FEMS Microbes

Microbial nitrogen (N) transformations in soil, notably denitrification, result in the production of the potent greenhouse and ozone depleting gas nitrous oxide (N2O). Soil chemistry and microbiome composition impact N2O emission potential but the relative importance of these factors as determinants of N2O emission in denitrifying systems is rarely tested. In addition, previous linkages between microbiome composition and N2O emission potential rarely demonstrate causality. Here, we determined the relative impact of microbiome composition (i.e. soil extracted cells) and chemistry (i.e. water extractable chemicals) on N2O emission potential utilizing an anoxic cell based assay system. Cells and chemistry for assays were sourced from soils with contrasting N2O/N2O+N2 ratios, combined in various combinations and denitrification gas production was measured in response to nitrate addition. Average directionless effects of cell and chemical extract on N2O/N2O+N2 (Cell: {Delta}0.16, Chemical extract: {Delta}0.22) and total N2O hypothetically emitted (Cell: {Delta}2.62 mol-N, Chemical extract: {Delta}4.14 mol-N) indicated chemistry is the most important determinant of N2O emissions. Independent pH differences of just 0.6 points impacted N2O/N2O+N2 on par with independent chemical extract differences, supporting the dominance of this variable in previous studies. However, impacts on overall N2O hypothetically emitted were smaller suggesting that soil pH manipulation may not necessarily be a successful approach to mitigate emissions over a fixed time period. In addition, we observed increased N2O accumulation and emission potential at the end of incubations concomitant with predicted decreases in carbon availability suggesting that carbon limitation increases N2O emission transiently with the magnitude of emission dependent on the both chemical and microbiome controls.

The application of environmental DNA analysis techniques to guide the bioremediation strategy for tetrachloroethene-contaminated groundwater is exemplified by the North Railroad Avenue Plume (NRAP) Superfund site located in New Mexico, USA. Enhanced reductive dechlorination (ERD) was selected as the remedy due to the presence of tetrachloroethene biodegradation byproducts, organohalide respiring genera Dehalococcoides and Dehalobacter, and associated reductive dehalogenase genes detected prior to remediation. DNA extracted from groundwater samples collected prior to remedy application and after four, 23 and 39 months was subjected to 16SrRNA gene amplicon and whole genome sequencing (WGS). The goals were to compare the potential of these methods as tools for environmental engineers and to highlight how advancements in DNA techniques can be used to understand ERD. The response of the indigenous NRAP microbiome to the injection and recirculation of electron donors and hydrogen sources is consistent with results obtained from microcosms, dechlorinating consortia, and other contaminated sites. WGS detects three times as many phyla and six times as many genera as 16S rRNA gene amplicons. Both techniques reveal abundance changes in Dehalococcoides and Dehalobacter that reflect organohalide form and availability. No methane was detected before remediation, its appearance after biostimulation corresponds to the increase in methanogenic Archaea. Assembly of WGS reads produced scaffolds containing reductive dehalogenase genes from Dehalococcoides, Dehalobacter, Dehalogenimonas, Desulfocarbo, and Desulfobacula. Anaerobic and aerobic cometabolic organohalide degrading microbes that increase in abundance at NRAP include methanogenic Archaea, methanotrophs, Dechloromonas, and Xanthobacter, some of which contain hydrolytic dehalogenase genes. Aerobic cometabolism may be supported by oxygen gradients existing at the aquifer-soil interface or by microbes that have the potential to produce O2 via chlorite dismutation. Results from next-generation sequencing-based methods are consistent with current hypotheses regarding syntrophy in environmental microbiomes and reveals novel taxa and genes that may contribute to ERD.

Global waste production is increasing rapidly, with the majority of waste destined for landfills. Microbial communities in landfills transform waste and generate methane in an environment unique from other built and natural environments. Previous work has largely considered landfill microbial diversity only at the phylum level, identifying complex and variable communities. The extent of shared organismal diversity across landfills or over time and at more precise levels of classification remains unknown. We used 16S rRNA gene amplicon and metagenomic sequencing to examine the taxonomic and functional diversity of the microbial communities inhabiting a Southern Ontario landfill. The diversity of microbial populations in leachate and groundwater samples was correlated with geochemical conditions to determine drivers of microbial heterogeneity. Across the landfill, 25 bacterial and archaeal phyla were present at >1% relative abundance within at least one landfill sample. The Patescibacteria, Bacteroidota, Firmicutes, and Proteobacteria had the highest relative abundances, with most other phyla present at low (<5%) abundance. Below the phylum level, very few populations were identified at multiple sites, with only 121 of 8,030 populations present at five or more sites. This indicates that, although phylum-level signatures are conserved, individual landfill microbial populations vary widely. Significant differences in geochemistry occurred across the leachate and groundwater wells sampled, with calcium, iron, magnesium, boron, meta and para xylenes, ortho xylenes, and ethylbenzene concentrations contributing most strongly to observed site differences. This study illustrates that leachate microbial communities are much more complex and diverse within landfills than previously reported, with implications for waste management best practices.

