Journal of Bacteriology最新文献

Bacterial membrane vesicles (BMVs) have attracted significant attention as highly efficient transport vehicles for molecules crossing biological barriers and as key mediators in infection processes. Based on this increasing interest, the need for standardized isolation protocols and comprehensive analytical approaches becomes apparent. Here, we evaluated BMVs from the human pathogen Pseudomonas aeruginosa, isolated at six distinct growth phases, using physicochemical assays, functional characterization, and Raman spectroscopy. Conventional analyses revealed growth phase-dependent differences in protein content, surface charge, and immunogenicity. Raman spectroscopy provided detailed molecular fingerprints, identifying shifts in protein-to-lipid ratios, increased lipid saturation, and alterations in protein secondary structure during later growth phases. Importantly, the absence of nucleic acid-specific spectral markers confirmed the outer membrane origin of the vesicles. Together, these findings demonstrate that the timing of BMV isolation critically determines their molecular composition and functional properties and establish Raman spectroscopy as a powerful label-free tool for semi-quantitative profiling of BMVs.IMPORTANCEPseudomonas aeruginosa is an opportunistic gram-negative pathogen and a leading cause of severe nosocomial infections. Its secreted bacterial membrane vesicles (BMVs) are increasingly recognized as mediators of pathogenicity and as potential therapeutic delivery systems. However, the lack of standardized and sensitive analytical techniques has hindered systematic characterization. Our study highlights the profound impact of the bacterial growth phase on BMV composition and immunogenicity. It introduces Raman spectroscopy as a chemically selective, label-free method for detecting subtle yet biologically relevant molecular changes. These insights provide a framework for improved standardization in BMV research and underscore the potential of Raman-based approaches in advancing both fundamental microbiology and translational applications.

The arginine-dependent acid resistance (Adi) system is a vital component that enables Escherichia coli and other enterobacteria to withstand the extreme acidity in the human gastrointestinal tract. It consists of the proton-consuming decarboxylation of arginine, catalyzed by AdiA, and the uptake of arginine, as well as the excretion of the more alkaline agmatine, catalyzed by the antiporter AdiC. The corresponding genes adiA and adiC are induced in E. coli under acidic conditions (pH < 5.5), a process that is tightly regulated by the AraC/XylS transcriptional activator AdiY. Here, we show that the pH-sensing mechanism of AdiY functions through the protonation of two histidines (His34 and His60) in the N-terminal domain. Replacing these histidine residues with alanine, glutamine, or aspartate abolishes the pH-dependent activation of AdiY, both in vivo, as demonstrated by promoter-reporter assays, and in vitro, as indicated by the loss of DNA-binding activity detected by surface plasmon resonance spectroscopy. Biochemical analyses of purified wild-type AdiY using size-exclusion chromatography and intrinsic tryptophan fluorescence revealed a pronounced and reversible pH-dependent conformational change that does not occur in the pH-sensing-deficient AdiY variant. A model is proposed in which AdiY forms a monomer at physiological pH. At a lower intracellular pH, the protonation of histidine in AdiY causes a conformational change that leads to the binding of AdiY as a tetramer to the DNA. This work elucidates the molecular mechanism of a one-component signal transduction system that combines both sensory and responsive functions.IMPORTANCEThroughout their life, Escherichia coli and other bacteria may encounter acidic environments, for example, when passing through the human stomach. Their chances of survival under these conditions depend on the number and efficiency of acid resistance systems. Although many acid resistance mechanisms have been extensively studied, the molecular mechanism by which bacteria sense low pH is not yet fully understood. This study demonstrates that the transcription factor AdiY acts as a direct pH sensor by using two histidines to detect intracellular acidification in E. coli. When these histidines become protonated, AdiY changes its conformation and activates genes that support cell survival under acid stress. These findings not only reveal a new way in which bacteria can perceive extremely low pH environments but also provide the basis for the development of AdiY as a pH reporter.

Leucyl-aminopeptidase (LAP) is a type of protease that targets peptides and the nitrogen terminus of protein molecules, playing a key role in the removal of amino acids. This function is not only significant but also enlightening, as it contributes to our understanding of microbial survival and persistence. The presence of M17-LAPs enzymes across various bacterial species indicates the possibility of creating selective inhibitors, offering new avenues for antimicrobial development amidst increasing antibiotic resistance. Additionally, understanding the relationship between the structure of these enzymes and their functions can aid in the development of more effective treatment methods and enhance current therapies. In this review, we unravel the structural blueprints, functional roles, and therapeutic promise of M17-LAPs, highlighting their relevance in the era of escalating antibiotic resistance. We also highlight future research avenues, emphasizing structural biology and protein-protein interaction mapping as keys to unlocking targeted therapeutic strategies. By bridging molecular structure with translational potential, we propose a new vision: harnessing the vulnerabilities of M17-LAPs to inspire next-generation antibacterial strategies.

