The ISME Journal

Aquatic environments can maintain and express antibiotic resistance genes even without antibiotic pressure, but the conditions that govern their activity remain poorly understood. Redox oscillations are pervasive in natural and engineered waters and strongly alter microbial metabolism, yet whether their frequency shapes resistance expression is unknown. We reanalyzed paired metagenomes and metatranscriptomes from a published chemostat experiment in which a sulfidic stream community was cycled between oxic and anoxic conditions at high, medium and low frequency (HF, MF, LF), with strain-resolved population genomics of the dominant Pseudomonas lineage. Resistance genes were broadly distributed across treatments, whereas transcription concentrated under fast cycling, reaching a median 168 TPM under HF against 38 under LF although no antibiotic was present, so genetic potential and expression diverged. Fast cycling induced ROS-generating flavoproteins, antioxidant defenses and transcription-coupled DNA repair, and Pseudomonas supplied 99% of resistance-gene transcripts under HF, chiefly through its intrinsic efflux systems. Within the dominant Pseudomonas genome, however, the four resistance genes carried no sequence variants under HF, where coverage was deepest, and no fixed difference from the reference in any sample. Genome-wide selection remained purifying throughout (pooled pN/pS 0.72 under HF against 0.12 under LF, both below 1), and the relaxation trended toward redox and central metabolism. Redox frequency therefore raises resistance expression by selecting a metabolically versatile, high-efflux lineage, while the resistance sequences themselves stay conserved. Resistance activity in aquatic systems responds to an abiotic driver, which argues for weighing expression alongside gene presence in environmental surveillance.

Microbial fermentations emerge from the collective activity of interacting species whose relative abundances, metabolic functions, and environmental constraints continuously change over time. Water kefir provides a tractable model to investigate how multispecies communities coordinate their metabolic activities during fermentation. Here, we combined time-resolved community profiling, physicochemical measurements, metabolite quantification, and controlled co-culture assays to characterize the dynamics of a water kefir consortium comprising 19 detected microbial species. Our results reveal that water kefir fermentation is not adequately described as a simple succession of independently acting microbial groups. Instead, the community undergoes a reproducible reorganization of its collective metabolism around 20 h of fermentation. This transition marks a shift from an early regime characterized by the accumulation of soluble sugars and ethanol to a later regime dominated by enhanced sugar consumption and lactate production. Controlled interaction assays further showed that bacterial partners can alter carbon-use behaviour in Saccharomyces cerevisiae, highlighting the capacity of interspecies interactions to reshape metabolic phenotypes within the consortium. Together, these results support an ecological view of water kefir fermentation in which community-wide metabolic reorganization emerges from the interplay between microbial interactions, resource availability, and environmental change.

Mangroves persist under strong environmental constraints with support from microbes. Although microbial communities differ among mangrove compartments, their assembly mechanisms and compartment-specific functional signatures remain poorly resolved. Here, we combine peptide nucleic acid-clamping coupled with 16S rRNA gene amplicon sequencing and genome-resolved metagenomics to investigate the microbial communities associated with the leaves and roots of gray mangrove trees in the Red Sea. Our results suggest homogeneous selection and homogenizing dispersal as key processes of microbial community assembly in leaves. This phyllosphere hosts novel prokaryotic lineages, with some of its members probably synthesizing rhodopsins, plant polysaccharide-degrading enzymes, and gamma-aminobutyric acid, a metabolite that increase tolerance to salinity stress. Genomes affiliated with Desulfobacterales and Sedimenticolaceae taxa are abundant belowground, suggesting complementary sulfur- and nitrogen-cycling capacities similar as occur in other blue-carbon ecosystems. Comparison with root-derived genomes from cordgrass revealed a host-driven selection of Sedimenticolaceae species with convergent metabolic profiles. Overall, this study provides an integrative view about the microbial biology of gray mangrove trees, offering foundational insights into the diversity, ecology, and predictive functionality of their aboveground-belowground microbiomes.

