Primer bias in 16S rRNA gene amplicon sequencing can distort microbial diversity estimates by underrepresenting key taxa. We introduce a modified primer pair (V4-EXT) targeting the hypervariable V4 region of bacterial and archaeal 16S rRNA genes, with improved in silico taxonomic inclusivity. To benchmark performance, we analyzed 938 samples from terrestrial, aquatic, and host-associated habitats, comparing microbial community profiles derived with V4-EXT and the currently most widely used V4-targeted primers. V4-EXT substantially improved the detection of Patescibacteria and other underrepresented lineages, such as Chloroflexota and Iainarchaeota, while enhancing recovery of novel amplicon sequence variants across sample types. Overall, V4-EXT provides broader taxonomic coverage and more inclusive microbial community profiles, particularly in high-diversity ecosystems such as groundwater and soils. We propose V4-EXT as a robust successor for comprehensive microbial community analysis across diverse habitats. ### Competing Interest Statement The authors have declared no competing interest. FWF Austrian Science Fund, Z383-B, 10.55776, COE 7, 10.55776, T 1218, ZK-74B Research Foundation - Flanders, G0A4821N, G000821N, 1174925N, BOF.MET.2021.0004.01

Vibrio natriegens is a Gram-negative, halophilic member of the Harveyi clade and Vibrio core group that inhabits marine and estuarine coastal waters and sediments. While generally considered nonpathogenic, this species shares this clade with serious human pathogens (e.g., Vibrio parahaemolyticus) and aquatic animal pathogens (e.g., Vibrio harveyi, Vibrio campbelllii, and Vibrio alginolyticus). Although it was discovered more than 65 years ago, most of our knowledge regarding this bacterium has been generated in the past decade.

Abstract. Bacterial flagella are known for facilitating motility to support nutrient acquisition and predator evasion, but can also serve as receptors for

Complex prophage integration dynamics, including low-level induction, cross-family host range and transposase-mediated mobilization, challenge existing paradigms and deepen our understanding of phage–bacterial interactions in the human gut microbiome.

Differences in genomic GC content skew species abundances in microbial communities. Here, the authors develop GuaCAMOLE, a computational method that quantifies this GC bias from metagenomic sequencing data and corrects abundances accordingly, showing its application increases abundances of some pathogenic bacteria up to twofold in human gut microbiomes.

Abstract. Microbes differ greatly in their organismal structure, physiology, and environmental adaptation, yet information about these phenotypic traits is

Abstract. Taxonomic classification underlies all biological science and is the basis for comparative analysis of biological organisms and therefore our und

This graphical abstract illustrates the integrated molecular dating workflow in PhyloSuite v2. The new Molecular Dating Suite unifies data configuration, analysis, and visualization into a single, st...

Daily dynamics in the composition and function of the human gut microbiota have been recognized since 2014, yet the molecular mechanisms underlying these rhythms and their impact on human health remain unclear. Disrupted microbial oscillations are increasingly linked to metabolic diseases such as obesity and type 2 diabetes, and to inflammatory conditions in the gut and beyond. We propose advancing from observational studies to experimentally targeting microbial rhythms and clocks to uncover causal relationships. In vivo and in vitro models offer opportunities to uncover how signaling cues and dietary patterns influence microbial oscillations and, in turn, host metabolic and immune functions. Manipulating microbial rhythmicity independent of host physiology represents a new frontier for microbiota-based strategies to promote health and prevent diseases.

