Environmental Microbiology Reports

Methane is a powerful greenhouse gas. Typically, a large fraction of the methane formed in coastal sediments is removed via anaerobic methane oxidation (AOM). Here, we demonstrate the potential for a range of AOM pathways in brackish coastal sediments by ANME-2a archaea. At our study site, geochemical profiles indicate that AOM is primarily restricted to a shallow, metal-oxide-rich sulfate-methane transition zone (SMTZ). ANME-2a were the sole methanotrophs detected, and metatranscriptomics showed the highest expression levels of the ANME-2a genes in the SMTZ. AOM activity was observed in sediment incubations with various electron acceptors, including sulfate, metal oxides, and the organic matter analogue graphene oxide. Highest potential rates were observed in sediments from below the SMTZ, pointing towards fast stimulation of the deeper methanotrophic community when alleviating the electron acceptor limitation. The variety of AOM pathways and persistence of methanotrophs below the SMTZ likely contribute to the resilience of the microbial methane filter in brackish coastal sediments.

Ammonia-oxidizing archaea (AOA) are frequently found in oxygen-depleted marine environments with permanent or temporal presence of sulfide (HS-). However, it remains unexplored how sulfide affects the activity of ammonia-oxidizing archaea. We studied the capability of Nitrosopumilus maritimus SCM1 to oxidize ammonia when exposed to HS-. Ammonia oxidation remained active even after exposure to sulfidic spikes in the lower micromolar range, albeit at reduced rates compared to the absence of HS-. However, 90 {micro}M HS- completely inhibited ammonia oxidation. We found no evidence of NO-dismutation under oxygen depletion and presence of HS- (20 {micro}M): the formation of O2, N2O and N2 did not occur. All in all, we confirmed ammonia oxidation in N. maritimus SCM1 under oxic conditions after sulfide additions, but no evidence of NO-dismutation under sulfidic conditions. Our findings suggest that AOA can recover ammonia-oxidation activity after oxygen re-exposure in regions with periodic sulfide accumulation. However, in permanently sulfidic areas, ammonia oxidation recovery seems unlikely, as NO-dismutation does not appear to be a viable mechanism.

Labyrinthulomycetes are a class of fungus-like heterotrophic protists from the Stramenopiles lineage, recognized for their ecological role as decomposers and contributors to nutrient cycling. They colonize various substrates, from seaweed to terrestrial environments, utilizing ectoplasmic networks for nutrient absorption. This study characterized a novel Labyrinthula strain associated with the marine diatom Biddulphia. Phylogenetic analysis of the full-length 18S rRNA gene positioned this strain as a new species, Labyrinthula merlionensis sp. nov. Scanning electron and light microscopy observations revealed bi-flagellated zoospores and spindle-shaped vegetative cells with ectoplasmic networks. Time-series observations of the interactions between L. merlionensis and Biddulphia were categorised into different phases: establishment, infection, and aggregation. Scanning electron and confocal microscopy observations during the infection phase established the use of ectoplasmic nets to target the marginal ridge regions between diatoms, and the detection of labyrinthulid cells within diatom frustules. These findings enhance the understanding of the diversity, morphology, and ecological roles of Labyrinthulomycetes, particularly their intra- and extra-cellular interactions with diatom hosts.

As key drivers of nitrification, ammonia-oxidizing archaea (AOA) play a central role in the global nitrogen cycle and contribute significantly to the emissions of the potent greenhouse gas nitrous oxide (N2O). However, the ecological implications of AOA growth as biofilms, remain poorly understood. Since nitrite production can be used to follow cellular activities directly we were able to compare biofilms with planktonic cells of the terrestrial model AOA Nitrososphaera viennensis at ecologically and agriculturally relevant conditions. Biofilms were more resistant across nearly all tested conditions and remained active at lower temperatures, acidic pH, and high ammonium concentrations. Collectively, activities in biofilm help reconcile discrepancies between earlier laboratory and environmental observations of soil AOA. Additionally, biofilms showed a high general resilience and lowered sensitivities to nitrification inhibitors. Although in situ biofilms grown in microrespiratory chambers exhibited activity and ammonia affinity similar to planktonic cells, biofilm cultures produced only half as much N2O. The enhanced fitness of biofilms across all tested conditions vastly expands the potential ecophysiological niche of AOA and supports the hypothesis that biofilm growth represents the in situ phenotype of AOA in soil environments.