High tunnels and open-field systems differ markedly in soil physicochemical properties, yet their effects on belowground microbiomes remain poorly understood. We characterized bacterial and fungal communities in paired high-tunnel and adjacent field soils from 100 small-scale vegetable farms across Minnesota, integrating amplicon sequencing of 16S rRNA and ITS2 regions with soil nutrient data, arbuscular mycorrhizal fungi (AMF) spore counts, and microbial co-occurrence networks. High-tunnel soils had higher pH, organic matter, and multiple macronutrients (notably P, K, and N forms) and lower bulk density than fields, reflecting intensive organic amendments and reduced leaching. Despite these differences, bacterial and fungal alpha diversity did not differ between environments, whereas beta diversity analyses revealed strong shifts in community composition. High tunnels were enriched in salt- and stress-tolerant bacterial phyla (Firmicutes, Deinococcota, Patescibacteria, Halanaerobiaeota, Halobacterota) and saprotrophic fungal groups (Mortierellomycota, Ascomycota, Basidiomycota, Mucoromycota), while several oligotrophic or symbiotic taxa, including Acidobacteriota and Glomeromycota, declined. Glomeromycota relative abundance was negatively correlated with high soil phosphorus, whereas AMF spore densities did not decline, suggesting suppression of active mycorrhizal symbioses rather than propagule loss under high-nutrient conditions. Co-occurrence network analyses showed that bacterial and fungal networks in high tunnels were less dense, more modular, and exhibited higher ratios of positive to negative associations than field networks, consistent with stress-induced shifts toward more facilitative interactions. Collectively, our results indicate that high-tunnel production homogenizes soil microbiomes and selects for stress- and high-nutrient-adapted taxa, with potential consequences for nutrient cycling, AMF function, and long-term agroecosystem outcomes.

