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PubMed2024

Rok from B. subtilis: Bridging genome structure and transcription regulation.

Bacterial genomes are folded and organized into compact yet dynamic structures, called nucleoids. Nucleoid orchestration involves many factors at multiple length scales, such as nucleoid-associated proteins and liquid-liquid phase separation, and has to be compatible with replication and transcription. Possibly, genome organization plays an intrinsic role in transcription regulation, in addition to classical transcription factors. In this review, we provide arguments supporting this view using the Gram-positive bacterium Bacillus subtilis as a model. Proteins BsSMC, HBsu and Rok all impact the structure of the B. subtilis chromosome. Particularly for Rok, there is compelling evidence that it combines its structural function with a role as global gene regulator. Many studies describe either function of Rok, but rarely both are addressed at the same time. Here, we review both sides of the coin and integrate them into one model. Rok forms unusually stable DNA-DNA bridges and this ability likely underlies its repressive effect on transcription by either preventing RNA polymerase from binding to DNA or trapping it inside DNA loops. Partner proteins are needed to change or relieve Rok-mediated gene repression. Lastly, we investigate which features characterize H-NS-like proteins, a family that, at present, lacks a clear definition.

Bacillus subtilis↗
PubMed2024

Natural variation of immune epitopes reveals intrabacterial antagonism.

Plants and animals detect biomolecules termed microbe-associated molecular patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multicopy MAMPs on immune induction is unknown. Here, we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy, and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple cold shock proteins, and 46% carry a nonimmunogenic form. We uncovered a mechanism for immune evasion, intrabacterial antagonism, where a nonimmunogenic cold shock protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

Epitopes↗
PubMed2026

VicMAG, an open-source tool for visualizing circular metagenome-assembled genomes highlighting bacterial virulence and antimicrobial resistance.

Bacterial pathogens spread in clinical and environmental settings, and mobile genetic elements (MGEs), such as plasmids and phages, mediate the transfer of virulence factor genes (VFGs) and antimicrobial resistance genes (ARGs) among bacterial communities. Metagenomic analysis of environmental and wastewater samples using highly accurate long-read sequencing technologies, such as Pacific Biosciences (PacBio) HiFi sequencing, provides valuable insights into monitoring the regional spread of VFGs and ARGs, including dissemination mediated by MGEs. No visualization tool is currently available for the comprehensive display of numerous resulting circular metagenome-assembled genomes (cMAGs) with functional gene annotations. Here, we developed visualization of circular metagenome-assembled genome (VicMAG), a visualization tool for highly complex cMAGs derived from long-read metagenome assemblies annotated using updated databases of VFGs, ARGs, and MGEs. Using 353 cMAGs from PacBio HiFi sequencing of a wastewater sample, we demonstrated the utility of VicMAG for metagenome visualization. VicMAG provides comprehensive, size-aware visualization of cMAGs representing bacterial chromosomes and plasmids, annotated with VFGs, ARGs, and phages. By simultaneously visualizing all cMAGs in a framework, VicMAG facilitates a holistic understanding of the distribution and genomic context of VFGs and ARGs across complex microbial communities. This tool supports integrated surveillance of bacteria associated with virulence and antimicrobial resistance across clinical, environmental, and One Health contexts.

Metagenome↗
PubMed2025

A comprehensive review of genomic-scale genetic engineering as a strategy to improve bacterial productivity.

Bacterial genome engineering has evolved to provide increasingly precise, robust and rapid tools, driving the development and optimization of bacterial production of numerous compounds. The field has progressed from early random mutagenesis methods, labour-intensive and inefficient, to rational and multiplexed strategies enabled by advances in genomics and synthetic biology. Among these tools, CRISPR/Cas has stood out for its versatility and its ability to achieve precision levels ranging from 50% to 90%, compared to the 10-40% obtained with earlier techniques, thereby enabling remarkable improvements in bacterial productivity. Nevertheless, like its predecessors, it still demands continuous refinement to reach full maturity. In this context, the present review addresses the lack of a unified overview by summarizing historical milestones and practical applications of genomic engineering tools in bacteria. It integrates diverse approaches to provide a comprehensive perspective on the evolution and prospects of these fundamental biotechnological tools.

