies to eradicate tuberculosis. Here, we characterize M. tuberculosis lacking the β-phosphoglucomutase encoded by rv3400. We show that deletion of rv3400 leads to accumulation of β-D-glucose-1-phosphate, impaired growth when trehalose is used as the sole carbon source, and increased susceptibility to oxidative stress. Taken together, these data provide evidence that Rv3400 is required for optimal catabolism of trehalose.
Background Cross-infection may occur if alginate impressions are not disinfected. Available chlorine (AC), though common, poses safety risks. Chlorogenic acid (CA), a natural compound, shows effective disinfection and, as we previously verified, inhibits pathogens without damaging impressions.Objective This study investigates the influence of CA and AC on the oral microbial community of alginate impressions from a community-ecology perspective.Methods The maxillary impressions of 60 participants were randomly assigned to four groups (n = 15). The NS-AC group received a normal saline (NS) rinse and a 2,000 mg/L AC spray. The NS-CA group got an NS rinse and a CA (10 mg/mL mix + 60 mg/mL spray). The CA-AC group obtained a 10 mg/mL CA rinse and a 2,000 mg/L AC spray. The CA-CA group acquired a CA (10 mg/mL rinse & mix + 60 mg/mL spray). An analysis of 16S rRNA sequencing was conducted on the samples that were collected.Results The NS-CA group exhibited substantially higher Chao1, Shannon and Simpson indices than the NS-AC group (p < 0.05), and the PCoA revealed a centralized clustering pattern. As the primary biomarkers in the NS-CA, Neisseria, Porphyromonas and Prevotella were identified through LEfSe analysis and a random forest model (LDA > 4, AUC = 0.911). A synchronous decrease in the NS-CA was predicted by FAPROTAX functional prediction in relation to aromatic_compound_degradation and human_pathogens_all. The NS-CA and CA-CA did not exhibit any significant differences in alpha diversity or FAPROTAX (p > 0.05), and the PCoA showed that the two groups were closely clustered.Conclusion The stabilization and maturation of oral microbiota on the surface of alginate impressions are facilitated by CA disinfection. Of all the groups, the NS-CA group demonstrates specific cost-effectiveness advantages and has the potential to serve as an alternative to conventional AC disinfection.
The actinomycete Actinoplanes missouriensis forms terminal sporangia that contain a few hundred spores. When immersed in water, the sporangium opens to release spores, which start swimming using flagella, via a process referred to as sporangium dehiscence. In this study, we conducted a functional analysis of genes encoding the components of the caseinolytic protease (Clp) complex, which comprises ATPase (chaperone) subunits (ClpX and ClpC) and proteolytic subunits (ClpP1-4). The clpX null mutant (ΔclpX) strain formed sporangia smaller than the wild-type strain. The small sporangia scarcely opened under conditions that induce sporangium dehiscence in the wild-type strain. Consistently, the number of released spores was three orders of magnitude lower in the ΔclpX strain than in the wild-type strain. S1 nuclease mapping determined two transcriptional start points of clpX (TSSU and TSSD for upstream and downstream, respectively). A housekeeping sigma factor-dependent promoter and a FliA-family sigma factor-dependent promoter were found upstream of TSSU and TSSD, respectively. A gene complementation test showed that apparently normal sporangium formation in the ΔclpX strain was restored by the introduction of clpX with either of the two promoters, whereas both promoters were required for sporangium dehiscence. Meanwhile, mutant strains that produced ClpC with T30S or I33F/L34F replacements, which presumably reduced their substrate-binding activity, produced sporangia with irregular shapes. Furthermore, gene disruption experiments of four putative proteolytic subunit genes indicated that clpP3 is conditionally involved in sporangium dehiscence. We concluded that sporangium formation and dehiscence are regulated at the post-translational level via proteolysis by Clp complexes in A. missouriensis. IMPORTANCE:Actinoplanes missouriensis has a complex life cycle, in which the sporangium containing a few hundred flagellated spores is the most characteristic structure. Spores are released from sporangia via a process called sporangium dehiscence. We have revealed that sporangium formation and dehiscence are regulated by several transcriptional regulators; however, post-transcriptional regulation of sporangium formation and dehiscence remains unknown. In the present study, we revealed that two ATPase components of the Clp complex, ClpC and ClpX, and a proteolytic component, ClpP3, are involved in sporangium formation and/or dehiscence. This study indicates that in addition to transcriptional regulation, proteolysis regulated by Clp complexes is another crucial factor in the morphological development of A. missouriensis.