Hydrostatic pressure is a defining feature of the deep ocean, yet how it structures microbial activity across the subseafloor biosphere is poorly understood. Here, we assess pressure effects on anabolic activity in seafloor and subseafloor communities across a margin-to-hadal-to-abyssal transect of the Puerto Rico Trench region (493-8,385 m water depth). Community-level responses to pressure reverse with burial, revealing a previously unrecognized shift toward piezophily in resident subseafloor communities down to 20 meters below seafloor (mbsf). In pressure-incubation experiments, atmospheric pressure yielded higher proportions of active cells in surficial sediments, whereas in situ pressure yielded higher proportions in the subseafloor. Pressure also structured active-community composition; in trench-axis sediments extending to 8.35 mbsf, JS1/Atribacterota populations belonging to geographically widespread hadal lineages exhibited a physiological response to in situ pressure, indicative of subseafloor piezophily. These findings suggest that hydrostatic pressure acts as an ecological filter after burial, favoring piezophilic traits that may support microbial persistence in the deep subseafloor biosphere.

Kiritimatiellia are a class of Verrucomicrobiota whose few cultured representatives are known for hosting large repertoires of carbohydrate-active enzymes and sulfatases. Many families of this class are restricted to metagenome-assembled genomes not yet discussed in literature, leaving their genome organization undescribed. Here, we survey 898 Kiritimatiellia genomes using similarity-colocalization networks (SCoNes), a data structure that captures conserved synteny among homologous genes across genomes. Using this approach, we investigate four metabolic adaptations to a glycan-rich environment: complex polysaccharide degradation, nitrogen cycling, hydrogenase activity, and natural product biosynthesis. Rather than unrelated functions, these capacities envelop a single anaerobic and saccharolytic strategy where sulfated glycan hydrolysis fuels fermentation, hydrogenases balance redox, and excess carbon supports nitrogen fixation.

A large fraction of microbes in natural environments are auxotrophs that rely on metabolite exchange for growth. The Black Queen hypothesis and related frameworks explain gene loss and reliance on shared metabolites. Yet it is not fully understood how metabolite cross-feeding emerges and gives rise to experimentally observable community performance. Here, we propose that exchange of biosynthetic pathway intermediates provides a mechanistic route for the emergence of metabolite cross-feeding. Building on prior work demonstrating intermediate exchange in E. coli auxotrophs, we investigated co-cultures between a methionine auxotroph ({Delta}metA) and a panel of histidine auxotrophs. Among these, only {Delta}hisD exhibited efficient co-culture growth with {Delta}metA, suggesting a unique role for the final step of histidine biosynthesis, which converts histidinol to histidine. We found that {Delta}metA-{Delta}hisD co-cultures accumulated high levels of extracellular histidinol (>200 M), suggesting a mechanism for metabolite exchange. Spent media from these co-cultures supported growth of all histidine auxotrophs except {Delta}hisD, confirming that histidinol, rather than histidine, was the dominant exchanged metabolite. Furthermore, we observed that {Delta}metA efficiently converted histidinol to histidine only under methionine-limited conditions, suggesting that nutrient limitation promoted overflow of converted histidine to the extracellular environment. To quantitatively assess whether histidinol exchange could account for the observed co-culture behavior, we constructed a mechanistic model that links extracellular histidinol dynamics to co-culture growth through independently measured conversion capacity of the {Delta}metA strain. Rather than fitting growth empirically, we used experimentally derived parameters to predict co-culture growth from the measured histidinol-to-histidine conversion flux. The model accurately recapitulated the timing and magnitude of growth and population dynamics, supporting the idea that accumulation of histidinol enabled the conversion-driven supply of histidine that sustained {Delta}hisD, while coupling growth between the two strains. Taken together, these results suggest that metabolite cross-feeding in syntrophic co-cultures can emerge from the accumulation of pathway intermediates and their conditional conversion under nutrient limitation, providing a plausible mechanism for the rapid establishment of mutualistic interactions.

