Fems Microbiology Letters最新文献

The peptidoglycan stem peptides of the hyperthermophile Thermotoga maritima contain an unusual D-lysine (D-Lys) alongside the usual D-alanine and D-glutamate. We identified a Lys racemase that catalyzes racemization between L-Lys and D-Lys, and a diaminopimelate (Dpm) epimerase that catalyzes epimerization between LL-Dpm and meso-Dpm. Herein, we characterized a Dpm decarboxylase (TM1517) that catalyzes the conversion of meso-Dpm to L-Lys. TM1517 displayed high decarboxylase activity toward meso-Dpm but no activity toward LL-Dpm. D-Lys was not detected in the decarboxylation of meso-Dpm. The pH and temperature dependencies and kinetic parameters of decarboxylase activity were determined. Although other amino acid metabolizing activities of TM1517 were investigated, TM1517 did not exhibit any activities. Therefore, TM1517 is a Dpm decarboxylase associated with L- and D-Lys biosynthesis in T. maritima.

Stenotrophomonas maltophilia is an emerging global opportunistic pathogen that causes nosocomial infections. We demonstrated that the superoxide stress-sensing transcriptional regulator SoxR directly modulated the expression of an operon encompassing sodA1 (encoding manganese-containing superoxide dismutase) and fre (encoding putative flavin reductase) by directly binding to the operator site, which was located between the -35 and -10 motifs of the sodA1 promoter. It is known that upon exposure to the superoxide generators/redox-cycling drugs, the SoxR, which is bound to the operator site, became oxidized. This oxidation causes a conformational change of SoxR to an active form, enabling the upregulation of sodA1-fre gene expression. A ΔsodA1 was constructed, and the mutant showed enhanced sensitivity to the redox-cycling drugs, including menadione, plumbagin, and methyl viologen (paraquat), relative to its parental strain K279a. Thus, sodA1 may play a role in the survival of S. maltophilia under superoxide stress during either its saprophyte stage (e.g. exposure to redox-cycling drugs) or host-pathogen interactions.

The GH11 xylanase XynCDBFV, derived from Neocallimastix patriciarum, is widely used in various industries. However, its relatively low thermostability limits its potential. In this study, two computational approaches-Rosetta Cartesian_ddG and the deep learning-based tool Pythia-were employed to identify key residues affecting XynCDBFV thermostability. Both methods highlighted residues D57 and G201 as promising targets. Site-saturation mutagenesis at these positions yielded 18 variants with improved thermostability. Notably, three D57 variants (D57N/S/T) exhibited a 10°C increase in optimal temperature and retained 3.4%-21.7% higher residual activity than the wild type after 1-h incubation at 80°C. Five G201 variants (G201A/C/F/I/V) showed 5°C/10°C enhancements in optimal temperatures, with 10.1%-22.6% improved residual activity. These findings validate D57 and G201 as pivotal sites influencing thermostability. However, combining beneficial mutations from both sites led to reduced thermostability due to negative epistatic interactions. Comparative analysis revealed that while Rosetta Cartesian_ddG offers broader screening, it suffers from a high false discovery rate. In contrast, Pythia provides a balanced trade-off between precision and speed. This study offers a robust framework for enzyme thermostability enhancement and underscores the value of integrating computational predictions with experimental validation in protein engineering.

Helicobacter pylori is a significant human pathogen associated with gastric diseases, yet the contribution of plasmids to its pathogenicity remains largely unexplored. In this study, we combined plasmid network analysis, dereplication, functional annotation, and phylogenetic approaches to provide a comprehensive genomic and functional characterization of the H. pylori plasmidome using publicly available plasmid sequences. Of 322 plasmids analyzed, we identified 158 high-confidence plasmid sequences, representing 76 non-redundant plasmids (NR-plasmids). Notably, several sequences previously annotated as plasmids were reclassified as Integrative and Conjugative Elements. NR-plasmids were enriched in genes encoding Filamentation induced by cAMP (Fic) family proteins, which clustered into two distinct phylogenetic groups. Conserved motif analysis suggests that these two Fic protein types may form a novel toxin-antitoxin (TA) system, with Type-2 proteins potentially suppressing Type-1 activity, analogous to the TA mechanism described in Campylobacter fetus subsp. venerealis. Additionally, we identified genes encoding ATP-binding cassette (ABC) and major facilitator superfamily efflux pumps, as well as the virulence-associated protein D (VapD), which may contribute to antimicrobial resistance and host colonization, respectively. Our findings reveal the genomic and functional diversity of the H. pylori plasmidome and highlight the need for experimental validation to clarify its role in pathogenicity, antimicrobial resistance, and bacterial adaptation.