Protistan predators are key regulators of microbial food webs, yet most are considered to occupy relatively narrow trophic niches. Here, we demonstrate that Mayorella spp. (Amoebozoa), isolated from marine and freshwater environments, exhibits exceptional trophic breadth spanning multiple trophic levels. Live-cell imaging revealed predation on bacteria, algae, dinoflagellates, diatoms, flagellates, ciliates, and multicellular prey including rotifers. Large or filamentous prey were engulfed whole or mechanically fragmented during ingestion. Notably, Mayorella consumed both trophozoites and cysts of free-living amoebae (Naegleria and Acanthamoeba), with clear digestion of cyst contents. Dense cultures showed aggregation around large prey and facultative cannibalism. Ingestion of microplastic-like particles occurred without evidence of digestion. Predator cell size and population density increased markedly when feeding on protist or mixed prey relative to bacterial diets, indicating pronounced trophic plasticity. These findings establish Mayorella as a broad-spectrum, cross-trophic predator with the capacity to exert top-down effects across microbial food webs and suggest a previously underappreciated role in the suppression of pathogenic free-living amoebae.

Despite decades of research, the factors determining the sinking velocity of marine biogenic particles remain poorly constrained, and growing evidence suggests that particle composition and morphology are as important as size in determining particle fate. We compared characteristics of suspended and sinking particles at three depths below the mixed layer and within the layer of maximal flux attenuation during the decline of a Phaeocystis pouchetii bloom in the Labrador Sea using marine snow catchers. Biochemical and morphological characteristics of suspended and sinking particles always differed, with differences depending primarily on bloom stage, and depth accounting for comparatively less variation. Exopolymer particles played a key role, with the relative concentrations of transparent exopolymer particles consistently higher in the suspended than in the sinking particle fraction. In contrast, the partitioning of coomassie-stainable particles changed with the bloom stage, as a function of the Phaeocystis life cycle. Ballast minerals played a negligible role during the late-bloom and bloom-decline stages, and their relative importance increased during the non-bloom stage. The C:N ratio was lower in suspended than sinking particles, with differences in morphological measures depending on bloom stage. Our findings emphasize that export potential is driven not only by particle size, but also by bloom stage, which is closely linked to plankton community composition and plays a key role in the timing and magnitude of carbon flux in the upper mesopelagic. Further, this work highlights the important and diverse roles of exopolymers in regulating carbon flux.

BackgroundAdult vestimentiferan tubeworms inhabiting hydrothermal vents and cold seeps lack a mouth and anus and rely entirely on organic matter produced by sulfur -oxidizing autotrophic bacterial symbionts in their trophosomes. These symbionts, which predominantly belong to the genus Proteobacteria, are acquired horizontally from the environment. However, the effects of rearing conditions that differ from natural habitats on the microbiome composition or abundance of these bacteria remain unclear. MethodsWe conducted a metagenomic analysis of Lamellibrachia satsuma reared in an aquarium under sulfide-supplemented and sulfide-free conditions. ResultsImmediately after collection, the microbiome was dominated by known symbionts within {gamma}-Proteobacteria, exhibiting low species diversity. After 6 months of rearing, the abundance of these symbionts significantly decreased under both conditions, whereas overall bacterial diversity increased. In particular, -Proteobacteria became more abundant under sulfide-supplemented conditions, while {delta}-Proteobacteria predominated in the absence of sulfide. Despite these changes, symbionts were not entirely lost, and the hosts survived for 6 months, likely due to their low metabolic rate. These findings suggest that the microbiome of L. satsuma can respond flexibly to changes in the rearing environment. They also indicate that the hosts metabolism can be maintained even with a smaller quantity of symbiotic bacteria.

Plant-associated bacteria can promote plant growth under saline conditions, but salinity-dependent changes in bacterial physiological traits remain insufficiently understood. Here, we isolated bacteria from seedlings of quinoa (Chenopodium quinoa Willd.) lines maintained under laboratory propagation for more than 30 years and evaluated their activity under saline conditions. A quinoa-associated Pantoea isolate, strain 6PN, promoted primary root elongation and whole-plant dry weight of Arabidopsis thaliana under salt stress, whereas no significant effect was observed under non-saline conditions. Comparative analyses with reference Pantoea agglomerans strains showed that strain 6PN exhibited salinity-responsive indole-3-acetic acid (IAA) production. Genome analysis identified a putative ipdC gene and additional genes related to stress responses, nutrient acquisition, polysaccharide biosynthesis and export, flagellar biosynthesis, and chemotaxis. Phylogenomic analysis indicated that strain 6PN was genomically distinct from representative Pantoea species examined here. In an Arabidopsis trench-plate assay, GFP-labeled strain 6PN was recovered from spatially separated plant tissues at higher levels than a GFP-labeled reference strain under saline conditions. These results identify strain 6PN as a quinoa-associated Pantoea isolate with salinity-responsive IAA production and plant growth-promoting activity under defined salt-stress conditions.