The presence of metaparalogs (related genes diverging in function) has been proven to be extensive in the pangenomes of streamlined Pelagibacterales genomes. Here by leveraging a large number of complete genomes from an Arctic population we have been able to establish their universality and mechanistic origins. The diversity of some metaparalog families was as high within a single population than comparing with another (Mediterranean) population/species, which points to an ancient diversification of metaparalog families predating genomospecies separation. Metaparalog families provided diversity for transport and envelope diversity which would provide vast capacities to degrade substrates within DOM and interact with other cells or viruses or other subcellular entities (like EVs). The pool does not saturate at fifteen genomes. Our results highlight the immense importance of the pangenome in understanding the biology of bacteria in natural environments and how critical is the study of population genomics for the future of microbiology.

Understanding the complex interactions between phage, antibiotics and bacteria is vital for the development of rationale phage-antibiotic therapies. These interactions have traditionally been investigated via bulk assays, thus masking eventual cellular heterogeneities and the impact of spatial refuges on phage and antibiotic efficacy. Using microfluidics-based time-lapse microscopy, we reveal large phenotypic heterogeneity in the response of Burkholderia thailandensis to both the bacteriophage {Phi}Bp-AMP1 and sub-inhibitory concentrations of two selected antibiotics ciprofloxacin and trimethoprim. We discover that simultaneous exposure to ciprofloxacin and phage decreases the size of individual B. thailandensis cells, whereas phage and trimethoprim together slow down cell doubling. Exposure to ciprofloxacin also causes extended phage lysis of B. thailandensis which is preceded by a distinguishable pause in cell elongation, whereas exposure to trimethoprim constrains lysis. As a consequence, exposure to ciprofloxacin further hinders the growth of B. thailandensis in the presence of phage, whereas exposure to trimethoprim enhances growth in the presence of phage. These findings help understand the effects of the interactions between antibiotics and phage on bacterial subpopulations in spatial refuges that are notoriously difficult to treat with antibiotics.

TonB-dependent transporters (TBDTs) play a central role in siderophore-mediated iron acquisition in the marine environment. However, assigning substrate specificity to TBDTs remains a major bottleneck in understanding the ecological and mechanistic roles of siderophore production and uptake. Here, we identify in Marinobacter nauticus two TBDTs, OptA and OptB, that share low sequence identity yet both mediate Fe-petrobactin uptake. Despite this functional overlap, they underlie distinct regulatory mechanisms, suggesting differential utilization under specific environmental conditions. Phylogenetic analyses indicate that OptA likely co-evolved with the petrobactin biosynthetic gene cluster, whereas OptB appears to have followed a distinct evolutionary trajectory, acquiring the capacity to import petrobactin as a xenosiderophore. Using resuscitated communities from the Southern Ocean, we further show that Fe-petrobactin stimulated prokaryotic growth. Based on amino acid similarity, we identified OptB-like transporters encoded in metagenomes and MAGs and show that taxa harbouring these transporters, such as Pseudoaltermonas, respond specifically to Fe-petrobactin addition. OptB-like transporters were also detected in environmental metagenomes from the Southern Ocean, whereas the petrobactin biosynthetic gene clusters were not, indicating that petrobactin primarily functions as a xenosiderophore in natural communities.

Fucoidan is the second most abundant polysaccharide in brown macroalgae, contributing up to 50% of the dissolved organic matter released daily from these algae into the ocean, substantially influencing the marine carbon cycle. Due to its complex chemical structure, fucoidan is highly resistant to microbial degradation and accumulates on the surface of macroalgae, where it contributes to regulating their microbiome. Here, we used a range of microfluidic assays to quantify the chemotactic and physiological responses of marine bacteria to fucoidan. Our results reveal that all the strains tested (14 out of 14) are attracted to fucoidan produced by two different species of brown macroalgae. Attraction occurred only at high fucoidan concentrations (0.1 to 1 mg/mL), such as those expected in the immediate vicinity of algal surfaces, whereas low concentrations elicited no response or modest chemorepulsion. Although fucoidan did not support bacterial growth under our experimental conditions, it did not impair the utilization of other carbon sources. Together, these findings suggest that fucoidan gradients can act as chemical cues guiding marine bacteria toward the nutrient-rich microenvironment of macroalgal surfaces, pointing to a potential ecological role of fucoidan in shaping macroalgal microbiota, bacterial behavior, and ultimately carbon cycling in the ocean.