Sporulation is a strategy employed by many bacteria to survive harsh environmental conditions. The genus Paenibacillus includes spore-forming species notorious for spoiling pasteurized dairy products and for causing American foulbrood in honeybee larvae, leading to colony collapse. Human pathogens within Paenibacillus are also a growing threat, causing fatal opportunistic infections. Here, we present a comprehensive survey of sporulation genes across 1,460 high-quality Paenibacillus genomes. We find that all members of the sporulation-initiating phosphorelay are well conserved, but that the Spo0B phosphotransferase contains a predicted transmembrane domain. We confirm that this domain localizes Spo0B to the cell membrane and therefore refer to this Spo0B variant as Spo0B-TM. Spo0B-TM is present in 92% of surveyed Paenibacillus genomes. Consistent with its high level of conservation, we find that the transmembrane domain is important for detecting its interaction with its phosphorelay partners Spo0A and Spo0F. Moreover, we find that Spo0B exhibits low sequence identity across Bacillota when compared with other members of the phosphorelay. Altogether, this work highlights the potential for diversity even within the highly conserved phosphorelay that initiates sporulation in Bacillota.IMPORTANCEThe spore is the most durable life form, and the sporulation process serves as a paradigm of cellular development and differentiation. Sporulation is well characterized in the model organism Bacillus subtilis, but we lack information about non-model spore formers. The genus Paenibacillus includes spore formers that negatively impact farming and food industries and public health. Here, we present the largest comprehensive search for sporulation genes in Paenibacillus and show that a unique membrane-localized variant of Spo0B is widespread throughout Paenibacillaceae and is present in other closely related families of Bacilli.

Fusobacterium nucleatum is a Gram-negative anaerobe associated with periodontitis and colorectal cancer. It secretes putrescine, a polyamine that promotes biofilm formation by oral co-colonizers and enhances the proliferation of cancer cells. However, the physiological importance of putrescine for F. nucleatum itself remains unexplored. Here, we show that putrescine biosynthesis, mediated by the ornithine decarboxylase gene oda, is essential for F. nucleatum viability. Deletion of oda was only possible when a functional copy was provided in trans, and CRISPR interference of oda expression resulted in complete growth defects. The essentiality of oda was conserved across multiple subspecies. Supplementation with exogenous putrescine enabled the isolation of a conditional oda mutant whose growth was strictly putrescine-dependent. Putrescine depletion caused filamentation, membrane disruption, detergent hypersensitivity, and lysis in hypoosmotic conditions, indicating a critical role in maintaining cell envelope integrity. RNA sequencing revealed broad transcriptional remodeling under putrescine-limited conditions, including upregulation of genes involved in lipid metabolism, osmoprotection, and cell wall remodeling. Notably, oda transcript levels increased when putrescine was depleted, suggesting a negative feedback mechanism. These findings demonstrate that putrescine is not only an extracellular communal metabolite but is also vital for the cellular integrity and survival of F. nucleatum under anaerobic conditions.

Importance: Fusobacterium nucleatum is a prominent member of the oral microbiota and has been linked to various human diseases, including periodontitis, preterm birth, and colorectal cancer. Despite its clinical significance, the metabolic requirements that support its growth and viability remain poorly understood. In this study, we identify the oda gene, which encodes ornithine decarboxylase, as essential for F. nucleatum survival due to its role in putrescine biosynthesis. We demonstrate that depletion of putrescine leads to severe growth and morphological defects, accompanied by widespread transcriptional changes. These findings reveal an underappreciated metabolic vulnerability and highlight the critical role of polyamine homeostasis in maintaining cellular integrity in this notorious anaerobe.

The alarmone (p)ppGpp (ppGpp) accumulates in response to starvation and other stress, leading to inhibition of multiple biosynthetic pathways and, at high concentrations, suppression of bacterial growth. Growth suppression by ppGpp is implicated in the formation of persister cells, which survive antibiotic challenge only to regrow once the drug is removed. However, there is also evidence that low levels of ppGpp contribute to resistance to certain cell wall-active antibiotics in actively growing cells. To characterize ppGpp's contribution to antibiotic resistance, we measured MICs of a panel of β-lactams in actively growing Escherichia coli cells overexpressing a ppGpp synthase (relA*). Cells engineered to modestly overproduce ppGpp exhibited up to 64-fold increases in resistance to PBP2-targeting β-lactams only, with mecillinam the most dramatically affected. Resistance required the transcription factor DksA and the class A penicillin-binding protein (PBP) PBP1B. PBP1B variants defective for transpeptidase activity, glycosyltransferase activity, or both were incapable of supporting resistance, suggesting the full enzymatic activity of PBP1B is required for resistance. Transcriptomics revealed that ppGpp overproduction leads to increased expression of lpoB, which encodes an activator of PBP1B. LpoB was required for mecillinam resistance, with an lpoB deletion mutant exhibiting a loss of ppGpp-dependent resistance. An lpoB deletion strain expressing an LpoB-bypass variant of PBP1B (mrcB*) exhibited an intermediate level of resistance. Together, these results suggest that ppGpp overproduction and the LpoB-dependent enzymatic activity of PBP1B function synergistically to promote survival in the presence of PBP2 inhibitors.