Background: Organized collections of bacterial strains can help bridge the gap between studying model organisms and communities, through comparative experiments between genetically and phenotypically diverse strains. We describe the establishment and initial characterization of a library of 62 marine heterotrophic bacteria, selected to represent a significant fraction of the genome-encoded functional diversity and a wide range of known phytoplankton-bacteria interactions. We focus on important but often undiscussed aspects of collecting and maintaining such a library, verifying strain identity, and applying classical microbiological methods across diverse strains. Results: Cultured strains contain up to hundreds of mutations compared with the reference genomes, with non-synonymous mutations in rpoB and/or rpoC genes observed in ~15% of the cultures. Most strains grow well at 25C, but the dependence of growth rate on temperature and the width of the temperature niche vary between strains in a systematic manner. We describe steps towards designing a universal, defined, minimal media for marine bacteria, revealing that growth inhibition on amino acids and peptides by carbohydrates is widespread. Cell counts obtained from flow cytometry and colony plating differ systematically, as do different methods to assess motility. Finally, we discuss traits potentially related to microbial interactions such as hemolysis, biofilm formation, and antibiotic resistance. Gammaproteobacteria such as Alteromonas, Pseudoalteromonas, and Vibrio reveal consistently robust growth, and activity, perhaps explaining why these clades are well-explored. Conclusion: Explicitly discussing the insights and challenges of working with strain libraries will pave the way to robust, reproducible, and generalizable mapping of bacterial traits across diversity. ### Competing Interest Statement The authors have declared no competing interest. Human Frontiers Research Program, RGP0020/2016 National Science Foundation - United States-Israel Binational Science Foundation, NSFOCE-BSF 1635070, NSF-BSF 2246707 NSF Center for Chemical Currencies of a Microbial Planet, publication #81 Israel Science foundation, 1786/20

Oxygenic phototrophs fix CO2 via the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) that shows relatively low CO2 affinity and specificity. To circumvent low and fluctuating CO2 concentrations in aquatic systems, cyanobacteria and algae have evolved sophisticated inorganic carbon (Ci) concentrating mechanisms (CCMs). Bicarbonate transporters such as SbtA play a crucial role in the cyanobacterial CCM and hence are tightly regulated at multiple layers. The control of sbtA gene expression and corresponding transporter activity involves the PII-like protein SbtB, whose gene is frequently co-transcribed with sbtA. Here we report on the discovery of a so far non-annotated gene in the model Synechocystis sp. PCC 6803, which is located upstream of the sbtAB operon and encodes the small protein SbtC, composed of 80 amino acids. Presence of SbtC was confirmed by immunoblotting after fusing the sbtC-coding sequence to a Flag-tag. Similar to sbtAB, transcription of the sbtC locus is induced by low CO2 availability but controlled independently. Mutation of the sbtC locus in a wild-type background showed only a mild phenotype even under low CO2, but the diurnal growth was impaired as found before in the mutant ∆sbtB. Biochemical analysis provided evidence for a trimeric SbtABC complex in the membrane. Recombinant Synechocystis strains harboring only SbtA as single Ci uptake system with either deleted sbtB or sbtC genes showed that bicarbonate leakage from the cell was strongly elevated in both mutants. Our results provide evidence that SbtC contributes to the formation of the SbtAB complex, thereby regulating bicarbonate exchange at the cytoplasmic membrane. Well-conserved SbtC-like proteins encoded in the neighborhood of sbtAB exist in many cyanobacterial genomes pointing towards an important role in the cyanobacterial CCM. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, HA2002/27-1, KL 3114/10-1, HE 2544/22-1 German

Bacterial oxidation of Mn(II) to Mn(III/IV) oxides is widespread in nature and proceeds markedly faster than abiotic oxidation. However, the physiological role of this process remains unclear. Building on recent evidence that environmental stress determines bacterial Mn(II) oxidation activity, we used Pseudomonas putida KT2440, a known Mn(II)-oxidizing bacterium, to investigate the potential drivers and benefits of this reaction. Using RNA-seq, we observed up-regulation of starvation-related two-component system genes during Mn(II) oxidation, supporting the idea that nutrient limitation acts as a trigger. This led us to hypothesize that introducing an external antagonistic stressor could further accelerate the onset and rate of Mn(II) oxidation. To test this, we established a synthetic microbial community in which KT2440 was confronted with Pseudomonas aeruginosa PAO1, which possesses both contact-dependent and diffusible antagonistic systems. Across multiple assays measuring oxidation initiation time, manganese oxide accumulation, and bacterial survival, the presence of PAO1 significantly accelerated Mn(II) oxidation by KT2440. More importantly, Mn(II) oxidation strongly enhanced KT2440 survival under antagonistic pressure. Correlation analysis indicated a positive relationship between survival rate and oxidation intensity. Fluorescence microscopy and agent-based modeling further confirmed that this protective effect is contact-dependent. By integrating these results with earlier findings, we propose that the physiological significance of bacterial Mn(II) oxidation lies in enhancing the survival fitness of the oxidizer under stressful conditions. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 52450009