BackgroundMetagenomic sequencing of wastewater (W-MGS) can in principle detect any known or novel pathogen in a population. We quantify the sensitivity and cost of W-MGS for viral pathogen detection by jointly analysing W-MGS and epidemiological data for a range of human-infecting viruses. MethodsSequencing data from four studies were analysed to estimate the relative abundance (RA) of 11 human-infecting viruses. Corresponding prevalence and incidence estimates were obtained or calculated from academic and public-health reports. These estimates were combined using a hierarchical Bayesian model to predict RA at set prevalence or incidence values, allowing comparison across studies and viruses. These predictions were then used to estimate the sequencing depth and concomitant cost required for pathogen detection using W-MGS with or without use of a hybridization-capture enrichment panel. FindingsAfter controlling for variation in local infection rates, relative abundance varied by orders of magnitude across studies for a given virus. For instance, a local SARS-CoV-2 weekly incidence of 1% corresponds to predicted SARS-CoV-2 relative abundance ranging from 3.8 x 10-10 to 2.4 x 10-7 across studies, translating to orders-of-magnitude variation in the cost of operating a system able to detect a SARS-CoV-2-like pathogen at a given sensitivity. Use of a respiratory virus enrichment panel in two studies dramatically increased predicted relative abundance of SARS-CoV-2, lowering yearly costs by 24-to 29-fold for a system able to detect a SARS-CoV-2-like pathogen before reaching 0.01% cumulative incidence. InterpretationThe large variation in viral relative abundance after controlling for epidemiological factors indicates that other sources of inter-study variation, such as differences in sewershed hydrology and lab protocols, have a substantial impact on the sensitivity and cost of W-MGS. Well-chosen hybridization capture panels can dramatically increase sensitivity and reduce cost for viruses in the panel, but may reduce sensitivity to unknown or unexpected pathogens. FundingWellcome Trust; Open Philanthropy; Musk Foundation Research In ContextO_ST_ABSEvidence before this studyC_ST_ABSNumerous other studies have performed wastewater metagenomic sequencing (W-MGS), with a range of objectives. However, few have explicitly examined the performance of W-MGS as a monitoring tool. We searched PubMed between database inception and September 2024, using the search terms "MGS OR Metagenomic sequencing OR Metagenomics OR Shotgun sequencing" AND "Performance OR Precision OR Sensitivity OR Cost-effectiveness OR Effectiveness" AND "Virus OR Viral OR Virome" AND "Wastewater OR Sewage". Among the 88 resulting studies, 17 focused specifically on viruses in wastewater. A 2023 UK study by Child and colleagues assessed hybridization-capture and untargeted sequencing of wastewater for genomic epidemiology, concluding that the former but not the latter provided sufficient coverage for effective variant tracking. However, they did find untargeted sequencing sufficient for presence/absence calls of human pathogens in wastewater, a finding supported by numerous other W-MGS studies. While several studies examined the effect of different W-MGS protocols on viral abundance and composition, none accounted for epidemiological or study effects, and none explicitly quantified the sensitivity and cost of W-MGS for viral detection. Added value of this studyTo our knowledge, this study provides the first quantitative assessment of the sensitivity and cost of untargeted and hybridization-capture W-MGS for pathogen surveillance. Linking a large corpus of public wastewater metagenomic sequencing with epidemiological data in a Bayesian model, we predict pathogen relative abundance in W-MGS data at set infection prevalence or incidence, and estimate concomitant read-depth and cost requirements for effective detection across different studies and viruses. Our flexible modelling framework provides a valuable tool for evaluation of sequencing-based surveillance in other contexts. Implications of all the available evidenceThe sensitivity of untargeted W-MGS varies greatly with pathogen and study design, and large gaps in our understanding remain for pathogens not present in our data. As untargeted W-MGS protocols undergo further improvements, our Bayesian modelling framework is an effective tool for evaluating the sensitivity of new protocols under different epidemiological conditions. While less pathogen-agnostic, hybridization capture can dramatically increase the sensitivity of W-MGS-based pathogen monitoring, and our findings support piloting it as a tool for biosurveillance of known viruses.

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent to increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. ORR Castellaniella strains showed a higher degree of genetic diversification than those originating from non-ORR sites, which we attribute to the multitude of extreme stressors faced in the ORR subsurface. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least two decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

We used a sensitive, specific PCR assay to detect mpox clade Ib DNA in over 3000 wastewater samples collected prospectively across the United States. Mpox clade Ib DNA was detected in one sample from a location with a confirmed case; it was not detected in locations with no confirmed cases.

Candida auris is an emerging, multidrug-resistant fungal pathogen that poses a significant public health threat in healthcare settings. Despite yearly clinical cases rapidly increasing from 77 to 8,131 in the last decade, surveillance data on its distribution and prevalence remains limited. We implemented a novel assay for C. auris detection on a nationwide scale prospectively from September 2023 to March 2024, analyzing a total of 13,842 samples from 190 wastewater treatment plants across 41 U.S. states. Assays were extensively validated through comparison to other known assays and internal controls. Of these 190 wastewater treatment plants, C. auris was detected in the wastewater solids of 65 of them (34.2%) with 1.45% of all samples having detectable levels of C. auris. Detections varied seasonally, with 2.00% of samples positive in autumn versus 1.01% in winter (p<0.0001). The frequency of detection in wastewater was significantly associated with states having older populations (p<0.001), sewersheds containing more hospitals (p<0.0001), and sewersheds containing more nursing homes (p<0.001). These associations are in agreement with known C. auris epidemiology. This nationwide study demonstrates the viability of wastewater surveillance for C. auris surveillance, and further highlights the value of wastewater surveillance when clinical testing is constrained.

