Meromictic lakes serve as natural laboratories for investigating microbially mediated biogeochemical cycling because their stable redox stratification supports diverse metabolic processes. Lake Suigetsu is a typical meromictic lake in Japan characterized by a permanent oxycline separating the oxic brackish mixolimnion from the anoxic saline sulfidogenic monimolimnion. Recently, Lake Suigetsu's vertical bacterial composition, especially of autotrophs, sulfide oxidizers, and sulfate reducers, has gained significant attention. However, little is known about microbial communities particularly the abundant heterotrophs that contribute to the biogeochemical cycling by degrading organic matter. To characterize biogeochemical cycling in Lake Suigetsu, we investigated the microbial community structures under the amplicon sequencing variants that distinguish species and/or intraspecies populations in each layer of Lake Suigetsu by using 16S rRNA gene amplicon sequencing analysis. We revealed the layer-specific microbial communities and there were abundant heterotrophs in each layer, in addition to the previously reported autotrophs. In the upper of monimolimnion sample, approximately 20% relative abundance was occupied by an uncultured Anaerolineaceae bacterium, that is probably an anaerobic heterotroph. In the mixolimnion, the most abundant one was SAR11 bacterium with approximately 6% relative abundance. In the oxycline, the most abundant, probably heterotrophic prokaryote was an uncultured Ectothiorhodospiraceae bacterium with approximately 5% relative abundance, while the four most abundant prokaryotes were comprised of autotrophic bacteria and occupied more than 43% relative abundance, apparently suggesting the primary production was predominant in the oxycline. This study provides insight into the layer-dependent, prokaryote-mediated biogeochemical cycling in Lake Suigetsu.

Giant endogenous viral elements (GEVEs) are ubiquitous in eukaryotic genomes, but how the virus adapts to the host and is capable of reactivation despite host defense mechanisms remains unclear. Here we examined this process in the green alga Chlamydomonas reinhardtii and its latent giant virus, punuivirus, which is integrated into the host genome. We reveal a chimeric genomic architecture in which punuivirus latency genes are enriched in spliceosomal introns and 6-methyladenine marks, mirroring host transcriptional regulation, while virulence genes are predominantly controlled by a conserved viral promoter. Using single-cell RNA-seq, we show that nearly 70% of cells express viral transcripts, but only a small subpopulation commits to viral production, expressing virulence genes with host transcriptional shutoff. Moreover, we find that the intact punuivirus GEVE remains refractory to host 5-methylcytosine (5mC) silencing, whereas a naturally occurring deletion variant is associated with widespread 5mC accumulation across the GEVE and elsewhere in the host genome. The deleted region encodes a highly expressed ten-eleven translocation dioxygenase (TET), and epigenetic profiling of intact and deletion strains implicates this GEVE gene in removing 5mC marks, thereby maintaining the virus in a transcriptionally permissive latent state. Our study provides novel insights into the molecular underpinnings of host-virus co-existence in a giant virus-alga interaction. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, 1R35GM147290 European Research Council, 950230

Abstract. Parasitic fungi of the phylum Chytridiomycota are known parasites of phytoplankton, yet little is known about drivers of their diversity and host

Abstract. Bacteriophages are not only the most ubiquitous biological entity on earth, but they also display remarkable genetic diversity across and within

In diverse natural communities, cells and phages interact with many potential partners, but the impacts of pairwise coevolution on the overall structure of phage-bacteria interaction networks (PBINs) is not fully understood. Here, we conducted multiple pairwise coevolution experiments and examined how coevolution with one partner could simultaneously influence interactions with other partners. We then analyzed the underlying genetic changes that alter interactions in a large cross-experiment PBIN. The results reveal that interactions at the cell surface as well as intracellular interactions can alter patterns across the PBIN. When phages utilized the same set of host cell-surface receptor genes, pairwise coevolution with one partner did alter interactions with other partners. In contrast, if different sets of host cell-surface receptor genes were used, modular patterns in the cross-experiment PBIN were generally observed. On the phage side, resistance-breaking mutations arose in many putative receptor-binding proteins, and these changes could alter interactions with other hosts. However, in both the hosts and phages, over half of the mutated genes were not associated with cell-surface interactions. Host mutations likely altering intracellular phage-host interactions, including translation, protein stability, and DNA replication often led to broad resistance across the PBIN. In phages, mutations occurring in genes encoding endolysins, DNA methylases, oxygenases, transcriptional regulators, and tRNAs were observed potentially also influencing intracellular interactions. Based on these results, we propose a model to illustrate how these two different types of genetic signatures (i.e. extracellular cell-surface interactions verses intracellular interactions) can impact the structure of PBINs. ### Competing Interest Statement The authors have declared no competing interest. U.S. National Science Foundation, OCE-1029684, OCE-1332782, OIA-1736030, OCE-2022895, OCE-1031783, OCE-1332740

