The ISME Journal

Microbial gene loss is hypothesized to be beneficial when gene function is costly, and the gene product can be replaced via cross-feeding from a neighbor. However, cross-fed metabolites are often only available at low concentrations, limiting the growth rates of gene-loss mutants that are dependent on those metabolites. Here we define conditions that support a loss of function mutant in a three-member bacterial community of: (i) N2-utilizing Rhodopseudomonas palustris as an NH4+ -excreting producer, (ii) N2-utilizing Vibrio natriegens as the ancestor, and (iii) a V. natriegens N2-utilizaton mutant that is dependent on the producer for NH4+. Using experimental and simulated cocultures, we found that the ancestor outcompeted the mutant due to low NH4+ availability under uniform conditions where both V. natriegens strains have equal access to nutrients. However, spatial structuring that separated the mutant from the ancestor, while maintaining access to NH4+ from the producer, allowed the mutant to avoid extinction. Counter to predictions, mutant enrichment under spatially structured conditions did not require a growth rate advantage from gene loss and the mutant coexisted with its ancestor. Thus, cross-feeding can originate from loss-of-function mutations that are otherwise detrimental, provided that the mutant can segregate from a competitive ancestor. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/634601v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1140808org.highwire.dtl.DTLVardef@1b72e8org.highwire.dtl.DTLVardef@14f70borg.highwire.dtl.DTLVardef@6707bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microscopic filaments of the siphonous green algae Ostreobium (Ulvophyceae, Bryopsidales) colonize and dissolve the calcium carbonate skeletons of coral colonies, in shallow-water reef environments of contrasted salinities. Their bacterial composition and plasticity in response to salinity remain unknown. Here, we analyzed the bacteria associated with coral-isolated Ostreobium strains from two distinct rbcL lineages, representative of IndoPacific environmental phylotypes, that had been pre-acclimatized (>9months) to three ecologically-relevant reef salinities: 32.9, 35.1 and 40.2 psu. Bacterial phylotypes were visualized at filament scale by CARD-FISH in algal tissue sections, localized to the surface, within filaments or in the algal mucilage. Ostreobium-associated communities, characterized by bacterial 16S rRNA metabarcoding of cultured thalli and corresponding supernatants, were structured by host genotype more than salinity and partly overlapped with those of environmental (Ostreobium-colonized) coral skeletons. Alphaproteobacteria dominated the thalli communities, enriched in Kiloniellaceae or Rhodospirillaceae depending on algal genotype. A small core microbiota composed of 7 ASVs ([~]1.5% of thalli ASVs, 19%-36% cumulated proportions), shared by multiple cultures of both Ostreobium genotypes and persistent across 3 salinities, included putative intracellular Amoebophilus and Rickettsiales bacteria. This novel knowledge on the taxonomic diversity of Ostreobium bacterial associates paves the way to functional interaction studies within the coral holobiont.

Virus-bacterial interactions are fundamental to microbial ecology and evolution, but they have been studied mostly under simplified laboratory conditions. To better understand how ecological complexity shapes these dynamics, we examined the extreme halophilic bacterium Salinibacter ruber strain M1 and the EM1 virus in the presence of additional Sal. ruber strains and viruses. In the short term, the presence of other strains delayed lysis and reduced EM1 virus production, indicating that community composition directly affects viral replication. In the long term, both Sal. ruber M1 and the EM1 virus persisted across all experimental conditions, but their evolutionary responses differed. The EM1 virus showed an increased mutation rate, reduced infectivity against the native host, and expanded host range when other viruses were present, suggesting a previously unrecognized form of virus-virus interaction, in which coexisting viruses influence each others evolutionary paths promoting viral diversification. In contrast, Sal. ruber M1 exhibited higher mutation rates evolving with other strains, indicating that in our system, intraspecific competition, rather than viral pressure, drives bacterial evolution. These findings demonstrate that incorporating biological complexity reveals distinct selective pressures acting on hosts and viruses, and is therefore essential for accurately predicting virus-host evolution in natural microbial ecosystems.