Salmonella contamination in chicken is a food safety problem that is widely concerned by all countries around the world. Based on the "One Health" concept, this study systematically collected samples from animals, the environment and workers across 5 stages of the broiler production chain (breeding farms, hatcheries, commercial broiler farms, slaughterhouses, retail) in China, to investigate the prevalence of Salmonella. Meanwhile, based on whole genome sequencing and risk assessment technology, combined with MLST, cgMLST traceability analysis was conducted to clarify the critical control points and transmission factors associated with Salmonella contamination. The results showed that the prevalence rate of Salmonella in the broiler production chain was 10.22% (469/4589). The broiler production chain encompassed 11 serotypes of Salmonella, with S.Enteritidis ran through the entire production chain. Fourteen types of ST were detected from 99 representative strains, and the dominant types were ST11, ST198 and ST1543. ST11 covered the samples from animals, environment and workers in all stages of broider production chain, and was further divided into 14 cgST types. Analysis using @RISK software revealed that the Spearman correlation coefficients for slaughterhouses and hatcheries were 0.54 and 0.26, respectively. These research findings are expected to comprehensively guide chicken production and provide effective strategies for preventing and controlling Salmonella contamination in the broiler production chain.

Phosphoenolpyruvate carboxylase encoded by ppc catalyzes the anaplerotic reaction of oxaloacetate in the tricarboxylic acid (TCA) cycle in Escherichia coli. Deletion of ppc does not prevent the cells from replenishing oxaloacetate via the glyoxylate shunt, but the ppc-deletion strain almost did not grow on glucose. In the present study, we obtained evolved strains by deleting both ppc and mutS to increase the mutation rate and investigated the mechanisms for improving growth by analyzing the mutated genes. Genome resequencing revealed that the evolved strains have non-synonymous mutations in icd encoding isocitrate dehydrogenase (ICDH). The introduction of icd mutations rescued the growth defects caused by ppc deletion. ICDH activity was strongly reduced by the amino acid substitutions G205D or N232S. The evolved strains appeared to suppress the competitive pathway for increasing the glyoxylate shunt flux. In metabolic engineering, the deletion of iclR, which encodes a repressor of the aceBAK operon, has been used to activate the glyoxylate shunt. The growth rate of the ΔppcΔiclR strain slightly increased, but it was still much lower than that of the Δppc + icdG205D strains. This finding suggests that iclR deletion is not sufficient to enhance glyoxylate shunt flux and that inactivation of the competitive pathway by icd mutations is more effective.

Hemolytic proteins are a major group of virulence factors in pathogenic Aeromonas hydrophila. Six genes encoding presumable hemolytic proteins were revealed from the genome of virulent A. hydrophila (vAh) that caused severe disease in channel catfish. The aim of this study was to assess the contribution of these hemolytic proteins to the virulence of this bacterium. Genes coding for following six proteins were investigated: aerolysin (Arl), 21-kDa hemolysin (Hly1), thermostable hemolysin (Hly2), phospholipase/lecithinase-related hemolysin (Hly3), membrane-associated hemolysin III (Hly4), and cytolysin-associated hemolysin (Hly5). Individual genes were deleted from the bacterium using CRISPR-Cas9 mediated methods. Assessment showed that deletion of Arl gene (Δarl) completely abolished hemolytic activity of this mutant while Δhly1-Δhly5 mutants had the same activity as the wild vAh. Extracellular proteins (ECPs) of the Δarl mutant caused significantly (p < 0.01) less cell death in vitro with viability increased by approximately 20%, compared to the wild vAh. ECPs of mutants Δhly1-Δhly5 remained the same cell toxicity as the wild vAh. A second deletion of hly5 from the Δarl mutant further lowered the cell toxicity of the ECP of the mutant (Δarl + Δhly5). Assays in vivo showed that both Δarl and Δhly5 mutants caused less fish mortality with reduction of 57% and 16%, respectively, compared to the wild vAh; the Δarl + Δhly5 mutant caused the least mortality with approximately 87% of reduction; and other mutants had the same virulence as the wild vAh. Analyses of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western blotting evidently indicate that both Arl and Hly5 proteins formed hexamer-like stable structures post secretion from the bacterium. Arl and Hly5 apparently had synergistic action in cytotoxicity and causing disease and were the major virulence factors among the six hemolytic proteins analyzed in this study.

