Journal of Bacteriology最新文献

Mycobacterium abscessus (Mab), a rapidly growing mycobacterial species with intrinsic and acquired resistance to multiple antibiotics, is an emerging public health concern. The rise in clinical cases of treatment-refractory infections of M. abscessus has propelled its research toward novel therapeutic approaches. The number of publications entitled "Mycobacterium abscessus" has increased by ~300% over the last decade, of which the majority of studies exploring the fundamental biology and pathogenesis of Mab have used the reference strain ATCC19977. However, whole-genome sequence analyses, combined with transposon-seq based functional genomics, reveal an open pan-genome with significant variations in the essential genes across ATCC19977 and clinical isolates. These new discoveries demand a careful selection of strains and growth conditions in experimental design. In this minireview, we discuss these challenges and propose a framework for future M. abscessus studies in silico, including a new web-based resource for pangenome analysis, in vitro, and in animal models.

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that causes both acute and chronic infections in vulnerable populations. Treatment of P. aeruginosa infections is increasingly challenging due to multi-drug resistance, and biofilm formation during infection further increases antibiotic tolerance. Iron, which is sequestered by the host innate immune system, is also a key nutrient that is required for P. aeruginosa biofilm formation. The iron-responsive PrrF small regulatory RNAs (sRNAs) are key to P. aeruginosa's iron starvation response, promote the production of the Pseudomonas quinolone signal (PQS) quorum sensing molecule, and are required for virulence in murine lung infection. Prior work showed that the PrrF sRNAs are dispensable for biofilm formation; however, these studies were performed using flow-cell biofilms grown at room temperature. Here, we demonstrate a temperature dependency for PrrF in P. aeruginosa biofilm formation: the genes for these sRNAs are required for optimal biofilm formation at 37°C but not 25°C. We further show that a ∆pqsA mutant, which lacks production of PQS and related metabolites, phenocopies the ∆prrF mutant. These studies demonstrate the importance of the PrrF sRNAs in P. aeruginosa biofilm formation at body temperature and reveal a previously underappreciated role of temperature in iron homeostasis and P. aeruginosa biofilm physiology.IMPORTANCEBiofilm formation is a critical virulence trait for many microbial pathogens that confers tolerance to the host immune system and antimicrobials. Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms resulting in treatment failure. Iron is a known requirement for P. aeruginosa biofilm formation, yet the precise role of iron in biofilm physiology remains unclear. Here, we show that temperature alters the requirement for the PrrF small regulatory RNAs, key components of P. aeruginosa's iron starvation response, for biofilm formation. Specifically, PrrF is required for the optimal formation of flow-cell biofilms at 37°C but not at 25°C, yet most flow-cell biofilm studies are conducted at 25°C. These results demonstrate a previously underappreciated role of temperature in P. aeruginosa biofilm physiology.

Yersinia pestis is a gram-negative bacterium and the causative agent of plague. The Y. pestis virulence factor plasminogen activator protease (Pla) is an outer membrane aspartic protease that facilitates the dissemination of bacteria from the site of inoculation to deeper tissue during bubonic plague. During pneumonic plague, Pla acts as an adhesin, which contributes to the suppression of early innate immune responses in the lungs, and as a protease that aids in resisting bacterial killing by neutrophils. Two-component regulatory systems (TCSs) are involved in bacterial adaptation to environmental stressors such as changes in pH, changes in ion concentrations, and the presence of cationic antimicrobial peptides. TCSs consist of a membrane-bound sensor kinase that detects environmental stressors and activates a response regulator to coordinately alter gene expression. The PhoP/PhoQ TCS regulates virulence factors and known Pla homologs in a variety of gram-negative pathogenic bacteria including Escherichia coli and Salmonella species. In the work described here, we evaluate whether pla is regulated by PhoP/PhoQ in Y. pestis. We identify a putative PhoP-binding site within the -10 box and the +1 transcription start site of pla that is bound by recombinant PhoP. Surprisingly, we show that the expression of pla is suppressed by PhoP/PhoQ under a variety of physiologically relevant PhoP/PhoQ-inducing conditions that are expected to be encountered during infection. This work demonstrates the regulation of an essential Y. pestis virulence factor by the PhoP/PhoQ TCS for the first time and highlights the importance of tightly regulating virulence factors that function as proteases.IMPORTANCEYersinia pestis causes plague, a highly lethal infection that results from inoculation via an infected flea (bubonic plague) or inhalation of contaminated respiratory droplets via person-to-person transmission (pneumonic plague). The plasminogen activator protease (Pla) is a critical Y. pestis virulence factor that is essential to the progression of infection via either route of inoculation. In this work, we show for the first time that the well-established two-component regulatory system PhoP/PhoQ regulates the expression of pla. Under conditions found during mammalian infection, PhoP/PhoQ suppresses pla expression, presumably to limit aberrant cleavage of Pla substrates during the critical early stages of infection. These results show interaction between two key virulence loci for the first time, and shed light on the regulation of a critical Y. pestis virulence determinant.

