Applied and Environmental Microbiology最新文献

Vibrionaceae are a diverse family of bacteria that contain pathogenic species, including those within the Vulnificus clade: Vibrio vulnificus, Vibrio navarrensis, and Vibrio cidicii. While V. vulnificus is a generally well-characterized environmental pathogen, V. cidicii and V. navarrensis are relatively rare, recently identified species that our current understanding of virulence and environmental adaptation is limited. Here, we investigate genetic relatedness across these three species to identify shared and species-specific genes, including markers of virulence. Using publicly available genome assemblies (n = 76), we evaluated phylogenetic and genomic diversity across this clade. We sampled all available V. navarrensis and V. cidicii genomes and a biodiverse curated set of four V. vulnificus ecotypes to ensure representative coverage. Our results indicate that all three species share 2,313 core genes, many of which are core bacterial functions in addition to pathways important to environmental response, host immune evasion, and iron acquisition. Moreover, V. cidicii and V. navarrensis have extensive genetic similarity between them, including average nucleotide identities >95% and 370 shared genes. Despite this similarity, they both remain more phylogenetically distant from V. vulnificus and lack key virulence genes, such as rtxA, indicating alternative pathogenic potential. Overall, these findings reveal distinct evolutionary strategies within the Vulnificus clade, with V. vulnificus specializing in enhanced pathogenesis, while V. navarrensis and V. cidicii have evolved enhanced environmental persistence capabilities.

Importance: Vibrio species are important environmental aquatic bacteria that pose a threat to human and animal health across the globe. This study applied comparative genomics to investigate the genetic relatedness of Vibrio vulnificus, Vibrio navarrensis, and Vibrio cidicii, with special focus on genes associated with environmental adaptation and virulence between and within each species. Results indicate V. navarrensis and V. cidicii share many genes, are phylogenetically close, and exhibit genomic signatures of enhanced environmental persistence and stress tolerance in addition to survival in anthropogenically impacted marine systems. Furthermore, V. vulnificus possesses an overall different virulence potential with the presence of RTX systems. This adds to our understanding of genetic diversity and pathogenic mechanisms within an important group of marine pathogens.

Sulfate-reducing bacterium Desulfovibrio piger is a common member of the human gastrointestinal microbiome, associated with inflammatory conditions but also prevalent in healthy individuals. This suggests that lifestyle factors may shape its ecological role. We investigated prophage carriage and release in three new D. piger strains from healthy donors and strain FI11049 from a patient with ulcerative colitis. Sequencing revealed a larger genome in strain FI11455 (3.096 Mb) compared to FI11311 (2.985 Mb) and FI11458 (2.838 Mb), including a 154 kb megaplasmid which contained an 87 kb section with high similarity to the chromosome of strain FI11311, suggesting horizontal gene transfer between chromosomes and plasmids. This section encoded genes involved in DNA replication, transcription, and recombination, as well as protein folding and modification, defense, and phage proteins. Strain FI11049 showed less than 95% similarity to other D. piger strains but shared similar prophages with them. Each strain carried four to five predicted prophages, ranging from 30 to 60 kb, which clustered into four groups, with at least three groups per strain. Although the prophages had no nucleotide similarity to known phages, genes for lysis, integration, regulation, and structural proteins were identified, and three groups contained Mu-like proteins. Electron microscopy and PCR of mitomycin C-induced supernatants confirmed the release of tailed bacteriophage particles and capsids of multiple prophages. Similar results were demonstrated from uninduced samples, indicating spontaneous prophage release. Host defense systems were widespread, and cross-infections failed to identify suitable hosts in related strains and species. This is the first evidence of prophage release in gut-associated Desulfovibrio, with implications for gene transfer in the gut.

Importance: Gastrointestinal health has a significant impact on quality of life, and increasing profiling of the gut microbiome is identifying key players involved in disease states. However, evidence of the association of sulfate-reducing bacteria with pathologies, such as inflammatory bowel disease and colorectal cancer, conflicts with their prevalence in healthy subjects. Investigating the ecology of D. piger in the gut may be key to answering if and why it can be harmful and could inform future interventions. Here, we show that gut-associated D. piger strains carry multiple prophages, some of which are spontaneously released as bacteriophage particles in culture. Our results pave the way for future work to understand prophage release in gut conditions and its effects on D. piger populations.