Bacteria↗
PubMed2026

A tiled amplicon protocol for culture-free whole-genome sequencing of M. tuberculosis from clinical specimens.

Whole-genome sequencing of Mycobacterium tuberculosis can be a valuable tool for TB surveillance and treatment, providing insights into transmission patterns and comprehensive drug susceptibility testing. However, the slow growth of M. tuberculosis means traditional culture-based sequencing methods can take weeks to return results, which has limited the widespread adoption of these techniques and limited their use in clinical decision-making. Tiled amplicon sequencing is a fast, reliable, and cost-effective method of whole-genome sequencing that can be done directly on clinical specimens and has been implemented at scale in academic and public health laboratories across the world; it was the cornerstone of SARS-CoV-2 sequencing and has been adapted for a wide range of viral pathogens. However, similar methods are not yet available for far larger bacterial genomes. Extending this approach to M. tuberculosis would significantly reduce the cost, labor, and turnaround time for whole-genome sequencing. We designed a tiled amplicon panel consisting of 5,128 primers that covers the entire M. tuberculosis genome, the largest tiled amplicon sequencing panel we are aware of to date. Applying our amplicon panels to clinical samples of sputum, we show the ability to recover whole-genome bacterial sequences without the need for culture. The resulting sequence data can be used to determine M. tuberculosis lineage and reliably identify markers of drug resistance. Using this approach in clinical settings could reduce the time needed for comprehensive drug susceptibility testing from weeks to days and enable genomic epidemiology to be performed at scale, even in resource-limited settings.IMPORTANCEWe have developed and tested an amplicon panel, TB-seq, for the priority pathogen Mycobacterium tuberculosis, demonstrating recovery of near-full genomes directly from patient sputum, including mixed and low-concentration samples. This approach significantly reduces the turnaround time for this slow-growing bacterium while maintaining high accuracy in detecting clinically relevant mutations, including those associated with drug resistance. Given the global burden of tuberculosis and the critical need for faster diagnostic solutions, we believe our method has the potential to improve clinical decision-making and public health strategies.

Mycobacterium tuberculosis↗
PubMed2026

Large-scale benchmarking of prokaryotic annotation tools across thousands of species.

BACKGROUND: Genome annotation is an important step in deriving functional meaning from prokaryotic sequencing data, yet systematic evaluations guiding tool selection are lacking. We present the first large-scale investigation of four prominent open-source annotation tools (Prokka, Bakta, EggNOG-mapper, and PGAP) across 156,033 diverse genomes. This includes Escherichia coli strains for baseline performance, thousands of archaea and bacteria genomes, as well as frameshifted and metagenome-assembled genomes. RESULTS: Bakta excels in annotating high-quality bacterial genomes, while PGAP was better for archaeal genomes and challenging bacterial assemblies, including metagenome-assembled, fragmented, or contaminated samples. For Gene Ontology annotation, PGAP consistently provides broader term coverage, whereas EggNOG-mapper offers more terms per feature. CONCLUSIONS: Our findings highlight tool-specific strengths crucial for selecting optimal solutions based on genome quality, taxonomy, and origin (e.g. MAGs). This study provides an evidence-based guide for users and informs future tool development.

Molecular Sequence Annotation↗
PubMed2026

Symbiotic interactions and climate change implications of the octocoral microbiome.

