Characterizing virus–host pairs and the infection state of individual cells is the major technical challenge in microbial ecology. We addressed these challenges using state-of-the-art single-cell genome technology (SAG-gel) combined with extensive metagenomic datasets targeting the bacterial and viral communities in Lake Biwa. From two water layers and two seasons, we obtained 862 single-cell amplified genomes (SAGs), including 176 viral (double-stranded DNA phage) contigs, which identified novel virus–host pairs involving dominant freshwater lineages. The viral infection rate, estimated by mapping the individual SAG’s raw reads to viral contigs, showed little variation among samples (12.1%–18.1%) but significant variation in host taxonomy (4.2%–65.3%), with copiotrophs showing higher values than oligotrophs. The high infection rates of copiotrophs were attributed to collective infection by diverse viruses, suggesting weak density-dependent virus–host selection, presumably due to their nonpersistent interactions with viruses resulting from fluctuating abundance. In contrast, the low infection rates of oligotrophs supported the idea that their codominance with viruses is achieved by genomic microdiversification, which diversifies the virus–host specificity, sustained by their large population size and persistent density-dependent fluctuating selection. Notably, we discovered viruses infecting CL500-11, the dominant bacterioplankton lineage in deep freshwater lakes worldwide. These viruses showed extremely high read coverages in cellular and virion metagenomes but were detected in <1% of host cells, suggesting a low infection rate and high burst size. Overall, we revealed highly diverse virus–host interactions within and between host lineages that were overlooked at the metagenomic resolution.

Halomonas species have recently emerged as promising chassis organisms for next-generation industrial biotechnology, due to their ability to thrive under high-salt conditions, where most microorganisms cannot survive. This feature minimizes contamination risks, thus enabling cultivation under open, unsterile conditions. In addition, many Halomonas species naturally produce large amounts of the bioplastic polyhydroxybutyrate PHB and the high-value osmolyte ectoine. This review explores the development of genetic manipulation tools and their pivotal role in establishing the genus Halomonas as an industrial chassis. Key additions to the synthetic biology toolbox, including cloning vectors, genetic parts, and genome editing systems are highlighted, along with challenges faced for their adoption, such as difficulties in transformation. In addition, we showcase how these tools have been employed for the development of more robust, high-producing strains through metabolic engineering, as well as for expanding the portfolio of target metabolites produced by Halomonas. Recent developments in synthetic biology tools and metabolic engineering highlighted in this review underscore the potential of Halomonas for large scale metabolite production and provide a promising outlook towards their role as a microbial chassis in industrial biotechnology.

Efficient secretion of heterologous proteins is crucial for applications in industrial and biomedical fields. Selecting appropriate signal peptides and bacterial strains is critical for successful protein expression and export. Bacillus thuringiensis, known for its robust secretion capabilities within the Bacillus genus, shows promise as an ideal host for this purpose. We performed genome-based bioinformatic analysis of B. thuringiensis HD73. A total of 525 proteins were predicted to contain signal peptides, exceeding those in other Bacillus species. The extracellular proteome of B. thuringiensis HD73 was analyzed via LC-MS/MS, identifying 100 secreted proteins. A library of 30 signal peptides was constructed by integrating genome-based predictions with experimental secretome data. Using this library, green fluorescent protein secretory expression systems were developed in the acrystalliferous mutant strain B. thuringiensis HD73−, and the strain carrying signal peptide S17 showed the highest secretion efficiency. Additionally, the top 10 performing signal peptides were used to express and secrete the convenient enzyme cutinase, with the S20 fusion strain exhibiting the highest cutinase activity (3.65 U/mL in the culture supernatant). This study provides the first combined bioinformatic and experimental characterization of the B. thuringiensis secretome. The developed secreted protein expression system and signal peptide library demonstrate effectiveness and offer potential for future heterologous protein secretion in B. thuringiensis.

Cas12a trans nuclease activity has been leveraged for nucleic acid detection, often coupled with isothermal amplification to increase sensitivity. However, due to the lack of highly efficient thermostable Cas12a orthologs, use of Cas12a in one-pot combination with high temperature (55–65°C) amplification, such as loop-mediated isothermal amplification (LAMP), has remained challenging. Here, we attempt to address this challenge by comparative study of the thermostable, but poorly trans-active YmeCas12a (from Yellowstone metagenome) with the mesophilic, highly trans-active LbaCas12a (from Lachnospiraceae bacterium ND 2006). Kinetic characterizations identified that poor trans substrate affinity (high Km) is the key limiting factor in YmeCas12a trans activity. We engineered YmeCas12a by structure-guided mutagenesis and fusion to DNA-binding domains to increase its affinity to the trans substrate. The most successful combinatorial variant showed 5–50-fold higher catalytic efficiency with the trans substrate depending on the target site. With the improved variant, we demonstrate efficient nucleic acid detection in combination with LAMP in a single reaction workflow, setting the basis for development of point-of-care tests.

