Probiotic-derived extracellular vesicles (EVs) serve as crucial mediators that alleviate intestinal barrier dysfunction. As a well-established probiotic, Clostridium butyricum (Cb) has been shown to ameliorate ETEC K88-induced intestinal injury. Nevertheless, the potential role of Cb-derived EVs (CbEVs) in ETEC K88-induced intestinal injury has not yet been elucidated. This study showed that CbEVs effectively ameliorated ETEC K88-induced ileal injury, as evidenced by improved ileal morphology and reduced levels of proinflammatory cytokines, diamine oxidase, and malondialdehyde. Mechanistically, CbEVs activated Wnt/β-catenin signaling, thereby accelerating the renewal of intestinal epithelial cell lineages, which is pivotal for maintaining intestinal homeostasis. In parallel, they also reshaped the ileal microbiota by restoring microbial balance. Collectively, these findings demonstrated that CbEVs alleviated ETEC K88-induced ileal barrier dysfunction by coordinating the restoration of intestinal stem cell (ISC) activity and gut microbiota composition, providing a theoretical basis for developing CbEV-based strategies to prevent ETEC-induced intestinal barrier dysfunction in both humans and animals.IMPORTANCEETEC infection-induced intestinal injury continues to pose a significant threat to human and livestock health, while effective preventive strategies remain limited. This study demonstrates that extracellular vesicles derived from the probiotic Clostridium butyricum accelerate intestinal epithelial regeneration by activating Wnt/β-catenin signaling and restoring gut microbiota homeostasis, thereby conferring protection against ETEC-induced intestinal injury. These findings provide a solid scientific basis for the development of Clostridium butyricum-derived extracellular vesicles as a promising non-viable probiotic (postbiotic) strategy to prevent intestinal infections, thereby offering a safer and more stable alternative to live bacteria in functional foods and feed additives.

Rock-inhabiting fungi thrive in subaerial oligotrophic environments, such as desert rocks, solar panels, and marble monuments, where organic carbon and nitrogen are scarce. We tested whether the rock-inhabiting fungus Knufia petricola showed a preference regarding nitrogen ([Formula: see text] or [Formula: see text]) and carbon (glucose or sucrose) sources, and whether it was sensitive toward carbon and nitrogen limitation. As this fungus produces the carbon-rich, nitrogen-free 1,8-dihydroxynaphthalene (DHN) melanin, we tested whether a melanin-deficient mutant would be less sensitive to carbon limitation. The carbon and nitrogen concentrations were the primary predictors of growth, with a broad optimum partially explained by an optimal fungal C:N ratio. Limiting carbon or nitrogen supply decreased biomass formation, [Formula: see text] production, and biofilm thickness, but promoted substratum penetration through filamentous growth. The nitrogen content of the biomass was flexible within limits, increasing with increasing nitrogen supply or decreasing carbon supply. The carbon use efficiency was fairly constant, whereas melanization correlated with a higher nitrogen content of the biomass, despite melanin being nitrogen-free. In conclusion, in vitro, K. petricola switches to explorative growth under nutrient limitations, like fast-growing fungi, revealing universal fungal resource-acquisition patterns.