Many plant endosymbionts are facultative, switching between host-associated and free-living stages. Extensive genomic and experimental studies suggest that adaptation during the saprophytic, off-host phase, rather than adaptation to hosts, primarily constrains the biogeographic distribution of these microbes. To test this hypothesis, we analyzed the growth capacities and genomic features of 38 Sinorhizobium strains nodulating Medicago lupulina (black medic), collected from two regions with distinct thermal environments. The warmer region is predominantly inhabited by S. meliloti, while S. medicae and non-symbiotic strains (lacking symbiosis genes, such as S. canadensis and S. adherens) are more common in the cooler region. Laboratory assays demonstrated that at 40{degrees}C, the upper temperature limit of their region of origin, S. meliloti remained viable, albeit with reduced growth, whereas S. medicae and non-symbiotic strains failed to grow under heat stress. Comparative genomics revealed isolation-by-distance in both the core and accessory genomes, particularly in S. meliloti in the warmer region, which exhibits less within-region thermal variation. This is consistent with an isolation-by-distance model where population divergence is governed by restricted gene flow. These findings suggest that metabolic constraints shape the regional distribution of this facultative microbial symbiont, while limited gene flow influences local population structure.

Vertical transmission of bacterial endosymbionts is accompanied by virtually irreversible gene loss that can provide insights into adaptation to divergent ecological niches. While patterns of genome reduction have been well described in some terrestrial symbioses, they are less understood in marine systems where vertical transmission is relatively rare. The association between deep-sea vesicomyid clams and chemosynthetic Gammaproteobacteria is one example of maternally inherited symbioses in the ocean. Differences in nitrogen and sulfur physiology between the two dominant symbiont groups, Ca. Ruthia and Ca. Vesicomyosocius, have been hypothesized to influence niche exploitation, which likely affects gene content evolution in these symbionts. However, genomic data are currently limited to confirm this assumption. In the present study we sequenced and compared 11 vesicomyid symbiont genomes with existing assemblies for Ca. Vesicomyosocius okutanii and Ca. Ruthia magnifica. Our analyses indicate that the two vesicomyid symbiont groups have a common core genome related to chemosynthetic metabolism, but differ in their potential for nitrate respiration and flexibility to environmental sulfide concentrations. Moreover, Ca. Vesicomyosocius and Ca. Ruthia have different enzymatic requirements for cobalamin and nickel and show contrasting capacities to acquire foreign genetic material. Tests for site-specific positive selection in metabolic candidate genes imply that the observed physiological differences are adaptive and thus likely correspond to ecological niches available to each symbiont group. These findings highlight the role of niche differentiation in creating divergent paths of reductive genome evolution in vertically transmitted symbionts.

Understanding how intra- and interspecific differentiation arises in natural microbial populations is central to explaining the processes that drive bacterial evolution. Motivated by the co-occurrence of several genospecies closely related to Shewanella baltica in Baltic Sea sediments, we investigated the genomic structure of this species complex across fine spatial scales. We analyzed 112 genome sequences from strains collected across multiple sediment cores and depths (0-6 cm) at Vaxon (Stockholm archipelago, Sweden), including sympatric isolates from this site as well as earlier isolates and allopatric strains from other locations in the Stockholm region obtained from both sediments and the water column. Using a reverse-ecology population genomics approach, we found that these strains form a species complex that resolves into three cohesive evolutionary lineages (G1, G2, and G3). Each lineage is characterized by extensive gene turnover, driven largely by horizontal gene transfer (HGT), and displays distinct genomic signatures of metabolic specialization. While G1 consists predominantly of a single species (S. baltica), G2 and G3 comprise a diverse set of divergent genospecies, many of which are repeatedly recovered from sediment samples. Patterns of homologous recombination indicate that speciation within G2 and G3 is primarily recombination-driven ( sexual), and that both groups derive from a common ancestor. Together, these results capture a snapshot of early-stage speciation within a shared ecosystem and provide insight into the mechanisms that diversify sympatric, recombining bacterial populations, with a sediment-associated lifestyle likely promoting this process.