Bacterial blooms are characterized by unusually high cell densities and exceptionally low diversity and can profoundly alter ecosystem function and services. Bacteriophages have long been considered an important cause of mortality in blooms, acting as a mechanism for control. Here, we characterize the viral ecology of a long-lasting estuarine bloom of green sulfur bacteria (Chlorobiota). We combined direct cell and viral counts with metagenomic and metaproteomic data to characterize host and phage activity at different time points. The abundance of virus-like particles (VLPs) decreased at high cell densities, suggesting reduced lytic infection rates. The dominant organism, GSB-TRL01 (genus Prosthecochloris), apparently contained a large conjugative plasmid encoding five different anti-phage defense systems. The organism's genome encoded 13 additional defense systems. Compared to the average of five defense systems per microbial genome, this enrichment suggests robust anti-phage defense capabilities. Proteins from ten different defense systems on GSB-TRL01's genome and four systems from the conjugative plasmid were expressed in the proteome. This suggests that GSB-TRL01 invests heavily in anti-phage defense, leading to reduced lysis at high cell densities and allowing blooms to persist for weeks to months. To determine whether this ability is widespread among bloom forming organisms, we compared genomes of putative bloomers to those of non-blooming organisms. We found that bloomer genomes were significantly enriched with anti-phage defense systems. This challenges traditional paradigms of phage ecology in bloom-forming systems and suggests that microbes adapted to high-density growth may have evolved mechanisms to reduce their susceptibility to phage attack.

Chemosynthetic symbioses underpin animal life at deep-sea hydrothermal vents, encompassing substantial variabilities in symbiont identity, metabolic capacity, and host dependence. Here, we characterise symbioses in two peltospirid snails from Mid-Atlantic Ridge vents, Peltospira gargantua and P. smaragdina, using 3D anatomical reconstruction, stable isotope analyses, fluorescence in situ hybridization, transmission electron microscopy, and metagenomics. Both species hosted abundant intracellular Campylobacterota within bacteriocytes of a hypertrophied oesophageal gland. Their dominant symbionts belonged to genus Sulfurimonas, but the two hosts were associated with distinct symbiont lineages. Symbiont genomes encoded carbon fixation through the rTCA cycle, sulfide oxidation, and Group I Ni-Fe hydrogenases, indicating combined thiotrophic and hydrogenotrophic capacities. Hydrogenase copy number varied across vent fields from one to three copies. The P. gargantua symbiont from the hydrogen-enriched Hydra vent field carried two divergent copies, including one with a truncated catalytic region, potentially reflecting local adaptation to vent geochemistry. Anatomical and isotopic comparisons further revealed contrasting host dependence, with P. gargantua having a proportionally larger oesophageal gland and ventricle coupled with a reduced radula, as well as a greater nitrogen-isotope enrichment between symbiotic and non-symbiotic tissues. The smaller oesophageal gland and larger feeding apparatus of P. smaragdina point to it being mixotrophic. Previously, all known peltospirid symbioses have partnered with Gammaproteobacteria, and we expand the known diversity of peltospirid symbioses. Our findings underscore how closely related hosts can exhibit different degrees of dependence on symbiosis, and that mixotrophic symbiosis may be prevalent in small vent species previously thought to be non-symbiotic.

Anoxygenic phototrophic green sulfur bacteria (GSB; Chlorobiia) are important primary producers in anoxic and sulfidic environments, whose significance in aquatic ecosystems will expand with coastal deoxygenation. Here, we employed a uniquely comprehensive analytical approach to investigate the formation, maintenance, and collapse of a GSB bloom. We combined multi-omics -- V4V5 and synthetic long read 16S rRNA amplicon sequencing, metagenomics, and metaproteomics -- with total and GSB-specific cell counts, biogeochemical measurements, and isotopic analysis. The GSB bloom exceeded 109 cells ml-1, among the highest environmental cell densities reported to date, with up to 96 % of the bloom consisting of a single strain-level Prosthecochloris lineage (GSB-TRL01). Extreme sulfide concentrations (> 17 mM) coincided with peaking cell density and a shift in isotopic composition. Bloom persistence is supported by tightly coupled sulfur cycling, high rates of nitrogen fixation, mechanisms to tolerate oxidative stress and maintain redox balance, and nutrient acquisition through outer membrane transport systems. A shift in sulfur oxidizing enzymes towards enzymes with higher sulfide affinity proceeded bloom demise. Prosthecochloris GSB-TRL01 differs from closely related lineages in several outer membrane transport systems, including porins to transport phosphate and tonB-dependent transporters. Collectively, these findings identify physiological and metabolic strategies that may enable near-clonal Prosthecochloris populations to attain extraordinary biomass while coupling the sulfur, carbon, and nitrogen cycles in coastal euxinic environments.

