Applied and Environmental Microbiology最新文献

Many thermophilic autotrophs in deep-sea hydrothermal vents are anaerobes that require H₂ for growth. However, our understanding of how non-methanogenic thermophilic hydrogenotrophs adapt to low H₂ conditions is nascent. In this study, the thermophilic autotrophic thiosulfate-reducing bacterium Desulfurobacterium thermolithotrophum HR11 was grown at 72°C in a chemostat at 2 and 43 µM aqueous H₂ concentrations to quantify changes in growth and H₂S production kinetics and in its proteome in response to H₂ availability. At 2 μM H₂, cultures showed a decrease in specific growth rate, maximum cell concentration, and H₂S production while cell yield (cells produced per mol of H₂S produced) was unchanged. Differential proteomic analysis identified 79 and 66 proteins having more than twofold higher and lower abundances (P_adj < 0.01), respectively, under 2 μM H₂ conditions out of 887 total detected proteins. Among these, a membrane-bound ferredoxin-dependent [NiFe] hydrogenase and a cytoplasmic NAD(P)⁺-dependent [NiFe] hydrogenase increased in cells grown on 2 μM H₂. Also, on 2 μM H₂, a membrane-bound menaquinone-dependent [NiFe] hydrogenase increased and likely generated a proton motive force and drove ATP synthesis, while a membrane-bound menaquinone-dependent [FeFe] hydrogenase and a membrane-bound NADH dehydrogenase decreased. Four reductive TCA cycle enzymes used for CO₂ fixation were more abundant on 2 µM H₂. These data suggest that hydrogenase and CO₂ fixation enzyme abundances are regulated by H₂ availability and support the idea of a shift in electron disposal toward CO₂ reduction and assimilation on low H₂.

Importance: Understanding how thermophilic, autotrophic sulfur-reducing bacteria such as Desulfurobacterium thermolithotrophum adapt to varying H₂ concentrations is important for understanding competition and survival strategies in energy-limited subseafloor hydrothermal environments. In this study, D. thermolithotrophum showed metabolic adaptations under low H₂ conditions that may provide them with a competitive growth advantage in hydrothermal vent ecosystems when H₂ is limited and other thermophilic hydrogenotrophs, such as methanogens, are also commonly found. This work also demonstrated the dynamic relationship between different types of hydrogenases and how they are coordinated with each other and other key pathways such as CO₂ fixation.

Shotgun metagenomic sequencing has emerged as a powerful tool for exploring microbial diversity and uncovering genes encoding novel biocatalysts from complex environments. Here, we report the discovery and characterization of a new FAD-dependent D-lactate dehydrogenase (PdG-D-LDH) from the gut microbiome of the isopod Porcellio dilatatus. The enzyme was identified through in silico screening using BLAST and AlphaFold3 and functionally characterized as a homodimeric, thermoactive, and thermostable protein, demonstrating the robustness required for biotechnological applications. PdG-D-LDH exhibits a strong catalytic preference toward D-lactate and preferentially reduces quinones over cytochrome c or molecular oxygen. X-ray crystallography revealed a VAO/PCMH-like fold with a solvent-accessible active site that harbors both a FAD cofactor and an Fe(II) ion. Molecular docking studies provided insights into the structural determinants of its stereoselective substrate recognition. Under mild conditions, the enzyme catalyzed the oxidation of D-lactate to pyruvate with a 90% yield after 24 h of reaction, using molecular oxygen as the electron acceptor.

Importance: This study illustrates how metagenomics, structural biology, and computational tools can jointly drive the discovery of new enzymes with valuable biotechnological applications aligned with circular economic principles. The newly identified D-lactate dehydrogenase, PdG-D-LDH, exhibits thermostability, stereoselectivity, and high catalytic efficiency, providing new insights into the structure-function relationships of lactate-metabolizing enzymes.

