Applied and Environmental Microbiology最新文献

Class IIa and IId bacteriocins are antimicrobial peptides with potential for combating antibiotic-resistant pathogens. However, their species-specific activity, dictated by recognition of the mannose phosphotransferase system (Man-PTS) receptor, often restricts their spectrum. Garvieacin Q/Garvicin Q (GarQ), a newly identified class IId bacteriocin, is unusual in that it targets both Lactococcus garvieae and the non-lactococcal pathogen Listeria monocytogenes, yet the structural basis for this cross-species activity has remained unclear. Using cryo-electron microscopy, we determined the structures of GarQ bound to Man-PTS receptors from Lactococcus garvieae and Listeria monocytogenes. In Lactococcus garvieae, the receptor contains a unique Tudor-like γ+ domain that sterically constrains the N terminus of incoming bacteriocins, thereby enforcing specificity for GarQ while excluding others such as lactococcin A (LcnA). In Listeria monocytogenes, GarQ engages the receptor through the same conserved binding mode, effectively bypassing the unusual species barrier. We further demonstrate that the C-terminal length of GarQ is a critical determinant of pore size and target specificity. Together, these findings uncover the structural mechanism underlying GarQ's atypical extended-spectrum activity and provide a framework for engineering bacteriocins with customized spectra to combat specific pathogens.IMPORTANCEThis study establishes a structural basis for how the extended-spectrum bacteriocin Garvieacin Q (GarQ) circumvents the canonical species-specificity of class II bacteriocins by engaging mannose phosphotransferase system receptors from different bacterial genera through both conserved and divergent binding modes. We identify a previously unknown Tudor-like γ+ domain in the Lactococcus garvieae receptor that sterically restricts the access of other bacteriocins, thereby defining bacteriocin specificity. Moreover, we demonstrate that the C-terminal length of GarQ critically determines pore size and bacterial targets, revealing an engineerable principle for designing synthetic bacteriocins with customized spectra against clinically relevant pathogens.

Septic arthritis (SA) is a cause of lameness in cattle attributed to bacterial infections. Mycoplasmopsis bovis is the best known and characterized etiological agent of SA; however, cases caused by diverse bacteria have been reported. Accordingly, we surveyed bacteria associated with septic and healthy joints from animals in western Canadian feedlots. Microbial community profiling showed that M. bovis was the most frequently detected pathogen in septic joints, followed by Metamycoplasma alkalescens and Trueperella pyogenes. In most cases, disease was ostensibly caused by a single pathogen, though polymicrobial infections and complex communities were also observed in DNA isolated from septic joints. The application of enhanced metagenomics by target DNA hybridization capture sequencing (CapSeq) provided more robust pathogen detection and characterization. CapSeq revealed resistance determinants that escaped detection using a conventional shotgun metagenomic approach. Notably, a series of nucleotide polymorphisms to M. bovis rrs, rrl, gyrA, and parC gene sequences were observed that confer resistance to macrolides and oxytetracycline-resistant T. pyogenes were also apparent in the CapSeq data. Complementary pathogen isolation, whole-genome sequencing, and phenotyping efforts were focused on the two most prominent pathogens, M. bovis and M. alkalescens, and corroborated metagenomic data sets.IMPORTANCEInformed antimicrobial use for the treatment of septic arthritis (SA) has been limited by overlooking the potential diversity of causative agents and our knowledge of their antimicrobial resistance (AMR) profiles. This survey begins to provide epidemiological insights, offering renewed appreciation of Metamycoplasma alkalescens as an etiological agent of SA and highlighting the prominence of important AMR determinants. Finally, the survey suggests that our knowledge of even the identities of the causative agents of SA is incomplete.

