Identification of the core microbiome from clear water lakes in the Arctic reveals non-unique members when compared with a conceptually similar lake from a more temperate region. Biogeographic assign...

We report the isolation and genomic sequencing of nine Actinomycetota obtained from both cave biofilms and trogloxenic Ceuthophilus (cave crickets). Strains were isolated from samples via actinomycetota-selective media and sequenced using nanopore sequencing. Provisional taxonomic inference of strains was performed by whole genome phylogenetic analysis, revealing a broad range of genera, with most strains clustering with known genera with digital DNA-DNA hybridization values less than 70%, and one strain not clustering with known genera. An analysis of secondary metabolism reveals the quantity and diversity of secondary metabolism in cave Actinomycetota. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, CA226833

Circadian rhythms, driven by endogenous clocks and synchronized with environmental cues, are fundamental to life on Earth. While extensively studied in diverse organisms, their influence on viral ecology remains largely unexplored. This study takes the temporal dynamics of viruses in Daya Bay as an example to uncover the rhythmic control of viral replication and activity. Using a high-resolution time-series dataset collected every 2 hours over a 3-day period, we identified a total of 22,151 viral operational taxonomic units (vOTUs) and 414 Nucleocytoplasmic Large DNA Virus (NCLDV) genomes. Our analysis revealed significant diel fluctuations, with 14.48% of vOTUs exhibiting diel patterns in metagenomic abundance and 1.97% showing diel transcriptional activity. We found that these abundant diel viruses infect hosts across the domains of life, including cyanobacteria, pelagibacteria, Marine Group II (MGII) archaea and protists, all known for their diel metabolism. The expanded spectrum of host diversity with diel viral interactions significantly broadens our understanding of virus-host rhythmic dynamics in natural environmental settings. A strong positive correlation was detected between the transcriptional activities of these diel viruses and their respective hosts. Contrary to bacteriophages, which mostly peaked during the day, we demonstrated that NCLDVs showed nocturnal diel abundance with a co-fluctuating diel transcriptomic activity pattern tightly hitched to their hosts, peaking at night and declining during the day. Furthermore, we identified a rich compendium of viral genes with significant diel expression patterns, including those related to structural protein production, DNA replication, and stress response. Notably, several essential viral genes involved in stress response and repair were found to be diel transcribed for the first time, including UV-endonuclease (UvdE), peroxidase, chaperones, and early light-induced protein (ELIP). Our findings suggest that viruses across host domains actively synchronize with environmental cues to optimize their replication and transmission, despite their dependence on host metabolism. This study provides novel insights into the rhythmic control of viral communities and their intricate interactions with hosts, with profound implications for microbial community succession and biogeochemical cycles. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 2476109, 42276163, 42406144 the Shenzhen Science, Technology and Innovation Commission Program, JCYJ20220530115401003 SUSTech Education Reform Programme, SJZLGC202437 grant from the Deutsche Forschungsgemeinschaft, SPP 2330 project number 464976318

Here the authors leverage a crossfeeding, engineered microbial community to demonstrate that strain abundance cycles are robust across environmental conditions. They pair this with a nonlinear dynamic model to elucidate population cycles.

AbstractSummary. Leveraging the Python/C API, eccLib was developed as a high-performance library designed for parsing genomic files and analysing genomic c

Intrinsically disordered proteins and regions (IDRs) lack stable 3D structures, posing challenges for interaction prediction. We present SpatPPI, a geometric deep learning model tailored for IDPPI prediction. SpatPPI leverages structural cues from folded domains to guide the dynamic adjustment of IDRs via geometric modeling, adaptive conformation refinement, and a two-stage decoding mechanism. It captures spatial variability without requiring supervised input and achieves state-of-the-art performance on benchmark datasets. Molecular dynamics simulations further validate its high adaptability to conformational changes in IDRs and strong capacity to generate distinct and structure-aware embeddings. A freely accessible server is available at http://liulab.top/SpatPPI/server .

R6K plasmids are commonly used for a wide range of genome engineering applications due to their ability to support transient delivery of genetic cargoes in many hosts. The maintenance of R6K plasmids requires specific strains. Unfortunately, many of these have obscure backgrounds, limited availability and were not built for efficient cloning. To address this issue, we present the construction and characterisation of a series of Pir E. coli strains called SHARK that are built from the DH10B derivative, Marionette-Clo. All SHARK strains have a genome encoded Pir gene for stable R6K plasmid maintenance and a λCI gene for tight unconditional repression of specific genes on plasmids. We show that SHARK strains are >100-fold more efficient than a commercial Pir strain for large and complex cloning reactions. SHARK is intended to help facilitate the cloning of large and complex R6K plasmids for challenging genome engineering projects, with all strains and genetic tools for their assembly being made publicly available. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, BB/Y007638/1 Royal Society, https://ror.org/03wnrjx87, URF\R\221008

Tertiary plastids derived from diatoms in “dinotom” dinoflagellates offer a rare view of organellogenesis in action, while the genomic and metaboli...