Long-term microbial population dynamics can reveal how ocean microbiomes respond to environmental variation, but resolving within-species change requires suitable genomic references. We integrated 30 PacBio HiFi long-read metagenome-assembled genomes (MAGs) from the northwestern Mediterranean Sea with monthly Illumina metagenomes from neighboring time series spanning 12 and 7 years. Population differentiation varied widely among genomes and often differed in magnitude between sites, while a subset showed increasing differentiation with temporal distance. Genetic diversity and population structure were frequently seasonal, with populations associated with warm or cold waters and others spanning both thermal regimes. Candidate adaptive genes accounted for 1.6-3.0% of coding sequences, and many were detected in both time series. Their pN/pS trajectories included regular seasonal fluctuations consistent with changing strain or ecotype contributions, alongside irregular temporal patterns. These results show that combining long-read MAG references with dense short-read time series can resolve seasonal population structure, site-specific differentiation, and candidate adaptive variation within marine microbial species, providing a framework for tracking contemporary population-genomic change in the ocean.

Variable phytoplankton resource demands can buffer productivity to climate-driven declines in nutrient supply, but the extent of this buffering depends on limits to cellular elemental plasticity. Elemental plasticity is driven by a combination of genomic adaptation and physiological regulation, but their mechanistic links remain unresolved. Here, we combine continuous culture experiments with global proteomics and elemental analyses to determine how genomic diversity shapes nutrient stress plasticity in the globally distributed cyanobacterium Synechococcus. By maintaining fixed growth rates in chemostats under nitrogen- or phosphorus-limiting conditions, we disentangle lineage-specific resource demands from growth effects. All strains show conserved induction of nutrient acquisition pathways, yet strain identity explains most pan-proteome variance, and variation in non-core protein expression predicts deviations in elemental plasticity. Strains from oligotrophic compared to mesotrophic regions exhibit broader proteomic adjustments and wider C:P and N:P ranges. Our findings show that genomic diversity, expressed through pan-proteome regulation, amplifies physiological plasticity and increases phytoplankton nutrient stress buffering capacity under future climate conditions.

Current environmental biosurveillance infrastructure excels at tracking known biological threats but is limited in its ability to proactively identify potential pre-emergent pathogens (PEPs). To address this gap, we developed a microfluidics-based Capture-Enrichment pipeline that isolates individual environmental cells via microdroplet encapsulation, preserving community biodiversity while enabling iterative selection against human sera. When applied to urban wastewater, this approach successfully recovered pathogens, commensals, and PEPs that fell below the detection limits of conventional amplicon sequencing. Genomic analysis revealed that wastewater-derived ESKAPE pathogens cluster closely with clinical isolates, showing that the pipeline captures clinically relevant threats. Furthermore, isolated PEPs exhibited known and emerging phenotypes associated with early-stage pathogenesis and antimicrobial resistance. These findings demonstrate that targeting PEPs can fundamentally expand the scope and quality of biosurveillance by providing a scalable strategy to identify, characterize, and close important gaps in our understanding of emerging microbial pathogens.

Microbial diversity is organised into operationally defined species. However, it remains unclear whether these, or finer-scale groups represent biologically meaningful units in natural communities. Here, we implement a scalable, cultivation-independent framework to identify 724 genomically discrete and ecologically cohesive subspecies across 270 species in the global ocean microbiome. Their diversification is underpinned by gene-specific, nucleotide-level selection on the core genomic backbone and by variable restructuring of gene content, both of which proceed in a subspecies-specific manner. In over half of the species tested, subspecies were significantly associated with distinct environmental conditions, most frequently temperature. This coupling of genomic discreteness and ecological cohesiveness positions these subspecies as biologically meaningful units, a fundamental level at which ecological and evolutionary processes jointly drive divergence and structure microbial diversity in the oceans.

