Applied and Environmental Microbiology

Manual intervention for concrete repair and replacement comes at high environmental and economic costs. Bioconcrete, which can be formed by bacteria via microbially-induced carbonate precipitation (MICP), is a sustainable method for concrete repair. Bioconcrete-forming bacteria can be incorporated into the concrete at mixing and then heal cracks where and when they occur. Bioconcrete is not intentionally made by bacteria; rather, it is a byproduct of alterations to the local environment that occur during their normal metabolic activities. Bacteria thus make bioconcrete by different metabolic mechanisms, and the environment plays a substantial role in the yield and physical properties of the bioconcrete produced by a given bacterium. The ureolytic bacterium Sporosarcina pasteurii is the most commonly used model organism for MICP, but it requires urea supplementation, which is not feasible for all applications because of nitrogenous waste. In particular, the marine environment is understudied for bioconcrete applications, yet there is a need for self-healing structures in this environment, wherein urea and nitrogenous waste would be detrimental to native biota. Here, we assessed the ability of S. pasteurii to form bioconcrete under marine-like media conditions with urea and calcium supplementation. We found that S. pasteurii generated higher bioconcrete yields in these media conditions compared to standard growth media. We then designed an enrichment protocol to isolate and characterize non-urea-requiring bioconcrete-forming bacteria from Atlantic seawater. We identified three isolates, from the Sulflitobacter, Marinobacter, and Bacillus genera, two of which yielded higher bioconcrete yields in seawater-mimicking media compared to non-ureolytic bacteria utilized in prior literature. Moreover, scanning electron microscopy (SEM)/energy dispersive spectroscopy (EDS) and Fourier transform infrared (FTIR) spectroscopy revealed distinct chemical and structural features of the bioconcrete produced by bacteria in seawater-mimicking medium and between ureolytic and non-ureolytic cultures. Overall, our work establishes a pipeline for the isolation and characterization of novel bioconcrete-forming bacteria from marine samples, with potential for application to marine self-healing materials.

The present-day martian surface is generally considered inhospitable to life because of low atmospheric pressure, intense surface radiation, global desiccation, and oxidizing chemistry which has been increasing since the late Noachian. However, shallow martian subsurface regions where mineralogy has shown groundwater movement may include localized hypersaline environments capable of retaining liquid water and supporting microbial metabolism. Haloferax volcanii, a model halophilic archaeon, has previously been shown to survive under low-pressure martian conditions (24 mbar) and to grow anaerobically supported by the Mars-relevant oxyanions nitrate and perchlorate under high-salinity conditions. Here, we investigated whether H. volcanii could actively grow under a combination of environmental and chemical conditions relevant to potentially habitable shallow subsurface martian lacustrine settings. Cultures were incubated for 160 days under anoxic, CO2-enriched, low-pressure conditions (24 mbar) in hypersaline liquid media supplemented with nitrate or perchlorate. Growth was observed in all low-pressure treatments and was confirmed by increases in optical density and biological reduction of nitrate and perchlorate. Scanning electron microscopy revealed extensive biofilm formation in low-pressure cultures, and Raman spectroscopy demonstrated the persistence of carotenoid biosignatures after prolonged incubation under martian conditions. Water loss remained below 4% across all treatments, indicating long-term stability of hypersaline brines throughout the experiment. These results demonstrate for the first time that a halophilic archaeon is capable of active growth and metabolism under a Mars-relevant combination of low pressure, high salinity, anoxia, and oxidizing chemistry, providing experimental support for the potential habitability of localized shallow subsurface martian environments. ImportanceThe search for cellular life is a major objective of future Mars exploration. While many studies have examined whether microorganisms can survive under martian conditions, far fewer have demonstrated active growth and metabolism. Here, we document Haloferax volcanii as the first halophilic archaeon capable of active growth under a defined combination of Mars-relevant low atmospheric pressure, high salinity, anoxia, and oxidizing chemical conditions. These findings expand the current understanding of the environmental limits of microbial growth and provide experimental evidence that localized brine environments in the shallow martian subsurface could support active microbial metabolism, if suitable organics and liquid water are present. In addition, this study establishes a practical framework for cultivating halophilic microorganisms under low-pressure martian conditions and may help guide future efforts to detect, cultivate, and characterize potential extant life on Mars.