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. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, ANR19-CE01-0012, ANR EDIR-22-0005, ANR 17-EURE-0015 CNRS - MITI interdisciplinary programs CNRS - LEFE actions Université de Pau et des Pays de l'Adour, https://ror.org/01frn9647, MeSMic

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. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, https://ror.org/00yjd3n13 Simons Foundation, https://ror.org/01cmst727

Microorganisms exert profound influences on both natural ecosystems and human society. As the cornerstone of microbial research, metagenomics relies on the precise analysis of large-scale, multi-source genomic data. However, discriminating among diverse sequence types remains a formidable challenge. To address this, we present CamFi (Codon-aware Multi-scale Feature Fusion), a unified framework integrating overlapping nucleotide-triplet representations with multi-scale dilated convolutions for classifying prokaryotic chromosomes, eukaryotic chromosomes, plasmids, and viruses. Benchmark evaluations demonstrate strong mean F1 scores of 97.70% for eukaryotic chromosomes and 94.13% for plasmids. On the CAMI II marine dataset, CamFi achieved a weighted F1 of 82.65%, ranking second among six methods based on reported aggregate metrics. In a balanced four-class benchmark, CamFi attained a macro-F1 of 93.77%, compared with DeepMicroClass (90.03%) and the XGBoost stage of 4CAC (64.66%). These results establish CamFi as a competitive approach for unified metagenomic contig classification under the evaluated conditions.

Flavobacterium spp. are rod-shaped, yellow- to orange-pigmented, Gram-negative bacteria that are ubiquitous in terrestrial and aquatic environments. Across these habitats, they are increasingly recognized as versatile members of environmental and host-associated microbiomes. This ecological breadth reflects the ability of Flavobacterium spp. to adapt to diverse environmental niches that support both beneficial and pathogenic lifestyles. Members of the genus are particularly widespread in aquatic ecosystems, including freshwater environments, sediments, and cold polar environments, while others occur in soils and marine-associated habitats.

Here, the authors show that protein design is a generalizable strategy to generate new antimicrobials by neutralizing the function of bacterial adhesins to combat two causative agents of urinary tract infections, E. coli and A. baumannii.

Bacteriophages exhibit staggering genomic diversity, yet phenotypic characterization remains bottlenecked by assays that generally yield simple binary infection outcomes. Quantitative traits can be extracted from standard liquid cultures, but many phages fail to produce a measurable effect in these settings, necessitating an improved quantitative approach on semi-solid media. While end-point plaque size is commonly used for quantitative phenotyping, it only provides a static snapshot that fails to disentangle phage performance across changing bacterial growth phases. Here, we report a cost-effective, scalable method to quantify the bacterial killing rates of diverse phages by continuously tracking plaque development using a consumer-grade flatbed scanner and custom open-source computer vision software. By fitting a minimal, five-parameter phenomenological model to the diverse BASEL collection of Escherichia coli phages, we accurately captured plaque expansion dynamics across all bacterial growth phases. We discovered that plaque expansion parameters vary significantly across individual phages and taxonomic families, with the initial expansion rate demonstrating an inverse correlation with viral genome size. Notably, these plaque expansion dynamics did not correlate with bacterial collapse times in liquid culture, indicating that they represent distinct physiological and kinetic processes. Finally, we demonstrated that phage taxonomic family can be predicted with 72.1% accuracy solely from dynamic plaque parameters using a random forest classifier, whereas conventional endpoint plaque sizes held no predictive power. This time-lapse phenotyping approach yields robust kinetic data superior to end-point measurements, establishing a scalable platform to accelerate phage discovery and genotype-phenotype mapping. ### Competing Interest Statement The authors have declared no competing interest.