Oil reservoirs are complex ecosystems where microorganisms play a vital role in hydrocarbon degradation, mostly with methanogenesis as the terminal electron-accepting process. Especially in offshore oil reservoirs, sulfate-containing seawater is injected during the oil production to maintain the reservoir pressure, resulting in increased sulfate reduction in the reservoir. Methanogenesis and sulfate reduction are typically thought to be mutually exclusive because sulfate reducers outcompete methanogens thermodynamically for hydrogen and acetate. However, coexistence is evident in environments like landfills, estuaries, marine, and polluted river sediments. Here, we studied methanogenesis in an adapted sulfate-reducing microbial community, enriched from an oil reservoir, using incubation experiments with metabolic inhibitors to assess microbial activity and degradation potential. The observed methane production rate accounted for 156.9 µM/a with a parallel carbon dioxide production rate of 67 mM/a and a sulfate reduction rate of 20 mM/a, suggesting a coexistence of sulfate reduction and methanogenesis even in a well-mixed system. The microbial community was predominantly composed of potential sulfate-reducing bacteria including the genera Desulfotignum, Desulfospira, and Geoalkalibacter. In addition, fermentative bacteria such as Mesotoga and Petrotoga were abundant and the methanogenic genera Methanolobus as well as the order Candidatus Methanofastidiosales were most prevalent among the archaea. These study results suggest that even with sulfate-adapted communities and high sulfate concentrations methanogenesis can co-occur to minor extents.IMPORTANCEThis study demonstrates the coexistence of two microbial processes-methanogenesis (methane production) and sulfate reduction-in sulfate-rich environments using a microbial community derived from an oil reservoir. Conventionally, these processes were considered mutually exclusive, as sulfate-reducing microbes are thought to outcompete methanogens for shared substrates. However, this research reveals that methane production can persist alongside active sulfate reduction, challenging established paradigms of microbial competition and metabolic exclusivity.

Pseudomonas alliivorans is an important emerging pathogen affecting numerous crops. The species is closely related to Pseudomonas viridiflava, with which P. alliivorans strains were often misidentified in the past. Here, we investigated the genetic and pathogenic characteristics of P. alliivorans strains isolated primarily from onions and weeds in Georgia, USA, using whole-genome sequencing, comparative genomics, and functional assays. We delineated the core genome and genetic diversity of these isolates, assessed their pathogenicity on onion foliage and red onion scales, and examined the roles of key virulence determinants (Hrp1-type III secretion system [T3SS], rhizobium-T3SS, type II secretion systems [T2SSs], and thiosulfinate [allicin]-tolerance alt cluster). Our results showed that the Hrp1-T3SS is pivotal for pathogenicity in P. alliivorans, whereas the rhizobium-T3SS, T2SSs, and alt cluster do not contribute to symptom development on red onion scales. Notably, the alt cluster confers in vitro thiosulfinate tolerance, supporting bacterial survival against onion-derived antimicrobial compounds. Additionally, homologous recombination in P. alliivorans occurs infrequently (at approximately one-tenth the rate of point mutations) and involves divergent DNA segments. The alt cluster is acquired through horizontal gene transfer, as evidenced by its lower GC content and the presence of adjacent transposases. In summary, our research provides valuable insights into the genetic diversity, evolutionary dynamics, and virulence mechanisms of P. alliivorans strains from Georgia, USA.IMPORTANCEPseudomonas alliivorans is an emerging plant pathogen that threatens onion and other plants of economic importance. This study identifies key traits that help this bacterium cause disease, such as a specific secretion system critical for infecting onions, and a gene cluster that aids bacterial survival in onion tissues. Beyond highlighting weed as a potential inoculum source and supporting better weed management, the findings of this research open avenues for more targeted disease menegement. By unraveling the genetics of this pathogen, we can develop improved ways to detect, prevent, and reduce its impact, protecting crop health and yields.

Viral remnants constitute approximately 8% of the human genome, reflecting extensive historical gene exchange between viruses and their hosts. Some viral genomes harbor genes acquired through horizontal gene transfer that are associated with potential benefits to human health, alongside genes associated with pathogenicity. However, their global distribution, functional characteristics, and coexistence patterns remain poorly understood. Here, using the Integrated Microbial Genomes and Virome (IMG/VR v4) database, we identified 4,556 viruses carrying gene segments associated with human health across eight habitat types spanning 13 regions and 76 countries worldwide. Among viruses with identifiable hosts, those distributed in humans (478) accounted for the highest proportion. The viral genes associated with human health included BCO1 (beta-carotene oxygenase 1), bioB (biotin synthase), COQ2 (4-hydroxybenzoate polyprenyltransferase), GPX1 (glutathione peroxidase 1), GSTs (glutathione transferases), GSTT1 (glutathione S-transferase theta 1), GULO (L-gulonolactone oxidase), and menA (1,4-dihydroxy-2-naphthoate polyprenyltransferase). These genes not only associate with human health but also function as auxiliary metabolic genes in viral genomes. Notably, four pathogenic genes were found in viral sequences carrying health-associated genes, with potential for transcription and expression, indicating functional interactions. Experimental transduction of the viral bioB gene into Escherichia coli altered the expression of host pathogenic genes GCH1 (GTP cyclohydrolase IA) and UGDH (UDP-glucose 6-dehydrogenase), supporting potential cross-regulatory interactions. Overall, this study incorporates health-associated genes into viral genomics, highlighting their coexistence with pathogenic genes, and provides new insights into virus-host coevolution and potential biotechnological applications.