Bacterial transcription terminator, Rho is an RNA (Ribonucleic Acid)-dependent ATPase that terminates transcription. Several structures of pretermination complexes of the Rho-transcription elongation complex (EC) revealed a static picture of components of the EC that come close to the nascent RNA-bound Rho, where many of the residues of EC reside ≤10 Å from the Rho residues. However, the in vitro-formed Rho-EC complexes do not reveal the in vivo Rho-EC dynamic interaction patterns during the termination process. Here we report synthetic defect analyses of various combinations of the mutations in RNAP β, β' and ω-subunits, NusA, NusG, and Rho proteins to delineate the functional network of this process. Several mutations in the β-flap and β'-Zn-finger and -Clamp helices domains of RNAP are synthetically defective in the presence of Rho mutants indicating functional involvement of these domains. Mutations in the NusA RNA-binding domains were synthetically defective with the Rho mutants suggesting its involvement. Our genetic analyses also revealed functional antagonisms between the ω-subunit of RNAP and the NusG-CTD (c-terminal domain) during termination. We concluded that the regions surrounding the RNA exit channel, the RNA-binding domains of NusA, the RNAP ω-subunit, and NusG-CTD constitute a functional network with Rho just before the onset of in vivo Rho-dependent termination.

Entomophagy, the practice of consuming insects, is a global tradition. In Mexico, one of the most notable and widely consumed insects is the larva of Aegiale hesperiaris. This insect feeds on the leaves of various Agave species with high polysaccharide content, suggesting their potential role as prebiotics for the intestinal microbiota, particularly lactic acid bacteria (LAB). LAB are recognized for their use as probiotics in foods due to their health-promoting capabilities. In this study, LAB from the intestinal microbiota of A. hesperiaris larvae were isolated and characterized, utilizing 16S rRNA gene identification. The analysis revealed three bacterial species from the Lactobacillaceae family, indicating a close symbiotic relationship with the insect. This suggests a significant impact on carbohydrate and protein metabolism, vitamin synthesis, and amino acid production, contributing to the high nutritional value of this edible insect. The study provides insights into the bacteria within the digestive tract of A. hesperiaris larvae and their role in enhancing the nutritional value of this edible insect. Additionally, it establishes a foundation for future research on the ecological roles and potential biotechnological benefits of these bacteria in the food industry and the development of therapies for various conditions and diseases.

The genus Bacillus features species with remarkable plant growth-promoting traits (PGPTs) and is widely recognized for its biotechnological potential in sustainable agriculture. Among them, Bacillus paramycoides has recently attracted attention for its versatility in green synthesis of biopolymers, metal-based nanoparticles, and inhibition fungal phytopathogens; however, its PGPTs remain poorly underexplored. In this study, an integrated genomic and physiological approach was applied to B. paramycoides RZ3MS14, isolated from the guarana rhizosphere in Amazonian rainforest, to explore and correlate its potential PGPTs through in vitro and in vivo assays. The genome of B. paramycoides RZ3MS14 harbors genes related to N/P/Fe mobilization, bacillibactin synthesis, exopolysaccharides and biofilm formation, plant signaling, stress tolerance, biocontrol, and antibiotic resistance. Functional validation through in vitro assays, confirmed the strain's ability to solubilize phosphate, mineralize phytate, and produce siderophores, auxins, exopolysaccharides, and biofilm. These findings point diverse plant-growth promoting (PGP) traits that contributed to significant improvements in sugarcane growth and root architecture in the greenhouse. Specifically, root dry mass, shoot dry mass, root length, root surface area, and root volume increased by 225.92%, 520.89%, 231.47%, 242.25%, and 252.92%, respectively. Bacillus paramycoides RZ3MS14 exhibited a low antagonistic effect against the phytopathogenic fungi Fusarium verticillioides and Ceratocystis paradoxa. In contrast, microbial volatiles defined synergistic interactions with beneficial fungi Trichoderma afroharzianum and Purpureocillium lilacinum. This is the first study to unveil the PGP attributes of B. paramycoides, underscoring RZ3MS14's potential as a sugarcane bioinput and providing insights into its combined application with other microorganisms.