Plant health is regulated by complex consortia of soil microbes with growth-promoting and pathogenic functions. In potato production, various soil management practices are undertaken to boost yields and suppress diseases, but connections between these practices, soil microbiomes, and tuber yields have not been characterized across diverse growing regions. To identify growing practices and microbes associated with increased yields, we established four-year field trials across eight US sites from Oregon to Maine that consisted of controls, fumigations, organic amendments, and mustard incorporations. Soil microbiomes consisted of 16S and ITS amplicon sequences from bacteria and microeukaryotes, respectively. While soil treatments influenced microbiomes differently across all field sites, eukaryotes were more sensitive than bacteria to all treatments. Soil treatments impacted proportions of distinct amplicon sequence variants (ASVs), and associations between ASVs and tuber yields varied within genus-level taxonomy and across field sites. Forty-five ASVs were identified as both treatment-impacted and yield-associated within any field site. Models identified three of thirteen compost amendment scenarios and one of thirteen fumigation scenarios that increased tuber yields by increasing proportions of these taxa within soil microbiomes. These ASVs were not influenced by treatment-associated changes in soil nutrients or organic matter, highlighting complex relationships within specific field sites that require further study to achieve the goal of implementing sustainable, microbiome-informed potato production techniques. ImportanceSoil microbes play diverse and interconnected roles in mediating plant health, growth, and disease, but understanding the specifics of how they work and applying them across different agricultural systems remains a challenge. To address this, we amassed a dataset from eight potato field sites across major US growing regions consisting of nearly two thousand soil bacterial and fungal microbiomes paired with soil chemical and tuber yield data. We describe how soil microbiomes are impacted by different soil treatments (compost amendments, chemical fumigation, and mustard incorporation), and identify treatments that boosted yields by up to 23% by increasing proportions of certain bacterial or fungal sequences. Compost amendments affected yield-associated taxa more often than other soil treatments, but these effects varied by rotation length and growing region. Changes to soil chemistry resulting from specific soil treatments did not influence abundances of yield-associated taxa, suggesting that the ways in which they may act to maintain plant vigor are field-specific and complex, calling for further study.

Non-typhoidal Salmonella is a common cause of gastroenteritis worldwide, but current non-typhoidal Salmonella surveillance is suboptimal. Here we evaluated the utility of wastewater monitoring to enhance surveillance for this foodborne pathogen. In June 2022, we tested composite raw sewage collected twice a week from two treatment plants in central Pennsylvania for non-typhoidal Salmonella and characterized isolates using whole genome sequencing. We recovered 43 Salmonella isolates from wastewater samples, differentiated by genomic analysis into seven serovars: 16 Panama (37.2%), 9 Senftenberg (20.9%), and 8 Baildon (18.6%), 3 or fewer for four other serovars. We assessed genetic relatedness and epidemiologic links between non-typhoidal Salmonella serovars from wastewater and isolates from patients with salmonellosis. All S. Baildon serovars from wastewater were genetically and epidemiologically associated with a known salmonellosis outbreak. S. Baildon from wastewater and 42 outbreak-related isolates in the national outbreak detection database had the same core genome multilocus sequence typing and code differed by no or one single polynucleotide polymorphisms. One of the 42 outbreak-related isolates was obtained from a patient residing in the wastewater sample collection catchment area, which serves approximately 17000 people. S. Baildon is a rare serovar (reported in <1% cases nationally over five years). Our study underscores the value of monitoring sewage from a defined population to supplement traditional surveillance methods for evidence of Salmonella infections and to determine the extent of outbreaks. Importance statementDuring the COVID-19 pandemic, surveillance for SARS-CoV-2 in wastewater was a powerful tool for assessing community burden days ahead of traditional reporting. Here, we show that domestic sewage monitoring is useful for surveillance for a foodborne pathogen. Salmonella enterica was detected in samples from two wastewater treatment plants in central Pennsylvania during June 2022. Using whole genome sequencing, we demonstrated that isolates of variant S. Baildon clustered with those from an outbreak that occurred in a similar time frame. Case reports were primarily from Pennsylvania, and one individual lived within the treatment center catchment area. This study provides support for the utility of domestic sewage surveillance in assisting public health agencies identify communities impacted by infectious diseases.