Here, we report the purification and characterization of a haloacid dehalogenase type II (HAD-II) enzyme capable of direct and cell-free enzymatic defluorination by cleaving the resilient C-F bond in perfluorooctanoic acid (PFOA). While conventional remediation strategies rely on energy-intensive chemical/thermal methods, biological alternatives are limited by long whole-microbiome incubations and poorly understood metabolic pathways. We discovered a novel HAD-II enzyme from Achromobacter mucicolens found in PFAS-contaminated lacustrine sediment, providing evidence of real-time microbial adaptation. Within 24-hour incubation, the system released approximately 0.55 ppm fluoride (17% yield) from a 5ppm PFOA (equivalent to maximum fluoride of 3.24 ppm) in recombinant enzyme assays. Structural and phylogenetic analyses reveal that the newly discovered HAD-II belongs to a deeply divergent lineage sharing only 25% sequence identity with the previously characterized Delftia homologue while preserving the core HAD-like catalytic fold. Comparative molecular docking further elucidated this functional divergence, demonstrating that PFOA adopts a productive binding orientation near the conserved catalytic Asp15 within the A. mucicolens active-site pocket, whereas the Delftia counterpart forces non-productive binding outside the catalytic site. Together, our work unveils a previously unrecognized Achromobacter-associated dehalogenase that mediates PFAS defluorination despite severe sequence divergence, offering a critical new paradigm for targeted biological remediation.

Coenzyme Q (ubiquinone, CoQ) is an essential component of the mitochondrial electron transport chain and a major lipid antioxidant in eukaryotic cells. Formation of its benzoquinone ring requires aromatic precursors whose metabolic origin remains incompletely defined. Here, we elucidate the biochemical link between tyrosine metabolism and the synthesis of the benzoquinone head group of coenzyme Q6 (Q6) in Saccharomyces cerevisiae through the 4-hydroxymandelate (4HMA) pathway. Using isotopic tracing with 13C6-tyrosine, 13C6-4-hydroxybenzoate, and 13C6-p-aminobenzoate (pABA), we demonstrate that tyrosine-derived 4-hydroxyphenylpyruvate is converted into 4-hydroxybenzaldehyde via benzoylformate decarboxylation, defining a functional 4HMA pathway in yeast. Chemical inhibition of benzoylformate decarboxylase with methylbenzoylphosphonate led to accumulation of pathway intermediates, which were identified by GCMS. Consistently, mutants lacking ARO10, DLD1, or DLD2 exhibited strongly decreased 4-hydroxybenzaldehyde formation. Despite disruption of the 4HMA pathway, the pABA route from chorismate compensated, demonstrating S. cerevisiae's metabolic flexibility to use pABA or 4 HB and maintain Q6 ring biosynthesis. Our results provide a mechanistic framework linking aromatic amino acid metabolism to respiratory quinone biosynthesis in eukaryotes and support the evolutionary conservation of the 4HMA-derived pathway as a source of 4-hydroxybenzoate for Q synthesis in higher organisms.

Background: Human activities such as mining generally lead to increased heavy metal concentrations in the environment. While traditional remediation techniques are often costly, the use of fungi as bioremediators, known as mycoremediation, is increasingly gaining attention as a sustainable approach for removal of heavy metals. Here, we evaluated heavy metal levels inside the Kiirunavaara iron ore mine in Northern Sweden and analysed fungal responses to various metal concentrations by comparing growth and metal uptake in mine-derived isolates and closely related control isolates. Results: Sediments inside the mine were enriched in heavy metals compared to those from the outlet of the mine to natural lakes. Six Fusarium isolates were recovered from contaminated mining environments: five isolates from inside the mine were identified as Fusarium oxysporum, and one isolate from the outlet was identified as Fusarium tricinctum. Isolates from the mine and outlet showed overall higher survival and biomass production in presence of copper, iron, and zinc across a range of concentrations (up to 1000 mg/L) compared to control isolates. At the same time, these isolates often exhibited reduced relative metal uptake. As a result, mycoremediation potential, assessed as total uptake in the grown mycelium, was isolate-dependent. Conclusions: Based on these results, we conclude that Fusarium isolates from the Kiirunavaara mine show increased growth in media enriched with heavy metals compared to closely related control isolates. We additionally show that mycoremediation potential is not necessarily associated with environmental origin. Instead, mycoremediation potential should be evaluated on a case-by-case basis for each isolate and based on specific needs for mycoremediation.