Prochlorococcus, the dominant cyanobacterium in the oligotrophic ocean, possesses a streamlined genome and depends on interactions with heterotrophic bacteria for survival under various stressors. While the role of 'helper' bacteria in mitigating oxidative stress is established, the mechanisms enabling its long-term survival under nitrogen (N) limitation remain poorly characterized. Here, we employ a multi-omics approach - integrating transcriptomics and proteomics - to investigate the physiological processes that facilitate the prolonged survival of Prochlorococcus in co-culture with the marine heterotroph Alteromonas during conditions of extreme N-deprivation. Our results demonstrate that, unlike axenic cultures which rapidly perish, Prochlorococcus in co-culture maintains viability for months following the depletion of initial N-sources. Molecular analysis identifies a shift in both organisms that underpins this persistence: Prochlorococcus strongly upregulates high-affinity N-scavenging pathways, while Alteromonas exhibits transcriptional and translational changes consistent with increased organic matter degradation and reduced motility. This suggests that Alteromonas functions as a key nitrogen recycler, providing a continuous, albeit low-level, supply of bioavailable NH4+ to its photoautotrophic partner through the remineralization of organic matter. These findings support and extend the Black Queen Hypothesis, illustrating that the benefits conferred by heterotrophs to genome-streamlined primary producers encompass not only the detoxification of reactive oxygen species but also the continuous provisioning of essential macro-nutrients under starvation conditions. This tightly coupled, mutualistic relationship represents a critical factor driving the resilience and productivity of microbial communities in oligotrophic marine ecosystems. ### Competing Interest Statement The authors have declared no competing interest.

The ecological role of viruses for CPR bacteria and DPANN archaea remains understudied. Here, the authors apply metagenomic approaches to study the biogeographic distribution of CPR and DPANN viruses in acid mine drainage sediments and elucidate their complex interplays.

Bacteriophages have genomes that span a wide size range, are densely packed with coding sequences, and frequently encode genes of unknown function. Classical forward genetics has defined essential genes for phage replication in a few model systems but remains laborious and non-scalable. Unbiased functional genomics approaches are therefore needed for phages, particularly for large lytic phages. Here, we develop a phage transposon sequencing (TnSeq) platform that uses the mariner transposase to insert an anti-CRISPR selectable marker into phage genomes. CRISPR-Cas13a-based enrichment of transposed phages followed by pooled sequencing identifies both fitness-conferring and dispensable genes. Using the Pseudomonas aeruginosa-infecting nucleus-forming jumbo phage ΦKZ (280,334 bp; 371 predicted genes) as a model, we show that ~110 genes are fitness-conferring via phage TnSeq. These include conserved essential genes involved in phage nucleus formation, protein trafficking, transcription, DNA replication, and virion assembly. We also isolate hundreds of individual phages with insertions in non-essential genes and reveal conditionally essential genes that are specifically required in clinical isolates, at environmental temperature, or in the presence of a defensive nuclease. Phage TnSeq is a facile, scalable technology that can define essential phage genes and generate knockouts in all non-essential genes in a single experiment, enabling conditional genetic screens in phages and providing a broadly applicable resource for phage functional genomics. ### Competing Interest Statement J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics, a consultant to LeapFrog Bio and a scientific advisory board member and cofounder of Acrigen Biosciences and ePhective Therapeutics. The remaining authors declare no competing interests. The Bondy-Denomy laboratory received past research support from Felix Biotechnology. National Institutes of Health, https://ror.org/01cwqze88, AI171041, AI167412