Saccharomyces cerevisiae is an important organism for basic research and applied biotechnology. Genome editing techniques, particularly CRISPR/Cas9 from Streptococcus pyogenes, have greatly facilitated saturation genome editing in yeast to understand mutant functions on a large scale. However, Cas9 is restricted by its targeting preference for G-rich protospacer-adjacent motif (PAM) sequences. To broaden the targeting scope, we established an efficient homology-integrated CRISPR/Cas12a system to install genetic variants through homologous recombination by targeting T-rich PAMs. We benchmarked the PAM compatibility of PAM-relaxed Cas12a variants and identified the improved LbCas12a (impLbCas12a) as the most efficient and PAM-relaxed variant in S. cerevisiae, showing high editing purity and an editing window centering the double-strand break. We show that our system can be used to perform targeted saturation mutagenesis to reveal functional variants not captured previously. By using a homology-integrated CRISPR RNA array, we utilized the multiplexing capability of CRISPR/Cas12a to realize multiplex genetic variant installation. Our system enriches the yeast genetic variant engineering toolbox, complementing the commonly used CRISPR/Cas9 system.IMPORTANCECRISPR/Cas9 has facilitated yeast functional genomics by generating a large number of precise genetic variants in a very short period of time. This enabled the interrogation of reconstituted natural genetic variants across different genetic backgrounds or entirely synthetic mutations to discover novel or improved functions. However, Cas9 only targets a limited genomic sequence space due to its preference for G-rich PAM sequences. In this study, we close this gap by developing a CRISPR/Cas12a-based system to engineer user-defined genetic variants targeting T-rich PAM sequences. Our system adopts a homology-integrated design and the most PAM-relaxed Cas12a characterized in yeast to date. These features collectively enabled the creation of genetic variant libraries and multiplex edited strains. This genome editing tool can be used together with Cas9-based tools to interrogate a greater genomic sequence space.

Plain river networks are becoming increasingly sensitive and vulnerable under the combined effects of climate change and landscape alteration. Understanding adaptive changes in bacterioplankton communities in response to the environment is important for improving the health of river networks. Still, little is known about the changes in bacterioplankton communities and their drivers during the rainy season. By applying co-occurrence networks, source-sink analyses, variance partitioning, and the MIKE11 model, we investigated changes in the composition, interaction, and source of the bacterioplankton community at 32 sampling sites along an anthropogenic pressure gradient (urban, suburban, and agricultural area) in the Taihu Lake plain river networks before and after the Plum rain (East Asian rainy season), as well as the drivers (water chemistry, land use, and hydrological factors) of this change. The results showed that the Chao1 diversity index and Shannon diversity index (richness) decreased significantly, and the inverse Simpson's diversity index (evenness) increased significantly after the plum rain, especially in the suburban area, indicating a decrease in the contribution of rare taxa. Related to the reduction of inputs by rare taxa after the plum rain, network analyses revealed that bacterioplankton communities were no longer tightly linked. The analysis demonstrated that water chemistry explained these changes in the cyanobacterial communities better than the hydrological indicators as evidenced by the substantially higher explanatory power of simulated data (pre-plum rain: 86%; post-plum rain: 50%) compared to measured data (pre-plum rain: 36%; post-plum rain: 22%). The use of models to obtain hydrological data was feasible when the integrity of the data analysis was ensured and the analytical process was optimized.

Importance: Unlike natural rivers, the microbial response mechanisms in human-dominated plain river networks remain poorly understood. These densely populated aquatic ecosystems, under intense anthropogenic influence, face dual pressures from climate change and landscape alteration. Their fragile hydrodynamics increase ecological vulnerability during the rainy season. Focusing on the Plum rain season, this study reveals that after the rainfall, community diversity significantly decreases, the contribution of rare taxa reduces, and bacterioplankton communities were no longer tightly linked. Combining MIKE11 modeling with multi-source environmental data, the study clarified the decisive role of water chemistry parameters in Cyanobacteria community shifts and validated the applicability of model data in ecological process research. These findings advance understanding of microbial adaptation in human-disturbed river networks and support ecosystem health assessment. The model-data fusion analysis method established in this study provides a technical framework for similar water ecosystem research.