BackgroundLarval habitat management is increasingly recognized as a complementary strategy for malaria vector control. While bacterial communities are abundant in mosquito breeding sites, their role in shaping oviposition behavior of Anopheles gambiae (Diptera: Culicidae) remains poorly understood. This study evaluated the relative attractiveness or repellency of water infused with bacterial isolates obtained from mosquito larval habitats to gravid Anopheles gambiae. MethodsWe isolated and taxonomically characterized bacteria from 30 larval habitats in coastal Kenya using 16S rRNA sequencing. Thirty-two representative isolates were tested in two-choice oviposition bioassays with gravid An. gambiae s.s., and volatile organic compounds (VOCs) emitted were analyzed by GC-MS. Statistical analyses included Students t-tests, Mann-Whitney U tests, and calculation of the Oviposition Activity Index (OAI). ResultsProteobacteria dominated larval habitats (78%), with frequent genera including Aeromonas, Acinetobacter, Pseudomonas, Enterobacter, and Cronobacter. Most isolates exerted strong deterrent effects, with Aeromonas hydrophila, Enterobacter hormaechei, and Pseudomonas mendocina completely inhibiting oviposition (OAI = -1; 100% repellency). Other isolates such as Neobacillus drentensis and Aeromonas veronii reduced egg deposition by >85%. In contrast, a small subset (Aquitalea pelogenes, Pseudomonas oleovorans, Cronobacter sakazakii) showed weak, non-significant attraction (OAI = 0.15-0.23). GC-MS revealed that highly repellent isolates emitted abundant benzenoids, ketones, phenols, ethers, oxygenated heterocycles, and aldehydes. ConclusionsBacterial volatiles strongly modulate oviposition site selection in An. gambiae, with most isolates acting as potent repellents. These findings highlight microbial VOCs as promising candidates for novel vector control strategies targeting gravid mosquitoes. Limitations include the laboratory-based bioassay design and lack of field validation of VOC activity. Future work should identify specific bioactive compounds and evaluate their efficacy under natural conditions. Research HighlightsO_LIBacteria from mosquito larval habitats influence oviposition of Anopheles gambiae. C_LIO_LISeveral bacterial isolates significantly repel gravid mosquitoes. C_LIO_LIGC-MS analysis revealed VOCs including benzenoids, ketones, phenols, and aldehydes. C_LIO_LIBacterial volatiles show potential for novel mosquito control strategies. C_LI

Course-based undergraduate research experiences (CUREs) are widely recognized as a high-impact practice in biology education, yet most existing CURE frameworks treat the research organism as an interchangeable teaching prop rather than a genuine scientific contribution. We argue that place-based, community-embedded CUREs - in which students isolate, characterize, and publicly deploy a locally meaningful wild organism - constitute a qualitatively distinct model warranting broader adoption. As proof of concept, we present the Declaration of Fermentation project at Indiana University Bloomington: graduate researchers isolated a wild Saccharomyces cerevisiae strain from the bark of a campus landmark tree, confirmed its wild provenance by whole-genome sequencing and phylogenomics, and partnered with local craft breweries to produce a colonial-era inspired ale released publicly for the 250th anniversary of the Declaration of Independence. Volunteer sensory panels at two independent public tasting events (combined n = 33-34 per attribute) confirmed a fruity-funky profile consistent with wild-strain fermentation, with no significant differences between events (Mann-Whitney U, Benjamini-Hochberg-corrected p > 0.05 for all 11 attributes). We describe three design principles - genomically confirmed strain identity, mandatory community partnership, and place-based historical narrative - that distinguish this model from prior wild yeast brewing CUREs, discuss how these principles generalize to other institutions and fermentation vehicles, and identify next steps for formal learning assessment. Complete implementation protocols are provided as supplemental Appendices 1-6, and the bioinformatics pipeline is freely available at https://doi.org/10.5281/zenodo.20679384.