Soil moisture and porosity regulate microbial metabolism by influencing factors such as redox conditions, substrate availability, and soil connectivity. However, the inherent biological, chemical, and physical heterogeneity of soil complicates laboratory investigations into microbial phenotypes that mediate community metabolism. This difficulty arises from challenges in accurately representing the soil environment and in establishing a tractable microbial community that limits confounding variables. To address these challenges in our investigation of community metabolism, we use a reduced-complexity microbial consortium grown in a soil analog using a glass-bead matrix amended with chitin. Long-read and short-read metagenomes, metatranscriptomes, metaproteomes, and metabolomes were analyzed to test the effects of soil structure and moisture on chitin degradation. Our soil structure analog system greatly altered microbial expression profiles compared to the liquid-only incubations, emphasizing the importance of incorporating environmental parameters, like pores and surfaces, for understanding microbial phenotypes relevant to soil ecosystems. These changes were mainly driven by differences in overall expression of chitin-degrading Streptomyces species and stress-tolerant Ensifer. Our findings suggest that the success of Ensifer in a structured environment is likely related to its ability to repurpose carbon via the glyoxylate shunt while potentially using polyhydroxyalkanoate granules as a C source. We also identified traits like motility, stress resistance, and biofilm formation that underlie the degradation of chitin across our treatments and inform how they may ultimately alter carbon use efficiency. Together our results demonstrate that community functions like decomposition are sensitive to environmental conditions and more complex than the multi-enzyme pathways involved in depolymerization. ImportanceSoil moisture and porosity are critical mediators of microbial metabolism by influencing factors such as redox conditions, substrate availability, and soil connectivity. However, identifying how microbial community metabolism shifts in response to varying levels of moisture and porosity remains a challenging frontier. This difficulty arises from challenges in accurately representing the soil environment and in establishing tractable microbial communities that limit confounding variables. Moreover, inferring phenotypes based on "key" genes often fails to predict complex phenotypes that arise from cellular interactions. Here, we establish a tractably complex microbial community in a soil analog system amended with chitin and leverage it to understand how microorganisms respond to changes in porosity and moisture. By using genome-resolved metagenomics, metatranscriptomics, and metaproteomics, we report on the microbial lifestyle strategies that underpin changes in community expression like carbon conservation, biofilm production, and stress response.

Wastewater surveillance is an effective approach for monitoring populations of antibiotic-resistant bacteria and tracking the spread of antimicrobial resistance (AMR) across clinical and environmental settings. In this study, hospital and municipal wastewater were collected monthly for 12 months from hospital and municipal locations in the greater Pittsburgh area to quantify the presence of antibiotic-resistant bacteria and investigate their genomic diversity. After quantitative culturing on six different selective medias, a total of 150 isolates were speciated by 16S rRNA sequencing, which revealed diverse pathogenic and non-pathogenic taxa, including Klebsiella spp., Pseudomonas spp. and Aeromonas spp. A subset of isolates belonging to clinically relevant species (n = 46) underwent whole-genome sequencing, which identified several antibiotic resistance genes of clinical concern, such as blaKPC, blaNDM, and blaIMP, and revealed genetic similarities between wastewater and clinical isolates collected from infected patients at a Pittsburgh-area medical center. In addition, analysis of plasmids carried by wastewater isolates revealed closely related plasmids present in isolates from different species and sampling locations. Overall, these findings demonstrate that both hospital and municipal wastewater act as interconnected reservoirs of antimicrobial resistance. Integrating wastewater surveillance with clinical and genomic data could enable the early detection of emerging resistance threats and support proactive infection-control strategies. IMPORTANCEAntibiotic-resistant bacteria persist within hospital environments and cause deadly infections. This study examined the prevalence of antibiotic-resistant bacteria in both hospital and municipal wastewater sampled from the Pittsburgh metropolitan area. Over a 12-month surveillance period, we observed a high abundance of antibiotic-resistant organisms, including pathogenic Gram-positive and Gram-negative species encoding diverse antibiotic resistance genes, many of which were plasmid-encoded. We also identified instances of closely related isolates sampled from wastewater and clinical sources, as well as closely related plasmids found in different species and sampled from different wastewater sites. Overall, this study demonstrates that wastewater surveillance is a viable method for non-invasive monitoring of resistant bacteria at a local scale.

Thermal recovery technologies for in-situ bitumen extraction can result in the heating of surrounding aquifers, potentially mobilizing arsenic naturally present in the sediments to the groundwater. The relative toxicity of dissolved arsenic is related to its speciation, with As(V) being less toxic than As(III). Microorganisms have various mechanisms of arsenic resistance, including efflux and methylation. Microorganisms may also perform reduction/oxidation of As(V)/As(III) as part of their detoxification and/or metabolic pathways. We characterized the microbial communities along two aquifer transects associated with thermally mobilized arsenic near Northeastern Alberta oil sands deposits. 16S rRNA amplicons and metagenomic sequencing data of biomass from filtered groundwater indicated major changes in the dominant taxa between wells, especially those currently experiencing elevated arsenic concentrations. Annotation of arsenic-related genes indicated that efflux pumps (arsB, acr3), intracellular reduction (arsC) and methylation (arsM) genes were widespread amongst community members but comparatively few organisms encoded genes for arsenic respiratory reductases (arrA) and oxidases (arxA, aioA). While this indicates that microbes have the capacity to exacerbate arsenic toxicity by increasing the relative concentration of As(III), some populations of iron oxidizing and sulfate reducing bacteria (including novel Gallionella and Thermodesulfovibrionia populations) show potential for indirect bioremediation through formation of insoluble iron/sulfide minerals which adsorb or coprecipitate arsenic. An unusually high proportional abundance of a single Paceibacteria population that lacked arsenic resistance genes was identified in one high-arsenic well, and we discuss hypotheses for its ability to persist. Overall, this study describes how aquifer microbial communities respond to thermal and arsenic plumes, and predicts potential contributions of microbes to arsenic biogeochemical cycling under this disturbance.