Calcium signaling is a fundamental cellular communication mechanism, well established in eukaryotes, that relies on calcium ions (Ca2+) acting both as first and second messengers to regulate diverse life-sustaining processes. In bacteria, although the significance of Ca2+ regulation is recognized, it is commonly attributed to its role as a first messenger, while the mechanistic basis for its capacity as a second messenger remains elusive. Previously, we have shown that Pseudomonas aeruginosa, an opportunistic pathogen causing life-threatening infections in humans, possesses the key prerequisites for intracellular Ca2+ (Ca2+in) signaling, including tightly controlled Ca2+in homeostasis, transient changes in Ca2+in levels in response to exogenous Ca2+, and Ca2+-regulated gene expression. In addition, we and others identified several key components of the Ca2+ signaling network, including Ca2+ sensors, transporters, and regulators. The remaining question is whether the levels of Ca2+in alter in response to any extracellular stimuli other than Ca2+. To determine this, we have used a cytoplasmic Ca2+ probe, R-GECO1, that produces a Ca2+-dependent fluorescence and tested a variety of extracellular stimuli, including ions, monosaccharides, amino acids, nucleotides, quorum-sensing signaling molecules, reactive oxygen species, and antibiotics. We observed changes in Ca2+in levels in response to several molecules, particularly glucose, asparagine, ATP, ADP, AMP, HOCl, and cycloserine. The results provide the first experimental evidence that, like eukaryotes, P. aeruginosa responds to select, chemically diverse host-related stimuli by altering its Ca2+in levels, supporting a potential signaling role of Ca²+in in bacteria.IMPORTANCECells use signaling to communicate and respond to their environment. Although the role of calcium ions (Ca²+) as a signaling molecule, regulating multiple essential processes, has been well established in eukaryotes, Ca²+ signaling in bacteria remains understudied. Previous research has indicated the role of Ca²+ in bacterial regulation as a first messenger; however, its role as a second messenger is not yet recognized. Here, we show, for the first time, that P. aeruginosa, an important human pathogen, alters its intracellular Ca²+ levels in response to various external stimuli commonly associated with the host environment. These findings provide the mechanistic basis for P. aeruginosa to use Ca²+ as a second messenger. Understanding these Ca2+ responses provides novel insights into Ca2+-mediated adaptation of invading pathogens to the host environment and may inform the development of new antimicrobial strategies.

Hepatitis E virus (HEV) exhibits genetic diversity associated with distinct public health outcomes and circulation patterns across interconnected human and environmental reservoirs. Most studies have focused on genome-wide diversity, revealing relationships between genotype, virulence, and dissemination routes. However, the role of localized mutations in the capsid protein (ORF2), which may influence the extracellular fate and ecology of HEV through changes in surface biological and physicochemical properties of virions, received limited attention. Here, we combined large-scale analysis of European HEV sequences, structural modeling, and physicochemical characterization to investigate such variations in ORF2. Analysis of ORF2 sequences on a genomic segment highly reported in GenBank, derived from patients and the environment, revealed recurrent mutation motifs (single H354Y and triple H354Y-G357S-V364I motifs) located in a conserved capsid hinge region connecting the middle (M) and protruding (P) domains of the protein. These motifs displayed distinct frequency patterns according to sequence sources and were associated with genotypes 3 and 1, respectively. AlphaFold-based modeling identified this region as a flexible interface between the M and P domains and indicated that mutations reduce predicted alignment error of the P domain relative to the overall protein model, consistent with altered exposure at the virion surface. In vitro analyses of synthetic peptides encompassing this critical region revealed increased hydrophobicity and structural rearrangements, supporting local modulation of capsid surface properties. Together, these results identify a structurally and physicochemically sensitive hinge region in the HEV capsid, suggesting a link between ORF2 sequence variations, genotype, virion surface properties, and environmental circulation.IMPORTANCEHepatitis E virus (HEV) is a major cause of viral hepatitis worldwide, which is transmitted through complex, genotype-linked dissemination routes between humans, animals, and environmental waters. Understanding the circulation dynamics of HEV virions is essential for improving environmental surveillance, risk assessment, and public health strategies within a One Health approach. By combining genetic, structural, and physicochemical analyses, this study highlights specific mutation motifs in a hinge region of the ORF2 capsid protein likely to link distinct distribution patterns of HEV across environmental reservoirs to potential changes in virion surface properties. Such mutation motifs may serve as molecular signatures for exploring virus persistence and dissemination in environmental contexts. Overall, this work provides a new framework for connecting viral genetic variation to environmental behavior beyond traditional genotype classification.