Chemically complex pharmaceutical mixtures in wastewater and sludge can affect microbial adaptation; however, the responses to different co-occurring compounds have not been elucidated well. In this study, the adaptation of a strain derived from hospital sludge, Klebsiella pneumoniae SS02, to 17-ethinylestradiol (EE2), warfarin sodium, and their combination has been studied. The organism grows under all three conditions, and pre-exposure experiments show induction and cross-induction to substrates. UHPLC MS/MS analyses demonstrated that there is conditional depletion of the parent compound EE2 by [~]15% at 36 h post-treatment compared to initial concentrations, but not for the abiotic and non-adapted controls. The rate of warfarin sodium depletion was approximately [~]30% within 36 h and was in accordance with first order kinetics (k = 0.0102 /h; t{square}/{square}= 67.9 h). Under the combined treatment regime, there was a delay in warfarin sodium depletion, suggesting staged substrate consumption. Growth inhibition with efflux inhibitors confirmed transport-driven tolerance. A genome-based study revealed the coordinated response strategy that involved a proposed flavin-dependent monooxygenase (RutA), an oxidative entry into the pathway; redox processing linked to Hpa; aromatic metabolism through {beta}-ketoadipate pathway; and RND efflux system. The structural study additionally supported ligand-mediated decrease in DNA binding affinity of RutR, which is in agreement with de-repression of the substrate-activated regulatory mechanism. All these findings lead to the development of a dual-strategy for adaptation model in which oxidative modification and efflux-mediated protection work together under the influence of a mixture of pharmaceuticals.

Biofilms on aquaculture infrastructure harbor diverse microbial communities that may influence water quality and fish health, yet the temporal dynamics of these communities remain poorly characterized. Here, we used 16S rRNA gene amplicon sequencing to profile biofilm communities on concrete raceway surfaces across an 80-day rainbow trout (Oncorhynchus mykiss) indoor hatch-house production period. One hundred twenty-three wall swab samples from 19 raceways at six time points (9, 23, 38, 53, 65, and 80 days) were analyzed after stringent quality control. Beta diversity analyses revealed that biofilm communities at each time point were significantly distinct (PERMANOVA, p < 0.001 for all pairwise comparisons), with early communities exhibiting greater variability than late-stage biofilms. Total bacterial load increased approximately 2.5-fold from early to late stages (qPCR, p < 0.001). Differential abundance testing (ANCOM-BC) identified 57 differentially abundant genera between early-and late-stage biofilms, and random forest classification distinguished early from late communities with over 93% test accuracy. A clear successional trajectory emerged: early biofilms were dominated by pioneer taxa including Pseudomonas, Caulobacter, and Flavobacterium; mid-succession communities featured predatory Bdellovibrio and the methylotroph Methylotenera; and mature biofilms were enriched in saprophytic Saprospiraceae and Haliscomenobacter, polysaccharide-degrading Verrucomicrobiaceae, and cooperative predatory myxobacteria. Flavobacterium columnare, a pathogen of concern in aquaculture, was detected at low levels throughout the production period. These results demonstrate predictable ecological succession in freshwater built environment biofilms and provide a foundation for understanding the role of surface-associated microbial communities in hatchery management.

Carbapenem-resistant Acinetobacter (CRA) has been associated with increased morbidity and mortality in clinical settings. In this study, we explored the transfer potential of a mobilizable plasmid-harboring blaOXA-72 gene between Acinetobacter species originating from patient, municipal wastewater, and pig farm wastewater. PCR-based evidence suggested putative transfer of blaOXA-72 from Acinetobacter pittii to Acinetobacter baumannii. In this pair, the apparent frequency of PCR-marker-positive putative transconjugants varied depending on temperature and meropenem supplementation, with higher number observed at 27{degrees}C compared to 17{degrees}C and 37{degrees}C. Likewise, the presence of antibiotic pressure yields to higher apparent conjugation frequency, however this observation was limited to a singled donor-recipient pair. Further, we revealed a phenotypic conversion in terms of meropenem susceptibility and a fitness cost in the putative transconjugants. While whole genome sequencing did not conclusively verify the presence of blaOXA-72 or fully resolved plasmid configuration, Oxford Nanopore read mapping consistently detected the chromosomal strA gene in all isolates. In contrast, only a limited number of reads aligned with blaOXA-72 gene, traC, or the complete plasmid sequences. Comparative analyses further revealed variations in the surface-associated factors and defense systems composition of the recipient strains, which could be considered as barriers in conjugation. Lastly, the persistence of PCR-detectable marker genes in putative transconjugants was variable and generally unstable over a 30-day period. Overall, these findings provide preliminary insights into the factors that may influence horizontal gene transfer and short-term maintenance of blaOXA-72.