Warming of the Arctic enhances microbial activity and the decomposition of large stocks of organic matter retained in permafrost soil. Resulting changes in the availability of sulfur may lead to increased emissions of volatile sulfur compounds (VSCs), which impact atmospheric particle and cloud formation, terrestrial and aquatic acidification, and malodor. Marine microbial production of dimethyl sulfide (DMS) has been studied for decades but other VSCs have been largely ignored, while VSC emissions from terrestrial ecosystems are even less studied. Currently, we lack fundamental understanding of the metabolic processes behind VSC production in permafrost soil bacteria, essential for estimating how emissions may change due to thawing. To fill this knowledge gap, we measured VSC emissions from thawing permafrost and three bacterial strains isolated from Greenlandic permafrost and biological soil crust. We show that the bacterial strains produced high levels of VSCs in vitro - including hydrogen sulfide, methanethiol, DMS, dimethyl disulfide, and dimethyl trisulfide. We further show that the same VSCs were also emitted from permafrost upon thaw. Metabolic pathway mapping of the bacterial strains revealed both inorganic sulfate reduction pathways and amino acid metabolism behind bacterial VSC production. High production of VSCs in the late-active and stationary phase suggests connection to secondary metabolism, except for DMS which was linked to early growth, and possibly, primary energy metabolism. Our findings suggest that thawing increases VSC emissions from permafrost soil, possibly leading to higher input of sulfur into the atmosphere from the warming Arctic in the future.

Endozoicomonas, a dominant symbiotic bacterium in coral holobionts, is noted for its ability to degrade dimethylsulfoniopropionate (DMSP) so as to generate acetate. While acetate is a well-known short-chain fatty acid in metabolic cross-feeding relationships, it remains unclear whether acetate derived from bacterial DMSP degradation is available to corals and their other symbionts. In this study, we employed Endozoicomonas ruthgatesiae strain 8E (herein referred as 8E) as a model to examine availability of DMSP-derived acetate for other symbionts. Using gas chromatography-mass spectrometry (GC-MS), we observed a significant increase in acetate excretion in 8E upon exposure to DMSP. Stable isotope labeling further confirmed that this elevated acetate efflux originated directly from DMSP, suggesting a complete cycle of DMSP-derived carbon among coral symbionts. Transcriptomic analysis revealed that DMSP exposure upregulated dddD expression and triggered a systemic reconfiguration of metabolism, characterized by down-regulation of the TCA cycle and the Pta-AckA pathway, with carbon flux redirected to the glyoxylate shunt. These findings suggest that upon exposure to DMSP, metabolism of 8E shifts from biomass production to DMSP catabolism, resulting in acetate efflux. Notably, we found that elevated temperature diminishes DMSP cleavage activity of 8E, indicating thermal sensitivity of this bacterial metabolic activity. ImportanceEndozoicomonas is known for its dominance in coral holobionts and its ability to degrade DMSP, an important compound in the marine sulfur cycle. Acetate is one resulting product in microbial DMSP metabolism and a common cross-feeding molecule. Whether DMSP-derived acetate in coral-associated DMSP-degrading bacteria is employed for cross-feeding stands a critical step in making a complete carbon cycle of DMSP metabolism within coral holobionts. In this study, we employed GC-MS and RNA-sequencing techniques to offer the first evidence of acetate excretion in Endozoicomonas while metabolizing DMSP, as well as its underlying genetic mechanism. Furthermore, we demonstrate reduced genetic response and DMSP-degrading capability under an elevated temperature in Endozoicomonas ruthgatesiae strain 8E, the model bacterium employed in this study. These findings provide the missing puzzle of DMSP metabolism in coral holobionts and suggest a potential role of DMSP in modulating symbiotic interactions within coral holobionts.

