Applied and Environmental Microbiology

Bacterial conjugation is the primary mechanism by which antibiotic resistance genes spread in microbial populations, yet our understanding of this process has been largely based on experiments conducted under aerobic conditions. This creates a fundamental disconnect: environments that are considered hotspots for gene exchange (e.g., the gut, abscesses, chronic wounds, and wastewater systems) are predominantly anaerobic. In this study, we investigate whether oxygen availability influences the transfer rate of a set of common ESBL-IncI1- and qnrS1-IncF plasmids in commensal Escherichia coli strains from chickens. We found that oxygen availability significantly shapes conjugation dynamics in a recipient strain-specific manner, with anaerobic conditions promoting higher ESBL-IncI1- plasmid transfer rates to commensal E. coli recipients. Conjugation rates of the ESBL-IncI1- plasmids to a laboratory strain of E. coli were several orders of magnitude higher and independent of oxygen level, while two qnrS1-IncF plasmids showed higher anaerobic rates. Our study reveals critical "oxygen blind spots" in conventional conjugation assays and suggests that conventional aerobic conjugation assays underestimate plasmid transfer rates in natural environments such as the chicken caeca. These findings highlight the importance of aligning experimental conditions with the physiological and ecological environments in which gene exchange naturally occurs. Tailoring these variables is essential for generating results that accurately reflect, predict, and potentially intervene in the horizontal spread of antimicrobial resistance.

Pre-harvest litter management is a key determinant of broiler production conditions, influencing NH3; generation, pathogen exposure, nutrient retention, and microbial reservoirs that accumulate across production cycles. Conventional chemical and physical management strategies can support flock health, but their effects on pathogen-associated bacterial populations are often transient and may not account for the microbial interactions that govern persistence, exclusion, and community succession. Here, we evaluated an alternative litter management strategy combining IndigoLT pre-/postbiotic with reduced-rate NaHSO4; across two broiler growouts, with litter sampled at the end of each flock to determine impact on prokaryotic microbiome structure, physicochemistry, and Enterococcus abundance. Alternative management influenced observed richness, phylogenetic diversity, community composition, and co-occurrence network structure while reducing the relative abundance of Enterococcus, including E. cecorum and E. hirae. Digital PCR corroborated sequencing-based Enterococcus abundance patterns, although 16S-based treatment effects were not always reflected as lower absolute copy number at terminal sampling, consistent with reduced proportional dominance rather than sustained absolute suppression. Complementary biofilm- and growth-inhibition assays performed with IndigoLT demonstrated context-dependent antibiofilm and bacteriostatic activity against reference and poultry-derived Enterococcus isolates, with stronger responses for E. cecorum than E. hirae and bactericidal-level reductions in viable recovery at higher exposure levels. These findings demonstrate that biologic-based litter management can alter microbiome structure and pathogen-associated taxa under commercial production conditions, providing a basis for microbiome-informed amendment strategies aimed at reducing pathogen load and supporting broiler health.

Syntrophic propionate oxidation in methanogenic environments depends on interspecies electron transfer through hydrogen and formate, yet the physical factors governing the relative use of these carriers remain poorly understood. Here, we examined how fluid motion alters electron-transfer energetics and pathway expression in the obligate syntrophic propionate oxidizer Pelotomaculum schinkii grown in coculture with Methanospirillum hungatei. A mass-transfer-constrained thermodynamic model was used to estimate H2 and formate concentrations at the P. schinkii cell surface and calculate the corresponding Gibbs free-energy change of H2- and formate-mediated propionate oxidation under different mixing conditions and growth stages. Transcriptomic analysis was used to assess expression of electron-transfer pathways. Under unmixed conditions, formate-mediated propionate oxidation was more thermodynamically favorable than the H2-mediated pathway, consistent with highly expressed genes involved in formate production. Mixing altered coculture activity and pathway energetics. H2 was more sensitive to mixing and certain conditions shifted the energetic advantage toward H2. Expression of the major hydrogenases and formate dehydrogenases generally tracked these pathway-specific energetic changes. These results show that fluid motion reshapes the near-cell thermodynamic favorability and enables condition- and growth-stage-dependent use of alternative electron-transfer pathways. Fluid motion should therefore be considered an ecological and engineering control on syntrophic metabolism.