Bacterial small-molecule messengers that function in diverse immune signaling pathways have been thought to derive strictly from biosynthesis using nucleotide and cofactor substrates. Here we report a nuclease-driven immune signaling pathway in which tRNA cleavage generates a specific small molecule that triggers cell death in response to RNA-guided transcript recognition in prokaryotes. We identify a class of type VI-B CRISPR systems in which the RNA-guided nuclease Cas13 and its accessory transmembrane protein, 2TMβ, are co-encoded in an operon and are jointly required for bacteriophage defense. Upon target RNA recognition, Cas13 activates 2TMβ by producing mono- and dinucleotide messengers bearing 2′,3′-cyclic uridine phosphate ends by tRNA cleavage. Activated 2TMβ destroys infected cells by forming a multimeric complex that disrupts cell membranes to mediate cell death. These findings demonstrate RNA-programmed tRNA cleavage for second-messenger biogenesis, revealing a new logic for prokaryotic immune signaling. ### Competing Interest Statement J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Evercrisp and Azalea; a scientific advisory board member of Caribou Biosciences, Scribe Therapeutics, Mammoth and Inari; and a Director at Johnson & Johnson, Tempus and Altos Labs. The regents of the University of California have patents issued for CRISPR technologies on which G.J.K. is listed as an inventor. M.R.O. is an inventor on patent applications related to CRISPR-Cas systems and uses thereof. M.R.O. is a member of the scientific advisory boards for Dahlia Biosciences and an equity holder in Dahlia Biosciences. F.S. heads research and development at BIOLOG, which sells cyclic nucleotide analogues. The other authors declare no competing interests. Howard Hughes Medical Institute, https://ror.org/006w34k90

Microbial sequencing data are subject to sample-specific and species-specific distortions that complicate inference about true microbial abundances. Recent work established assumptions under which relative fold-differences in microbial abundances can be estimated from sequencing data, accompanied by an estimation and testing method called radEmu. While radEmu has excellent error rates, its computational cost can be prohibitive for large-scale analyses. Here we introduce fastEmu, a scalable approximation to radEmu's testing methodology. fastEmu uses a small, data-driven subset of taxa to approximate the computationally expensive test statistic. In a metagenomic analysis, we show a reduction in per-taxon runtime from 4-8 hours to 0.5-45 seconds, and simulations illustrate no adverse impact on estimation error or Type I error rate control. fastEmu enables testing differential abundance for thousands of microbial taxa while retaining clear parameter interpretation and error rate control. ### Competing Interest Statement The authors have declared no competing interest. National Institute of General Medical Sciences, https://ror.org/04q48ey07, R35 GM133420

Bacterial species, even the strains within a bacterial species, can display very different gene inventories (1, 2). Bacterial genomes are tightly packed with genes, and there is an almost uniform association between genome size and gene number across species—from the 0.1-Mb genome of Nasuia deltocephalinicola to the 16-Mb genome of Minicystis rosea—with an average size of about one gene per kilobase (3).

Harada et al. describe an archaeon, recovered from a single marine plankton cell, whose 238-kb genome is the smallest known for the domain. It has lost nearly all metabolism but keeps the machinery to copy and express its own DNA, marking a distinct limit of cellular simplification.

Wang et al. map >1,000 interactions between Salmonella isolates and bacteriophages to measure the relative contributions of surface features and intracellular defense to phage resistance across a major human pathogen. They further identify AppA, which blocks DNA packaging by targeting the large terminase and restricts phage replication within mammalian hosts.

Most microbial species are represented by a single genome in public databases. The lack of genomes for these ‘singleton’ organisms limits our understanding of their pan-genome diversity and evolution. Although the Sequence Read Archive (SRA) contains millions of sequencing datasets that could be used to expand our understanding of many species, the extent to which under-represented microbial species are present at levels sufficient for genome recovery is unclear. Here, we show that the pre-computed taxonomic profiles generated by the National Center for Biotechnology Information SRA Taxonomy Analysis Tool (STAT) can be used to identify SRA datasets containing recoverable genomes for under-represented microbes. Across >28 million SRA datasets, tens of thousands of singleton archaeal and bacterial species were detected, often at abundances consistent with successful genome recovery. Applying targeted genome recovery to 804 singleton species, we recovered genomes representing new strains for 472 species. The success rate of genome recovery correlated with STAT-derived estimates of genome coverage, demonstrating that genome recovery from the SRA is both predictable and scalable. Using the single available genome of Clostridium tarantellae as a case study, SRA data mining recovered seven additional C. tarantellae genomes, correcting assembly gaps in the reference genome, expanding its pan-genome and increasing its known host range by seven additional fish species. These findings reveal that many microbial species currently represented by a single genome are in fact widely distributed across existing sequencing data and highlight a major opportunity to systematically expand strain-level genomic diversity and pan-genomic representation for under-sampled microbial species without additional sequencing.