Microbial interaction and their evolution is vital for shaping the structure and function of microbial communities. However, the mechanisms governing the directionality and stability of the evolution of interactions within microbial communities remain poorly understood. Here, we used a syntrophic two-species biofilm consortium composed of Bacillus velezensis SQR9 and Pseudomonas stutzeri XL272 that promotes plant growth through their metabolic interactions and investigated how the interactions within the consortium change over evolutionary timescale to characterize the phenotypic and genetic diversification. The focal species B. velezensis SQR9 rapidly diversified into diverse colony morphotypes, both in the presence and absence of its interactor, P. stutzeri XL272, with variable frequencies. These morphotypes displayed phenotypic differentiation among biofilm formation, planktonic growth, and spore formation. The evolved P. stutzeri altered the fitness landscape of B. velezensis morphotypes, allowing the weaker rough morphotype to outcompete the biofilm-enhanced slimy morphotype. Whole genome re-sequencing correlated these phenotypic changes with mutations in specific genes encoding regulators of B. velezensis, including ywcC, comA, comP, degS, degQ and spo0F. The coevolutionary partner, P. stutzeri increased its exopolysaccharide production that could be explained by a frame shift mutation in cpsA gene encoding capsular polysaccharide (CPS) biosynthesis protein. Compared with the mono-evolution, co-evolved B. velezensis populations showed greater mutation accumulation in intergenic regions, which led to greater genetic parallelism. Furthermore, the dissimilarity between mono-evolved and co-evolved populations increased over time. Our study reveals intricate genetic diversification and fitness differentiation within a biofilm consortium, shaped by both abiotic conditions and biotic interactions.

Glycolate is a major product of phytoplankton photorespiration, but its fate in the microbial food web is not well constrained. Here, we used stable isotope probing and mass spectrometry combined with genomic analyses and microscopy to quantify glycolate metabolism by a taxonomically diverse set of heterotrophic marine bacteria. We found that 9 of 16 tested strains with the genomic capability to metabolize glycolate directly assimilated and respired glycolate carbon in monoculture. We next co-cultivated glycolate-incorporating strains with non-incorporating strains and found that several cross-feeders incorporated more glycolate carbon into their biomass than direct incorporators. Carbon use efficiency, reflecting proportional differences in movement of glycolate carbon into biomass versus into carbon dioxide, were distinct across co-cultures and ranged from 0.01 -3.15% depending on the strain mixtures. These results suggest that the fate of glycolate carbon is not limited to microbial taxa with the genetic capability for direct assimilation, and that bacterial metabolic interactions via cross-feeding play a critical role in influencing the efficiency of carbon transfer. Such information is critical to refine conceptual and numerical models of heterotrophic processing and transfer of organic carbon in an era of global change with predicted increases in photorespiration.

Marine bacteria-phytoplankton interaction ultimately shapes ecosystem productivity. The biochemical mechanisms underlying their interactions become increasingly known, yet how these ubiquitous interactions drive bacterial evolution has not been illustrated. Here, we sequenced genomes of 294 bacterial isolates associated with 19 coexisting diatom cells. These bacteria constitute eight genetically monomorphic populations of the globally abundant Roseobacter group. Six of these populations are members of Sulfitobacter, arguably the most prevalent bacteria associated with marine diatoms. A key finding is that populations varying at the intra-specific level have been differentiated and each are either associated with a single diatom host or with multiple hosts not overlapping with those of other populations. These closely related populations further show functional differentiation; they differ in motility phenotype and they harbor distinct types of secretion systems with implication for mediating organismal interactions. This interesting host-dependent population structure is even evident for demes within a genetically monomorphic population but each associated with a distinct diatom cell, as shown by a greater similarity in genome content between isolates from the same host compared to those from different hosts. Importantly, the intra- and inter-population differentiation pattern remains when the analyses are restricted to isolates from intra-specific diatom hosts, ruling out distinct selective pressures and instead suggesting coexisting microalgal cells as physical barriers of bacterial gene flow. Taken together, microalgae-associated bacteria display a unique microscale metapopulation structure, which consists of numerous small populations whose evolution is driven by random genetic drift.

Ammonia-oxidising archaea and nitrite-oxidising bacteria are common members of marine sponge microbiomes. They derive energy for carbon fixation and growth from nitrification - the oxidation of ammonia to nitrite and further to nitrate - and are proposed to play essential roles in the carbon and nitrogen cycling of sponge holobionts. In this study, we characterise two novel nitrifying symbiont lineages, Ca. Nitrosokoinonia and Ca. Nitrosymbion in the marine sponge Coscinoderma matthewsi using a combination of molecular tools, single-cell imaging techniques, and physiological rate measurements. Both represent a new genus in the ammonia-oxidising archaeal class Nitrososphaeria and the nitrite-oxidising bacterial order Nitrospirales, respectively. Furthermore, we show that larvae of this viviparous sponge are densely colonised by representatives of Ca. Nitrosokoinonia and Ca. Nitrosymbion indicating vertical transmission. In adults, the representatives of both symbiont genera are located extracellularly in the mesohyl. Comparative metagenome analyses and physiological data suggest that ammonia-oxidising archaeal symbionts of the genus Ca. Nitrosokoinonia strongly rely on endogenously produced nitrogenous compounds (i.e., ammonium, urea, nitriles/cyanides, and creatinine) rather than on exogenous ammonium sources taken up by the sponge. Additionally, the nitrite-oxidising bacterial symbionts Ca. Nitrosymbion may reciprocally support the ammonia-oxidisers with ammonia via the utilisation of sponge-derived urea and cyanate. Interestingly, comparative analyses of published environmental 16S rRNA amplicon data revealed that Ca. Nitrosokoinonia and Ca. Nitrosymbion are widely distributed and predominantly associated with marine sponges and corals, suggesting a broad relevance of our findings.