Octocorals are vital components of tropical, temperate, and cold-water benthic marine ecosystems. Their associated microbiomes, comprising microeukaryotes, prokaryotes, and viruses, are increasingly recognised as central to host health, nutrient cycling, and chemical defence. Metagenomics and amplicon sequencing have uncovered taxonomic and functional complexity within these microbial communities, revealing patterns of host specificity and health status, along with seasonality and geographic structuring. However, anthropogenic stressors, particularly those associated with global climate change, exert intense pressure on coral-dominated ecosystems, leading to complex and poorly understood local and regional patterns of octocoral expansion and mortality. Microbial interactions may be a main driver of these contrasting outcomes by mediating the ecological resilience of octocorals to environmental stress. We synthesise the current state of research on the diversity, organisation, and function of the octocoral microbiome, and identify critical knowledge gaps on octocoral holobionts relative to scleractinian corals. Our meta-analysis of 79 publicly available bacterial genomes from octocorals reveals group-specific specialisation in denitrification and nitrate assimilation, along with widespread capacities for essential amino acid, cofactor, and vitamin production, suggesting important contributions to nutrient cycling in the holobiont. While sampling efforts between cultured and uncultured lineages are even, our genomic survey reveals strong sampling bias toward the Atlantic Ocean, temperate gorgonians, and healthy host states, whereas bacterial genomes representing the pathobiome, tropical and/or deep-sea regions, and other octocoral taxa remain underrepresented. Accordingly, we propose future research directions to advance understanding of octocoral microbiome ecology and its role in the resilience of tropical, temperate and cold-water coral reefs.

Endozoicomonadaceae↗
PubMed2025

Pitfalls of bacterial pan-genome analysis approaches: a case study of Mycobacterium tuberculosis and two less clonal bacterial species.

SUMMARY: Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety in methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. Using Mycobacterium tuberculosis, a clonally evolving bacterium with a small accessory genome, as a model system, we systematically evaluated sources of variability in pan-genome estimates. Our analysis revealed that differences in assembly type (short-read versus hybrid), annotation pipeline, and pan-genome software, significantly impact predictions of core and accessory genome size. Extending our analysis to two additional bacterial species, Escherichia coli and Staphylococcus aureus, we observed consistent tool-dependent biases but species-specific patterns in pan-genome variability. Our findings highlight the importance of integrating nucleotide- and protein-level analyses to improve the reliability and reproducibility of pan-genome studies across diverse bacterial populations. AVAILABILITY AND IMPLEMENTATION: Panqc is freely available under an MIT license at https://github.com/maxgmarin/panqc.

Genome, Bacterial↗
PubMed1975

Temperature dependence of strand separation of the DNA molecules containing integrated SV40 DNA in transformed cells.

It has previously been demonstrated that the DNA molecules containing a genetic marker from one region of a bacterial genome undergo complete strand separation at temperatures usually different from the molecules containing a genetic marker from another region of the genome. The experiments also showed that if a group of molecules undergo complete strand separation over a narrow temperature range, of the order of one to three degrees, it is highly likely that they all come from one region of the bacterial genome. The purpose of the work reported here was to establish appropriate procedures for doing a similar analysis of the DNA molecules containing the integrated SV40 DNA in transformed mouse cells. One result of interest is that in the 11A8 cell line about 40 per cent of the integrated SV40 DNA detectable by an RNA-DNA hybridization assay can be accounted for by a group of molecules which undergo complete strand separation within a 1.0 degree C interval.

Binding Sites↗
PubMed2024

Natural variation of immune epitopes reveals intrabacterial antagonism.

Plants and animals detect biomolecules termed Microbe-Associated Molecular Patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multi-copy MAMPs on immune induction is unknown. Here we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple Cold Shock Proteins and 46% carry a non-immunogenic form. We uncovered a new mechanism for immune evasion, intrabacterial antagonism, where a non-immunogenic Cold Shock Protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

comparative genomics↗
PubMed2025

Why do bacteria accumulate antiphage defence systems?

While it is well established that bacterial genomes encode multiple and diverse antiphage systems, the reasons for their co-occurrence and their heterogeneous distribution remain debated. This review examines why bacteria accumulate antiphage systems and how this influences phage-bacteria interactions, particularly in the context of phage therapy. Two main hypotheses may explain this phenomenon: (i) the pan-immunity hypothesis, which suggests that defence system accumulation provides protection against phage predation at the community level, and (ii) mobile genetic element (MGE) competition, where defence systems primarily protect intra-bacterial MGEs against other ones rather than the bacterial host itself. The ecological context also influences the distribution of antiphage systems, with defencee accumulation shaping phage-bacteria interactions in diverse communities but playing a lesser role at the species level, potentially explaining why multiple defences do not strongly limit phage host range in therapeutic settings. Finally, we address the challenges in understanding the drivers shaping the distribution of defence systems across bacterial genomes (expressions, costs, etc.) and their implications for elucidating the ecological role of defence systems and optimizing phage therapy strategies.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.