Abstract. Diel light cycles profoundly influence estuarine biogeochemical processes, yet the mechanistic responses of planktonic prokaryotic communities to

Abstract. Bathyarchaeia, among the most ancient and abundant microbial lineages on Earth, dominate diverse anoxic subsurface ecosystems and play a pivotal

Abstract. Ribosomes are essential for protein synthesis and require ribosome biogenesis factors for assembly. To uncover the evolutionary diversity of ribo

Serpentinization drives abiotic synthesis of organics (e.g., hydrocarbon) potentially conducive to the emergence of life, making serpentinite hosted systems and associated microbial community key windows into nature of life's origin. Although cultivation independent studies uncovered the Candidatus Bipolaricaulota widely distributed in serpentinizing environments, cultivation of this phylum has been unsuccessful. Here we cultured the first pure strain, J31, of Ca. Bipolaricaulota from the Lost City hydrothermal field, a well characterized marine serpentinite system, using hydrocarbons as the primary nutrient source. As an early-branching bacterial lineage, strain J31 exhibits an unusual morphology composed of a central rod and elongated Toga like extensions at both ends, and divides by binary fission. Strain J31 absorbs hexadecane through Toga ends via coordinated processes resembling inhalation and swallowing, after which hexadecane is efficiently transported to the central rod in the vesicle like structures and subsequently converted into membrane lipids to support Toga synthesis and cellular growth. Hydrocarbon degrading capability is widespread among the globally distributed members of Ca. Bipolaricaulota. Strain J31 colonizes serpentine minerals, facilitating the utilization of hydrocarbons derived from serpentines and promoting the release of soluble iron and silicon, thereby linking microbial activity to geochemical cycling. Thus, our study presents a novel strategy for cultivating deep-branching bacteria and offers insights into the metabolic foundations of early life on Earth-and potentially on other rocky planets undergoing serpentinization. ### Competing Interest Statement The authors have declared no competing interest. NSFC Innovative Group Grant, 42221005

The deep sea is home to a vast and largely unexplored microbial biosphere, along with significant amounts of complex organic matter (COM). However, the ability of deep-sea microbes to metabolize complex organic matter across diverse regions remains poorly understood. Here, we combine 16S rRNA gene amplicon sequencing, metagenomics, and metatranscriptomics to comprehensively characterize prokaryotic communities across different years and habitats (cold seeps, hydrothermal vents, and seamounts). Our results reveal spatio-temporal community heterogeneity driven by geochemical gradients, alongside widespread genomic and transcriptomic potential for COM metabolism. Notably, the PVC (Planctomycetota-Verrucomicrobiota-Chlamydiota) superphylum exhibits extensive polysaccharide degradation capabilities, with Planctomycetota strain WC338 and Lentisphaerota strain WC36 isolated via laminarin enrichment. Growth and transcriptome data confirm their obligate laminarin dependence and robust degradation capacity, employing distinct enzymes (GH16 and GH2), whose broad distribution across diverse PVC superphylum lineages underscores their prevalence. Furthermore, we demonstrate that laminarin acts as a highly effective selective substrate for enriching and isolating the deep-sea PVC superphylum bacteria. Collectively, these findings delineate specialized adaptations within the PVC superphylum for polysaccharide degradation, significantly expanding our understanding of deep-sea microbial roles in global carbon cycling. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 42406104 and 42530409

Abstract. Microorganisms, including bacteria, archaea, fungi, and viruses, are the most taxonomically diverse and ecologically dominant life forms on Earth

Abstract. PROSITE (https://prosite.expasy.org/) is a database of entries documenting protein domains, families, and functional sites, along with the associ

Abstract. The AlphaFold Protein Structure Database (AFDB; https://alphafold.ebi.ac.uk), developed by EMBL–EBI and Google DeepMind, provides open access to