The genomic and epigenomic complexity of microbiomes with low species richness remains poorly characterized. Here, we integrated 111 non-redundant isolate genomes with deep long-read metagenomics to investigate an undefined mesophilic dairy starter. Closely related isolates harboured distinct combinations of features associated with fermentation traits and phage defence, including surface polysaccharide genotypes, plasmids, prophages, defence systems, and methylation profiles. Metagenomics revealed additional elements and inferred host associations. However, the limited overlap between approaches across core-genome lineages and accessory repertoires underscored complementary but incomplete recovery, to the extent that the dominant metagenomic cell wall polysaccharide genotype (29.6% abundance) was absent from isolates. Metagenomic methylation signatures supported 36 species-level plasmid-host assignments, corroborated by isolate genomes in nine of ten testable cases. Our findings revealed extensive combinatorial diversity hidden from either approach alone, providing a plausible molecular basis for phage resilience, while likely representing only a lower bound on population complexity.

Colistin (CST) is a critical last-line antibiotic for treating multidrug-resistant Pseudomonas aeruginosa infections, especially in chronic respiratory disease. Despite its clinical importance, the P. aeruginosa CST resistome remains incompletely characterised, with most studies focusing on the PAO1 prototypic strain. Here, we used experimental evolution to apply stepwise CST selective pressure to nine P. aeruginosa lineages representing different clinical presentations and diverse genetic backgrounds. Population-level whole-genome sequencing of broth cultures exposed to 2-fold increasing CST (0.0625 to 512 g/mL) was undertaken at multiple concentrations. We identified several known and novel mutational drivers of low- and high-level CST resistance, alongside associated compensatory mutations at allele frequencies (AFs) as low as 5%. Despite diverse clinical and genetic backgrounds, all lineages convergently evolved mutations within the two-component system pmrAB, with most accompanied by phoPQ variants. When canonical TCS remodelling plateaued, populations acquired novel secondary driver mutations, most notably in the lipid A-modifying gene, lpxO2. In addition to AMR driver mutations, strains evolved complex adaptive strategies to mitigate severe CST-induced oxidative and metabolic stress. Adaptations included the emergence of hypermutators and alterations to targeted DNA and protein repair networks. Furthermore, we observed convergent regulatory rewiring of the cyclic-di-GMP network and central metabolism pathways, likely acting as compensatory mechanisms to promote biofilm formation and offset the fitness costs of CST resistance. Using a mixture-aware population genomics approach, we captured intense clonal interference and transient tolerance mutations that failed to reach fixation, illustrating dynamic evolutionary complexities that would be missed with traditional end-point analysis methods.

A diverse array of photosynthetic phytoplankton drives primary production in equatorial surface waters. Among these, the cyanobacterium Prochlorococcus is an important contributor to net primary production in these typically iron-limited, high-nutrient and low-chlorophyll (HNLC) regions. Here, we explore the diversity of these organisms, in part, through targeted enrichment of Prochlorococcus cells using field-based high-speed cell sorting techniques. We demonstrate that the genomes of Prochlorococcus belonging to the low-light adapted LLI clade, and isolated from the surface of the Equatorial Pacific Ocean, are depleted in functions related to the assimilation of urea, nitrite, and amino acids. These are the first examples of LLI Prochlorococcus that have lost the ability to use nitrite, a trait considered to be a core feature of this clade. All new equatorial cultures of LLI Prochlorococcus appear to use a distinct isoform of protoporphyrinogen IX oxidase (HemG), for the biosynthesis of a chlorophyll precursor, that does not require the use of iron-containing heme. In contrast, the heme-dependent HemJ isoform is typically used by Prochlorococcus found outside equatorial HNLC waters. Together, these findings suggest that low-light adapted Prochlorococcus in the equatorial ocean possess accessory gene content that reflects adaptation to the generally iron-limited but nitrogen-replete conditions of surface waters.