The maximum growth rate of microbes plays a central role in shaping ecological outcomes and is a major target of bioengineering efforts. Previous data compilations suggest single cell maximum growth rates mostly decrease with cell volume across species, except for an initial increase from the very smallest microbes to an intermediate size. Here we hypothesized that this unimodal relationship is shaped by the volumetric requirements of ribosomes and the surface area-dependence of nutrient supply. We developed a mechanistic model relating ribosomal protein mass fraction to growth rate and cell size. Predictions from this model were empirically supported by proteomic data from 97 prokaryotes ranging over 3 orders of magnitude in cell volume. Our analysis suggests that the smallest cells' growth rates are restricted by the volumetric constraints on ribosomes per cell and rely on compact, heterotrophic metabolisms. In contrast, growth rates of the largest prokaryotes face geometric constraints on specific resource acquisition rates, capping growth rates and investment in ribosomal protein, but allowing investment in protein-expensive metabolisms including phototrophy and sulfur oxidation. Prokaryotic cells of intermediate size face neither constraint and exhibit a higher maximum growth rate potential, which, we hypothesize, is associated with elevated metabolic diversity. ### Competing Interest Statement The authors have declared no competing interest. Damon Runyon Cancer Research Foundation, DRG-2470-22 Simons Foundation, 549931

SUMMARYThe concept of the pangenome has emerged as the approach of choice for targeted comparative genomics across groups of organisms, from strain or population levels within a species to broader taxonomic or phenotypic categories. Pangenome analyses employ diverse procedures that inevitably introduce reproducibility challenges. Nevertheless, they have already yielded important insights into the structure, function, and evolution of archaeal, bacterial, and eukaryotic genomes across space, time, biogeography, and phylogeny. There is a wide range of applications from vaccine design to synthetic biology that are expected to yield additional technological and biomedical tools for genome biology and biotechnology. We review here the basic concepts underpinning pangenome research, including the estimation of gene and protein family numbers, the modeling of open and closed pangenomes, and key definitions relevant to reporting guidelines. We list and compare computational platforms, algorithms, and data structures, along with their salient features. We present key findings from 249 taxa covering 17,858 genome sequences, subjected to such analyses, and discuss major patterns observed, such as novel families per genome and pangenome size estimates for this validated data set. We propose revised criteria for pangenomes, standard operating procedures, and further targets to enhance the reproducibility of analysis and expand opportunities for experimental and computational comparative genomics. This framework attempts to clarify current methodologies and chart a path forward for integrating pangenome analyses.

Abortive infection (Abi) systems can stop phage replication by forcing infected cells to arrest growth or die, but this strategy is risky if it is triggered in the absence of phage. Here, we show that two Hachiman-family Abi systems—AbpAB and HamAB—require Escherichia coli DNA polymerase I (PolA) to block phage growth. PolA is also required for AbpAB-dependent growth arrest triggered by DNA damage (mitomycin C) or by expression of the T4 single-stranded DNA-binding protein gp32. In contrast, deleting rnhA (encoding RNase HI) makes AbpAB expression toxic even in the absence of phage. These results show that host replication and repair enzymes can both support anti-phage defense and prevent Abi-associated growth inhibition during uninfected growth.

Closely related bacteriophages often display strikingly different infection phenotypes, yet the genetic basis of this variation remains poorly understood. Here, we uncover a previously unrecognized feature within the genus Teseptimavirus: the HNH endonuclease can occupy two distinct genomic positions, either associated with virion assembly or DNA packaging modules, suggesting functional specialization. We further demonstrate that this enzyme is closely linked to infection phenotypes, as even a single nucleotide substitution in the HNH gene significantly alters the efficiency of plating. These findings identify the HNH endonuclease as a key determinant of phage infectivity and reveal that its genomic context may shape functional outcomes. This work provides new insight into how subtle genetic differences drive phenotypic divergence among closely related phages and highlights the HNH endonuclease as a promising target for rational phage optimization in therapeutic and biocontrol applications.