Optimizing nitrogen (N) utilization in ruminant production systems holds both economic and environmental significance. However, traditional paradigms of N metabolism, derived primarily from well-studied model rumen bacteria, cannot fully capture the diverse and complex N metabolic dynamics within the rumen ecosystem. To address this gap, we utilized comparative genomics and genome-resolved multi-omics analyses using a curated set of microbial genomes to investigate N assimilation and regulation in rumen microbes. We discovered that canonical mechanism of ammonia assimilation and regulation, such as the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathways and its regulatory proteins, are absent in the genomes of many key and predominant rumen microbes, which likely utilize alternative pathways for ammonia assimilation. These findings challenge the applicability of E. coli-based N regulation models to rumen bacteria. We further linked polysaccharide utilization and ammonia assimilation across hundreds of rumen microbial species. Furthermore, we identified specific microbial species involved in ureolysis and denitrification, as well as phages carrying auxiliary metabolic genes that perform N assimilation. Using an animal trial involving 11 pairs of lamb twins in a crossover design, we demonstrated that dietary crude protein (CP) concentrations had minimal impact on rumen microbiome composition and expression of N assimilation genes. Instead, shifts in concentrate levels triggered alterations in N assimilation, including increased expression of amino acid biosynthesis pathways. These findings indicate a nuanced, species-specific microbial response to dietary interventions, highlighting the limitations of traditional N metabolism models applied to rumen microbes and the need for more granular studies of rumen microbial ecosystems.

Plant-associated microorganisms, particularly endophytes, are essential for plant health and development. Endophytic microbiota is intimately associated with host plants colonizing various tissues, including seeds. Seed endophytes are particularly noteworthy because of their potential for vertical transmission. This pathway may play a role in the long-term establishment and evolution of stable bacteria-host interactions across plant generations. Hundreds of seed-bacteria associations have been recently uncovered; however, most seem to be transient or unspecific. While it is known that microorganisms can be transmitted from plant tissues to seeds and from seeds to seedlings, the experimental confirmation of bacterial transfer through successive plant generations remains unreported. In this study, we identified Pantoea as the unique core endophytic bacteria inhabiting the endosperms of 24 wheat seed samples originally harvested in different worldwide locations. Remarkably, Pantoea is the genus with the highest relative average abundance in wheat seeds (61%) and also in germinated seedlings grown under gnotobiotic conditions (30%). In the field, it was the only genus dwelling roots, shoots, spikes and seeds of 4 different wheat varieties tested and its abundance progressively increased across these tissues. This genuine pattern of vertical enrichment, which was not found in other common wheat-associated taxa, suggests a role in the transfer of these endophytic bacteria through the seeds. To confirm intergenerational transmission, parental plants were inoculated with labelled Pantoea isolates, which specifically colonized the next generations of Poaceae plants, experimentally demonstrating bacterial vertical inheritance to the offspring generations and suggesting transmission specificity.

