Applied and Environmental Microbiology最新文献

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.

Poultry house fine particulate matter (PM_2.5) poses significant respiratory risks to poultry by penetrating deep into the lung and triggering inflammatory cascades. In this study, 21- to 28-day-old broilers were exposed to total suspended particulates enriched in PM_2.5 (2 mg/m³, 2 h/day) to investigate pulmonary injury and gut-lung axis perturbations. PM_2.5 exposure induced collapse of the hexagonal lobular architecture, elevated pulmonary expression of IL-1β, IL-2, IL-6, IL-8, and IL-10, and activated NF-κB signaling. Concurrently, cecal microbiota α-diversity increased while the community shifted toward pro-inflammatory taxa (Alistipes, Rikenellaceae) and away from SCFA-producing species (Bacteroides uniformis, Parabacteroides). Oral supplementation of B. uniformis restored its abundance, replenished acetate and propionate levels, and attenuated lung injury by reducing APC activation (CD40, CCL4) and Th1 polarization (T-bet, IFN-γ, IL-18), while promoting regulatory T cell markers (FoxP3). Dietary sodium propionate supplementation in feed (0.4%) similarly mitigated pulmonary inflammation and Th1 skewing, albeit without enhancing Treg responses. These findings demonstrate that PM_2.5-induced lung damage is intricately linked to gut dysbiosis and SCFA depletion and that restoration of B. uniformis or its metabolite propionate can recalibrate the gut-lung axis to suppress innate and adaptive inflammatory pathways. This work highlights microbiota- and metabolite-based interventions as promising strategies to protect poultry respiratory health and performance under air-polluted conditions.IMPORTANCEThis study reveals that poultry house-derived PM_2.5 not only causes direct lung inflammation but also perturbs the gut-lung axis by depleting beneficial SCFA-producing bacteria. The resulting gut dysbiosis amplifies respiratory immune injury, highlighting a previously underappreciated systemic effect of airborne pollutants in livestock environments. Our findings suggest that microbiota- and metabolite-targeted dietary strategies can mitigate air pollution-induced health risks in poultry. This work provides new insights into the broader ecological and agricultural consequences of PM_2.5 exposure and supports sustainable, non-antibiotic interventions to enhance animal welfare and productivity under deteriorating air quality conditions.

Despite the increasing number of reports on hypervirulent and extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae infections, data on the distribution of these pathogens in the community are limited. To address this knowledge gap, we investigated the carriage rates of K. pneumoniae complex in the stools of community-dwelling individuals in Japan. From 627 stool samples submitted to a commercial diagnostic laboratory, 407 Klebsiella strains were identified from 368 samples, corresponding to a colonization rate of 58.7%. Based on whole-genome sequencing, K. pneumoniae was the most prevalent species (n = 218, 53.6%), followed by Klebsiella variicola (n = 137, 33.7%). The detection rate of K. variicola was higher than previously reported in studies from other Asian countries. The overall distribution of sequence types (STs) was similar to those observed in previous studies of clinical isolates. However, hypervirulent K. pneumoniae clones, specifically ST23-K1 and ST412-K57, and ESBL-producing strains were rare, each accounting for less than 1% of the strains. These findings suggest that, while carriage of K. pneumoniae complex species is common in the community, healthcare settings may represent a more significant reservoir of hypervirulent and ESBL-producing K. pneumoniae strains in this epidemiological setting.IMPORTANCEKlebsiella pneumoniae complex species are bacteria that can cause serious infections, especially in hospital settings. Some types have become more dangerous because they are resistant to antibiotics or highly virulent. To better understand where these harmful clones come from, this study looked for Klebsiella species in healthy people living in the community in Japan. The results showed that these bacteria are commonly found in the gut, particularly K. pneumoniae and K. variicola. While some strains with traits linked to antibiotic resistance or severe infections were identified, they were rare. These findings suggest that most people carry Klebsiella strains as commensals and that the more dangerous forms of Klebsiella are likely spreading mainly in healthcare settings.