Resistance emergence is a major challenge to antibiotic development, often undermining promising drug candidates and negating years of research and pharmaceutical investment. In this Review, Pál and colleagues propose a resistance-based classification system for antibiotic development that integrates early resistance predictions into the prospective development of new antibiotics.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is foundational to life on Earth, catalyzing carbon dioxide (CO2) fixation to generate biomass. However, Rubisco is a slow and inefficient enz...

Ultrasmall episymbiotic bacteria grow on the surface of their obligate bacterial hosts. These episymbionts have a restricted host range and likely rely on host-derived receptor molecules to identify and associate with cognate hosts. Nanosynbacteria from the oral microbiome, like Nanosynbacter lyticus, utilize a pair of distinct type 4 pili (T4P) for host surface interactions, T4P-1 and T4P-2. In this study we identify the adhesin components of both T4P, demonstrate that these adhesins localize to the cell wall of the cognate host Actinobacterium Schaalia odontolytica, and demonstrate that each adhesin binds distinct components of the host cell wall. The adhesin from the motility driving T4P-1, termed PilX1, showed a wide range of affinities in microarray screening and bound peptidoglycan directly, but could not bind purified cell wall polysaccharides (CWPS) from host surfaces. Conversely, the adhesin from T4P-2, termed PilX2, bound host-derived CWPS and preferentially bound episymbiont-naïve hosts over episymbiont-exposed hosts. Within the bacterial host we identified two CWPS biosynthesis pathways that altered adhesin binding and episymbiont susceptibility. Disruption of a rhamnose-CWPS pathway left hosts susceptible to episymbiont binding and disruption of a fucose remodeling pathway made hosts more resistant to episymbiont binding. Analyzing polysaccharide compositions revealed multiple CWPS, potentially including a rhamnose- and mannose-rich rhamnomannan. Consistent with this finding, exogenously added rhamnose and mannose were able to block binding by N. lyticus, supporting a role for these sugars in episymbiont defense. Our study reveals that Nanosynbacteria rely on the complex sugars that coat their host bacteria for recognition and attachment. ### Competing Interest Statement The authors have declared no competing interest. National Institute of Dental and Craniofacial Research, 1R01DE031274, 1R01DE023810, T90DE026110-07 The Darwin Trust of Edinburgh, Studentship Wellcome Trust, 226644/Z/22/Z

Spatial organization during bacterial fission is critical to ensure that division results in identical, viable progeny cells. In Escherichia coli, one important system that accomplishes this is the Min system, comprising MinC, MinD, and MinE proteins. The Min system regulates assembly of the FtsZ-ring to ensure proper placement of the division machinery at midcell. We explored the dynamics of the Min system and demonstrate that Min oscillation and release of MinD from the membrane is modulated by MinC. Deletion of the minC gene from a strain expressing minD fused to the gene encoding green fluorescent protein (Gfp) resulted in faster pole-to-pole oscillation of Gfp-MinD in vivo, leading to a shorter period. This was also observed in a strain expressing the MinC(R133A), a variant mutated at the MinC-MinD interface. In phospholipid binding and release assays in vitro, we observed that the concentration of MinE required to release MinD was lower with MinC, suggesting that MinC activates MinD for MinE-dependent release. Oscillation is produced by a diffusion-capture-release mechanism occurring at the membrane with proteins proceeding toward a pole. To determine if an increase in the diffusive proportion of MinD leads to faster oscillation, we constructed MinD membrane targeting sequence (MTS) variants to reduce the membrane associated pool of MinD. MTS variants induced a range of oscillation defects, with MinD(KR265AA) oscillating faster in vivo. Together our results show that MinC is an effector that modifies Min oscillation by priming MinD for MinE-dependent membrane release and fine-tunes the Min oscillation rate in dividing cells. ### Competing Interest Statement The authors have declared no competing interest. National Institute of General Medical Sciences, https://ror.org/04q48ey07, R01GM158901