Generalist non-obligate organochlorine-degrading bacteria (ODB) play an important role in the bioremediation of γ-hexachlorocyclohexane (γ-HCH), yet their ecological roles and functional mechanisms remain underexplored. In this study, we used both "top-down" and "bottom-up" strategies to construct two functional consortia (M and Y13) based on non-obligate ODB. Both consortia demonstrated efficient degradation in sediment microcosms, reducing γ-HCH (C/C₀) to 63.0% (M) and 56.1% (Y13). Simultaneously, functional microbial consortia inoculation significantly suppressed the CO₂ reduction methanogenesis pathway compared with the non-inoculated group (P < 0.05), as evidenced by a marked decline in the CH₄/CO₂ ratio in inoculated groups. The well-known degradation gene pceA was enriched in the Y13 group, and the co-localization of pceA-encoding contigs with mobile genetic elements was identified. Notably, Enterococcus was found to have successfully colonized inoculated groups. It has strong environmental evolutionary adaptability and might acquire organochlorine-degrading genes through horizontal gene transfer under pollution stress. The broad applicability of non-obligate ODB (e.g., Enterococcus) makes them a promising candidate for future environmental remediation efforts, breeding new avenues of "One Health" win-win solutions in carbon reduction during pollution remediation for wetlands.

Microplastics are increasingly detected in water and food-associated environments, yet how particle morphology influences microbial persistence during inactivation remains poorly understood. This study aims to develop polyvinyl chloride (PVC) microplastic particles with distinct morphologies and controlled size ranges, and to evaluate how particle morphology influences bacterial attachment and survival during thermal and ultraviolet-C (UVC) inactivation in aqueous systems. First, we developed a PVC microplastic model system with distinct morphologies and controlled size fractions. Original PVC particles were relatively smooth and rounded, whereas irregular particles were generated by compounding, pelletizing, and cryogenically grinding the same PVC material, followed by sieving into defined size ranges. Second, using this model system, we evaluated how PVC particle morphology affected bacterial attachment and survival during thermal and UVC inactivation in aqueous systems. Escherichia coli O157:H7 and Listeria innocua were used as model gram-negative and gram-positive bacteria. Confocal microscopy showed attachment of E. coli to both particle types, with stronger association on ground PVC particles. In the presence of PVC particles, thermal log reductions decreased to approximately 4.0-5.1 log CFUs/mL, compared with about 5.9 log CFU/mL in microplastic-free controls, while UVC log reductions decreased to about 3.9-4.6 log CFU/mL, compared with about 5.2-6.0 log CFU/mL in microplastic-free controls. Ground PVC provided greater protection during thermal treatment, whereas both particle types similarly reduced UVC inactivation. These findings show that PVC microplastics can interfere with microbial control processes and highlight particle morphology as an important factor influencing bacterial survival in aqueous environments.IMPORTANCEMicroplastics are increasingly present in water and food-related environments, but their effects on microbial control processes remain poorly understood. This study demonstrates that PVC microplastics can reduce the effectiveness of both thermal and UVC inactivation of foodborne pathogens in aqueous systems. By developing PVC particles with distinct and defined morphologies, we further demonstrate that particle shape and surface characteristics influence bacterial attachment and survival, particularly during thermal treatment. These findings provide new mechanistic insight into how microplastics may alter bacterial persistence under disinfection or food-processing conditions. This work highlights microplastic morphology as a previously underappreciated factor affecting microbial inactivation and establishes a useful PVC microplastic model system for future studies on microplastic-microbe interactions in environmental and food-associated settings.