BackgroundMicrobial taxonomic diversity declines with increased environmental stress. Yet, few studies have explored whether phylogenetic and functional diversities track taxonomic diversity along the stress gradient. Here, we investigated bacterial communities within an aquifer in Oak Ridge, Tennessee, USA, which is characterized by a broad spectrum of stressors, including extremely high levels of nitrate, heavy metals like cadmium and chromium, radionuclides such as uranium, and extremely low pH (<3). ResultsBoth taxonomic and phylogenetic -diversities were reduced in the most impacted wells, while the decline in functional -diversity was modest and statistically insignificant, indicating a more robust buffering capacity to environmental stress. Differences in functional gene composition (i.e., functional {beta}-diversity) were pronounced in highly contaminated wells, while convergent functional gene composition was observed in uncontaminated wells. The relative abundances of most carbon degradation genes were decreased in contaminated wells, but genes associated with denitrification, adenylylsulfate reduction, and sulfite reduction were increased. Compared to taxonomic and phylogenetic compositions, environmental variables played a more significant role in shaping functional gene composition, suggesting that niche selection could be more closely related to microbial functionality than taxonomy. ConclusionsOverall, we demonstrated that despite a reduced taxonomic -diversity, microbial communities under stress maintained functionality underpinned by environmental selection.

Enhanced rock weathering (ERW) is increasingly recognized as a way to sequester atmospheric carbon dioxide (CO2) to slow global warming, but its effectiveness needs to be optimized. Carbonic anhydrase (CA), an enzyme capable of accelerating rock weathering both in vitro and in soil, offers a valuable target due to its ability to convert CO2 into carbonic acid. This conversion promotes rock dissolution, enabling immediate CO2 absorption through cation release and charge-balance mechanisms. Studies have shown that bacteria grown in axenic, rock-amended media increase CA gene expression, but the influence of bacterial CA on rock dissolution rates remains unclear. To investigate this, we used a reverse genetics approach with the phosphate-solubilizing bacterium Burkholderia thailandensis E264. We examined three CA-inactivated mutants alongside the wildtype, growing them in minimal media with basalt rock dust (0-10% w/v) at an initial pH of 6.0. After 7 days, we measured weathering potential through elemental concentrations, pH, and dissolved inorganic carbon. In 1% basalt medium, inactivation of the CA1 gene (BTH_I1052) significantly reduced base cation weathering by 41% compared to the wildtype, whereas inactivation of CA2 (BTH_I0345) and CA3 (BTH_I1199) had no significant effect. In the highly buffered, 10% basalt medium, CA1 had a minor role in weathering, and both CA2 and CA3 had no effect. These findings suggest that CA genes in B. thailandensis operate differently and that CA1s effect is pH-dependent. Surprisingly, CA1 was localized intracellularly, raising questions about how intracellular CAs might influence mineral dissolution, potentially through acidity export or abiontic enzyme activity after cell lysis. ImportanceWhile purified carbonic anhydrase (CA) protein has been shown to increase mineral dissolution rates in mineral-amended media in vitro, it remains unclear if the bacterial CA gene directly drives this process. This study used CA-inactivated mutants of the soil bacterium Burkholderia thailandensis in basalt-amended liquid media and found that only one of the three CA genes influenced mineral dissolution rates. This finding supports prior evidence that bacterial CAs may contribute to mineral dissolution in soils. Importantly, it also showed that not all CA genes in a bacterium may activate under the same conditions, which could impact how soil bacterial CAs are leveraged to enhance weathering. Furthermore, cellular localisation predictions indicated that all three CA genes in B. thailandensis are cytosolic, challenging the common focus on extracellular CAs and suggesting that CA proteins may influence the external environment without needing to be actively exported from the cell.