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.

Anaerocellum (formerly Caldicellulosiruptor) bescii, an anaerobic, extremely thermophilic (Topt [~]78 {degrees}C) lignocellulolytic bacterium, is a promising chassis for metabolic engineering and next-generation bioprocessing. Yet, a lack of well-characterized genetic parts in A. bescii has hampered metabolic engineering efforts. Here, using a previously developed hyperthermophilic {beta}-galactosidase reporter system, we screened a diverse panel of putative A. bescii promoter sequences, identifying promoters that drove reporter output across a broad range. For a select subset, we mapped their transcriptional start sites (TSSs) and evaluated ribosome binding site (RBS) regions using chimeric promoter constructs. By constructing truncated promoter variants, we defined functional regions within the widely used, high-expression S-layer protein promoter (Pslp) and engineered a compact 99 bp variant that retained substantial reporter activity. Finally, we demonstrated that these new promoters can be used for metabolic engineering by using two newly characterized promoters to express an established thermostable alcohol dehydrogenase from Thermoclostridium stercorarium to drive ethanol production in A. bescii. Together, this work expands and diversifies the A. bescii genetic toolkit, opening doors to future metabolic engineering efforts in this species.

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.

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.

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.

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.

Endophytic biocontrol agents may contribute to grapevine health, but their ability to establish, persist, and move within woody tissues remains poorly understood. In this study, a stem injection method was developed to introduce Pseudomonas chlororaphis M71 and Trichoderma atroviride SC1 into rooted and grafted grapevine plants, and their spatial and temporal colonization patterns were compared with the movement of a dye tracer. The dye tracer moved rapidly through xylem tissues, whereas both microorganisms showed more restricted early distribution. Over time, M71 and SC1 displayed distinct colonization patterns. M71 persisted after injection, but remained localized near the inoculation site, with limited movement toward roots or distal aerial tissues. In grafted plants, M71 recovery depended on the injection site and declined more markedly after rootstock injection than after scion injection. In contrast, SC1 showed broader and more persistent colonization. In rooted cuttings, SC1 was recovered from stem and root tissues up to 56 days post-injection, and in grafted plants it was recovered across the graft union, particularly after scion injection. Microscopy supported internal localization of both microorganisms. GFP-labelled M71 and SC1 hyphae were observed mainly within xylem vessels, and viable microorganisms were recovered from corresponding wood tissues. No contamination was observed in control plants. These results show that beneficial microorganisms can be introduced into grapevine tissues by stem injection and that bacterial and fungal biocontrol agents differ markedly in their internal movement and persistence. IMPORTANCEIntroducing beneficial microorganisms directly into plant tissues could help in establishing protective endophytic populations, but little is known about how such microorganisms move and persist inside grapevine. This study shows that stem injection can deliver Pseudomonas chlororaphis M71 and Trichoderma atroviride SC1 into grapevine tissues without visible phytotoxicity. The two microorganisms followed different colonization patterns. M71 remained mostly localized near the injection site, whereas SC1 spread more broadly and persisted in both rooted and grafted plants. These findings provide a basis for developing targeted endophytic biocontrol strategies in grapevine propagation and early plant establishment. This approach may be particularly relevant for grapevine trunk diseases and other vascular disorders, in which pathogens colonize internal woody tissues.

Caldimonas thermodepolymerans is a thermophilic polyhydroxyalkanoate (PHA)-producing bacterium with strong potential for sustainable bioplastic production. Besides serving as intracellular carbon and energy storage compounds, PHAs are increasingly associated with bacterial stress resistance and cellular robustness. This study aimed to investigate the physiological and transcriptomic response of C. thermodepolymerans to osmotic stress induced by elevated NaCl concentrations. Growth analysis demonstrated tolerance up to a supplementation of 2% NaCl, while moderate salt concentrations enhanced PHA accumulation, reaching 65% cell dry weight at 1.5% NaCl supplementation. To better understand the bacterial response to osmotic stress, RNA sequencing was performed under sublethal salt stress conditions. Differential expression analysis revealed major changes in genes related to osmoprotection, trehalose metabolism and type VI secretion systems, whereas motility and chemotaxis genes were strongly repressed. Phenotypic assays confirmed increased biofilm formation and reduced swarming motility under salt-induced osmotic stress. Although canonical PHA biosynthesis genes were not significantly differentially expressed, increased polymer accumulation suggests other underlying mechanisms linked to osmoadaptation. Together, these findings demonstrate that osmotic stress induces metabolic, physiological and regulatory responses in C. thermodepolymerans, highlighting the importance of PHA in stress adaptation besides its industrial applicability.