Extracellular matrices that scaffold microbial biofilms offer a rich source of polymeric materials with a range of potential applications. However, our understanding of these matrices is largely restricted to model organisms that form biofilms with specific architectures. Examining biofilms from understudied niches has the potential to identify extracellular matrices with novel properties. Here, we targeted a resource-rich boundary at the top of the water column known as the air-liquid interface (ALI). ALIs lack a solid substrate for cellular attachment, suggesting that microbes utilize specialized biofilm architectures to persist at this boundary. We used samples from a lake to enrich for microbes that colonize the ALI. Mixed-species pellicle biofilms formed rapidly in these enrichments and displayed a pronounced ecological succession. We isolated 31 members of the genus Pararheinheimera from early stages of pellicle maturation that formed five phylogenetically distinct clades. We used representative isolates to show that only one Pararheinheimera clade formed adherent films resembling classical pellicles. Isolates from the remaining clades formed floating structures that could be categorized either as non-adhesive films or viscous masses (VMs). VM pellicle formation was a polyphyletic trait that correlated with a mucoid appearance on agar plates, suggesting that the process is driven by copious extracellular matrix secretion. Matrices from VM biofilms were largely non-adhesive, contained a mixture of acidic polysaccharides and proteins, and formed thermally stable, shear-thinning hydrogels. Our results demonstrate that ALI colonization strategies vary widely even among closely related aquatic bacteria and identify VM pellicles as a promising platform for biomaterials development.

Thermophilic filamentous fungi are widely used for cellulase production at elevated temperatures; however, efficient recombinant protein production in these organisms remains limited because construction and screening of large transformant populations remain difficult. Here, Myceliophthora thermophila was engineered into a thermophilic fungal chassis through combinatorial deletion of genes associated with proteolysis, vesicle trafficking, and secretion, resulting in a hyper-secretory strain. An optimized expression cassette coupled with a laccase reporter enabled activity-based identification of productive transformants. Iterative genome engineering combined with QPix-assisted colony picking enabled parallel recovery and screening of fungal transformants, with 552 colonies recovered after transformation and dilution plating and 96 transformants further evaluated by ABTS-based plate screening. Targeted integration at the MtLacA locus enabled identification of productive recombinants through loss of laccase activity, and transformants frequently carried multicopy integrations. Through iterative screening and strain improvement, extracellular protein production reached ~1.1 g/L in shake-flask cultures and 5.9 g/L in a preliminary 5-L fermentation after 144 h using glucose as the carbon source. The chassis also supported the secretion of recombinant fungal enzymes and porcine growth factors. Collectively, these results establish a practical semi-automated workflow for recombinant protein production and strain engineering in thermophilic filamentous fungi.

Listeria monocytogenes can persist in niches of meat processing environments despite routine cleaning and disinfection. Its persistence may depend not only on stress tolerance but also on interactions with resident microbial communities, which may promote or inhibit survival. However, these ecological relationships remain poorly understood. We combined 16S rRNA V3/V4 amplicon sequencing, culture-based detection, and multilocus sequence typing (MLST) to characterize microbial communities in six German meat processing facilities over one year. We examined associations among community structure, sampling sites, and the occurrence of Listeria spp., including L. monocytogenes. Microbial communities were dominated by core genera typical of food-processing environments, particularly Pseudomonas spp. and Acinetobacter spp., but differed significantly among facilities (PERMANOVA, P = 0.001; pairwise R² = 0.023-0.079), indicating facility-specific communities. Culture-based analyses detected Listeria spp. in 51 of 370 environmental samples (13.8%), mainly from drains (44/51, 86.3%). L. monocytogenes was detected in five of six facilities, with 19 of 21 isolates originating from drains (90.5%). MLST of 74 typeable L. monocytogenes isolates revealed high diversity, comprising 21 sequence types across 15 clonal complexes, with lineage II predominating (86.5%). Overall microbial community composition was significantly associated with Listeria spp. and L. monocytogenes presence (PERMANOVA, P = 0.001; R² = 0.0137 and 0.0083). In drains, amplicon sequence variants (ASVs) assigned to the genera Acinetobacter, Rhizorhapis, and Vagococcus showed positive associations with Listeria spp.-positive samples. Together, these findings suggest that drains are key ecological niches for Listeria spp. and that associated taxa may indicate drain communities linked to Listeria spp. recovery.