Background:Oral squamous cell carcinoma accounts for over 90% of oral neoplasms. Despite therapeutic advances, the lack of reliable, non-invasive biomarkers and delayed diagnosis continues to impede effective clinical management. By combining paired lesion and non-lesion sampling with predictive metagenomics analysis, our study addresses this gap and advances the current understanding of microbiome‒tumor interactions. Methods:We analyzed 92 buccal swab samples from 39 OSCC patients and 14 healthy controls using 16S rRNA gene (V3-V4) sequencing. Taxonomic profiling was conducted using QIIME2 and SILVA/eHOMD databases, functional pathways were predicted using PICRUSt2, and hub taxa were identified through co-abundance network analysis. Results:Microbial community structure differed significantly across lesion, non-lesion, and healthy sites (PERMANOVA, p = 0.001). Lesions were enriched with Selenomonas infelix and Treponema vincentii, while healthy controls harbored Streptococcus oralis and Gemella haemolysans. Co-abundance network analysis revealed lesion-specific hub species, notably T. vincentii, strongly correlated with predicted activation of pyrimidine biosynthesis pathways (r = 0.69, q < 1E-6), suggesting predicted metabolic alterations in the tumor microenvironment. Non-lesion sites were also characterized by two hub species, Prevotella melaninogenica and Segatella oulorum. Conclusion:Our findings define a lesion-specific microbial signature of OSCC characterized by the depletion of health-associated taxa, enrichment of pro-inflammatory pathobionts, and predicted associations with metabolic pathways implicated in carcinogenesis. These alterations reflect a predicted functionally altered tumor microenvironment.
Objective:To investigate the bidirectional causal associations between genetically predicted oral microbial taxa and allergic rhinitis (AR) in East Asian populations. Methods:We used summary-level genome-wide association study (GWAS) data from East Asian populations to conduct a bidirectional two-sample Mendelian randomization (MR) analysis. The inverse-variance weighted (IVW) method was the primary method. Bayesian weighted Mendelian randomization (BWMR) was used to assess the robustness of taxa that remained significant after IVW-based FDR correction. Cochran's Q test, the MR-Egger intercept test, and MR-PRESSO were used to assess heterogeneity and horizontal pleiotropy. Results:In the forward MR analysis, 16 tongue dorsum and 22 salivary microbial taxa were associated with AR risk. BWMR supported associations for 15 tongue dorsum and 17 salivary taxa, with effect directions generally consistent with those obtained using IVW. Representative associations included a positive association between genetically predicted salivary Campylobacter_A_mgs_1095 abundance and AR risk (OR = 1.381), and an inverse association between tongue dorsum Streptococcus_mgs_1057 abundance and AR risk (OR = 0.767).Sensitivity analyses indicated no substantial heterogeneity or horizontal pleiotropy for most taxa. Conclusions:Genetically predicted abundances of several tongue dorsum and salivary microbial taxa were associated with AR risk in East Asian GWAS datasets.These findings require validation in larger independent cohorts and experimental studies.
Backgroud Dental fluorosis is a prevalent endemic condition, yet its impact on the oral microbiota structure and resistance in children remains understudied.Objective To assess the microbial composition, diversity, functional pathways and co-occurrence patterns among urinary fluoride (UF), bacterial taxa and antibiotic resistance genes in relation to fluorosis severity. Design: Metagenomic analysis of dental plaque was conducted on 96 school-aged children, who were grouped into normal, dubious, very mild, mild, moderate and severe base on fluorosis severity.Results Microbial diversity increased with fluorosis severity. Actinomyces sp. HMT 175, Actinomyces oris and Corynebacterium matruchotii, Fusobacterium nucleatum and Rothia dentocariosa were significantly enriched in the moderate and severe groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed a reduced relative abundance of genes involved in carbohydrate metabolism and genetic information processing in moderate and severe groups. Network analysis revealed positive correlations among Actinomyces sp. HMT 175, UF levels and GRD33_1 (a carbapenem resistance gene).Conclusions These findings highlight severity-dependent shifts in the oral microbiota and resistance, warranting further investigation into fluoride's public health implications.