Rinsing-dependent dental hygiene presents a significant public health challenge in water-scarce environments. This study investigated combinations of xylitol (Xyl), chitosan (Chi), glycyrrhizin (Gly), epigallocatechin gallate (EGCG), dicalcium phosphate (DCP), and nano-hydroxyapatite (nHA) on the primary bacteria behind dental caries, S. mutans. These combinations were assessed for markers of dental caries by biofilm reduction, bacterial killing, and acid buffering against S. mutans when applied to an in vitro simulated enamel model using glass bead surfaces for biofilm formation, and gene expression was subsequently examined via RT-qPCR. Separately, mineral retention was also quantified. The EGCG-DCP-Xyl film demonstrated the highest overall efficacy, achieving a significant reduction in biofilm concentration compared to the untreated control and performing similarly in magnitude to the positive toothpaste control. Dead fluorescence staining confirmed that the EGCG-DCP-Xyl film induced the highest rate of non-viable cells, followed by the Chi-Gly film and the Gly-Xyl film. During 10-day pH cycling, the EGCG-DCP-Xyl and DCP-Xyl formulations buffered pH the most, consistently maintaining mean pH levels safely above the demineralization threshold of pH 5.5. The EGCG-DCP-Xyl also optimized mineral stability with the highest retained calcium concentration, significantly outperforming the Chi-Xyl film. At the transcript level, the EGCG-DCP-Xyl film induced substantial downregulation of key virulence genes, yielding decreases in expression for glucosyltransferase B (gtfB), associated with biofilm synthesis, collagen-binding protein (cnm), associated with tissue invasion, and lactate dehydrogenase (ldh), associated with lactic acid production, compared to the untreated control, with effects comparable in magnitude to the positive toothpaste control. This research suggests that targeting bacterial pathways and mineral loss through a portable film may have potential for preventing dental caries, especially in environments where water is limited. However, additional studies are necessary to evaluate real-world effectiveness.

Black soldier fly larvae (BSFL) are generalist decomposers with promise for converting agricultural and food-processing by-products into value-added bioproducts, but BSFL performance on lipid-rich waste oil streams and the role of the gut microbiome in this process remains unclear. Here, we evaluated BSFL bioconversion of a standard chicken feed diet supplemented with three chemically distinct waste oils: acidulated vegetable oil (AVO), pork grease (PG), and used cooking oil (UCO). Larval performance, bioconversion rate, gut microbiome composition, total protein and fat content, and fatty-acid profiles were measured across bioconversion. Larval age was a major driver of gut microbiome structure, but waste oil supplementation further reshaped community membership and structure, with the strongest diet-associated effects occurring during early-to-intermediate bioconversion. Most differentially abundant taxa were members of the baseline core gut community, suggesting that oil supplementation primarily altered dominance patterns among resident taxa. PG and UCO supported larval growth and bioconversion performance comparable to the chicken feed control, whereas AVO reduced bioconversion rate and showed weaker growth outcomes. Oil supplementation also increased larval fat content, reduced protein content, and shifted fatty-acid profiles toward the corresponding oil feedstocks, although larval biomass composition remained shaped by basal diet and host or microbial metabolism. These findings show that selected lipid-rich waste streams can support efficient BSFL bioconversion while restructuring resident gut microbiome members that may tolerate, metabolize, or indirectly respond to oil-associated conditions, contributing to substrate-dependent changes in larval lipid accumulation and fatty-acid composition. IMPORTANCEAgricultural and food-processing systems generate large amounts of lipid-rich by-products that are difficult to manage using conventional waste-valorization approaches. Black soldier fly larvae (BSFL) offer a biological route for recovering nutrients from these materials, but efficient conversion depends on interactions among substrate chemistry, larval physiology, and the gut microbiome. This study shows that selected waste oil streams can support larval growth while restructuring resident gut microbial communities and altering larval fatty-acid composition. These findings are important for agricultural biotechnology because they frame BSFL production as a host-microbiome bioconversion system rather than simply an insect-based waste-reduction process. Understanding how gut microbes respond to chemically distinct lipid wastes can guide substrate selection, pretreatment, and microbiome-informed optimization strategies for converting underutilized agricultural and food-processing residues into value-added bioproducts for circular agricultural systems.