Truffle fungi, such as Tuber melanosporum and T. aestivum, are prized luxury food products that have been historically cultivated in Mediterranean regions of Europe. Recent advances in truffle cultivation have led to the establishment of productive truffle orchards in North America, South America, China, and Australia. Despite these advances, a consistent issue has been the introduction and accidental cultivation of non-target truffle species. Non-target truffle species (e.g., T. indicum and T. brumale) often morphologically resemble target species (e.g., T. melanosporum) and may unintentionally be included as an inoculum and introduced to seedlings. Detecting inoculum and seedling contamination is difficult and often relies on inconsistent morphological analyses or labor-intensive and time-consuming PCR-based methods. Furthermore, quantitative PCR (qPCR) assays have only been developed for T. melanosporum and T. aestivum, leaving truffle growers without rapid tools to detect important contaminants such as T. brumale. Here, we develop a rapid loop-mediated isothermal amplification (LAMP) assay to detect three globally cultivated Tuber species (T. melanosporum, T. borchii, and T. aestivum) and the most common introduced contaminant species (T. brumale). The assays presented here are sensitive to at least 10-3 ng of DNA and are species-specific. These assays should be of special interest to both truffle orchard owners and truffle testing facilities given the low time commitment, high sensitivity, high specificity, and cost comparable to PCR and qPCR-based approaches.

Legionella pneumophila populations within environmental reservoirs can exhibit genomic diversity, complicating source attribution during outbreak investigations. Core genome single-nucleotide variant (cgSNV)-based approaches are widely used for identifying closely related lineages during molecular epidemiological investigations; however, they primarily focus on nucleotide-level variation and may overlook structural variation among closely related isolates. In this study, we investigated genomic diversity among clinical and environmental L. pneumophila isolates obtained during an epidemiological investigation of a legionellosis case. By analyzing multiple colonies isolated from individual samples, we integrated cgSNV analysis with comparative genome structural analysis to characterize insertion sequence (IS)-mediated structural variation within a highly related population. A total of 138 serogroup 1 isolates belonging to sequence type 138 were analyzed. cgSNV analysis revealed that clinical isolates formed a clonal lineage, whereas environmental isolates from the same bath system exhibited substantial diversity despite differing by no more than 20 cgSNVs. Complete genome analysis of 48 representative isolates demonstrated nearly identical genome synteny with limited large-scale rearrangements. In contrast, IS insertion patterns showed considerable variability, particularly for IS256, which exhibited extensive diversity in insertion sites across isolates. Differences in IS insertion patterns were observed even among isolates sharing identical cgSNV genotypes, indicating that IS-mediated structural variation can occur independently of core genome SNV variation. More than half of the coding sequence differences relative to the reference genome were associated with IS insertion sites, predominantly IS256-associated events. These findings suggest that closely related L. pneumophila populations within environmental reservoirs undergo fine-scale diversification associated with dynamic IS activity.IMPORTANCEIn this study, we analyzed a large collection of Legionella pneumophila isolates obtained from a patient and a bath-associated environmental source and identified extensive insertion sequence-associated structural variation even among isolates sharing highly similar core genome single-nucleotide variant genotypes. Despite this structural diversification, the closely related isolate population retained highly conserved genome organization and broadly similar insertion sequence distribution patterns, supporting the interpretation that it remained highly clonal rather than undergoing large-scale genomic divergence. These findings highlight the importance of analyzing multiple colonies from individual samples to better characterize within-source population diversity. They further indicate that structural genome variation, particularly mobile insertion sequences such as IS256, provides complementary resolution beyond conventional nucleotide-based approaches for resolving fine-scale relationships among closely related isolates. Our study provides new insight into the microevolution of L. pneumophila populations within environmental reservoirs relevant to public health investigations.