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.

The respiratory microbiome plays important roles in poultry health, immune regulation, and pathogen resistance, yet its development throughout the broiler production cycle remains poorly understood. This study investigated temporal changes in the tracheal microbiome of broiler chickens across production phases. Tracheal samples were collected during the starter (day 12), grower (day 21), early finisher (day 26), and late finisher (day 35) phases and analyzed using 16S ribosomal RNA gene sequencing. Tracheal microbial richness, diversity, community structure, and taxonomic composition changed significantly across broiler production stages, including starter, grower, early finisher, and late finisher feeding phases. Alpha diversity increased progressively throughout production, with significant increases in richness, diversity, and phylogenetic diversity during later stages. Beta diversity analysis revealed distinct microbial communities associated with each production phase, with starter-phase samples clearly separated from later phases. Taxonomic profiling showed dominance of Proteobacteria during the starter and grower phases, with enrichment of Methylobacterium-Methylorubrum and Pseudomonas during the starter phase and of Escherichia-Shigella during the grower phase. In contrast, the finisher phases exhibited reduced Proteobacteria abundance and increased Firmicutes and Actinobacteriota, including Lactobacillus, Ligilactobacillus, Faecalibacterium, Streptococcus, Staphylococcus, Romboutsia, and Corynebacterium. Overall, the tracheal microbiome underwent progressive maturation, shifting from a Proteobacteria-dominated community to a more diverse, complex, Firmicutes-rich ecosystem. These findings provide new insights into the development of the respiratory microbiome in broiler chickens and may support strategies to improve poultry respiratory health. Because dietary transitions occurred concurrently with age progression, the observed microbiome shifts should be interpreted as production-stage-associated changes rather than diet-specific effects.

Marine dissolved organic carbon is a chemically complex substrate pool that fuels heterotrophic bacteria, yet it remains difficult to determine which compounds are used by specific microbes. Bacterial transcriptomes offer a potential biosensor of substrate availability, but the reliability of this approach in chemically mixed substrates remains uncertain. Here, we evaluated the reliability of this transcriptional sensor approach using the model marine bacterium Ruegeria pomeroyi DSS-3 grown on either glucose or a defined mixture containing glycerol, benzoate, succinate, leucine, dimethylsulfoniopropionate, and trimethylamine N-oxide. Genome-wide transcription differed strongly between treatments, with the mixed-substrate treatment enriched in genes associated with C1 metabolism, sulfur oxidation, benzoate degradation, and motility. Across substrates, the most diagnostic transcriptional responses occurred at pathway entry points and first committed reactions, including glucose transport and Entner-Doudoroff metabolism, trimethylamine N-oxide transport and catabolism, and early steps of aerobic benzoate oxidation. In contrast, downstream metabolic genes were less substrate-specific, likely because multiple pathways converged on shared central metabolic intermediates. Transporter transcription was also less consistently diagnostic than expected, although substrate-binding subunits often showed the strongest responses within transporter complexes. Comparisons with previous single-substrate studies indicated that some transcriptional markers, particularly benzoate oxidation genes, remained detectable in the substrate mixture, whereas glycerol and succinate responses were weakened or lost. These findings show that transcriptomics can provide useful insight into bacterial substrate use, but interpretation is most robust when focused on experimentally validated transporters and early pathway genes, and when evaluated in the context of pathway connectivity, cellular physiology, and substrate mixture complexity.