Growing evidence suggests that interactions among heterotrophic microbes influence the efficiency and rate of organic matter turnover. These interactions are dynamic and shaped by the composition and availability of resources in their surrounding environment. Heterotrophic microbes inhabiting marine environments often encounter fluctuations in the quality and quantity of carbon inputs, ranging from simple sugars to large, complex compounds. Here, we experimentally tested how the chemical complexity of carbon substrates affects competition and growth dynamics between two heterotrophic marine isolates. We tracked cell density using species-specific PCR assays and measured rates of microbial CO2 production along with associated isotopic signatures (13C and 14C) to quantify the impact of these interactions on organic matter remineralization. The observed cell densities revealed substrate-driven interactions: one species exhibited a competitive advantage and quickly outgrew the other when incubated with a labile compound while both species seemed to coexist harmoniously in the presence of more complex organic matter. Rates of CO2 respiration revealed that co-incubation of these isolates enhanced organic matter turnover, sometimes by nearly twofold, compared to their incubation as mono-cultures. Isotopic signatures of respired CO2 indicated that co-incubation resulted in a greater remineralization of macromolecular organic matter. These results demonstrate that simple substrates promote competition while high substrate complexity reduces competitiveness and promotes the partitioning of degradative activities into distinct niches, facilitating coordinated utilization of the carbon pool. Taken together, this study yields new insight into how the quality of organic matter plays a pivotal role in determining microbial interactions within marine environments.

Nitrification occurs widely from the deep sea to animal holobionts, but the eco-evolutionary forces shaping the niches and dynamics of the lineages of the chemoautotrophic bacteria and archaea responsible remain largely unknown. To make strides towards this goal in a rapidly changing, exemplar marine ecosystem, the Baltic Sea, we studied basin-scale nitrifier spatio-temporal dynamics, coupled with enrichment-enabled comparative genomics. Based on metagenomes and rRNA gene sequencing, we found nitrifiers to be persistently relatively abundant throughout deep depths (>25 m), and from late-fall to spring in surface waters, as revealed by twice-weekly sampling across two years in the southwest Baltic Sea surface waters. In these surface waters, we observed time-lagged dynamics between ammonia- and nitrite-oxidizers, which were positively correlated with nitrite, nitrate, and diverse other prokaryotes, and negatively correlated with day length, light, and chlorophyll. For the dominant nitrifiers, ammonia-oxidizing archaea (AOA), we enriched five novel species including the dominant deep Baltic Sea species, and obtained genomes from all dominant AOA phylotypes. Among these genomes, which enabled fine-scale niche-differentiation, we observed a high degree of gene conservation, with most differences related to genes associated with interactions with the external environment, including genes involved in signal transduction, cell wall/membrane biogenesis, and inorganic ion transport, indicating these may be the primary drivers of strain-variability. We also observed differences in nitrogen and phosphorus metabolism between two dominant surface types. Together our study provides key insights into the niche of nitrifiers, and begins the process of understanding the mechanisms and functional implications of these patterns.

Phages are generally described as species- or even strain-specific viruses, implying an inherent limitation for some to be maintained and spread in diverse bacterial communities. Moreover, phage isolation and host range determination rarely consider the phage ecological context, likely biasing our notion on phage specificity. Here we identified and characterized a novel group of promiscuous phages existing in rivers by using diverse bacteria isolated from the same samples, and then used this biological system to investigate infection dynamics in distantly related hosts. We assembled a diverse collection of over 600 native bacterial strains and used them to isolate six podophages, named Atoyac, from different geographic origin and capable of infecting six genera in the Gammaproteobacteria. Atoyac phage genomes are highly similar to each other but not to those currently available in the genome and metagenome public databases. Detailed comparison of the phages infectivity in diverse hosts and trough hundreds of interactions revealed variation in plating efficiency amongst bacterial genera, implying a cost associated with infection of distant hosts, and between phages, despite their sequence similarity. We show, through experimental evolution in single or alternate hosts of different genera, that plaque production efficiency is highly dynamic and tends towards optimization in hosts rendering low plaque formation. Complex adaptation outcomes observed in the evolution experiments differed between highly similar phages and suggest that propagation in multiple hosts may be key to maintain promiscuity in some viruses. Our study expands our knowledge of the virosphere and uncovers bacteria-phage interactions overlooked in natural systems. ImportanceIn natural environments, phages co-exist and interact with a broad variety of bacteria, posing a conundrum for narrow-host-range phages maintenance in diverse communities. This context is rarely considered in the study of host-phage interactions, typically focused on narrow-host-range viruses and their infectivity in target bacteria isolated from sources distinct to where the phages were retrieved from. By studying phage-host interactions in bacteria and viruses isolated from river microbial communities, we show that novel phages with promiscuous host range encompassing multiple bacterial genera can be found in the environment. Assessment of hundreds of interactions in diverse hosts revealed that similar phages exhibit different infection efficiency and adaptation patterns. Understanding host range is fundamental in our knowledge of bacteria-phage interactions and their impact in microbial communities. The dynamic nature of phage promiscuity revealed in our study has implications in different aspects of phage research such as horizontal gene transfer or phage therapy.