Ribosome hibernation helps cells survive stress by reversibly silencing translation and preserving ribosomal complexes. Although well characterized in bacteria and eukaryotes, archaeal hibernation remains poorly understood. Using cryoEM of archaeal lysates, we identified AHA (AMPKγ-HPF from Archaea), a broadly conserved ribosome-associated factor composed of two modules. AHA bound across ribosomal subunits, occluding the mRNA channel and tRNA binding sites, supporting its role in ribosome hibernation. ΔAHA cells displayed reduced viability, loss of ribosomal proteins in the stationary phase, and impaired growth reentry in rich media. Phylogenetic analyses revealed that AHA’s C-terminal domain is homologous to the bacterial Hibernation Promoting Factor (HPF), consistent with inheritance from the last universal common ancestor and thereby identifying HPF as a universal hibernation module in prokaryotes. Strikingly, we observed two AMP molecules bound to AHA’s N-terminal CBS-tetrad, which showed both sequence and structural similarity to the eukaryotic energy sensor AMPKγ, supporting a shared evolutionary origin of the archaeal CBS-tetrad and the AMPKγ family. Together, these findings uncover a widespread archaeal ribosome hibernation factor and reveal an evolutionary connection between prokaryotic ribosomal hibernation and eukaryotic energy sensing.

Microcystis aeruginosa partitions metabolism into daytime anabolism and nighttime catabolism via two negatively correlated modules. This diel strategy optimizes resource allocation, enhances competit....

The mgtC gene encodes an inner membrane protein that has been horizontally acquired by multiple bacterial species, including several mammalian pathogens. In Salmonella enterica, MgtC promotes replication in mammalian macrophages and allows this bacterium to survive cytoplasmic magnesium (Mg2+) starvation. These phenotypes are thought to result from MgtC’s inhibition of Pi metabolism and adenosine triphosphate (ATP) production, which prevents the accumulation of toxic levels of Mg2+-chelating ATP and disrupts other physiological processes that depend strictly on Mg2+, such as ribosome assembly and translation. In the current study, we show that processes that are controlled by MgtC vary with the genetic and biochemical contexts in which this protein is expressed. While establishing a broader role for MgtC as a regulator, our findings illustrate how horizontally acquired regulatory genes can potentiate regulatory interactions, facilitating the evolution of new traits.

Bacteria deploy diverse immune systems to counter phage infection, yet how these systems sense invasion remains incompletely understood. This Review categorizes phage-derived triggers and outlines emerging principles that govern immune recognition of phage infection.

Multiomic experiments using defined and native gut communities reveal that complex dietary glycans mediate the dominance of Prevotellaceae in non-industrialized gut metagenomes through signalling-mediated cross-feeding with Enterobacteriaceae.

Viral-induced DNA damage triggers both eukaryotic and prokaryotic innate immunity. Here, we report that self-DNA ends generated routinely during normal termination of bacterial replication suffice to trigger the P2-encoded OLD antiphage system. Normally, these physiological triggers are cleared by the dual helicase-nuclease activities of the conserved RecBCD enzyme, which acts as an immune safeguard analogous to mammalian TREX1 exonuclease and plant DBR1 debranching enzyme. However, when this clearance fails, the transient self-DNA ends persist and are recognized by ATP-bound OLD dimers via a positively charged channel across their ATPase subunits. ATP hydrolysis then activates OLD to cleave tRNAs, causing cytotoxicity. Single-cell microscopy reveals that dozens of OLD molecules associate with kilobases of adjacent DNA, amplifying the sparse physiological DNA end signal. Our findings reveal how an endogenous byproduct poses a constant autoimmune threat in bacteria, highlighting a universal molecular conflict across kingdoms and the convergent solutions evolved to resolve it. ### Competing Interest Statement A.A.G., I.U., M.S., C.T. and H.C.L. are employees of New England Biolabs, a manufacturer and vendor of molecular biology reagents. This affiliation does not affect the impartiality of the authors, adherence to journal standards and policies, or availability of data. The remaining authors declare no competing interests. National Institute of General Medical Sciences, R35GM143203, R35GM118120 U.S. National Science Foundation, https://ror.org/021nxhr62, 2243257 Burroughs Wellcome Fund, 1186087

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. ### Competing Interest Statement The authors have declared no competing interest. National Science Foundation, OCE-2049515 National Aeronautics and Space Administration (NASA), 80NSSC24K1832