Importance: Antimicrobial resistance is an increasing global health threat, but its underlying molecular mechanisms remain incompletely understood. This work clarifies ppGpp's role in mediating antibiotic resistance in Escherichia coli. Elevated levels of ppGpp caused resistance to β-lactam antibiotics targeting the cell wall synthesis enzyme PBP2. Resistance required transcriptional regulation by ppGpp and enzymatic activity of the cell wall enzyme PBP1B. ppGpp overproduction was found to increase expression of the PBP1B activator lpoB. Because ppGpp levels are controlled by nutritional conditions, this work suggests that nutritional availability may impact antibiotic efficacy.

High-temperature requirement A (HtrA) aids in protein homeostasis by playing a key dual role as a chaperone and protease. HtrA ensures protein folding quality control during secretion and protects cells against protein aggregation by degrading misfolded proteins. HtrA proteins are typically composed of a protease domain and at least one PDZ domain, proposed to help regulate their activity and interactions with substrates. In gram-positive bacteria, HtrA contributes to critical cellular functions and has been linked to processes such as maintaining envelope integrity, stress resistance, and virulence. In addition, HtrA has been shown to contribute to the modulation of competence and biofilm dynamics as well as the degradation of host proteins in infection models. In some gram-positive bacteria, HtrA expression is regulated by two-component systems, but many HtrA upstream signals and downstream targets remain unclear. As antibiotic resistance continues to rise, HtrA is gaining attention as a promising target of inhibition for new antibacterial strategies. However, a lack of structural information, unclear regulatory mechanisms, and unknown substrates make designing effective HtrA inhibitors challenging. This review highlights these knowledge gaps and aims to spark more focused research on HtrA in gram-positive species.

Aeromonas caviae, Gram-negative bacteria ubiquitous in the environment, are an emerging human pathogen associated with various infectious diseases, particularly gastroenteritis. Despite recent studies demonstrating A. caviae is the most predominant Aeromonas species underlying human infection, A. caviae remains understudied, and no A. caviae-specific virulence factors associated with human disease have been identified. To identify A. caviae-specific putative virulence factors, we conducted comparative genomic analyses among clinical Aeromonas isolates (n = 431), which identified a variant of flgB, predicted to encode a polar flagellum machinery protein, as over-represented in A. caviae isolates. To examine the role of flgB in virulence and host-pathogen interactions, we generated an A. caviae flgB deletion mutant and genetic complementation constructs. Swimming motility and polar flagella assembly were abolished in the mutant and functionally rescued with genetic complementation. As it remains unknown where A. caviae infects the human gastrointestinal tract, we assessed host-pathogen interactions in HT-29 and Caco2 human intestinal cell lines, representative of the large and small intestine, respectively. Deletion of flgB significantly decreased bacterial adherence in only HT-29 cells and also decreased production of proinflammatory cytokines, IL-8, IL-13, IL-1β, and IL-6, by both cell types. Given the lack of relevant mammalian models for studying most enteric pathogens in vivo, we characterized in vivo virulence in a Galleria mellonella larval survival model, where the flgB deletion modestly attenuated virulence. Deletion of flgB altered aspects of virulence and host-pathogen interactions, and this study provides a framework for identification and characterization of A. caviae-specific putative virulence factors.IMPORTANCEAeromonas caviae is an emerging human bacterial pathogen associated with gastroenteritis, wound infections, and numerous other infectious diseases. Recent studies demonstrate that A. caviae accounts for the greatest burden of human Aeromonas infections. Despite this, A. caviae is understudied as a human pathogen. To address this gap in knowledge, this study characterizes A. caviae-specific virulence genes. We examined 431 clinical Aeromonas isolates using comparative genomics and identified and functionally characterized a putative A. caviae-specific virulence factor, flgB. Genetic deletion of flgB in A. caviae resulted in deficiencies in bacterial motility, adherence, host-cell proinflammatory cytokine production, and in vivo virulence in an invertebrate model. This work establishes the foundation for further study of additional A. caviae-specific virulence factors.