Soil virus communities are diverse and dynamic but contributions to specific processes, such as nitrification, are largely uncharacterised. Chemolithoautotrophic nitrifiers perform this essential component of the nitrogen cycle and are established model groups for linking phylogeny, evolution and ecophysiology due to limited taxonomic and functional diversity. Ammonia-oxidising bacteria (AOB) dominate the first step of ammonia oxidation at high supply rates, with ammonia-oxidising archaea (AOA) and complete ammonia-oxidising Nitrospira (comammox) often active at lower supply rates or when AOB are inactive, and nitrite-oxidising bacteria (NOB) completing canonical nitrification. Here, the diversity and genome content of dsDNA viruses infecting different nitrifier groups were characterised after in situ enrichment via differential host inhibition, a selective approach that alleviates competition for non-inhibited populations to determine relative activity. Microcosms were incubated with urea to stimulate nitrification and amended with 1-octyne or 3,4-dimethylpyrazole phosphate (AOB inhibited), acetylene (all ammonia oxidisers inhibited), or no inhibitor (AOB stimulated), and virus-targeted metagenomes characterised using databases of host genomes, reference (pro)viruses and hallmark genes. Increases in the relative abundance of nitrifier host groups were consistent with predicted inhibition profiles and concomitant with increases in the relative abundance of their viruses, represented by 200 viral operational taxonomic units. These included 61 high-quality/complete virus genomes 35-173 kb in length and possessing minimal similarity to validated families. Most AOA viruses were placed within a unique lineage and viromes were enriched in AOA multicopper oxidase genes. These findings demonstrate that focussed incubation studies facilitate characterisation of host-virus interactions associated with specific functional processes.

Plant growth-promoting rhizobacteria benefit plants by stimulating their growth or protecting them against phytopathogens. Rhizobacteria must colonise and persist on plant roots to exert their benefits. However, little is known regarding the processes by which rhizobacteria adapt to different plant species, or behave under alternating host plant regimes. Here, we used experimental evolution and whole-population whole-genome sequencing to analyse how Bacillus subtilis evolves on Arabidopsis thaliana and tomato seedlings, and under an alternating host plant regime, in a static hydroponic setup. We observed parallel evolution across multiple levels of biological organisation in all conditions, which was greatest for the two heterogeneous, multi-resource spatially-structured environments at the genetic level. Species-specific adaptation at the genetic level was also observed, possibly caused by the selection stress imposed by different host plants. Furthermore, a trade-off between motility and biofilm development was supported by mutational changes in motility- and biofilm-related genes. Finally, we identified several condition-specific and common targeted genes in different environments by comparing three different B. subtilis biofilm adaptation settings. The results demonstrate a common evolutionary pattern when B. subtilis is adapting to the plant rhizosphere in similar conditions, and reveal differences in genetic mechanisms between different host plants. These findings will likely support strain improvements for sustainable agriculture. Data summarySequencing data associated with this article are available in the CNGB Sequence Archive (CNSA) [1] of the China National GeneBank DataBase (CNGBdb) [2] under accession numbers CNP0002416 and CNP0003952. Strain data for the DK1042 ancestor are available under accession number CNP0002416. Impact statementFor rhizobacteria to benefit plant growth and protect against phytopathogens, bacteria must colonise and persist on plant roots. Understanding how rhizobacteria adapt to different plant species will assist strain development in sustainable agriculture. To explore the rhizobacterial adaptation process for different plant species and alternating host regimes, B. subtilis was experimentally evolved on A. thaliana or tomato roots, or an alternating host regime. Both parallel and species-specific adaptation was revealed at the genetic level. Analysis of the trade-off between motility and biofilm formation revealed several condition-specific and commonly targeted genes based on experimentally evolving B. subtilis biofilms.