Indigoidine is a blue pigment synthesized by several bacteria, including Vogesella indigofera. Industrial production of indigoidine has been a research focus, but less is known about why bacteria make this pigment or how its biosynthesis is regulated. We isolated V. indigofera strain OSW_575 and investigated the basis for its indigoidine production using genomic and genetic approaches. Mutation of the indigoidine synthase gene igiD eliminated pigment production, and complementation restored it. A transposon mutagenesis screen uncovered 34 mutations across 20 genes that affect pigment production, including some involved in metabolism, translation, protein homeostasis, and regulation. Three chaperones that combat misfolded proteins, dnaK, dnaJ, and grpE, were required for indigoidine production, while mutations affecting the clpAP proteasome resulted in hyperpigmentation. These results are consistent with prior studies and suggest a role for the protein homeostasis system in regulating indigoidine. We also found that the alternative sigma factor rpoN contributes to indigoidine production. Finally, one transposon mutation affected a predicted sensor histidine kinase, which we dub tciK. Our genetic characterization of tciK and its cognate response regulator tciR suggests that they function in the same pathway to regulate indigoidine. TciR is of interest due to its non-canonical domain architecture that combines an N-terminal REC domain and C-terminal RsbW-like anti-sigma factor domain. Further investigation of this system may reveal novel regulatory mechanisms. To our knowledge, this is the first study to employ genetic tools in V. indigofera, and we propose it as a useful experimental system for studying the regulation and function of indigoidine.IMPORTANCEDespite being known to science for more than a century, Vogesella indigofera has been the focus of few studies. We isolated a strain of this bacterium, sequenced its genome, and investigated which genes contribute to its production of the blue pigment indigoidine. We found that mutations in genes involved in metabolism, protein homeostasis, and regulation can affect pigment production. One locus required for indigoidine production encodes a novel two-component regulation system. We conducted a preliminary genetic characterization of this system, which includes a non-canonical response regulator. Based on the results, we propose our strain as a model organism for studying indigoidine production and regulation.

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.

Methanogenic archaea (methanogens) are microorganisms that obligately produce methane as a byproduct of their energy metabolism. While most methanogens grow on CO₂+H₂, isolates of the genera Methanosarcina and Methanothrix can use acetate as the sole substrate for methanogenesis. Methanogenic growth on acetate, i.e., acetoclastic methanogenesis, is hypothesized to require two distinct genetic modules: one for the activation of acetate to acetyl-CoA and another for producing a chemiosmotic gradient using electrons derived from ferredoxin. In Methanosarcina spp., the activation of acetate to acetyl-CoA is mediated by acetate kinase (Ack) and phosphotransacetylase (Pta), whereas Methanothrix spp. encode AMP-forming acetyl-CoA synthetases (Acs). The Rhodobacter nitrogen fixation complex (Rnf) or energy-converting hydrogenase (Ech) is critical for energy conservation in Methanosarcina spp. during growth on acetate, and a F₄₂₀:methanophenazine oxidoreductase-like complex (Fpo') likely plays an analogous role in Methanothrix spp. Here, we tested the proposed modularity of these pathways to facilitate acetoclastic methanogenesis. First, we surveyed over 100 genomes within the class Methanosarcinia to show that the genomic potential for acetoclastic methanogenesis is widespread. We then used the genetically tractable strain, Methanosarcina acetivorans, to build all modular combinations that might support acetoclastic methanogenesis. Our results indicate that Acs, while functional, cannot replace Ack+Pta to rescue acetate growth in M. acetivorans. Similarly, the Fpo' bioenergetic complex cannot replace Rnf. As such, our work suggests that, in addition to horizontal gene transfer of core catabolic modules, acetoclastic metabolism in methanogens requires changes to core energy metabolism too.

Importance: A large fraction of biogenic methane is derived from acetate, yet acetoclastic methanogens, i.e., methanogens that grow on acetate, remain poorly characterized due to their slow growth. Two groups of methanogens, Methanosarcina spp. and Methanothrix spp., perform acetoclastic methanogenesis using distinct sets of genes for acetate activation and energy conservation. It is widely hypothesized that these genetic modules from Methanosarcina spp. and Methanothrix spp. are functionally analogous and would thus be interchangeable. To test this hypothesis, we engineered different combinations of modules for acetoclastic growth in Methanosarcina acetivorans. Our results challenge this hypothesized paradigm of modularity, and we posit that other changes to the carbon and electron transfer pathways are crucial for the emergence of acetoclastic methanogenesis.