Bacteria↗
PubMed2025

Genome size estimation from long read overlaps.

MOTIVATION: Accurate genome size estimation is an important component of genomic analyses such as assembly and coverage calculation, though existing tools are primarily optimized for short-read data. RESULTS: We present LRGE, a novel tool that uses read-to-read overlap information to estimate genome size in a reference-free manner. LRGE calculates per-read genome size estimates by analysing the expected number of overlaps for each read, considering read lengths and a minimum overlap threshold. The final size is taken as the median of these estimates, ensuring robustness to outliers such as reads with no overlaps. Additionally, LRGE provides an expected confidence range for the estimate. We validate LRGE on a large, diverse bacterial dataset and confirm it generalizes to eukaryotic datasets. On bacterial genomes, LRGE outperforms k-mer-based methods in both accuracy and computational efficiency and produces genome size estimates comparable to those from assembly-based approaches, like Raven, while using significantly less computational resources. AVAILABILITY AND IMPLEMENTATION: Our method, LRGE (Long Read-based Genome size Estimation from overlaps), is implemented in Rust and is available as a precompiled binary for most architectures, a Bioconda package, a prebuilt container image, and a crates.io package as a binary (lrge) or library (liblrge). The source code is available at https://github.com/mbhall88/lrge and an archive at https://doi.org/10.5281/zenodo.17183812 under an MIT license.

Genome Size↗
PubMed2025

PMkbase (version 1.0): an interactive web-based tool for tracking bacterial metabolic traits using phenotype microarrays made interoperable with sequence information and visualizing/processing PM data.

Bacteria showcase remarkable metabolic diversity and traits, even among strains of the same species. In recent years, a large number of bacterial genomes have been sequenced, leading to the elucidation and documentation of genomic differences and commonalities across and within species. Genome-scale metabolic reconstructions, which are often defined and curated using data from phenotype microarrays, elucidate the differences in metabolic traits resulting from genomic diversity. These microarrays measure cellular respiration on a variety of carbon, nitrogen, phosphorus, and sulfur sources and various stressors and inhibitors over a period of time to determine the metabolic activity of a given strain. Despite their popularity in measuring bacterial metabolic activity and traits, no public databases that allow researchers to warehouse, access, and analyze this information currently exist. Additionally, there are no publicly available tools that allow researchers to view the variance of these metabolic traits across bacterial strains. To address this need, we present Phenotype Microarray Knowledgebase (PMkbase [version 1.0], https://pmkbase.com/), an interactive database that acts as a repository of phenotype microarray (PM) data with integrated sequence information. Binarized activity calls, along with associated kinetic parameters, are made for all metabolic substrates and inhibitors. Users can upload their own data for analysis and visualization and to perform quality checks on their experiments. PMkbase will address an unmet need to track and view bacterial metabolic traits and provide researchers with valuable information to develop metabolic models, enrich pangenomic analyses, and design new experiments.IMPORTANCEBacterial species can be differentiated by their metabolic profiles or the type of nutrients they consume. Interestingly, strains within the same species also display differences in nutrient consumption. Phenotype microarrays are a high-throughput, widely used technology to measure which substrates can be metabolized by various microbial strains and the extent to which inhibitors can affect it. Despite their widespread use, public databases to parse and access this data type at scale do not exist. PMkbase, which contains 9,024 data points for nitrogen substrate utilization, 41,664 data points for carbon substrate utilization, 8,448 data points for phosphorus/sulfur substrate utilization, and 27,264 data points on various antibiotics across three species (Escherichia coli, Pseudomonas putida, and Staphylococcus aureus), has been developed to allow researchers to freely access PM data, along with enriching the data with sequence information.