Desiccated cells lose over 85% of their water content and experience widespread biomolecular dysfunction. Romero-Pérez et al. show that the yeast proteome contains a subset of proteins that are desiccation tolerant and possess specific surface chemistries. Moreover, resistant proteins show a functional bias that can facilitate recovery upon rehydration.

Transcription attenuation fine-tunes biosynthetic gene expression in bacteria via premature termination upon metabolic signals. In transcription initiation-controlled bacterial systems, promoter architecture and transcription factor binding sets the size of transcriptional bursts at σ70 promoters, while distal enhancer elements and associated transcriptional activators modulate burst frequency at σ54 promoters. Using the tryptophan biosynthesis operon as a model, we show that transcription attenuation, acting post-initiation and alongside transcriptional repression, simultaneously modulates both burst size and frequency from a σ70 promoter. This challenges the view that frequency modulation requires distal enhancer input and reveals that post-initiation mechanisms can shape divergent transcriptional bursting. We also uncover that bacteria use cross-feeding as a previously unrecognised strategy for controlling cell-to-cell variation in gene expression, with implications for metabolic coordination among cells. These findings redefine transcription dynamics within cell populations and suggest new principles by which bacteria regulate gene expression to adapt to environmental change. ### Competing Interest Statement The authors have declared no competing interest. Leverhulme Trust, RPG-2021-050 Biotechnology and Biological Sciences Research Council, BB/W019698/1

Thioredoxins are ubiquitous redox proteins that are found in all domains of life. These conserved proteins are also found in many phages, including marine cyanophages that infect the ecologically important marine cyanobacteria. However, their role in phage infection is not known. Cyanophages also carry many small genes lacking homology to known functional domains. Whether these have a functional role or not remains unknown. Here, we explore the distribution and role of a cyanophage thioredoxin (trxA), and that of a small gene directly downstream of it (g26), in phage infection. For this we used the T7-like cyanophage, Syn5, which infects an open-ocean marine Synechococcus strain, WH8109. We found that thioredoxin genes are common in phage genomes, including in cyanophages. The g26 gene, however, is restricted in it distribution to the cyanophages. The cyanophage thioredoxin is catalytically active and it increases phage DNA replication, progeny production and competitive fitness. It also negatively impacts host growth. The g26 gene product is translationally coupled to, and thus dependent on, translation of the thioredoxin gene. This gene itself significantly increases phage virulence and fitness, yet reduces burst size. Our findings demonstrate that cyanophage thioredoxins impact phage fitness and infection physiology and that small viral genes with no homology to known genes can play an important role in the infection process. These findings provide insights into the importance of unusual genes in phage genomes and show that they are likely to play an important role in the interactions between abundant cyanobacteria and cyanophages in ocean ecosystems. ### Competing Interest Statement The authors have declared no competing interest.

The structural integrity of bacterial cells is traditionally attributed to the peptidoglycan cell wall, and more recently to the outer membrane, with the cytoplasmic membrane assumed to be mechanically passive. Cells lacking filaments of the actin homolog MreB are more bendable, suggesting a role for the cytoskeleton in cell stiffness. Here, we show that MreB does not stiffen the envelope directly, but instead mechanically couples the cell wall to the cytoplasmic membrane through its role in peptidoglycan synthesis, increasing resistance to bending. Under hyperosmotic stress, MreB relocalized to the poles, forming linkages that prevent membrane detachment from the cell wall and attenuate cytoplasmic contraction. Disruption of MreB filament formation, nutrient starvation, or inactivation of glycan elongation factors abolished or reduced this coupling, revealing that peptidoglycan biosynthesis actively mediates stress distribution across surface layers. Our findings redefine the bacterial envelope as a mechanically integrated composite, with the cytoplasmic membrane having substantial load-bearing capacity. ### Competing Interest Statement The authors have declared no competing interest.

Abstract. Species are a fundamental unit of biodiversity. Yet, the existence of clear species boundaries among bacteria has long been a subject of debate.