Nitrification inhibitors are applied to reduce nitrogen losses and greenhouse gas emissions from fertilized agricultural ecosystems. However, their characterization is typically focused on determining effective inhibitor concentrations from growth or substrate conversion assays that are time-intensive and provide limited mechanistic resolution. Here, we present a microrespirometry (MR)-based workflow for rapid mechanistic characterization of nitrification inhibitors using oxygen consumption as a real-time readout for metabolic activity. The workflow enables the simultaneous assessment of inhibitor efficacy, competitiveness, and enzyme specificity within a single experimental setup, as sequential substrate and inhibitor additions enable direct discrimination between competitive and non-competitive inhibition and between ammonia monooxygenase-specific and broader respiratory inhibition. As a proof of concept, we evaluated three known nitrification inhibitors phenylacetylene (PA), nitrapyrin (NP), and dicyandiamide (DCD) using the ammonia-oxidizing bacteria Nitrosomonas europaea and Nitrosospira multiformis, the complete ammonia oxidizer Nitrospira inopinata, and the nitrite oxidizer Nitrospira moscoviensis. We also compared the results from the MR-based inhibition workflow with those from a conventional growth-based approach and observed a poor correlation between results for inhibitors that are not fully enzyme specific. In conclusion, this work establishes MR as a rapid and versatile platform for the mechanistic screening of novel potential nitrification inhibitors. MR assays reproduce known inhibitory responses while substantially reducing experimental time and increasing mechanistic resolution compared to other assays types. Additionally, we provide the first pure-culture characterization of PA, NP, and DCD efficacy and inhibition mechanisms in a complete ammonia oxidizer, N. inopinata.

This study investigated the genomic and biochemical characteristics of two amylolytic microbial strains, NATIVEDY160T (= JE7B6T = NRRL B-68523T) and NATIVEDY161T (= JL13D9T, = NRRL B-68524T) isolated from the rumen of healthy Holstein dairy cattle. Both strains are obligately anaerobic, non-motile, Gram positive, catalase-negative, and oxidase-negative. Morphologically, NATIVEDY160T grows in long coccoid chains while NATIVEDY161T grows in short chains or pairs. NATIVEDY160T can catabolize amygdalin, esculin/ferric citrate, and starch, compared to NATIVEDY161T which utilizes amygdalin, arbutin, esculin/ferric citrate, glycogen, and D-maltose as determined by API 50 CH carbon panels. Starch degradation ability was verified for both strains, but neither showed cellulolytic activity as confirmed by starch agar and Congo red agar assays, respectively. HPLC analysis revealed that lactate was the primary end product of both strains carbohydrate fermentation, while strain NATIVEDY161T also produced small amounts of acetate. 16S rRNA sequences from both strains cluster with the Oscillospiraceae (formerly Ruminococcaceae) lineage Ruminococcus species, but average nucleotide identity of either strain compared to closely related Ruminococcus members was under the species threshold (95%). Genomic, phylogenetic, and phenotypic interrogation support NATIVEDY160T and NATIVEDY161T as novel species. Each strain was isolated from the rumen of dairy cows located within the central valley of southern California, which has a rich history of Dutch and Basque dairy farm ownership and is still the case today in the region. In recognition of the contributions and heritage of the central and southern California dairy industry, the names Ruminococcus hollandia and Ruminococcus vasco are proposed with NATIVEDY160T and NATIVEDY161T as their respective type strains.

Combined sewer overflows (CSOs) are a major pathway for untreated wastewater into urban streams, yet their role in shaping antimicrobial resistance (AMR) dynamics remains poorly understood. Here, we used high-frequency, time-resolved sampling during two storm-triggered CSO events across two monitoring locations and one stormwater-only control site in an urban stream to quantify how these disturbances affect microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) in an urban stream. CSO events caused rapid, up to two orders of magnitude, increases in bacterial, pathogen, and ARG abundance, with multiple transient peaks occurring within single overflow episodes. However, these increases were largely proportional to the total bacterial load, and most ARGs and MGEs did not change in relative abundance, indicating that CSOs primarily act as mass-transfer events rather than drivers of in situ selection. Downstream attenuation was governed by hydrological dilution despite additional CSO inputs: Both microbial and resistance signals largely returned to baseline within short time frames. This demonstrates that CSOs function as hydrologically driven pulse disturbances that generate acute but transient AMR exposure. Because CSO events lack the sustained pressure associated with continuous wastewater discharges, rapid washout prevents the long-term establishment of sewage-derived resistance. These findings highlight that AMR risk in CSO-impacted systems is driven primarily by short-term exposure rather than by persistent ecological transformation, with important implications for urban water management under increasingly extreme rainfall conditions.