Microbial fuel cells (MFCs) technology offers sustainable electricity production. Current research largely focuses on few select model organisms, therefore the true prevalence of exoelectrogenesis amongst bacteria remaining largely unknown. We present a broad-scale survey of monomicrobial electricity production among environmental bacterial isolates inoculated in MFCs, using model organism Shewanella oneidensis MR-1 as a benchmark. Of the assessed taxa, 11-22% displayed exoelectrogenic activity, exceeding current predictions and identifying a further three novel exoelectrogenic species. Phylogenetic analysis based on the 16S sequences enabled the evolutionary relationship between isolates to be visualised, revealing that exoelectrogenesis is non-randomly distributed and phylogenetically conserved. Polarisation studies were implemented, revealing that numerous electron transfer mechanism were being utilised to perform exoelectrogenesis. The results of this study imply that bacterial electricity production is more widespread amongst culturable bacteria than previously estimated, with implications for bioprospecting novel exoelectrogens and predicting electrogenic activity in diverse microbial communities.

Microbial weathering of minerals represents a key biogeochemical process through which microorganisms access essential nutrients locked within rock and ore substrates. In this study, we investigated the metabolic response of a model fungus during growth on lithium (Li) ore to elucidate the mechanisms underpinning biologically mediated mineral weathering and subsequent metal mobilization. Our results revealed a distinct metabolic shift in the fungus when cultivated on Li ore, characterized by altered patterns of organic acid production and energy metabolism. This shift coincided with measurable weathering of the Li ore matrix and the consequent release of soluble Li into the surrounding environment, demonstrating a direct link between fungal metabolic reprogramming and mineral breakdown. Importantly, these observations are consistent with metabolic shifts documented in previous studies of mineral weathering by this fungus, suggesting that such responses constitute a conserved and reproducible strategy employed during the colonization of mineral substrates. Although the present work was conducted using a single model microorganism under controlled conditions, the findings carry broader ecological implications. Natural microbial communities inhabiting mineral-rich environments may undergo analogous metabolic shifts when weathering minerals to acquire limiting nutrients, thereby contributing to large-scale elemental cycling and metal release. Understanding these processes not only advances fundamental knowledge of microbe-mineral interactions but also informs emerging applications in biomining and bio-based recovery of critical metals such as lithium. Collectively, this study highlights the central role of microbial metabolism in driving mineral weathering and offers a framework for predicting and harnessing similar processes within complex microbial communities. IMPORTANCEThe model fungus exhibits a distinct, reproducible shift in metabolism--particularly in organic acid production and energy pathways--when grown on Li ore, directly linking cellular metabolism to mineral breakdown. This metabolic shift promotes weathering of the ore matrix and the release of soluble lithium, demonstrating a biological route for liberating a critical metal from its mineral host. Because similar shifts occur across different minerals, natural microbial communities likely employ comparable strategies to weather minerals and acquire limiting nutrients, contributing to global elemental cycling. These findings provide a foundation for sustainable biomining and bio-based recovery of lithium and other critical metals, offering a lower-impact alternative to conventional extraction methods.

Reactive nitrogen species (RNS), particularly peroxynitrite generated from the reaction of superoxide and nitric oxide, are implicated in thermally-induced oxidative stress but remain difficult to resolve in live coral cells. We optimized fluorescent dye strategies to directly quantify superoxide, nitric oxide, and peroxynitrite production in thermally stressed Pocillopora acuta cell suspensions. Thermal stress was associated with an increase in intracellular peroxynitrite concentration, but not in its precursors, nitric oxide and superoxide, highlighting challenges with the application of fluorescent probes and their controls to live coral cells. Compounds developed for mammalian systems often translate poorly to non-model systems such as corals: strong endogenous fluorescence and multiple membrane barriers within the coral symbiocyte, for instance, limited the function of the nitric oxide probe, DAF-2DA. Despite these limitations, the detection of peroxynitrite in live, thermally stressed P. acuta cells represents a step forward in understanding the mechanism of coral bleaching. We also outline strategies for improving the performance of commercial dyes in non-model systems, including media optimization with EDTA treatment to preserve both cell viability and probe performance.