Genes that remain hypothetical, uncharacterized, and unannotated comprise a substantial portion of metagenomic datasets and are likely to be particularly prevalent in soils where poorly characterized taxa predominate. Documenting the prevalence, distribution, and potential roles of these genes of unknown function is an important first step to understanding their functional contributions in soil communities. We identified genes of unknown function from 50 soil metagenomes and analyzed their environmental distributions and ecological associations. We found that genes of unknown function are prevalent in soils, particularly fine-textured, higher pH soils that harbor greater abundances of Crenarchaeota, Gemmatimonadota, Nitrospirota, and Methylomirabilota. We identified 43 dominant (abundant and ubiquitous) gene clusters of unknown function and determined their associations with soil microbial phyla and other "known" genes. We found that these dominant unknown genes were commonly associated with microbial phyla that are relatively uncharacterized, with the majority of these dominant unknown genes associated with mobile genetic elements. This work demonstrates a strategy for investigating genes of unknown function in soils, emphasizes the biological insights that can be learned by adopting this strategy, and highlights specific hypotheses that warrant further investigation regarding the functional roles of abundant and ubiquitous genes of unknown function in soil metagenomes.

Competition for limited carbon resources in the soil can drive complex microbe-microbe dynamics including selection for antagonistic resource competitors. Lignin, one of the main components of plant residue, is one source of carbon input into soil. However, the highly recalcitrant nature of lignin requires degrading organisms to invest in extracellular enzymes, whose breakdown products are susceptible to poaching by opportunistic competitors. Here, we tested two hypotheses: 1) lignin-degrading Streptomyces are more inhibitory than non-degrading isolates, and 2) competition for lignin contributes to the frequency and intensity of antagonistic phenotypes. We collected 133 Streptomyces from the soil of 55-year corn monocultures that had been managed in a +/- fertilizer by +/- residue removal factorial design. Using a subset of 24 lignin-utilizing and 24 non-utilizing isolates we measured the inhibition of each isolate against ten Streptomyces standards. Lignin-degrading Streptomyces had larger mean zones of inhibition than non-lignin-degrading populations, but only in plots where residue was retained. Among non-lignin-degrading Streptomyces, there was no difference in the frequency or intensity of antagonistic phenotypes with fertilizer or residue practices. Our results suggest that competition for byproducts of lignin degradation by lignin-degrading Streptomyces imposes selection for antagonistic phenotypes in habitats with regular residue inputs, but not in non-lignin-enriched soils. This work has potential implications for the development of antibiotic-mediated disease suppressive soils, the survival of plant pathogens within residue, and the structure and function of the soil microbial community.

Reducing the environmental impact of Canadian field crop agriculture, including the reliance on conventional synthesised fertilisers, are key societal targets for establishing long-term sustainable practices. Municipal biosolids (MSB) are an abundant, residual organic material, rich in phosphate, nitrogen and other oligo-nutrients, that could be used in conjunction with conventional fertilisers to decrease their use. Though MBS have previously been shown to be an effective fertiliser substitute for different crops, including corn and soybean, there remain key knowledge gaps concerning the impact of MBS on the resident soil bacterial communities in agro-ecosystems. We hypothesised that the MBS fertiliser amendment would not significantly impact the structure or function of the soil bacterial communities, nor contribute to the spread of human pathogenic bacteria, in corn or soybean agricultural systems. In field experiments, fertiliser regimes for both crops were amended with MBS, and compared to corn and soybean plots with standard fertiliser treatments. We repeated this across four different agricultural sites in Quebec, over 2021 and 2022. We sampled MBS-treated, and untreated soils, and identified the composition of the soil bacterial communities via 16S rRNA metabarcoding. We found no indication that the MBS fertiliser amendment altered the structure of the soil bacterial communities, but rather that the soil type and crop identities were the most significant factors in structuring the bacterial communities. Moreover, there was no evidence that the MBS-treated soils experienced a shift in functions, nor contributed potential human bacterial pathogens over the two years of our study. Our analysis indicates that not only can MBS function as substitutes for conventional, synthesised fertilisers, but that they also do not disrupt the structure, or function, of the resident soil bacterial communities in the short term. Finally, we suggest that the use of MBS in agro-ecosystems poses no greater concern to the public than existing soil bacterial communities. HighlightsO_LIMunicipal biosolids may represent a sustainable fertiliser substitute C_LIO_LIBut, the impact of biosolids on soil bacteria in agricultural fields is unknown C_LIO_LIUsing 16S rRNA metabarcoding we analysed community structure and functions C_LIO_LIWe found no disruption of soil bacterial communities fertilised with biosolids C_LIO_LIBiosolids are safe, sustainable fertilisers with little impact on soil bacteria C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/571735v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1b9ca2corg.highwire.dtl.DTLVardef@8818d2org.highwire.dtl.DTLVardef@1158864org.highwire.dtl.DTLVardef@ad952f_HPS_FORMAT_FIGEXP M_FIG C_FIG