Spontaneous vegetable fermentation is driven by the indigenous microbiota associated with the raw ingredients. The process often involves an initial heterofermentative phase followed by a predominantly homofermentative phase, both ensured by lactic acid bacteria (LAB). While microbial dynamics during fermentation are well characterized, phage communities remain poorly understood, despite evidence linking phage activity to LAB succession. We monitored viral and bacterial succession during sauerkraut and carrot fermentations using a metagenomics approach to characterize community dynamics. To further assess the impact of phage PLE001 on bacterial community dynamics, we developed a simplified carrot fermentation model comprising representative strains of Rahnella aquatilis, Leuconostoc mesenteroides and Lactiplantibacillus plantarum. Two consortia were assembled: one containing the PLE001-susceptible strain L. mesenteroides CIM3004, and the other containing also its phage-resistant derivative, CIM3004 BIM. We observed major shifts in the viral community during the first week of fermentation, coinciding with changes in bacterial structure during hetero- and homofermentative phases transition. In the simplified model, phage PLE001 reduced the population of the host during the heterofermentative phase but did not affect overall fermentation dynamics nor metabolite production. These results suggest that bacteriophages contribute to bacterial succession during fermentation, while communities remain resilient to phage infection, preserving fermentation stability. ### Competing Interest Statement The authors have declared no competing interest. French National Research Agency (ANR), ANR-21-CE21-0003

Microbial communities underpin ocean productivity and biogeochemistry, but their functional adaptation to environmental gradients remains poorly understood. Codon usage bias (CUB), a marker of translational efficiency, constrains growth rate potential, yet its role in functional adaptation across natural communities is underexplored. By analysing 589 metagenome-derived microbial communities from the global ocean, we demonstrate that CUB in 320 metabolism-related gene groups capture genomic adaptation to distinct temperature and nutrient concentrations. We identified four adaptation signatures shaped by distinct temperature-nutrient regimes, revealing how microbes optimize protein synthesis in their ecological niche. This genome-centric approach deepens our understanding of functional adaptation in uncultured marine microbes and provides a framework to inform ocean biogeochemical models and predict microbial responses to climate-driven changes. ### Competing Interest Statement The authors have declared no competing interest. Priority Research Program Ocean & Climate (CNRS, IFREMER), ANR n°21-POCE-0001 BlueRemediomics, 101082304 TaxCy, ANR-23-CE02-0007 SEASONING, ANR-24-CE02-7681

Cell surface lipoproteins (SLPs) play crucial roles in the pathogenesis and cell biology of gram-negative bacteria, yet the route by which they reach the cell surface has remained unknown except in special cases. Here we show that in Bacteroidota bacteria, a phylum that includes prominent human commensals and anaerobic pathogens, SLP export is carried out by the outer membrane lipopolysaccharide (LPS) translocon. Isolation and structural characterisation of LPS translocon complexes from two Bacteroidota species reveals that they are massively enlarged and elaborated relative to the Escherichia coli paradigm with up to eight additional subunits located within, and on either side of, the outer membrane. We establish a role for the universally conserved core LptD subunit and four of the additional Bacteroidota-specific subunits in SLP export. Expansion of the LPS translocon in the Bacteroidota may allow export of the unusually complex SLPs found in this phylum. Our discoveries raise the possibility that SLP export is a conserved second function of the LPS transport pathway across gram-negative bacteria. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 101201241, 833713 Wellcome Trust, 317933/Z/24/Z St. Jude Children's Research Hospital, https://ror.org/02r3e0967

Marine microbial communities vary seasonally and spatially, shaped by physiochemical gradients and biotic interactions. Spatiotemporal variability of seawater microbiomes in tropical coastal regions is comparatively understudied, especially in Southeast Asia with high pressures from development and anthropogenic activities. Here, we examined the microspatial and temporal variability of bacterioplankton in the Singapore Strait, which is a tropical system in Southeast Asia influenced by monsoons. By applying total community amplicon sequencing to three particle size fractions reflecting microspatial habitats of seawater (0.22-1 µm ″free-living″, 1-10 µm ″large cells and small aggregates″ and 10-150 µm ″particle-associated″), we show that the seawater microbial community was structured by their microspatial habitats, monsoon seasons and physiochemical variables. Monsoon-driven alternation of water masses originating from the South China Sea to the East and from off the Sumatran coast to the West resulted in a distinct community during the Southwest monsoon across all three size fractions. Total DNA (biomass) was lowest in the 10-150 µm fraction, but these particle-associated sequences had the highest diversity and were surprisingly enriched with picocyanobacterial and some pathogenic taxa, potentially reflecting aggregate formation and/or host-derived particle association. This fraction also exhibited greater temporal variability than the two smaller fractions, which could be related to a more heterogenous environment in particle microhabitats. Overall, this study demonstrated compositional variability in the bacterioplankton across space and time in an equatorial region influenced by monsoons. ### Competing Interest Statement The authors have declared no competing interest. National Research Foundation, https://ror.org/03cpyc314, NRF-NRI-2020-MESN Nanyang Technological University, https://ror.org/02e7b5302

To develop gene therapy tools based on CRISPR-associated transposons (CASTs), it is essential to define how transposon ends are recognized and paired during transposition. Transposon 7 (Tn7)–like tran...