Importance: Viruses are the most abundant biological entities on Earth and key drivers of microbial evolution through horizontal gene transfer. While often studied for their pathogenic effects, viruses can also carry genes that influence host metabolism and health. Genes associated with human health have been identified in viral genomes, yet their global distribution, functions, and coexistence with pathogenic genes remain largely unexplored. This study integrates datasets of health-associated genes into viral genomic analyses, revealing for the first time the coexistence of viral health-associated genes with those linked to pathogenicity. This dual genetic potential is observed across diverse habitats, highlighting viruses as multifaceted reservoirs of both beneficial and harmful genes. The study findings advance understanding of viral functional diversity and open new avenues for exploring viral roles in microbial ecology, biotechnology, and human health.

Botrytis cinerea is a significant postharvest fungal pathogen responsible for substantial losses in a variety of fresh produce. Ultraviolet-C (UV-C) irradiation is a potential intervention that could reduce postharvest losses due to fungal disease. However, the diversity of fungal reproductive forms and lack of standardization in antifungal assessment methods complicate the evaluation of UV-C efficacy. This study evaluated the in vitro antifungal efficacy of UV-C against B. cinerea conidia and hyphal fragments (HF) treated in liquid suspension and on agar surfaces. B. cinerea viability was assessed using several methods: colony counts (from agar surface and suspension treatments), growth kinetics (suspension treatment), and conidial germination assays (suspension treatment). UV-C treatment caused significant, dose-dependent reductions in viability across all methods and for both reproductive forms. Surface treatments yielded the highest log reductions (>3.5 log reduction of conidia at 543.6 mJ/cm²), while suspension treatments showed comparatively modest reductions (~1-2 log reduction of conidia at 543.6 mJ/cm², depending on the method). Growth kinetics showed extended lag phases following UV-C exposure. Conidial germination was reduced by up to 95%, with diminishing efficacy at higher doses. While conidia generally exhibited greater sensitivity to UV-C than HF, the magnitude of inactivation varied by assay. A mixed-effects model confirmed that UV-C dose, reproductive form, and assessment method had a significant impact on fungal inactivation.

Importance: This study systematically compared UV-C effects on conidia and HF using a multi-method in vitro approach, underscoring the utility of UV-C for fungal control and providing critical insights for designing effective, context-aware antifungal strategies. The results have practical implications for improving postharvest sanitation protocols and minimizing produce spoilage in storage and packing environments.

The Mesorhizobium nre (nickel resistance) operon has previously been shown to mediate Ni tolerance in serpentine soils with naturally high concentrations of this transition metal. In most serpentine-derived strains evaluated, the putative efflux genes nreX and nreY are conserved, along with a small gene (nreA) encoding a CsoR/RcnR-family transcriptional regulator. CsoR/RcnR-family regulators are small (around 100 amino acids in length); they bind transition metals; and they use an unconventional and poorly understood DNA-binding mechanism. NreA is 93 amino acids in length and belongs to a poorly characterized clade within the CsoR/RcnR family. This investigation is focused on regulatory DNA elements that functionally interact with Mesorhizobium NreA, as well as amino acid residues in NreA that influence its regulatory activity. We show that NreA is a transcriptional repressor that is responsive to exogenous Ni. The Ni-responsive promoter is immediately upstream of the nreAXY operon, resulting in a leaderless transcript. An operator sequence with dyad symmetry occupies the spacer region between the -35 and -10 promoter elements. Mutational analysis of this operator highlights functionally crucial base pairs occupying opposite ends of the 19 bp element. Changes to conserved residues in the NreA polypeptide result in varying effects, with changes predicted to prevent Ni binding leading to a super-repressor phenotype. Structural modeling of the NreA-operator complex provides a plausible mechanism for DNA binding by a tetrameric form of NreA, with DNA contact sites along a positively charged surface. The modeled contact sites agree well with the mutational analysis.

Importance: Bacteria employ diverse mechanisms for maintaining optimal intracellular levels of bioactive metals. Isolates of Mesorhizobium bacteria from Ni-rich serpentine soils possess a small genetic region controlling Ni efflux. This region is subject to transcriptional regulation via the small repressor protein NreA. In this work, the essential components of the NreA protein and the DNA operator with which it interacts are defined, enabling potential adaptation of this system for metal sensing or other technologies requiring a compact inducible gene expression module.