The type VI secretion system (T6SS) is a contact-dependent contractile nanomachine widely distributed among Gram-negative bacteria, essential for interbacterial competition, host interactions and environmental adaptation. The T6SS encoded by Serratia is known to play roles in bacterial competition and contribute to niche adaptation. Yet, the diversity and distribution of T6SS loci and relevant proteins across different Serratia species remain underexplored. In this study, we conducted bioinformatic and comparative genomic analyses of the T6SS to expand our understanding of T6SS in Serratia and their distribution within the genus. A dataset comprising 2,337 Serratia genomes was analysed, identifying 4 distinct T6SS loci classified into 3 main types based on TssB sequence variation, with varying distributions across specific Serratia haplogroups. T6SSserratia-1b subtype was found to be the most prevalent. By integrating homology searches and positional information, we expanded the known database of T6SS proteins in Serratia, identifying a range of effectors likely involved in interbacterial interactions and host adaptation. Additionally, statistical analysis reveals a strong correlation between the presence of T6SS and the diversity of associated genes, including not only known T6SS effectors and immunity proteins but also other co-occurring gene families, enabling the identification of multiple candidate loci potentially involved in bacterial competition and pathogenicity. These findings shed light on the complex distribution, evolutionary dynamics and functional diversity of T6SS and associated proteins in Serratia, advancing our understanding of their role in bacterial competition and environmental adaptation.

Lignocellulosic wine grape waste (WGW) is a cheap medium for Escherichia coli growth and H2 production. The current study investigated the effect of initial redox potential (oxidation–reduction potential (ORP)) on the growth, H2 production, ORP kinetics, and current generation of the E. coli BW25113 parental and a mutant strain optimized for hydrogen evolution when fermenting WGW (40 g L−1) hydrolysate. Bacteria were cultivated anaerobically on pre-treated WGW hydrolysate with dilutions ranging from undiluted to fourfold dilution, at pH 7.5. Notably, a twofold diluted medium, with pH adjustment using K2HPO4, exhibited reduced acidification, prolonged H2 production, and enhanced biomass formation (OD600, 1.5). The addition of the redox reagent DL-dithiothreitol (DTT) was found to positively influence the H2 production of both the E. coli BW25113 parental and mutant strains. H2 production started after 24 h of growth, reaching a maximum yield of 5.10 ± 0.02 mmol/L in the wild type and 5.3 ± 0.02 mmol/L in the septuple mutant strain, persisting until the end of the stationary growth phase. The introduction of 3 mM DTT induced H2 production from the early-exponential phase, indicating that reducing conditions enhanced H2 production. Furthermore, we assessed the efficacy of using intact E. coli cells (1.5 mg cell dry weight) as anode catalyst in a bio-electrochemical fuel-cell system. Whole cells of the septuple mutant grown under reduced ORP conditions yielded the highest electrical potential, reaching up to 0.7 V. The results highlight the potential of modifying medium buffering capacity and ORP as a tool to improve biomass yield and H2 production during growth on WGW for biotechnological biocatalyst applications.

Defining the minimal genetic requirements for cellular life remains a fundamental question in biology. Genomic exploration continually reveals novel microbial lineages, often exhibiting extreme genome reduction, particularly within symbiotic relationships. Here, we report the discovery of Candidatus Sukunaarchaeum mirabile, a novel archaeon with an unprecedentedly small genome of only 238 kbp —less than half the size of the smallest previously known archaeal genome— from a dinoflagellate-associated microbial community. Phylogenetic analyses place Sukunaarchaeum as a deeply branching lineage within the tree of Archaea, representing a novel major branch distinct from established phyla. Environmental sequence data indicate that sequences closely related to Sukunaarchaeum form a diverse and previously overlooked clade in microbial surveys. Its genome is profoundly stripped-down, lacking virtually all recognizable metabolic pathways, and primarily encoding the machinery for its replicative core: DNA replication, transcription, and translation. This suggests an unprecedented level of metabolic dependence on a host, a condition that challenges the functional distinctions between minimal cellular life and viruses. The discovery of Sukunaarchaeum pushes the conventional boundaries of cellular life and highlights the vast unexplored biological novelty within microbial interactions, suggesting that further exploration of symbiotic systems may reveal even more extraordinary life forms, reshaping our understanding of cellular evolution.

Bacterial swimming is mostly powered by the bacterial flagellar motor and the number of proteins involved in the flagellar motor can vary. Quantifying the proteins present in flagellar motors from a range of species delivers insight into how motility has changed throughout history and provides a platform for estimating from its genome whether a species is likely to be motile. We conducted sequence and structural homology searches for 54 flagellar pathway proteins across 11 365 bacterial genomes and developed a classifier with up to 95% accuracy that could predict whether a strain was motile or not. We then mapped the evolution of flagellar motility across the GTDB bacterial tree of life. We confirmed that the last common bacterial ancestor had flagellar motility and that the rate of loss of this motility was four-fold higher than the rate of gain. We showed that the presence of filament protein homologues was highly phylogenetically correlated with motility and that all species classified as motile contained at least one filament homologue. We calculated the rate of gain and loss for each flagellar protein and that the filament protein FliC was highly correlated with motility across the tree of life. We then measured the correlation of each flagellar motor protein with FliC and showed that the filament, rotor, and rod and hook proteins were all highly correlated with FliC, and thus with motility. We calculated the differential rates of gain and loss for each flagellar protein and quantified which genomes encoded for partial sets of flagellar proteins, indicating potential pathways by which motility could be lost. Overall, we show that filament, rod and hook and rotor proteins are conserved when flagellar motility is preserved and that the presence or absence of a FliC homologue is a good, simple predictor of whether or not a species has flagellar motility.