Filamentous phages infecting Gram-positive bacteria remain largely unexplored. Notably, only two filamentous phages, B5 and Philemon infecting Propionibacterium freudenreichii, have been described to date in the phage-rich dairy ecosystem. Although both were genomically characterized, only B5 was confirmed to be an infective filamentous single-stranded DNA phage. The aim of this study was to isolate and characterize new filamentous phages from Swiss-type cheese to investigate their diversity, structural features, host specificity, and potential adaptation to the dairy environment. Thirty raw and pasteurized milk cheeses from France were screened for phages infecting P. freudenreichii strains. Eleven phages were isolated, nine of which displayed a filamentous morphology. Named MINOG1 to MINOG9, these filamentous phages exhibited genomic features typical of this morphotype, including small single-stranded DNA genomes with collinear genes organized into functional modules. Comparison with B5 and Philemon revealed sequence divergence ranging from 0.1% to 7%. These phages also exhibited a diverse host range. To further explore phage-P. freudenreichii interactions, we screened the genomes of the strains used in this study, as well as additional genomes retrieved from the NCBI database, for CRISPR spacers predicted to target these filamentous phages. Numerous strains contained CRISPR spacers showing 79 to 100% identity to genomic regions of these phages. Two P. freudenreichii strains displayed markedly different phage resistance levels despite exact spacer-protospacer matches with phages B5, MINOG1, MINOG2, and MINOG8. Conversely, several strains were resistant to nearly all tested phages despite lacking CRISPR spacers targeting them suggesting the presence of additional defense systems in P. freudenreichii. IMPORTANCEFilamentous phages can play important roles in bacterial ecology by modulating host physiology, population dynamics, and bacterial adaptation to specific environments. However, filamentous phages infecting Gram-positive bacteria remain among the least explored bacterial viruses, and their diversity, ecology, and interactions with their hosts are still poorly understood. This knowledge gap is particularly relevant in dairy ecosystems, where phages are abundant and can influence microbial communities and fermentation processes. In characterizing nine new filamentous phages infecting Propionibacterium freudenreichii from Swiss-type cheeses, this study expands the known diversity of filamentous phages associated with Gram-positive bacteria and provides new insights into phage-host interactions and bacterial defense strategies in dairy-associated bacteria.

NicA2 is a flavin-bound amine dehydrogenase from Pseudomonas putida S16 that converts nicotine to the pharmacologically inactive N-methylmyosmine. In animal models of nicotine addiction, injection of NicA2 can decrease nicotine-seeking behavior 10-fold. Accordingly, NicA2-related enzymes have been investigated as smoking-cessation therapeutics. However, efficient catalysis by NicA2 in Pseudomonas putida relies on electron transfer to CycN, a cytochrome c, and not directly to O2. Impractically high amounts of NicA2 are thus necessary to achieve a pharmacological effect in the absence of CycN. Directed evolution has improved the ambient-O2 value of kcat from 0.007 s-1 to 1 s-1 for NicA2, but further improvements have been challenging. Here, we identify a strain of Peribacillus frigoritolerans NIC8 which encodes two flavin amine oxidoreductases, Ncox and Pnox. In the presence of oxygen, Ncox and Pnox act on nicotine and pseudooxynicotine respectively with apparent kcat values of 7.7 s-1 and 3.9 s-1. Transient kinetics establishes bimolecular rate constants of 51100 M-1s-1 and 81000 M-1s-1 for the half-reactions between Ncox and O2 and between Pnox and O2 respectively, consistent with Ncox and Pnox being bona-fide oxidases. Transcriptomics shows enhanced expression of Ncox and Pnox under nicotine-dependent growth as well as supporting the identification of downstream enzymes. Phylogenetic analysis suggests that Ncox and Pnox arose out of repurposing of homologous enzymes found in Bacillus species. The enzymes we describe may be useful for the development of nicotine addiction therapeutics and for bioconversion of nicotine in waste streams.