Aminopeptidases are widely distributed in bacteria, but outside of a few model strains, their function is largely unexplored. Focussing on beta-alanine aminopeptidase activity, a new series of selectively-activatable, caged fluorescent probes were designed and synthesised. A beta alanine amino acid was coupled to resorufin or 7-hydroxycoumarin via a self-imolative linker, such that amino acid removal led to gain of fluorescence. These were used to probe selectivity and specificity of probe activation, against a range of priority drug-resistant pathogens. When added to bacterial growth curves run in Muller Hinton broth, these probes allowed essentially real time fluorescence measurement of activation by bacterial species, modelled on the standard microbroth dilution method. Activation was observed for all Pseudomonas aeruginosa and Burkholderia spp strains tested. Selective activation was seen for Ochrabactrum species, with the probe activated by O.anthropii (2/4 strains) but not O.intermedium and strain-specific activation was seen for some isolates of Serratia marcescens (2/4 strains). No activation was observed in any isolates of Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii or Staphylococcus aureus or Eneterocccus faecium/faecalis PAO1 transposon mutants in the putative beta-alanine aminopeptidase gene (annotated as bapF or dmpA; PW3678) showed no activation of the probe in growth assays, confirming the specificity of the probe for beta-alanine aminopeptidase. Transposon mutants in other aminopeptidase genes, including those encoded by pepN, PepP and the prolyl aminopeptidase gene had no effect on probe activation in PAO1. Based on the operon structure in PA01, transposon mutants in two adjacent genes were also tested for probe activation. Mutants in both a putative transcriptional regulator (PW3674) and a predicted amino acid permease (PW3676) retained their ability to activate the beta-alanine probes with activation significantly higher than the wild type, when assessed by the total fluorescence yield after 10 hours growth. This points to both redundancy in permease function and perhaps the presence of a feedback regulatory mechanism controlling beta alanine aminopeptidase activity in P.aeruginosa. Given that the operon structure is conserved in other species, this may point to a common mechanism of beta alanine aminopeptidase function, perhaps related to exploiting beta-alanine containing peptides in certain environmental niches.

Endosymbiosis is widespread throughout the tree of life. Understanding how the transition of a bacterial endosymbiont from facultative to host-dependent obligate life occurs is an important question for defining the origin of endosymbiosis. A novel gram-positive bacillus, Brevibacillus sp. TJ4 was isolated from the nitrogen-fixing yeast Rhodotorula mucilaginosa JGTA-S1, which houses several endobacteria within its cells. TJ4 can survive independently of yeast but exhibits genomic and metabolic features characteristic of an evolving endosymbiont, slowly assuming a host-dependent, obligate lifestyle. The TJ4 genome contains several incomplete pathways for carbohydrate, amino acid, vitamin, and cofactor metabolism, which is reflected in its increased reliance on host-derived nutrients and auxotrophy compared with that of other Brevibacillus spp. Comparative genomics revealed widespread genome rearrangements, loss of synteny, and multiple cross-genus and inter-kingdom horizontal gene transfer (HGT) events in TJ4 compared to other Brevibacillus spp. These HGTs include the acquisition of genes from bacteriophages and co-resident endobacteria of JGTA-S1. One such horizontally acquired gene, Type II 3-dehydroquinate dehydratase (AroQ), appears to have originated from the Rhodotorula host itself. This acquisition functionally restores the shikimate pathway in strain TJ4, as evidenced by the phylogenetic placement of AroQ from TJ4 within the clade of fungal AroQ homologs. Potential exploitation of the host JGTA-S1 appears to be a probable mode of endosymbiosis of TJ4, an evolving endosymbiont that we named Brevibacillus rhodotorulae sp. nov.

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.