Microbial growth in both natural environments and artificial media is strongly influenced by metal speciation, which can be quantitatively modeled for a given chemical composition. Despite its importance, metal speciation is rarely incorporated into the design of microbial growth experiments, often leading to misinterpretations of metal bioavailability and toxicity. In this study, we revisit two historical microbial investigations: one that drew inaccurate conclusions due to the absence of speciation calculations, and another that relied on flawed assumptions about metal speciation. Through targeted recalculations, we demonstrate how these oversights impacted the interpretation of metal–microbe interactions including the applicability of the free ion activity model (FIAM). Additionally, we perform metal speciation analyses for a bacterial growth medium to illustrate how speciation can clarify distinctions between "stimulatory" and "non-essential" metals. Further simulations were conducted for six DSMZ–listed microbial media and six chemical variants of a representative medium, using estimated stability constants where experimental data were unavailable. Collectively, this work underscores the value of integrating metal speciation calculations into microbial research to improve the accuracy of conclusions regarding metal bioavailability and toxicity. ### Competing Interest Statement The authors have declared no competing interest. DCO

Only a fraction of bacterial genomes encode CRISPR-Cas systems but the selective causes of this variation are unexplained. How naturally virulent bacteriophages (phages) select for CRISPR immunity has rarely been tested experimentally. Here, we show against a panel of genetically and functionally diverse virulent phages that CRISPR immunity was not universally beneficial, and its fitness effect varied strongly between phages in predictable ways. In addition to mechanisms known to alter the effectiveness of CRISPR immunity, such as encoding a matching spacer or a protective nuclear shell, we show that the fitness effect of CRISPR immunity negatively correlated with the probability of evolving receptor-based resistance to the phage via spontaneous mutation. Supply of resistance mutations differed strongly between very closely related lipopolysaccharide-binding phages and was associated with variation at the C-terminus of the tail fibre protein altering residues involved in hydrogen bonding and the predicted binding site. Our results show that CRISPR immunity is more beneficial against virulent phages that are harder to evolve resistance to via receptor mutations, suggesting that virulent phage community composition and diversity will be important drivers of the prevalence of CRISPR immunity. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/T014342/1, BB/Y007743/1, BB/X003051/1

Metabolic processes underpinning ocean biogeochemistry are powered by molecular machines, proteins, that require various elements to function. Yet, the allocation of elements to these proteins, and subsequent implications for biogeochemical processes, remain poorly characterized. Here we integrate elemental measurements with metaproteomics to quantitatively examine elemental use in Southern Ocean microbial proteins and metabolic processes. We demonstrate that iron availability influences elemental allocation, including decreased iron allocation to photosynthesis and compensatory incorporation of non-iron metals into metalloproteins under iron scarcity. Manganese was primarily allocated to photosynthesis in iron-replete conditions, and reallocated to other metabolic roles under low iron. Photosystem I:II protein mass ratios impacted both iron and manganese allocation, and appeared to be driven by iron availability. Approximately half of biogenic copper was found in plastocyanin, likely substituting for iron-containing cytochromes in photosynthesis. Moreover, biogenic nitrogen to phosphorus ratios were decoupled from ribosomal abundance, contrary to prevailing assumptions about ribosomal influence on stoichiometric regulation in the ocean. Instead, our results suggest that community composition and intracellular storage are important regulators of N:P in the Southern Ocean. Together, our findings identify key molecular mechanisms that modulate elemental demand and limitation, and provide a foundation for quantitatively connecting molecular measurements with biogeochemical models. ### Competing Interest Statement The authors have declared no competing interest. NSERC Discovery Grant, RGPIN-2015-05009 Simons Foundation, https://ror.org/01cmst727, 504183 Simons Foundation CBIOMES Award, 1001702 Canada Research Chair Dutch Research Council, ALWPP.2016.020 Nova Scotia Graduate Scholarship

Synthetic one-carbon assimilation could contribute to a more sustainable and circular carbon economy, but much work in this field has focused on model microorganisms. Here the authors provide their perspective on the potential value of non-model microbes, and how that potential could be realised.

Enterololin is a narrow-spectrum antibiotic that selectively kills Enterobacteriaceae in vitro and in a mouse model of adherent-invasive Escherichia coli gut infection. AI-guided mechanism-of-action studies identified LolCDE as its molecular target.

Chromosome architecture influences Agrobacterium fitness, competitiveness, and virulence, shaping its adaptation and evolution.