Abstract. Homologous recombination is ubiquitous across evolution, generating variation through genetic reshuffling. Although bacteria lack meiotic recombi

Abstract. One of the biggest misconceptions about clonal bacterial populations is that all members act identically. In fact, microbiologists have known for

Abstract. The archaeal symbiotic Nanobdellales have been isolated with their hosts from a shallow hot marine vent off Iceland and from terrestrial hot spri

Reverting waste carbon into chemicals with minimal freshwater use is essential for a sustainable future. Here, the authors engineer the marine bacterium R. nubinhibens, by decoupling bioproduction from central metabolism to convert lignin into chemicals with seawater as a water source.

The genus Paulinella represents a rare, independent case of primary endosymbiosis, providing a unique system to study the early stages of organelle evolution. Here, we expand current understanding of...

The viral contributions to deep-sea polymetallic ecosystems, rich in valuable mineral nodules, remain unclear. Here, the authors created the first large-scale catalog of viruses from the Clarion-Clipperton Fracture Zone and found extensive viral diversity that may influence nutrient cycling, microbial survival, and adaptation to metal-rich environments.

Proviruses are widespread components of prokaryotic genomes, yet their characterization is impeded by border identification errors. We developed ProBord (Provirus Border Delimiter), a tool achieving single-base-precision border mapping via cross-genome ...

Abstract. Viruses are increasingly recognized as critical regulators of microbial ecology, yet their roles in pollutant degradation remain largely unexplor

Reverting waste carbon into chemicals with minimal freshwater use is essential for a sustainable future. Here, the authors engineer the marine bacterium R. nubinhibens, by decoupling bioproduction from central metabolism to convert lignin into chemicals with seawater as a water source.

Cyclic oligonucleotide–based antiphage signaling systems (CBASS) are bacterial immune pathways evolutionarily related to cGAS-STING (cyclic GMP-AMP synthase–stimulator of interferon genes) in humans. ...

Background/Objectives: Carotenoids play essential roles in light harvesting, photoprotection, and oxidative stress defense in photosynthetic organisms. The filamentous anoxygenic phototroph Chloroflexus aurantiacus synthesizes diverse carotenoids; however, the biosynthetic basis and regulation of these pigments are unclear. Methods: Carotenoid compositions of Cfx. aurantiacus grown under various conditions were analyzed by HPLC. Furthermore, candidate carotenoid biosynthesis genes from Cfx. aurantiacus were hetero-logously expressed in Escherichia coli to analyze their functions. Results: We examined carotenoid composition in cells grown under anaerobic light, aerobic dark, and aerobic light conditions, and found that the major carotenoid species changed markedly in response to oxygen availability. Under anaerobic photosynthetic conditions, γ-carotene, β-carotene, and hydroxylated γ-carotene derivatives were predominant, whereas aerobic growth induced the accumulation of keto-carotenoids, including canthaxanthin and echinenone-related compounds. Genome mining revealed candidate carotenoid biosynthesis genes, including three crtI paralogs and genes encoding enzymes involved in cyclization, hydroxylation, glucosylation, esterification, and ketolation. Heterologous expression in E. coli revealed that CrtI1 acts as a phytoene desaturase, CrtY catalyzes cyclization of lycopene to β-carotene via γ-carotene, CruF functions as a 1,2-hydratase, CruC as a carotenoid glucosyltransferase, CruD as an acyltransferase involved in esterification, and CrtO as a β-carotene ketolase. Conclusions: These results establish a carotenoid biosynthetic pathway in Cfx. aurantiacus and show that oxygen-dependent transcriptional regulation, particularly of CrtO, contributes to the selective production of keto-carotenoids under aerobic conditions. Our findings provide new insight into carotenoid metabolism in filamentous anoxygenic phototrophs and the adaptive physiological strategies that support growth under contrasting environmental conditions.