Killer toxins are proteinaceous antifungal molecules produced by yeasts, with activity against a wide range of human and plant pathogenic fungi. Fungus gardens of attine ants in Brazil were surveyed to determine the presence of killer toxin-producing yeasts and to define their antifungal activities and ecological importance. Our results indicate that 10 out of 59 yeast species isolated from fungal gardens are killer yeasts. Killer yeasts were less likely to inhibit the growth of yeasts isolated from the same environment but more effective at inhibiting yeasts isolated from other environments, supporting a role for killer yeasts in shaping the community composition. All killer yeasts had genome-encoded killer toxins and lacked cytoplasmic toxin-encoding elements (i.e., double-stranded RNA satellites and linear double-stranded DNAs). Of all the killer yeasts associated with attine ants, Candida sinolaborantium (strain LESF 1467) showed a broad spectrum of antifungal activities against 39 out of 69 (57%) of yeast strains tested for toxin susceptibility. The complete genome sequence of C. sinolaborantium LESF 1467 identified a new killer toxin, Ksino, with similarities in the primary sequence and tertiary structure to the Saccharomyces cerevisiae killer toxin named Klus. Surveys of publicly available genome databases identified homologs of Ksino in the genomes of yeast strains of Saccharomycetes and Pichiomycetes, as well as other species of Ascomycota and Basidiomycota filamentous fungi. This demonstrates that killer yeasts can be widespread in attine ant fungus gardens, possibly influencing the fungal community composition and the importance of these complex microbial communities in the discovery of novel antifungal molecules.

Importance: Attine ants perform essential ecosystem services through the harvesting of substrates for fungiculture. The cultured fungi are a food source for attine ants. Characterizing antifungal toxin-producing yeasts (killer yeasts) is vital to understanding how they might protect gardens from invasion by unwanted fungal species. This study also describes a new toxin named Ksino from the yeast Candida sinolaborantium, a member of a new group of toxins found across many different species of fungi. This work supports the role of killer yeasts in the ecology of fungicultures and competition between fungi. The observed high prevalence of killer yeasts in fungal gardens also enables the discovery of novel antifungal molecules with the potential to be applied against disease-causing fungi.

The ability of Listeria to show reduced susceptibility to sanitizers commonly used in fresh produce packing and processing environments continues to be mentioned as a concern. We assessed the survival of 501 produce-associated Listeria isolates (328 Listeria monocytogenes [LM] and 173 Listeria spp. [LS]) after 30 s of exposure to benzalkonium chloride (BC, 300 ppm) and peroxyacetic acid (PAA, 80 ppm). A subset of 108 isolates was also exposed to sodium hypochlorite (NaOCl, 500 ppm) for 30 s. Isolates showed a range of log reductions, including 2.76-5.73 log for BC, 0.15-6.16 log for PAA, and 1.34-7.02 log for NaOCl; the variance of log reductions was significantly lower for BC compared to PAA and NaOCl. Cluster analysis on log reduction data identified four clusters, including one cluster of five LM isolates that showed reduced susceptibility to all three sanitizers. Log reductions of LS were significantly lower than LM after exposure to PAA, indicating reduced PAA susceptibility among LS. Whole genome sequence (WGS)-based characterization of all isolates revealed that the presence of known BC resistance genes (i.e., bcrABC, mdrL, and sugE1/2) was not significantly associated with log reductions to BC, and the presence of stress survival islet SSI-2 was not significantly associated with log reductions to PAA and NaOCl. Genome-wide association studies did not reveal any association of pangenome genes with phenotypic sanitizer susceptibility but identified several SNPs in core genes as associated with sanitizer susceptibility.IMPORTANCEDespite frequently stated concerns about LM and LS with reduced susceptibility to sanitizers (which could facilitate persistence and increase risk of product contamination), there are limited data available on Listeria susceptibility to sanitizers used in produce packing and processing environments at their recommended use-level concentrations. Importantly, our data showed that reduced sanitizer susceptibility of Listeria is not linked to the presence of any previously reported sanitizer resistance genes. However, we identified a group of five LM isolates that showed reduced susceptibility to all three sanitizers tested; these isolates represented lineages I, II, and III. Combined, these data suggest that there are no distinct "sanitizer-resistant" clonal Listeria groups and that WGS data may not be particularly valuable for predicting sanitizer susceptibility at use-level concentrations. Moreover, the high variability of log reductions observed across all three sanitizers highlights the importance of considering log reduction variability, in addition to average log reduction, when assessing different sanitizers.