Many environmental bacteria do not readily grow under laboratory conditions and population establishment often occurs stochastically. Although the scout hypothesis has been proposed to explain stochastic population establishment in environmental bacteria, how stochastic population establishment is shaped by individual cell growth behaviors in environmental isolates remains unclear. In the present study, we focused on the ammonia-oxidizing bacterium Nitrosomonas sp. PY1 and showed that environmentally responsive individual cell growth behavior, incorporating time-dependent stochastic growth initiation, shapes both deterministic and stochastic population establishment dynamics. Using single-cell observation, we revealed that PY1 altered cell growth behavior in response to surrounding biomass production ({Delta}Vt). These {Delta}Vt-dependent changes in growth behavior were suppressed by the addition of its own cell-free supernatant (CFS), indicating the presence of a growth regulation mechanism via cell-cell communication. Replicate cultures under the same conditions showed that the population establishment of PY1 was stochastic, whereas the model strain Nitrosomonas europaea exhibited synchronized population establishment, consistent with previous reports. This stochasticity in PY1 was also eliminated by the addition of CFS. Finally, a simulation model based on {Delta}Vt-dependent cell growth behavior of PY1 successfully reproduced synchronized population establishment in the presence of CFS. By contrast, the stochastic population establishment observed in the absence of CFS was successfully reproduced by a model incorporating {Delta}Vt-independent growth initiation following a Weibull distribution. Such environmentally responsive changes in population establishment dynamics may contribute to the low isolation success of environmental bacteria and sudden blooms of the rare biosphere.

Antarctic soils represent one of the more extreme environments for microbial life on Earth, yet they harbor heterogeneous and diverse microbial communities. Biologists have long hypothesized that Antarctic microorganisms are unique from those found on other continents due to the extreme geographic isolation and the cold, dry, and challenging conditions typical of Antarctica. To test this hypothesis, we focused on a cosmopolitan bacterial genus, Arthrobacter, that is widely distributed across global soils. We first profiled a global metagenomic dataset from both Antarctic and non-Antarctic surface soils to quantify the distributions of Arthrobacter strains. Despite high strain-level diversity, 90% of the strains found in the Antarctic soils were only found on the continent. We then used cultivation-based phenotypic analyses and strain-level genomic comparisons to assess how Antarctic strains and non-Antarctic strains differ in their traits and environmental preferences. Not only did we find evidence of endemism, but Antarctic Arthrobacter also have genomic characteristics and environmental tolerances that suggest they are uniquely adapted to Antarctic conditions. Significance StatementAntarctic soils are among the most extreme environments on Earth, yet they host diverse microbial communities whose adaptations are poorly understood. To test whether Antarctic microbes are distinct from those elsewhere, we examined Arthrobacter, a bacterial genus common in soils worldwide. Analysis of global metagenomic data revealed Arthrobacter strains in Antarctic soils are found exclusively on the continent. Cultivation experiments and comparative genomics further showed that Antarctic strains differ from nonAntarctic relatives in genomic features and environmental tolerances. Together, these results demonstrate that geographically isolated and extreme conditions can drive microbial endemism and local adaptation, even within globally distributed bacterial lineages.

Syngas fermentation by carbon monoxide (CO)-utilising acetogens offers a sustainable route for converting gasified waste materials into value-added chemicals. In this study, we isolated a novel thermophilic CO-utilising bacterium, strain AZ2, from marine hydrothermal sediment collected on the island of Sao Miguel, Azores, Portugal. Strain AZ2 is an obligately anaerobic, spore-forming bacterium. Average nucleotide identity (ANI; 78.4-86.7%) and digital DNA-DNA hybridization (dDDH; 23.4-32.5 %) analyses indicate that strain AZ2 represents a novel species within a previously uncharacterised lineage represented by the GTDB placeholder genus UBA2545 in the Neomoorellaceae family. Strain AZ2 was able to grow fermentatively on CO, producing acetate. We further demonstrated that its closest isolated relatives - Thermanaeromonas toyohensis, T. burensis, and Thermanaeromonas sp. strain 9S - are capable of growing on CO, producing either acetate or hydrogen gas (H2). Additionally, we unveiled the genomic potential for CO utilisation within other members of the GTDB placeholder class DSM-521 (previously Moorellia) to which our isolate belongs, expanding the list of possible thermophilic CO-utilising acetogens. We propose that strain AZ2T represents the type strain of a novel genus and species, named Thermobium azorense gen. nov., sp. nov. (= DSM 121889T = JCM 39698T).