1.Structured Abstract1.1 AbstractSoybean red crown rot, caused by the soil-borne fungus Calonectria ilicicola, causes substantial yield losses, but the response of the root-associated bacterial microbiome remains poorly understood. Here, we combined 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to characterize bacterial communities in soybean root-associated soils. 16S rRNA gene sequencing showed that diseased plants had rhizosphere and, more strikingly, rhizoplane microbiomes distinct from those of healthy plants, often with increased Enterobacterales. Shotgun metagenomics further revealed enrichment of genes associated with antibiotic resistance, particularly cationic antimicrobial peptide resistance, in diseased rhizoplane samples. Single-cell genomics recovered seven nonredundant Enterobacterales genomes and showed that plant pathogenicity-related genes were broadly distributed across these lineages. In contrast, dlt genes, which are associated with cationic antimicrobial peptide resistance, were detected only in the Enterobacterales lineages enriched in diseased rhizoplane soils. These results support a model in which soybean red crown rot is accompanied by microbiome restructuring and opportunistic enrichment of specific Enterobacterales lineages carrying putative cationic antimicrobial peptide resistance genes. More broadly, this study highlights the value of strain-resolved single-cell genomics for linking disease-associated community shifts to specific bacterial traits. 1.2 ImportanceUnderstanding crop disease requires resolving not only the primary pathogen but also the root-associated bacteria that respond to infection. Here, we used 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to examine the soybean rhizoplane microbiome under red crown rot. Diseased plants showed reproducible shifts in bacterial composition, including frequent enrichment of Enterobacterales and antimicrobial resistance-related functions. Strain-resolved genomes further revealed that the Enterobacterales lineages enriched in diseased rhizoplane soils specifically carried putative dlt-mediated resistance to cationic antimicrobial peptides, whereas general pathogenicity-related genes were broadly shared. These findings suggest that host defense-associated selection, rather than pathogenicity genes alone, may help shape disease-associated root microbiomes. This study demonstrates how single-cell genomics can uncover strain-level traits hidden within bulk community data and thereby clarify plant-pathogen-microbiome interactions.

Antibiotic resistance genes (ARGs) are ubiquitous in marine environments, yet whether their distribution primarily reflects anthropogenic pollution or intrinsic ecological functions remains unresolved. We used genome-resolved metagenomics to characterize resistomes in 371 genomic operational taxonomic units (gOTUs) across a gradient of human impact: the heavily impacted Baltic Sea, the moderately impacted North Sea, and the minimally impacted West Greenland shelf. ARG density was distinctly elevated in the Baltic Sea (3.20 ARGs Mbp-1) relative to the North Sea (1.90) and West Greenland (1.67), which did not differ significantly from each other, suggesting a relatively uniform oceanic baseline. Variance partitioning revealed that taxonomic identity explained 20.1% of ARG density variation, with environment contributing 11.4%; critically, Baltic gOTUs carried 35.1% more ARGs than predicted from taxonomy alone, indicating environment-driven enrichment beyond baseline taxonomic carriage. Lifestyle-dependent ARG partitioning between particle-attached and free-living prokaryotes emerged only under anthropogenic pressure: free-living bacteria were enriched in multiple resistance classes in the Baltic Sea but showed no differentiation in West Greenland. Only 0.85% of detected ARGs showed [≥]70% amino acid identity to clinically characterized sequences in the CARD database, showing that marine ARGs are highly divergent from clinical resistance determinants. Virulence factor annotations were widespread but weakly coupled with ARG abundance, suggesting independent ecological selection. Our results suggest that marine resistomes integrate an intrinsic baseline of ecological functions with selective enrichment of specific resistance mechanisms under anthropogenic pressure, and that genome-resolved approaches are able to quantify the relative contributions of each.

The role of the microbiome in shaping the host phenotype has emerged as a critical area of investigation, with implications in ecology, evolution, and host health. The complex and dynamic interactions involving plants and their diverse rhizosphere microbial communities are influenced by a multitude of factors, including but not limited to soil type, environment, and plant genotype. Understanding the impact of these factors on microbial community assembly is key to yielding host-specific and robust benefits for plants, yet remains challenging. Here we ran an artificial ecosystem selection experiment, over eight generations, in Arabidopsis thaliana Ler and Cvi to select soil microbiomes associated with higher or lower biomass of the host. This resulted in divergent microbial communities, shaped by a complex interplay between random environmental variations, plant genotypes, and biomass selection pressures. In the initial phases of the experiment, the genotype and the biomass selection treatment have modest but significant impacts. Over time, the plant genotype and biomass treatments gain more influence, explaining [~]40% of the variation in the microbial community composition. Furthermore, a genotype-specific association of a plant growth-promoting rhizobacterial taxa, Labraceae with Ler and Rhizobiaceae with Cvi, is observed under selection for high biomass.