The type VI secretion system (T6SS) is a nanomachine utilized by Gram-negative bacteria to secrete toxic effectors. The T6SS is employed against both prokaryotic and eukaryotic targets, allowing for competitive advantage and virulence of the attackers in a wide variety of environments. Recent work has revealed the great diversity of T6SS systems. Beyond the canonical, conserved architectural genes of the T6SS, diverse T6SS-associated genes (TAGs) are employed to optimize T6SS activity, compensate for variations in architecture, and dictate T6SS use. Although various TAGs have been discovered in T6SS gene clusters, they display unique activities in modifying their cognate T6SS. In this review, we discuss several of the better-studied TAGs, as well as highlight under-researched TAGs that are likely of great significance. As a means to better understand TAG roles, identify common principles and unifying themes, we present the following six functional categories: Firing Coordinators, Target Recognition Factors, Structural Factors, Transcriptional Regulators, Secretion Regulators, and Unknown TAGs.
Penetration of the peptidoglycan (PG) layer by the nascent flagellar rod is a critical step in basal body assembly and has long been attributed to the acetylglucosaminidase activity of the flagellar rod cap protein FlgJ. Previous work in Salmonella enterica suggested that occasional preexisting openings in the PG layer allow some flagella to assemble in the absence of the FlgJ enzymatic activity. More recently, studies in Bacillus subtilis demonstrated that membrane mobility of nascent flagellar structures enables rod penetration without dedicated PG hydrolysis. Here, we revisited the requirement for FlgJ acetylglucosaminidase activity in S. enterica by testing whether inhibition of class 3 flagellar gene expression by the anti-σ28 factor FlgM contributes to the flagellation defect of FlgJ catalytic mutants. Consistent with previous studies, loss of FlgJ acetylglucosaminidase activity did not abolish flagellar assembly but instead reduced its efficiency, resulting in a heterogeneous population in which many cells assembled functional basal bodies and flagella. Deletion of flgM significantly increased both the proportion of flagellated cells and the number of flagellar filaments per cell, indicating that reduced class three gene expression contributes substantially to the observed defect. These findings support a model in which FlgJ enzymatic activity enhances the efficiency of local PG remodeling but is not essential for rod penetration or basal body assembly. Our results demonstrate that acetylglucosaminidase-independent flagellar rod growth occurs in S. enterica, although less efficiently than in organisms that lack FlgJ-like PG hydrolases, highlighting the evolutionary diversity of mechanisms that accommodate flagellar assembly through the bacterial cell wall.IMPORTANCEBacterial flagella allow pathogens, such as Salmonella, to navigate complex environments and invade host cells. During flagellar assembly, the basal body must traverse the peptidoglycan layer, a step-long thought to require the dedicated cell wall-degrading activity of FlgJ. Here, we show that Salmonella can assemble functional flagella, even when the acetylglucosaminidase activity of FlgJ is genetically inactivated. Although these mutants produce fewer flagella per cell, the assembly pathway remains active, and enhanced σ28-dependent gene expression partially restores flagellation. These findings reveal that early steps in flagellar assembly are more diverse than previously thought and that differences in cell wall structure between gram-positive and -negative bacteria likely determine whether a cell wall hydrolyzing activity is required for flagellum assembly.
Background:Periodontal pathogens are associated with cardiovascular disease, but clinical evidence linking Porphyromonas gingivalis (Pg) detection with coronary heart disease (CHD) and gut microbiota features remains limited. This study evaluated associations between oral and peripheral blood Pg detection status, CHD prevalence and exploratory gut microbiota characteristics in participants undergoing coronary angiography (CAG). Materials and methods:This analytical cross-sectional study included 165 CAG participants, including 124 patients with CAG-confirmed CHD and 41 non-CHD controls. Oral and peripheral blood Pg were detected by TaqMan quantitative polymerase chain reaction. Modified Poisson regression with robust standard errors was used to estimate adjusted prevalence ratios (PRs). Gut microbiota profiles were assessed by 16S rRNA gene sequencing. Results:Oral Pg positivity was more frequent in CHD participants than in non-CHD controls and remained associated with CHD in the primary adjusted model (PR = 1.237, 95% CI 1.044-1.465). The oral+/blood+ group showed the strongest association with CHD prevalence (PR = 1.362, 95% CI 1.099-1.688). Five FDR-significant ASVs were enriched in oral Pg-positive participants, whereas predicted pathways were not significant after FDR correction. Conclusions:Oral Pg positivity, especially combined oral and peripheral blood Pg nucleic acid positivity, was associated with CHD prevalence in this CAG-defined cohort.