Keratin-rich by-products from the poultry, textile, and leather industries pose a significant challenge for sustainable waste management due to their highly recalcitrant nature. While microbial degradation of keratin has been studied and may offer a viable solution, the complex enzyme machineries that are potentially needed to degrade these recalcitrant by-products remain partly unknown. In this study, we employed a high-resolution proteogenomic approach to characterize the keratinolytic machinery of Onygena corvina, a non-pathogenic saprophytic fungus. Using a membrane agar plate method with insoluble substrates, we obtained secretomes enriched in secreted and substrate-bound proteins during growth on α- and β-keratin-rich substrates, specifically wool and feather meal. Our findings reveal that O. corvina has a richer proteolytic machinery than previously reported, including enzymes that are used across keratin types, as well as enzymes that are specifically targeted to either α- or β-keratin. In addition to proteases, the secretomes contain numerous other proteins, including cell wall-modifying enzymes, oxidoreductases, esterases, phosphatases, and sialidases that are involved in the deconstruction of keratin. We propose that these additional enzymes destabilize keratin through a combination of mechanical keratinolysis, removal of post-translational modifications, reduction of disulfide bonds, and cleavage of isopeptide bonds, thereby enhancing proteolytic accessibility. Interestingly, keratin degradation by O. corvina was most efficient when using mixed substrates containing both feather and wool meal. These novel insights into the keratinolytic system of O. corvina underscore the importance of considering synergistic enzyme interactions when developing biotechnological approaches for the valorization of keratin-rich byproducts.IMPORTANCEKeratin-rich by-products from agro-industrial processes are generated in large volumes and present a significant environmental burden due to their recalcitrant nature. Microbial degradation offers a promising solution, but the mechanisms involved in keratin decomposition remain elusive. In this study, we show that the saprophytic fungus O. corvina secretes a diverse and specialized enzymatic arsenal when grown on keratin-rich substrates, such as feather (β-keratin) and wool (α-keratin) meal. Its secretome includes both shared and keratin-type specific proteases, along with accessory proteins, such as oxidoreductases, esterases, phosphatases, and sialidases, that aid in substrate destabilization. Our findings uncover the complex enzymatic system driving keratinolysis in this fungus and provide a foundation for developing sustainable, enzyme-based strategies to valorize keratin-rich waste.

Bacterial selenate reduction is a key microbial process that affects the speciation and mobility of selenium in the environment. In this study, we identified the selenate reductase gene in the facultative anaerobe Enterobacter cloacae SLD1a-1. Genome sequencing revealed a membrane-bound, twin-arginine translocation (TAT) exported molybdoenzyme operon designated as srnABCD, under the regulation of the fumarate and nitrate reductase regulator (FNR) transcription factor. The srnA gene encodes a molybdenum-containing subunit; srnB and srnC encode iron-sulfur and membrane anchor subunits, respectively; and srnD encodes a TAT chaperone. Targeted mutagenesis of the srnA gene resulted in a mutant defective in selenate reduction. Complementation with the wild-type srnA sequence restored the abolished phenotype. Heterologous expression of srnA in an Escherichia coli ΔynfEF mutant conferred selenate reduction activity, demonstrating cross-species functionality. Protein structure modeling of the selenate reductase using Boltz-1 showed a funnel-shaped active site involved in selenate binding and reduction. These findings provide new molecular insights into the genetics and mechanism of bacterial selenate reduction.

Importance: Selenium pollution poses risks to ecosystems and human health, largely due to the mobility and toxicity of selenate, a common form found in soil and water. Diverse bacterial species are able to convert soluble selenate into insoluble elemental selenium, but the genes and enzymes that underpin this process are not fully understood. In this study, we identified a gene in Enterobacter cloacae SLD1a-1 that enables the bacterium to catalyze selenate reduction. We showed that this gene produces a functional enzyme even when it is transferred into a different species, Escherichia coli. Protein structure modeling revealed features of the enzyme that help it recognize and reduce selenate. This information advances our understanding of how selenium is enzymatically cycled in the environment.