The maintenance of endosymbiosis in cnidarians depends on the tight regulation of host immunity, cell cycle, and nutrient exchange, yet how these processes are impacted by interacting environmental stressors remains largely unknown. To address this, we employed physiological metrics, gene expression analysis, microbiome characterization, imaging (NF-{kappa}B localization, endoplasmic reticulum ultrastructure, EdU labeling), and stable isotope tracing in the model sea anemone Exaiptasia diaphana to examine the effects of heat and nitrate on these regulatory processes, individually and in combination. Heat treatment led to NF-{kappa}B activation, proteostatic stress, suppression of nutrient exchange, decreased cell-cycle progression, and microbiome restructuring, with all effects more pronounced in symbiotic than aposymbiotic anemones. In symbiotic anemones, nitrate partially offset these heat-induced responses through sustained carbon translocation, suggesting that the presence of symbionts, in conjunction with elevated nitrate, can temporarily buffer host thermal stress. However, prolonged combined exposure resulted in holobiont failure. These findings reveal that while nitrate enrichment can transiently delay the onset of bleaching, it does not preserve the regulatory networks required for symbiotic stability -- underscoring the vulnerability of cnidarian holobionts to the compounding effects of warming and nitrate pollution.

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.

A facultatively anaerobic, hydrogen-oxidizing, thermophilic bacterium (strain KUI-RBT) was isolated from a geothermal spring biofilm in Rotorua, New Zealand. Strain KUI-RBT is a motile, straight rod, measuring approximately 0.7 {micro}m by 1.0 to 1.5 {micro}m with a diderm cell wall. Growth of KUI-RBT occurred from 39 to 74 {degrees}C (Topt 64.5 {degrees}C), pH 5.0 to 7.5 (pHopt 6.5), and 0 to 1% (w/v) NaCl (NaClopt 0.4-0.7%, w/v). KUI-RBT utilizes carbon dioxide and various organic carbon substrates as carbon sources and hydrogen as an electron donor. KUI-RBT can use oxygen (0-21%, v/v), elemental sulfur, thiosulfate, sulfite, nitrate, arsenate, and selenate as terminal electron acceptors. Major fatty acids of strain KUI-RBT include C20:1, C18:1, and C18:0 and the primary quinone is MTK-7. The whole genome G+C content is 34.23 mol%. Phylogenetic analyses indicate KUI-RBT to be a member of the family Hydrogenothermaceae, with Sulfurihydrogenibium azorense Az-Fu1T its closest characterised relative (94.51% 16S rRNA gene sequence similarity, 78.01% whole genome ANI, 61.34% whole genome AAI). Based on phylogenetic and phenotypic analyses, we propose KUI-RBT represents a novel genus and species within the family Hydrogenothermaceae, for which we propose the name Reysenbachia aerophila gen. nov., sp. nov. The type strain is KUI-RBT (=KCTC accession =JCM accession). The GenBank accession number for the 16S rRNA gene sequence of strain KUI-RBT is PZ052650. The GenBank accession number for the whole genome of strain KUI-RBT is JBVODP000000000.

From Brazilian limestone caves, we isolated 29 bacteria utilizing phenol (23 bacteria), toluene (all bacteria), and/or benzene (all bacteria) as sole carbon sources. One isolate showed phosphate solubilization, while lipase/esterase activity occurred in two isolates; no amylase activity was detected, but 16 isolates ([~]55%) exhibited protease activity. Among them, Nocardioides sp. SF1 was selected for whole-genome sequencing due to its aromatic compound tolerance and protease activity. Additionally, catechol cleavage assays yielded unexpected purple pigmentation, suggesting non-canonical aromatic metabolism. Its high-quality draft genome (4.25 Mbp, 16 contigs, N50 of 887 kb) lacks canonical phenol hydroxylase but encodes alternative oxidation systems, phenylacetyl-CoA pathway, besides, desferrioxamine siderophore, biosurfactants, and phosphate solubilization, key adaptations for oligotrophic caves and biotechnologically interesting activities. Whole-genome comparisons (TYGS/GGDC, OrthoANI and k-mer) suggest potential new species. Lacks acquired antimicrobial resistance genes (ResFinder) and pathogenicity potential (PathogenFinder). Nocardioides sp. SF1 emerges as a non-pathogenic candidate for aromatic bioremediation and plant growth promotion in contaminated, nutrient-poor environments, highlighting cave actinobacterias unexplored biotechnological potential.