Prevotella species are an extremely common and abundant bacteria detected within the low microbial biomass of the lungs and are a core component of the oral microbiome. Clinical studies have drawn associations between Prevotella abundance and lung homeostasis, indicating potential relationships between Prevotella and lung inflammation, infection defense, and lung function. Across several studies in critically ill patients, the depletion of Prevotella and other obligate anaerobes is linked to significantly reduced survival, leading to calls for anaerobe preservation in empiric antibiotic therapy regimens. In recent years, mechanistic studies have provided new information regarding Prevotella-host relationships, highlighting several processes by which Prevotella exposure activates epithelial, innate, and adaptive immune responses. Prevotella species also have direct and indirect effects on important lung bacterial pathogens, including Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa, with Prevotella species-dependent consequences for pathogen infection and regulation of pathogen-induced inflammation. This review summarizes our current understanding regarding how Prevotella regulate lung immune homeostasis, with a discussion of key knowledge gaps necessary for the translation of these insights into new therapeutic approaches to reduce the burden of lung infection and disease.
The flagellar motor of Pseudomonas aeruginosa operates in a near-unbiased manner, extending the classical paradigm established by Escherichia coli. Rather than altering rotational bias, P. aeruginosa achieves chemotaxis by symmetrically modulating the dwell times of the two rotational states. This strategy is associated with a mechanically adaptive dual-stator architecture (MotAB and MotCD) and is further modulated by c-di-GMP signaling and auxiliary proteins such as FliL and MotY. At the molecular level, the distinctive reversal behavior is thought to arise from the unique response of the switch complex (located on the rotor) to phosphorylated CheY, which alters switching kinetics rather than rotational bias. These features collectively give rise to distinct swimming behaviors, including a "wrap" state that facilitates three-dimensional reorientation. This system reveals general principles of how molecular machines balance speed, torque, and switching in complex environments.
Toxin-antitoxin (TA) systems are genetic modules widely distributed across bacterial chromosomes and mobile genetic elements. Initially described as plasmid addiction systems that ensure vertical inheritance through post-segregational killing, they are now recognized as versatile regulators of bacterial adaptability. In Helicobacter pylori, a gastric pathogen infecting nearly half of the global population, multiple TA systems have been experimentally validated, including type I RNA-RNA modules and type II protein-protein pairs. These systems contribute to growth arrest, morphological transition from spiral to coccoid forms, biofilm formation, intracellular survival, and responses to environmental stressors, such as oxidative stress, metal availability, and antibiotic exposure. Type I AapA/IsoA modules have been linked to membrane targeting and dormancy induction, whereas certain Vap-like type II toxins act as ribonucleases and likely contribute to colonization and persistence during infection. Although hundreds of putative TA loci have been identified through bioinformatic analyses, only a small fraction has been functionally characterized, highlighting substantial knowledge gaps. This minireview summarizes experimentally validated TA systems in H. pylori, discusses predicted candidates, and examines their potential roles in pathogen adaptability, emphasizing their relevance to H. pylori persistence and pathogenicity.