Background Deep ocean blue holes are characterized by distinct physicochemical gradients and complex biological processes, and Yongle Blue Hole (YBH) in the South China Sea (SCS) is the world’s deepest (301 m) underwater cavern with unique environmental characteristics. So far, studies investigated the bacterial community structure with different lifestyles of the YBH; however, our understanding of viruses in the YBH remains limited. Here, we utilized a metagenomic approach to investigate viral communities in both the “viral fraction” and “cellular fraction” of seawater samples in oxic and anoxic zones within YBH. Results A total of 1,730 viral operational taxonomic units (vOTUs) were identified, with over 70% affiliated with the classes Caudoviricetes and Megaviricetes, particularly within the families Kyanoviridae, Phycodnaviridae and Mimiviridae. Gene-sharing network analyses indicated that the deeper anoxic layers contain a high proportion of novel viral genera, while the oxic layer’s viral genera overlap with those found in the open water samples from SCS. Virus-linked prokaryotic hosts predominantly belong to the phyla Patescibacteria, Desulfobacterota, and Planctomycetota. Notably, the detected putative auxiliary metabolic genes (AMGs) suggest that these viruses may influence photosynthetic and chemosynthetic pathways, as well as methane, nitrogen, and sulfur metabolisms, especially with several high-abundance AMGs potentially involved in prokaryotic assimilatory sulfur reduction. Conclusions Together, these findings highlight the potential ecological roles and diversity of viral communities within YBH and shedding light on niche-separated viral speciation.

Microbiota play crucial roles in host health, including protection against pathogens through competitive interactions between commensal and pathogenic bacteria that are mediated by direct contact or secreted factors. We previously demonstrated that Chryseobacterium massiliae, a zebrafish commensal, protects larvae against infection by Flavobacterium covae (formely named F. columnare). Here, we investigated whether interference interactions contribute to this protective effect. We found that C. massiliae culture supernatant inhibits F. covae growth and a transposon mutagenesis screen identified mutants lacking this activity. All identified mutants carried insertions in a gene encoding a protein homologous to Bacteroidales BSAP pore-forming toxins, characterized by a Membrane Attack Complex/Perforin (MACPF) domain. We showed that this protein, which we named CSAP-1 (for Chryseobacterium Secreted Antimicrobial Protein) displays bactericidal, pore-forming activity that lyses F. covae cells. Unlike BSAP proteins from Bacteroides spp., CSAP-1 displays broader antagonistic activity, targeting multiple species across the Flavobacterium and Chryseobacterium genera - thus mediating interspecies and intergenus inhibition within Bacteroidetes. Although CSAP-1 is not essential for the in vivo protective effect of C. massiliae, administration of purified CSAP-1 alone confers significant protection to zebrafish larvae against sensitive F. covae infection. This study therefore identifies CSAP-1 as the first MACPF protein from C. massiliae with broad-spectrum inhibitory activity against members of the order Flavobacteriales. These findings highlight CSAP-1 as a promising candidate for the development of novel antimicrobial strategies and warrant further mechanistic investigation. ### Competing Interest Statement The authors have declared no competing interest.

Nutrient scarcity is a common environmental stressor encountered by bacteria. Despite its significance in bacterial virulence and physiology, the underlying epigenetic mechanisms of bacterial adaptation to prolonged starvation remain poorly understood. In this study, we utilized the latest Nanopore R10.4.1 sequencing technology to comprehensively characterize the genome-wide methylation landscape of the freshwater fish pathogen Flavobacterium columnare following long-term starvation at different temperatures. Our results revealed significant methylation plasticity under starvation conditions, characterized by distinct motif-dependent patterns. Notably, demethylation of the 6mA-modified CAYNNNNNRTG motif emerged as a robust epigenetic signature of starvation adaptation, with functional enrichment of genes involved in pathways for translation and metabolism, suggesting the role of this methylase in regulating essential cellular functions under starvation stress. Additionally, another 6mA-modified GCAGA motif exhibited temperature-dependent variation, indicating its potential role in temperature-specific response. Together, this study provides novel insights into the epigenetic mechanisms underlying the bacterial adaptation to nutrient deprivation and establishes a valuable methodological reference for bacterial epigenetics analysis utilizing advanced Nanopore sequencing technologies. ### Competing Interest Statement The authors have declared no competing interest. Hong Kong Research Grant Council, 9048294

Kubota et al. show that bacteria can use their filamentous phage to outcompete other bacteria, but increased phage production enables the evolution of cheater miniphages with truncated genomes. Miniphages exploit full-length phages and lower total phage production, causing some bacteria to lose their prophages and any associated fitness benefits.

Phylogenetic mining and protein language models uncover improved PiggyBac transposase sequences.

Panoptes, an anti-phage defence system against virus-mediated immune suppression, is revealed.