Streptomyces are abundant soil inhabitants with extensive secondary metabolism and antibiotic resistance traits. Yet, their ecological role in shaping soil antibiotic resistome dynamics remains understudied. Here, we investigated how two different bio-based fertilizers harbouring Streptomyces shaped soil resistome and mobilome by combining genome analysis of eight Streptomyces isolates to metagenomic profiling of soils before fertilization, within 48 hours after fertilizer application, and six weeks after. Streptomyces genomes showed linkages among antibiotic resistance genes, carbohydrate-active enzymes, and antibiotic-production-associated biosynthetic gene clusters, connecting resistance and biosynthesis to broader metabolic strategies. Relationships between carbon degradation and biosynthesis associated with specific enzyme families, indicating that carbon availability shapes secondary metabolism. We confirmed experimentally that antibacterial potential varied with carbon source, suggesting that microbial activity during manufacturing of the bio-based fertilizers may create localized selection pressures before fertilizers enter the soil. Fertilization with the studied materials induced modest but consistent shifts in resistome and mobilome without major changes in dominant taxa or overall bacterial abundances, indicating functional reorganization within soil communities. Diversity of antibiotic resistance genes and mobile genetic elements increased, whereas abundance changes were small. Mobile genetic element composition showed stronger responses that were associated with fertilizer inputs, Streptomyces abundance, and taxa linked to faecal and resistance sources. Together, our results show that bio-based fertilizers shape soil resistome primarily through ecological restructuring of resident soil communities, while carbon-dependent microbial activity within fertilizers may enrich resistance. These factors should be considered in manufacturing of bio-based fertilizer as well as in designing agricultural practices.

Microbes play a vital role in plant development, health, and resilience, yet relatively little is known about the specific metabolic mechanisms driving interactions in these host-associated communities. Systems biology models enable a computational approach to understanding metabolic interactions, which can be difficult to pinpoint experimentally; however, these methods cannot yet accommodate the large number of species in natural communities. Synthetic communities (SynComs) provide a more tractable alternative to explore targeted interactions. Here, we investigated metabolite exchange in a seven-member maize root-associated SynCom, specifically accounting for plant host context by designing a customized exudate medium. We constructed metabolic models for each bacterial species and curated them with in vitro phenotyping data to reflect experimentally based carbon uptake potential. Flux balance analysis of individual species demonstrated that integrating phenotype data and changing medium type had substantial impacts on predicted growth rates, which in turn shaped potential interspecies interactions. In silico community growth optimization of the seven-member community model showed that the exudate medium supported a more diverse community composition compared to minimal medium, with predictions of community member abundance closely aligned to literature-derived experimental results. Predicted metabolite exchange in the root exudate environment showed Enterobacter ludwigii as a community hub, and cross-feeding of indole suggested a potential effect of bacterial community interactions on the plant host. Our in silico findings indicate the host plays an important role in structuring microbial interactions and cross-feeding at the metabolic level, underscoring the importance of considering environmental context from both theoretical and experimental perspectives. IMPORTANCETrue understanding of a system is marked by the ability to predict its behavior. The complexity of natural host-microbe systems represents a frontier of knowledge that scientists are working to understand, and elucidating principles of interactions within multi-partite microbial communities remains a challenge in microbial ecology. Synthetic communities provide a tractable starting point for investigating interaction mechanisms, and computational approaches complement laboratory experiments by systematically evaluating multiple possibilities for metabolic pathway processing, thereby allowing us to comprehensively study the interconnected metabolic networks of host-associated microbiota. The model we developed for the seven-member maize root-associated bacterial community presents a step toward predicting plant-microbe behavior, providing hypotheses for future experimental testing and serving as a template for expanding model complexity to more members and other systems.

The contribution of predation between bacteria to microbial community dynamics in agricultural fields has hardly been investigated. Here. dynamics of general prokaryotes (GEP) and of the predators Bdellovibrionales (Bd) and Bacteriovoracales (Bac) (Bdellovibrio-and-Like Organisms, BALOs) were studied in two agricultural fields differing in organic and mineral input regimes, for one year. Season, but not fertilization, affected absolute sizes of GEP and of BALO communities. 16S rRNA gene community analysis identified numerous novel Bd and Bac lineages, with none of the dominant BALOs related to characterized isolates. A few dominant BALO amplicon sequence variants (ASVs) persisted year-round, whereas others showed seasonal- or treatment specific responses. GEP, Bd, and Bac ASV a-diversity was mostly influenced by season, with some changes due to fertilization in Bd, and Bac communities. Seasonal changes, site, and fertilization regimes influenced {beta}-diversity of GEP, Bd and Bac communities and determined the structure of BALO-gram-negative bacteria interaction networks, signaling that niche segregation acts at the microbiome-BALO interface. Accordingly, we suggest that shifts in GEP community structure triggered by environmental changes and agricultural practices cascade to BALO predators, in turn affecting BALO-microbiome interactions. These dynamics may be harnessed to manipulate the soil microbiome to benefit sustainable environmental and agricultural outcomes.