In this work, we investigated the chemical process underlying the interplay between two rice sheath pathogens: Pseudomonas fuscovaginae, the causal agent of sheath brown rot, and Rhizoctonia solani AG 1-IA, which causes sheath blight. Specifically, we studied the fate of the bacterial cyclic lipopeptides (CLiPs) syringotoxin and fuscopeptin in this interaction. Both compounds exhibit potent antifungal activity against R. solani and induce phytotoxic effects. Ultra-performance liquid chromatography-tandem mass spectrometry analysis demonstrated that R. solani AG 1-IA likely secretes two distinct enzymes: an esterase that hydrolyzes the ester bond in syringotoxin, producing a linear derivative, and a protease that cleaves the glycine-alanine bond within the peptide backbone of fuscopeptin. The degradation products lack antifungal and phytotoxic activities, nullifying the competitive advantage of P. fuscovaginae. These enzymatic effects showed increased activity at 28°C. In contrast, R. solani AG 2-2 did not degrade CLiPs. Further analysis with a broader range of R. solani isolates revealed that CLiP degradation is a common trait among AG 1-IA isolates. This study provides the first evidence that R. solani AG 1-IA actively neutralizes the antifungal and phytotoxic activities of P. fuscovaginae through targeted enzymatic degradation.IMPORTANCERice is a global staple crop that is susceptible to various pathogens, including Pseudomonas fuscovaginae, causing sheath brown rot, and Rhizoctonia solani AG 1-IA, which causes sheath blight. Notably, P. fuscovaginae primarily occurs at lower temperatures, whereas R. solani AG 1-IA is more prevalent under warmer conditions. Previous research demonstrated that P. fuscovaginae produces higher levels of the virulence-associated cyclic lipopeptides (CLiPs) syringotoxin and fuscopeptin at 18°C, potentially explaining its pathogenicity on rice plants grown at high altitudes. Conversely, R. solani AG 1-IA, which is sensitive to these CLiPs, was found to secrete CLiP-degrading enzymes, with degradation activity enhanced at 28°C. When combined with the reduced CLiP production by P. fuscovaginae at higher temperatures, this enzymatic degradation may confer a competitive advantage to R. solani in warmer environments. The absence of reports documenting the co-occurrence of both pathogens in field conditions may, at least in part, be explained by this temperature-dependent antagonism.

Microbially induced carbonate precipitation (MICP) provides a sustainable approach for the autonomous repair of microcracks in concrete. However, its practical application is limited by the poor long-term survival of microorganisms in the highly alkaline environment of cement matrices. This study used expanded perlite as an immobilization carrier to systematically investigate the effects of pH, temperature, and aging on microbial spore survival. Under non-immobilized conditions, acclimatized spores showed optimal long-term activity at pH = 10 and 0°C. After 180 days, the spore survival rate reached 12.31%, and urease activity achieved 0.74 mmol/(L·min)-approximately twice and nine times higher, respectively, than those recorded at 30°C over the same period. Although environmental factors reduced microbial mineralization capacity under immobilized conditions, mineral precipitation stabilized at around 5.60 g, representing a 28-fold increase compared to non-immobilized results over the same duration. These findings confirm that the carrier effectively alleviates the adverse effects of high alkalinity and temperature variations. The expanded perlite-based immobilization strategy significantly extended microbial service life, improved remediation efficiency, enhanced engineering feasibility, and reduced long-term maintenance costs. This research offers critical technical support for the development of durable and high-efficiency self-healing concrete systems.IMPORTANCEMicrobially induced carbonate precipitation (MICP) has gained significant attention as a promising technology in architecture and civil engineering. However, the understanding of microbial long-term activity and mineralization capacity within cement-based materials remains limited. This study investigated the influence of environmental factors on microbial spore survival in such materials by monitoring key indicators, including microbial concentration, urease activity, and mineral precipitation. Furthermore, it identified specific environmental conditions that support prolonged microbial viability. The use of expanded perlite as a carrier material aimed to mitigate external environmental stresses on microorganisms, thereby extending their mineralization capability over extended periods. These findings provide a scientific basis for the rational design of microbially mediated self-healing concrete systems.