Ulvan is a major polysaccharide in marine green algae. Its oligosaccharide degradation products possess diverse bioactivities and hold considerable potential for various applications. Ulvan lyases, the key enzymes responsible for cleaving ulvan glycosidic bonds, generate bioactive oligosaccharides and play an essential role in ulvan degradation. However, studies on ulvan lyases remain limited, particularly for the poorly characterized polysaccharide lyase (PL) 40 family. Here, we identified Uly1040, a novel PL40 ulvan lyase, from the marine bacterium Alteromonas macleodii isolated from the intestine of an Aplysia sea slug. Uly1040 displays a unique two-domain architecture not previously reported in ulvan lyases. Mechanistically, Uly1040 employs a distinct His/Tyr catalytic strategy, divergent from known ulvan lyase mechanisms. During catalysis, Trp246 and Asn245 neutralize the negative charge of the carboxyl group at the +1 subsite. Concurrently, Mn²⁺, His487, and Asp358 activate His485 to serve as the catalytic base, while Tyr305 functions as the catalytic acid. Bioinformatic, phylogenetic, and biogeographic analyses further demonstrated that this catalytic mechanism is conserved across PL40 lyases and that Uly1040-like enzymes are widespread in marine environments. Collectively, these findings expand our understanding of PL40 ulvan lyases and provide new insights into the enzymatic basis of marine biomass utilization.

Importance: Ulvan is a major structural polysaccharide in marine green algae, and its enzymatic degradation releases bioactive oligosaccharides with promising biotechnological potential. Ulvan lyases are key to this process, yet most characterized enzymes belong to only a few polysaccharide lyase families, leaving the PL40 family largely unexplored. Here, we identify and characterize Uly1040, a novel PL40 ulvan lyase from Alteromonas macleodii, revealing an unprecedented two-domain architecture and a distinct His/Tyr catalytic mechanism. Structural and biochemical analyses show that Mn2+, His487, and Asp358 cooperatively activate His485 as the catalytic base, while Tyr305 acts as the catalytic acid-representing a mechanistic innovation in ulvan cleavage. Bioinformatic and phylogenetic analyses indicate that the PL40 lyases are widespread in marine environment, and this catalytic strategy is conserved among PL40 enzymes. This work uncovers a previously unknown enzymatic paradigm for ulvan degradation, deepening our understanding of marine polysaccharide utilization and microbial carbon cycling.

A recent study by R. Cheng, T. Lv, P. Ji, B. Ma, et al. (Appl Environ Microbiol 91:e01685-25, 2025, https://doi.org/10.1128/aem.01685-25) used multi-omics analysis to reveal the molecular map of pathogen virulence differentiation driven by natural variation. Building on this work, this article examines how natural variation shapes pathogen virulence and disease prevalence and explores the use of multi-omics approaches to uncover associated molecular mechanisms. The opportunities and challenges of applying multi-omics technologies in plant disease management are also discussed in this article.