Natural products are central mediators of microbial interactions. However, once released into the environment, they also become available for neighboring microorganisms capable of degrading and modifying them through biotransformation. These biotransformations may fundamentally reshape metabolomes and influence community behavior, yet our understanding of these processes remains limited. Ribosomally synthesized peptides are particularly compelling in this context because their structural complexity and potent antimicrobial activity coexist with the potential to yield essential nutrients and reduced bioactivity through biotransformation. Identifying the pathways underlying these biotransformations is essential for understanding mechanisms that support microbial coexistence and nutrient recycling in soil microbiomes. Here, we used nisin as a model peptide to investigate biotransformation by soil bacteria. Selective isolation under nisin-rich, carbon-limited conditions yielded two Gram-negative isolates, Burkholderia stabilis and Pseudomonas fragi. Using growth assays and liquid chromatography-mass spectrometry, we found that both isolates grow in the presence of nisin while biotransforming and depleting the peptide. Burkholderia stabilis completely converted nisin through sequential cleavage of the C-terminus, hinge region and lanthionine ring C, whereas Pseudomonas fragi showed more limited processing restricted to the C-terminal region. Although these biotransformations dismantled structural features required for nisins antimicrobial activity, the intrinsic resistance of both isolates suggests a role beyond detoxification. We further detected nisin biosynthetic genes in the source environment, supporting nisins ecological relevance and suggesting that these bacteria may participate in its turnover in soil. Together, these findings reveal extensive microbial processing of nisin and support a role for antimicrobial peptide recycling in soil microbiomes.

Simultaneous removal of organic waste and industrial gypsum was assessed in continuous flow-through bioreactors that treat sulfate-rich sewage sludge. Metabolic fluxes, the composition of microbial communities, and profiles of organic matter in the presence of different organic loads were tracked over [~]190 days. The addition of a pre-enriched microbial community enhanced the rates of sulfate reduction during the establishment of the sludge blanket, but microbial diversity in established reactors depended primarily on organic loading. Organic removal rates were comparable to those in standard anaerobic digesters, but methane production accounted for [~]1% of electron flow compared to >70% in traditional systems. Stoichiometric analyses revealed that molar COD: sulfate ratios below [~]1 favored complete oxidation of acetate by sulfate-reducing bacteria (SRB) and those above [~]2.1 permitted either complete or incomplete oxidation, allowing sulfate reduction and methanogenesis to co-occur. Sequencing of the 16S rRNA confirmed these trends by revealing that the faster-growing SRB that do not oxidize acetate were more abundant at higher organic loads and during the establishment of the sludge blanket, whereas complete oxidizers became more abundant when the molar COD: sulfate ratio was [&le;]3.2. In reactors that had been seeded with the pre-enriched communities, acetate-oxidizing SRB became prevalent over the incomplete oxidizers 25-50 days earlier. These results enable targeted design and control of microbial processes and bioreactors that remove waste organics and gypsum while producing less methane due to the competition for acetate between methanogenic archaea and SRB that oxidize acetate.

Municipal wastewater constitutes a major reservoir of unutilized reactive nitrogen, representing a significant opportunity for biological valorization. The biopolymer cyanophycin is promising as a means of nitrogen capture and recovery, but current production strategies are not optimized for the physicochemical constraints of municipal wastewater systems. Here, we engineered the naturally competent soil bacterium Acinetobacter baylyi ADP1 ISx to synthesize cyanophycin from carbon and nitrogen sources prevalent in municipal wastewater and over a range of wastewater-relevant temperatures. To overcome the recurring problem of arginine availability limiting cyanophycin synthesis, we engineered an arginine-producing strain (AP1) which accumulated cyanophycin when grown on acetate and ammonium (19% CDW), nitrate (9% CDW), or urea (29% CDW) and without arginine supplementation. During this work, we observed that conditions associated with reduced cell fitness correlated with increased intracellular cyanophycin content. As temperature strongly influences cell growth but cannot be realistically modulated in wastewater contexts, we investigated the potential of induced fructose-auxotrophy to modulate cell growth independently from temperature. This intervention, accomplished with a single knockout (gap), expanded the effective range of cyanophycin accumulation from 12 C up to 30 C. Collectively, these results establish the relevance of arginine-producing strains for cyanophycin biosynthesis and position A. baylyi as a promising chassis for continued development under real-world wastewater conditions.