Deep-sea hydrothermal vents emit geothermal light, yet its ecological role in shaping microbial adaptation remains largely unexplored. To investigate this, we designed a custom cultivation system that simultaneously applies high hydrostatic pressure (HHP) and light and used it to isolate 12 bacterial strains from active chimney fragments collected at the Kairei hydrothermal field. All 12 isolates exhibited enhanced growth under light exposure. Among them, strain SY138, identified as a novel Microbacterium species, was selected for mechanistic investigation. Our results demonstrate that light exposure directly induces carotenoid biosynthesis in SY138, which in turn reduces intracellular reactive oxygen species (ROS) levels. Inhibition of carotenoid synthesis using fosmidomycin abolished both the ROS-lowering effect and the light-dependent growth promotion, confirming that carotenoids are essential for this physiological response. Notably, while HHP severely inhibited growth by elevating intracellular ROS, light-induced carotenogenesis effectively counteracted this oxidative stress and alleviated HHP-induced growth inhibition. Collectively, these findings uncover a previously unrecognized adaptive strategy, wherein deep-sea hydrothermal vent bacteria exploit geothermal light as an environmental signal to activate carotenoid-based antioxidant defenses, thereby enhancing their survival under HHP conditions. This study highlights a previously underappreciated role of light in microbial stress tolerance in extreme deep-sea ecosystems.IMPORTANCEDeep-sea hydrothermal vents harbor thriving ecosystems under extreme pressure and perpetual darkness, yet they have long been considered to be driven solely by chemosynthesis. The discovery of geothermal light has raised fundamental questions regarding its ecological role. Using a specialized cultivation system that simulates in situ high pressure and illumination, we isolated multiple light-responsive bacterial strains from a deep-sea vent, substantially expanding the known diversity of such microorganisms. Using Microbacterium sp. SY138 as a model, we uncovered a previously unrecognized adaptive strategy: these bacteria exploit light not as an energy source but as an environmental cue to preemptively activate carotenoid-based antioxidant defenses. This light-induced mechanism counteracts oxidative stress imposed by high pressure, a ubiquitous deep-sea stressor. Our findings reveal an unexpected interplay between light and pressure in the deep-sea environment, suggesting that light sensing could represent a more widespread and ecologically significant adaptive trait than previously recognized.

Bacteria integrate environmental signals through chemosensory systems to coordinate chemotaxis and make lifestyle decisions, such as surface attachment and biofilm formation. The core sensory complex consists of three essential components: chemoreceptors, histidine kinases, and adaptor proteins. Together, these proteins form extended arrays that result in efficient signal transduction. The Chp chemosensory system of Pseudomonas aeruginosa regulates two outputs: direct actuation of type IV pili resulting in twitching motility and modulation of cyclic-AMP production, which regulates pilus biogenesis and acute virulence. A fully functional Chp chemosensory system requires two adaptor proteins, PilI and ChpC. In this study, we investigated the distinct contributions of these adaptors to chemosensory array organization. Using bacterial two-hybrid and fluorescence microscopy, we show that PilI interacts with the histidine kinase and is essential for polar signaling array formation. In contrast, ChpC fails to show direct interaction with the histidine kinase, and the loss of ChpC reduces the number of detectable Chp signaling arrays. Bioinformatic and AlphaFold modeling support the concept of distinct interactions within the array. We integrated these results with previously published data on the roles of the adaptors in signal transduction to propose a model for extended array architecture in which PilI acts as the primary adaptor, while incorporation of ChpC appears to enhance array organization and signaling efficiency.IMPORTANCEBacteria rely on chemosensory systems to detect environmental cues and regulate behaviors such as surface attachment and motility. While many of these systems use a single adaptor protein, others encode multiple adaptors whose roles in array organization and signaling are not well understood. In this study, we show that the two adaptor proteins of the Pseudomonas aeruginosa Chp system are distinct, with PilI required for assembly of signaling arrays, and ChpC appears to extend the array and its performance. These findings provide a framework for understanding how multi-adaptor chemosensory systems are structured and how array architecture influences signal integration. More broadly, this work highlights how variations in core signaling components can shape bacterial responses to environmental conditions.