The spaceflight environment imparts unique selective pressures on the plants and microbes of plant growth chambers on the International Space Station (ISS), which generally manifests through genetic signatures associated with a heightened response to stress. Terrestrially, a baseline understanding of the gene fitness response for any plant growth-promoting microbe when in a tripartite relationship with host and pathogen is currently unknown and is important to characterize before closed-environment spaceflight implementation. To that end, this study evaluated the behavior of an ISS plant habitat isolate of Burkholderia contaminans as tomato seeds transitioned to seedlings and assessed gene fitness during challenge with Fusarium oxysporum f. sp. lycopersici (FOL), the causal agent of Fusarium wilt. Using a seed film delivery method vetted for spaceflight, B. contaminans was applied to Solanum lycopersicum cv. Red Robin seeds. Green fluorescent protein (GFP)-tagged B. contaminans was primarily found to localize at the shoot-root junction and was detected on shoots. Upon FOL challenge, B. contaminans population levels remained stable, and despite harboring antifungal and plant growth-promoting capacity, these properties were not conferred in response to FOL in the tissue culture environment. To probe mechanisms underlying the bacterial-fungal interaction between B. contaminans and FOL in the tomato root zone, a genome-wide transposon mutant library was developed for the B. contaminans isolate. Transposon sequencing (Tn-Seq) analysis revealed that the type II secretion system (T2SS) was critical for root zone establishment, whereas a Nudix hydrolase was specifically important for responding to FOL infection and provided further confirmation that antifungal and siderophore-producing gene clusters were not.IMPORTANCEThis study is the first to evaluate the genetic fitness of a Burkholderia contaminans International Space Station (ISS) isolate in the plant root zone in association with the obligate pathogen Fusarium oxysporum f. sp. lycopersici (FOL). This isolate of B. contaminans establishes in the tomato root zone, does not confer plant growth promotion in tissue culture, but is persistent in the tomato root zone when challenged with FOL through stress-adaptation mechanisms rather than direct antifungal antagonism. The response of B. contaminans in the host root zone when in the presence of the pathogen suggests the microbe is primed to counter stress, which may further confer an advantage in the spaceflight environment.

Laccases are present as isozymes in white-rot fungi, yet their evolutionary history and functional role in lignin degradation remain controversial. Trametes hirsuta, a ubiquitous fungus in forest ecosystems, can completely break down cellulose, hemicellulose, and lignin in wood. Based on bioinformatic and biochemical characterization, we have shown that five laccase isozyme genes (lacA-E) in Trametes hirsuta AH28-2 were derived from a single ancestral laccase gene, lacF, with lacA and lacB originating from disparate evolutionary branches. The syringyl-type (S-type) lignin model compounds significantly induced the expression of lacA-lacE at both transcriptional and expression levels. Furthermore, in vitro and in vivo analyses demonstrated that the later-emerging laccases, LacA and LacB, primarily contribute to oxidizing S-type lignin present in hardwood, whereas laccase LacF plays a primary role in oxidizing guaiacyl-type (G-type) lignin found in softwood. Finally, evolutionary analysis of ancestral laccases from Agaricomycetes fungi also revealed a shift from better oxidation of G-type lignin in softwood by earlier ancestral laccases to better oxidation of S-type lignin in hardwood by later ancestral laccases. These findings indicate that laccase evolution in Agaricomycetes fungi is consistent with lignin synthesis. We have demonstrated the direct involvement of laccases at different evolutionary stages in preferentially oxidizing different types of lignin.IMPORTANCELaccases in white-rot fungi always exist in the form of isozymes. However, the evolutionary history and functional diversification of laccase isozymes remain controversial. Our study demonstrates that the six laccase isozymes in Trametes hirsuta AH28-2 belong to three distinct evolutionary branches. Among them, LacF represents an earlier-emerging branch and primarily contributes to oxidizing the G-type units of gymnosperm lignin. In contrast, LacA and LacB, which are later-emerging, primarily contribute to oxidizing the S-type units in angiosperm lignin. Interestingly, ancestral laccases reconstructed at different evolutionary nodes also display distinct lignin oxidation preferences. This suggests that the evolution of laccases in Agaricomycetes fungi is closely linked to the emergence of S-type lignin units in angiosperms. These findings reveal the co-evolutionary relationship between lignin structural changes and fungal laccase diversification, providing new insights into the evolutionary mechanisms and biological functions of laccase isozymes.