Trichodiene is a sesquiterpene hydrocarbon and the precursor of trichothecene mycotoxins produced by Fusarium and other fungi. Interestingly, utilizing trichodiene as a volatile treatment has been shown to reduce mycotoxin production in Fusarium graminearum-infected wheat heads. This research developed a pilot-scale fungal fermentation method to produce trichodiene as a biofumigant to mitigate mycotoxin contamination. A TRI4 mutant strain of Fusarium sporotrichioides was used to mass-produce trichodiene. Xanthotoxin, a furanocoumarin produced by parsnips, was used to enhance trichodiene production in this mutant. Xanthotoxin treatments significantly increased trichodiene yield and were found to promote lipid droplet release from the fungal germlings. Benchtop scale evaluations were conducted to determine the impacts of xanthotoxin concentration, fermentation time, extraction solvent, and filtration on overall yield. After optimal conditions were identified, the fungal cultures treated with xanthotoxin were fermented for 1 week in a 30 L bioreactor. After an organic extraction of the fungal culture and concentration of the extract, trichodiene was isolated using silica gel column chromatography. Purified trichodiene reduced mycotoxin production in F. graminearum in a dose-dependent manner. This research will allow the production of trichodiene in sufficient quantities to further evaluate its efficacy as a biofumigant to suppress mycotoxin production in F. graminearum.IMPORTANCEFood contamination from microbial toxins is a threat to human and animal health. Globally, the pathogen Fusarium graminearum causes annual losses in billions of dollars for cereal farmers and producers. Previous studies have shown that the fungal terpene trichodiene can suppress the production of vomitoxin (deoxynivalenol) by F. graminearum. We developed a way to scale production of trichodiene and use it to inhibit F. graminearum toxin contamination. A mutant strain of Fusarium sporotrichioides that produces trichodiene was grown in large 30 L fermenters and treated with xanthotoxin, a natural compound made by parsnips. Xanthotoxin caused lipid droplet release from the fungus and increased trichodiene yield. The purified trichodiene effectively reduced toxin production by F. graminearum by direct contact or as a volatile. Based on these research findings, trichodiene can be produced using common large-scale fermentation methods. Field formulations can now be developed to suppress mycotoxin contamination in food and feed.

The development of sustainable biocatalytic processes for pharmaceutical synthesis represents a major goal in green chemistry. Ene-reductases (ERs) are attractive biocatalysts for asymmetric hydrogenation of activated alkenes, yet their industrial application is often constrained by limited substrate scope and stability. In this study, we explored the deep-sea sediment metagenome of the South China Sea and identified 41 putative ER genes, with 22 successfully solubly expressed in Escherichia coli. Biochemical characterization revealed broad substrate specificity, achieving up to 90% conversion for diverse α,β-unsaturated compounds. Notably, three enzymes (S2gene2614772, S2gene1139, and S2gene22028) exhibited exceptional adaptability, maintaining high activity over a wide pH range (5.5-8.5) and at low temperatures (15°C). However, none of the wild-type ERs showed significant activity toward the prochiral substrate 2-oxo-4-phenyl-3-butenoic acid, a key intermediate for angiotensin-converting enzyme inhibitors (ACEIs). Through directed evolution, we obtained a mutant (S2gene22028-G102S) with 30-fold enhanced activity, reaching 90% conversion at 10 mM substrate. Scale-up synthesis (5 mmol substrate) afforded 2-oxo-4-phenylbutyric acid (OPBA) at 11 mg/mL, demonstrating industrial potential. This study highlights marine metagenomes as valuable sources of novel ERs and provides an efficient biocatalytic route to ACEI precursors.IMPORTANCEThe development of sustainable biocatalysts for pharmaceutical synthesis is a pivotal goal in green chemistry. This study leverages the untapped enzymatic diversity of the South China Sea deep-sea sediment metagenome to discover novel ene-reductases (ERs). We not only identified robust ERs with broad substrate promiscuity and exceptional adaptability to low temperature and pH fluctuations but also successfully engineered a variant to overcome the key biocatalytic challenge in the synthesis of 2-oxo-4-phenylbutyric acid (OPBA), a critical precursor to angiotensin-converting enzyme inhibitors. Our work underscores marine metagenomes as a valuable reservoir for discovering industrially relevant biocatalysts and demonstrates the power of combining metagenomic mining with protein engineering to enable greener manufacturing routes for high-value pharmaceuticals.