The geochemical process of serpentinization releases energy and organic carbon: two of the basic requirements needed to support life. Sites of active serpentinization in the deep subsurface provide the intriguing possibility of a non-photosynthetically-supported biosphere. However, serpentinization also creates conditions, such as high pH and limited electron acceptors, which may limit microbial growth and diversity. Gaining an understanding of the identity and metabolic potential of microbes that thrive in these environments may provide insight as to whether serpentinization is sufficient to independently support life. Tablelands Ophiolite in Gros Morne National Park, Newfoundland, Canada is a continental site of serpentinization where serpentinite springs form surface pools. These pools provide easy sampling access to subsurface fluids and may allow for sampling of the subsurface microbial community. However, identification of members of the subsurface community in these pools is complicated by both surface contamination and contamination by organisms that inhabit the transition zone where hydrogen-rich subsurface fluids meet oxygen-rich surface fluids. This study was designed to distinguish among these potential sources of microorganisms by using a sampling technique that more effectively samples subsurface fluids. Community dissimilarity comparisons using environmental 16S rRNA gene sequencing indicate that the sampling design led to more direct access to subsurface fluids. These results are supported by metagenomic analyses that show metabolic pathways consistent with non-photosynthetic carbon fixation in the samples expected to represent subsurface fluids and that show hydrogen oxidation pathways in samples associated with the surface sources. These results provide a clearer picture of the diversity and metabolic potential of microbial communities potentially inhabiting subsurface, serpentinite-hosted habitats.

Soil bacteria are a major source of clinically useful antibiotics, yet the majority of soil-dwelling microorganisms remain uncultivable by standard laboratory methods. To access this untapped microbial diversity, we employed microbial diffusion chambers to isolate bacteria from ten Australian soil samples. A total of 1,218 bacterial isolates were recovered, representing a diverse collection spanning 61 genera from 32 families, covering the major known phyla of soil bacteria. Antibiotic activity screening revealed that 16% of isolates inhibited the growth of at least one of E. coli or S. aureus, with 120 isolates displaying activity against multidrug-resistant pathogens including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Mass spectrometry-based dereplication using GNPS identified known antibiotics in 33% of bioactive strains, including actinomycin D, nonactins, and valinomycin. Genomic analysis confirmed the presence of corresponding biosynthetic gene clusters (BGCs), while targeted analysis of selected strains uncovered production of additional antibiotics such as nigericin and streptothricin that were not initially detected by mass spectrometry. Our results demonstrate that diffusion chambers enhance bacterial recovery from soil and show the benefits of a combined pipeline including bioactivity screening, mass spectrometry, and genomics for effective antibiotic dereplication and discovery. Impact StatementThis study delivers a significant advance in natural product discovery by demonstrating that microbial diffusion chambers can dramatically improve the recovery of diverse soil bacteria with antibiotic-producing potential from Australian soil samples. By integrating in situ cultivation with high-throughput screening, mass spectrometry-based dereplication and genome mining, this study yielded more than 1,200 bacterial isolates--including many with activity against multidrug-resistant pathogens--and confirms the production of both known and previously undetected antibiotics. The discovery of streptothricin production via genomics, despite its absence from mass spectrometry data, underscores the power of a multi-layered dereplication strategy. This work not only validates the utility of diffusion chambers for unlocking the "rare biosphere" of uncultivable microbes but also highlights critical methodological refinements to diversify antibiotic-producing strains beyond Streptomyces. The findings will inform and accelerate future efforts to discover urgently needed antibiotics from environmental microbiomes. Data SummaryThe whole-genome sequences were deposited in the National Center for Biotechnology Information National Library of Medicine under BioProject accession number PRJNA1272227.