The current understanding of carbohydrate substrate degradation is largely derived from incubation experiments involving specific substrates. In estuaries, carbohydrates are often grouped together with other sources of carbon, for analytical purposes, and measured as total and fractional organic matter. Here, we describe putative carbohydrate degradation at the polysaccharide level by the prokaryotic community in an estuary. Samples were collected along a freshwater-to-marine salinity gradient from both the water column and underlying benthic sediments. Metagenomic and metatranscriptomic data were used to determine carbohydrate-active enzyme (CAZyme)-encoding metagenome-assembled genomes and associated transcriptional activity across the gradient. Previous work demonstrated assimilation of xylan (a component of hemicellulose) in estuaries. We show the genetic mechanisms associated with the degradation of xylan, as well as arabinogalactan (also from hemicellulose), and various other glycans were widespread among estuarine taxa and actively expressed. In addition, results show different carbohydrate degradation strategies between planktonic and benthic organisms. For example, results indicate that sediment communities harbored a greater variety and density of CAZyme-encoding genes and capacity to degrade complex plant biomass (cellulose and hemicellulose) and dedicated more gene transcription overall to CAZymes than planktonic communities. In contrast, planktonic prokaryotes tended to express a greater fraction of their CAZyme-encoding gene repertoires. The transcription of gene clusters associated with degrading beta-1,3-glucans such as laminarin was prevalent in the water column. Microbial activity to degrade alpha-glucans such as glycogen was predicted to be ubiquitous but was greatest in planktonic communities. Taken together, results highlight differences in the capacity of planktonic and benthic communities to degrade carbohydrates, which reflect differences in substrate availability and complexity.IMPORTANCEEstuaries are productive ecosystems that combine various forms of organic carbon from autochthonous (e.g., algal primary producers and mangroves) and allochthonous (e.g., terrestrial plant) sources. The degradation and recycling of this organic carbon is driven by heterotrophic bacteria that are expected to harbor diverse genetic mechanisms for carbohydrate degradation to match the diversity and complexity of organic carbon encountered in the environment. Results here illustrate the diversity of carbohydrate-active enzymes (notably glycosyl hydrolases) encoded by estuarine communities and the different substrate prioritizations of planktonic and benthic communities.

Despite the promise of phages as antibiotic alternatives, their efficacy is often undermined by the rapid emergence of bacterial resistance. Phage-derived enzymes, particularly depolymerases, offer a compelling strategy to overcome this limitation and enhance antibacterial therapy. Focusing on Vibrio pathogens, the major threats to global aquaculture, our bioinformatic analysis revealed that 79.4% of cultured and 46.2% of uncultured Vibrio phages encode putative depolymerases, underscoring a vast but underexploited antibacterial resource. We further isolated and characterized VnaP, a depolymerase-encoding phage (novel genus, Caudovircetes) that forms distinctive halo plaques indicative of depolymerase activity. Genome analysis identified ORF193, encoding a novel polysaccharide depolymerase lacking sequence or structural homology to any characterized depolymerases. Heterologously expressed Dep193 efficiently degraded Vibrio surface polysaccharides and exhibited potent antibiofilm activity. While Dep193 exhibits modest standalone antibacterial activity, its synergistic combination with VnaP significantly enhances bacterial clearance and delays resistance emergence across multiple Vibrio species. As the first biochemically validated Vibrio phage depolymerase, Dep193 broadens the known diversity of these enzymes and establishes an effective strategy for Vibrio control in aquaculture.IMPORTANCEThe rapid emergence of antibiotic-resistant Vibrio strains threatens global aquaculture sustainability, necessitating alternative antimicrobial strategies. This study identifies and characterizes Dep193, a novel phage-encoded depolymerase with polysaccharide-degrading and antibiofilm activities that enhances phage therapy efficacy through a previously unreported mechanism. The Dep193-phage VnaP combination exhibits broad-spectrum activity against multiple Vibrio species, demonstrating strong potential as a therapeutic strategy for aquaculture. Notably, Dep193 lacks any recognizable functional domains found in characterized depolymerases, representing the first validated member of a novel evolutionary clade. These findings expand the known diversity of phage depolymerases and provide a promising avenue for the targeted control of Vibrio infections in aquaculture.