Understanding biological sources of methane is critical for elucidating the global methane cycle and addressing climate change. While traditional research has predominantly focused on prokaryotic methanogens, emerging evidence suggests that eukaryotes, particularly fungi, can produce methane through both enzymatic and non-enzymatic pathways. However, no eukaryotic organism has been documented to possess both pathways simultaneously. Here, we provide preliminary evidence suggesting that the wood-decaying fungus Schizophyllum commune strain 20R-7-F01, isolated from sediment approximately 2 km below the seafloor, employs dual mechanisms for methane production. By comparing wild-type strains with mct1 (methyl chloride transferase 1) and dh3 (dehalogenase 3) deficient strains under iron-replete (50 μM Fe³+, to facilitate Fenton reactions) and iron-free conditions, we estimated that enzymatic pathways account for roughly 82%-85% of total methane production. The remaining ~15%-18% of methane output is consistent with a contribution from Fenton-type chemistry, and this quantitative partitioning of pathway contributions was derived from cross-condition yield comparisons. Environmental factors such as oxygen, temperature, and substrate type significantly influence enzymatic efficiency, whereas the efficacy of the non-enzymatic pathway depends on the concentration of methyl donors, particularly dimethyl sulfoxide. Additionally, both pathways are activated by hydrogen peroxide, indicating that reactive oxygen species positively regulate methane synthesis. This study reveals a novel co-existing "enzymatic-Fenton" mechanism for methane production in fungi, highlighting their crucial role in the global methane budget and carbon cycling.IMPORTANCEMethane is a key driver of climate change, yet our understanding of its biological sources has long been limited to bacteria and archaea. Here, we reveal that a fungus, Schizophyllum commune 20R-7-F01, isolated from deep below the seafloor, produces methane through two distinct mechanisms operating simultaneously: an enzymatic pathway and a Fenton reaction-driven non-enzymatic pathway. This dual system, previously unreported in any eukaryote, redefines the potential contribution of fungi to the global methane budget. Our findings challenge the traditional prokaryote-centric view of methanogenesis and open new avenues for understanding methane cycling in oxygen-rich and iron-rich environments, with implications for climate modeling and carbon cycle research.

Microbial steroid metabolism represents an underappreciated extension of the vertebrate endocrine system, with growing evidence that host-associated microbes contribute to the diversity and bioavailability of sex steroids to human tissues. Emerging studies have linked microbial androgen metabolism to urinary microbiome composition and to resistance to androgen deprivation therapy (ADT) in prostate cancer. While microbial pathways capable of converting steroid precursors such as cortisol to androgens, via the steroid-17,20-desmolase pathway, such as DesG-mediated interconversion of androstenedione to testosterone, have been reported, the diversity of enzymes mediating downstream androgen interconversion remains incompletely defined. Here, we investigate the androgen-forming capabilities of anaerobic bacteria from the male genitourinary microbiome, focusing on NADPH-dependent 17β-hydroxysteroid dehydrogenases (17β-HSDHs) that catalyze the interconversion of androstenedione and testosterone. We isolated androgen-forming bacterial strains from human male urine and identified a previously uncharacterized 17β-HSDH in Peptoniphilus obesi, demonstrating that this enzyme catalyzes the NADPH-dependent reduction of androstenedione to testosterone and the reverse oxidation reaction. Sequence similarity searches further identified a similar 17β-HSDH in Anaerococcus, which was synthesized and functionally validated, revealing conserved activity despite low sequence identity to the previously characterized urinary tract enzyme DesG from Propionimicrobium lymphophilum. The enzymes were found to have broad substrate specificity for C19 and C18 17-keto- and 17β-hydroxysteroids. Together, these findings expand the known diversity of microbial 17β-HSDHs and identify previously unrecognized androgen-forming activities within the genitourinary microbiome.IMPORTANCEMicrobial steroid-transforming pathways may provide a mechanism by which commensal anaerobes contribute to androgen availability in the genitourinary tract. By identifying novel 17β-hydroxysteroid dehydrogenases from Peptoniphilus and Anaerococcus, genera repeatedly associated with prostate cancer, this study provides mechanistic insight into how microbial steroid metabolism may influence hormone-driven disease.