Hyperlipidemia complications caused by obesity are a hot issue threatening human health worldwide, and there is an urgent need to explore low-toxicity health foods for intervention. Sargassum fusiforme polysaccharides (SFPS) display cholesterol-lowering properties, but the underlying mechanism has not been elucidated. This study investigates the mechanisms by exploring changes in gut microbiota composition, gene expression, and metabolites in mice fed a high-fat diet after intervention with S. fusiforme fucoidan (SFF) and SFPS through 16S rRNA sequencing, transcriptomics, and non-targeted metabolomics. The experimental findings indicated that SFPS markedly enhanced the abundance of Akkermansia while concurrently reducing the levels of Actinobacteria, Erysipelotrichaceae, Bifidobacteriaceae, and Peptostreptococcaceae. This was achieved by upregulating the expression of Mt1, Prlr, and slc25a21 and downregulating SREBP-1, thereby inhibiting the cholesterol metabolism pathway. These changes resulted in increased hepatic production and fecal excretion of bile acids and reduced hepatic cholesterol. Our results shed light on the mechanisms behind the cholesterol- and lipid-lowering effects of SFPS, suggesting its potential as a therapeutic agent for hypercholesterolemia.

Importance: Obesity and its associated metabolic disorders pose a global health challenge, necessitating safe and scalable interventions. This study demonstrated that polysaccharides from Sargassum fusiforme (SFPS), extracted via a novel non-alcoholic precipitation method, effectively ameliorate high-fat diet (HFD)-induced obesity by remodeling gut microbiota and restoring metabolic homeostasis. Integrating multi-omics approaches, we reveal that SFPS enriches beneficial taxa like Akkermansia while suppressing obesity-linked bacteria like Actinobacteria, Erysipelotrichaceae, and Bifidobacteriaceae; modulates cholesterol metabolism through gene regulation (e.g., downregulating Srebf1 and upregulating Mt1); and enhances bile acid excretion. Notably, SFPS exhibits efficacy comparable with the well-studied fucoidan (SFF); however, its cost-effective extraction method offers superior scalability for functional food development. These findings underscore the potential of SFPS as a prebiotic agent targeting the gut-liver axis, providing mechanistic insights into natural product-based strategies for metabolic disease management. This work advances our understanding of how polysaccharides interact with the host microbiome and metabolism, advancing dietary interventions for obesity management one step further.

Puerarin is C8-glycosylated daidzein and an active ingredient in the Chinese herbal medicine "Kakkonto." It is used in the early stage of cold treatment. A previous study reported that intestinal bacteria can remove the sugar moiety from puerarin. Despite their potential roles, C-glycosylated flavonoid-metabolizing microorganisms, including those acting on puerarin, have only been found in the gut but not been identified in natural environments. In this study, we identified a puerarin-catabolizing microorganism by screening from soil and clarified two-step deglycosylation reaction. The first step in the metabolism of puerarin was the oxidation of a sugar moiety, catalyzed by a FAD-dependent oxidase named PurA. PurA is the first example of a C-glycoside oxidase that showed activity toward isoflavone-C-glycosides. Notably, PurA exhibited a broader substrate specificity, acting on both C6- and C8-glycosylated flavonoids. This feature was not found in previously reported C-glycoside oxidases and could be useful to synthesize aglycone compounds from C-glycosides in the future. The second step involved the cleavage of the C-C bond between the sugar moiety and daidzein, which was catalyzed by a C-glycoside deglycosylation enzyme named PurBC. Surprisingly, the initial enzyme in puerarin catabolic pathway in this strain differs from that found in the intestinal bacteria. These findings indicate that, in natural environments, bacteria distinct from intestinal microbiota are responsible for puerarin degradation. Our findings provide a novel insight into microbial C-glycoside metabolism in soil environments.

Importance: Plants, fungi, bacteria, and insects synthesize various kinds of unique compounds. Those synthesized compounds are generally degraded by microorganisms; otherwise, they would accumulate on the surface of the Earth. However, in many cases, their fate in the natural environment remains poorly understood. In this study, we isolated a bacterium from soil by the enrichment culture method and identified it as Paenarthrobacter sp. No. 37. This strain was able to grow in the medium containing puerarin as the sole carbon source. It revealed that strain No. 37 can utilize puerarin as a source of carbon and energy. Furthermore, a puerarin degradation activity was induced upon the addition of puerarin to the culture medium. Thus, we successfully identified a bacterium that physiologically catabolizes puerarin and potentially other C-glycosylated compounds. By uncovering C-glycoside metabolism in natural environments, our findings shed light on the part of microbial contribution to the degradation and regeneration of plant-derived natural compounds in biogeochemical cycles.