Biofilm dispersion is a regulated process that enables bacteria to escape as free-living cells. This response coincides with matrix degradation, reduced cyclic di-GMP levels, heightened antibiotic susceptibility, and restored flagellar motility. While dispersed cells have been reported to be motile, the mechanisms enabling motility upon induction of dispersion remain unclear. Here, we explored the regulatory mechanism of how Pseudomonas aeruginosa biofilm cells switch their motility phenotype to disperse from biofilms. Our findings reveal that changes in motility gene expression are initiated at the periphery of biofilms during induced dispersion, in which increased expression of the flagellar gene fliC is controlled by the phosphodiesterase (PDE) DipA and the chemosensory protein BdlA. In response to the dispersion signal, BdlA forms dynamic clusters and relocates to the flagellated cell pole, where it interacts with and stimulates the PDE activity of DipA, ultimately leading to elevated fliC expression. These findings establish a mechanistic link between signal sensing and motility reversion, demonstrating that the spatiotemporal coordination of BdlA and DipA governs the motility switch during P. aeruginosa biofilm dispersion.IMPORTANCEOur findings reveal a new regulatory mechanism in which dispersion signals drive BdlA relocation, activate DipA's phosphodiesterase activity, lower cyclic di-GMP, and elevate fliC expression. Moreover, by showing that BdlA foci formation and co-localization with DipA occur in response to dispersion signals, we demonstrate that the spatiotemporal regulation of BdlA and DipA extends the Touch-Seed-and-Go model beyond surface attachment to encompass dispersion and, thus, the reversion from a sessile to a motile growth state.
Biofilms are the predominant lifestyle for bacteria. Biofilm abundance makes understanding their behavior important for different research domains. These domains can have different information and reporting standards. Information reporting standards have been proposed for some biofilm research methods. Standard methods and consensus standards have also been promulgated. We propose dimensional analysis to encourage abundant information reporting and facilitate comparative analysis. Dimensional analysis utilizes physical system parameters to generate generalizable model data. We review dimensionless quantities that can be applied to biofilm studies. We suggest dimensionless quantities for existing standards, with three examples using dimensional analysis to quantitatively compare literature data.
Pathogenic and persistent biofilms are a major challenge in medicine and industry, acting as resilient microbial communities that resist antibiotics and cause persistent infections. In this review, we discuss pulcherriminic acid, a promising iron-chelating molecule produced by antagonistic Bacillus subtilis as a natural weapon against persistent biofilms, particularly those formed by pathogens such as Candida albicans. Drawing on recent research, we explore pulcherriminic acid biosynthesis, its native mechanism of depriving fungi of essential iron to disrupt their growth and virulence, and innovative methods to enhance its production through metabolic engineering. We also highlight its potential applications, from fighting infections to serving as a biocontrol agent in agriculture and even as a natural preservative in food. By harnessing this Bacillus-derived metabolite, we suggest sustainable ideas for tackling antibiotic resistance and persistent biofilm-related problems.
The genus Neisseria includes two human pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, in addition to commensal species that also inhabit human and animal hosts. One of the features that occurs during neisserial host colonization is biofilm formation. In other bacteria, biofilm formation enables persistence as well as evasion of the host immune system. Extracellular DNA (eDNA) forms a major component of Neisseria biofilms and contributes to initial steps in their formation, as well as the structure and stability of the biofilm extracellular matrix. Here, we review the potential roles and sources of eDNA in Neisseria biofilms and its relevance to both pathogenic and commensal Neisseria spp. We also highlight the paucity in research on biofilm formation by commensal Neisseria and hope this will stimulate further research on the role of eDNA in nonpathogenic Neisseria.
Since its isolation from a Yellowstone hot spring in 1972, Sulfolobus acidocaldarius has become one of the most important model organisms for archaeal biology. Initially studied for its remarkable adaptation to high temperature and low pH, it has evolved into a genetically tractable system that has contributed substantially to our understanding of archaeal physiology, molecular biology, and evolution. Here, we summarize more than 5 decades of research on S. acidocaldarius, highlighting key developments in genetic and microscopy tools and their impact on our understanding of archaeal cell biology. Studies in this organism have improved our understanding of archaeal metabolism, chromosome organization, DNA replication and segregation, cell division, protein glycosylation, biofilm formation, and the assembly and regulation of archaeal surface structures, including archaella and type IV pili. Beyond fundamental biology, S. acidocaldarius has also served as a valuable source of thermostable enzymes and other biomolecules with biotechnological potential. Today, S. acidocaldarius is still one of the main model organisms for archaeal biology. Ongoing advances in genetics, imaging, and structural biology continue to expand its experimental potential, while its phylogenetic position within the Thermoproteota makes it a powerful system for investigating the evolutionary origins of eukaryotic cellular complexity.