The German cockroach, Blattella germanica, can harbor and transmit enteric human pathogens, including Salmonella enterica serovar Typhimurium. German cockroaches are omnivores that subsist on highly varied diets in the field, in contrast to most arthropod vectors. Diet plays an important role in shaping the gut microenvironment across a range of animals, which can in turn affect numerous aspects of physiology, including the ability to resist infection. Yet, the impact of diet on the ability of cockroaches to maintain and transmit pathogens had not been investigated previously. Here, we tested the hypothesis that dietary differences among otherwise identical populations of B. germanica could lead to differences in vector competence for S. Typhimurium. Cockroaches were maintained on three defined formulated diets with distinct macronutrient profiles for 10 days. Food consumption was monitored during this period, and the gut microbiome was profiled by 16S rRNA amplicon sequencing. The cockroaches were then orally infected with S. Typhimurium, and pathogen loads in the gut and excreta were quantified. Cockroaches equally consumed formulated high-protein, high-fat, and high-carbohydrate diets in no-choice assays. Furthermore, as expected, some significant differences in microbiome composition and diversity were observed between groups of cockroaches maintained on different diets. However, despite the effects on the microbiome, no significant diet-dependent differences in the load of S. Typhimurium maintained in the gut or shed in the excreta were observed. Although the results provide evidence that the dietary macronutrient profile is not a major contributor to vector competence, the possibility that other natural diets could influence pathogen infection and transmission dynamics is not ruled out by this study.

Importance: German cockroaches are one of the most common structural pests worldwide, while Salmonella enterica serovar Typhimurium is an emerging human pathogen accounting for a significant portion of the global burden of enteric disease. Understanding the factors that contribute to the ability of cockroaches to transmit pathogens is important for infection prevention, but these remain almost entirely unknown. Here, we provide new insight into the variables involved in the vector competence of cockroaches.

Controlled environment agriculture (CEA) offers protection from external contaminants but introduces unique food safety challenges, particularly related to equipment sanitation. This study evaluated the occurrence and persistence of Listeria monocytogenes and related species in a commercial soil-based CEA facility through a 1-year environmental monitoring program (EMP). A total of 169 samples, including baby leaves, soil, water, and swabs from equipment surfaces, were collected across three visits. L. monocytogenes was detected in only one sample (0.59% prevalence), whereas Listeria innocua was isolated from harvesting crates and structural surfaces (3/169). Molecular confirmation and characterization were conducted using PCR (hly, iap, sigB), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and whole genome sequencing (WGS). To assess the potential for cross-contamination, L. innocua isolates were used for inoculation studies on reusable plastic crates. Viable cells survived up to 24 h post-inoculation despite water rinsing under 25°C and 60%-70% relative humidity. These findings suggest a low risk of L. monocytogenes contamination in this soil-based CEA system but underscore the importance of targeted sanitation, particularly for harvesting equipment, to prevent harborage and transfer of Listeria spp.IMPORTANCEControlled environment agriculture (CEA) is a rapidly expanding approach to producing fresh food year-round with greater resource efficiency. However, it presents unique challenges for managing foodborne pathogens. This study demonstrates that in this soil-based indoor system, contamination risks persist, particularly via inadequately cleaned harvesting equipment. The boot cover finding is interpreted as an indicator of environmental contamination, but it does not constitute evidence of footwear-to-crop transfer risk. Although Listeria monocytogenes was rarely detected, related species like Listeria innocua persisted on reusable plastic crates and structural surfaces, highlighting weaknesses in current sanitation protocols. The use of boot covers as environmental monitoring tools proved valuable for detecting contamination sources. These findings underscore the need for tailored sanitation strategies and comprehensive environmental monitoring programs to enhance food safety in CEA systems and prevent pathogen harborage and spread.