Bacteria↗
PubMed2026

Plasmid-mediated dissemination of blaKPC-3 and multidrug resistance genes among different species of Klebsiella.

Carbapenem resistance is a serious threat to public health because carbapenems are used as last-resort antibiotics. Carbapenem resistance gene KPC (Klebsiella pneumoniae carbapenemase) inactivates a broad range of β-lactam substrates. In this manuscript, we examined intra-host transmission of blaKPC-3 via interspecies gene transfer. Two carbapenem-resistant Klebsiella pneumoniae isolates and one Klebsiella michiganensis isolate were identified from two patients. Genetic relations of these isolates were investigated with whole-genome sequencing (WGS). Hybrid assembly of bacterial genomes showed the three isolates carried plasmids that harbor common antimicrobial resistance (AMR) gene clusters that confer multidrug-class resistance, including carbapenems. Our results suggest that AMR gene clusters are disseminated across the species as fragments rather than as complete, intact plasmids.IMPORTANCEAn antimicrobial resistance gene cluster encompassing multiple drug classes on plasmids could lead a drug-susceptible pathogen to gain multidrug resistance. Interspecies gene transfer enables K. michiganensis to become multidrug-resistant through the acquisition of clustered, plasmid-encoded resistance genes spanning multiple antibiotic classes.

Plasmids↗
PubMed2026

Applications of transposon-insertion sequencing for understanding bacterial physiology.

Transposon-insertion sequencing (Tn-seq) couples transposon mutagenesis with next-generation sequencing to identify the transposon insertion site for thousands of mutants in parallel. It is a powerful technology with a myriad of uses beyond the identification of essential genes required for a cell to grow and divide. Tn-seq is particularly useful as a high-throughput method to assign function to function-unknown genes, which have increased steadily with the abundance of newly sequenced bacterial genomes. Tn-seq has now been adapted for use in over 100 bacterial species. Here, we summarize the applications of Tn-seq for querying bacterial physiology and discuss some of the possible applications for the future.

DNA Transposable Elements↗
PubMed2025

Effectidor II: a pan-genomic AI-based algorithm for the prediction of type III secretion system effectors.

MOTIVATION: Type III secretion systems are used by many Gram-negative bacteria to inject type 3 effectors (T3Es) directly into eukaryotic cells, promoting disease or provoking immune response. Because of these opposing evolutionary forces, T3E repertoires often vary within taxonomic groups. Identifying the full effector gene repertoire in genomes of related individuals is crucial for determining core and specialized effectors, understanding the disease dynamics, and developing appropriate management strategies against pathogens. It can also help uncover novel T3Es that have recently emerged in a population. Our previously published Effectidor web server successfully addressed the challenge of identifying T3Es in a single bacterial genome. Here, we enriched the web server with various novel capabilities, including the identification of T3Es from multiple genome sequences simultaneously. RESULTS: We present Effectidor II, a web server that relies on machine learning to predict T3E-encoding genes within bacterial pan-genomes. We demonstrate the benefit of learning based on features extracted from the entire sequences comprising the pan-genome and report a novel T3E discovered by it in Xanthomonas euroxanthea. AVAILABILITY AND IMPLEMENTATION: Effectidor II is available at: https://effectidor.tau.ac.il and the source code is available at: https://github.com/naamawagner/Effectidor. A stand-alone version of Effectidor II is available at: https://github.com/naamawagner/Effectidor/tree/StandAlone. The source code for the standalone version and the data used in this work are also provided in https://doi.org/10.5281/zenodo.15081636.

Type III Secretion Systems↗
PubMed2026

Global lessons from antibiotic resistance: Metformin-hydrolysing genes in transposable elements, a new threat for type II diabetic patients?

OBJECTIVES: To investigate the evolutionary origin, genomic mobility, and potential dissemination of metformin-hydrolysing genes (mfmAB), and to assess whether environmental selection by metformin pollution may drive the emergence of transferable pharmaceutical-degrading traits analogous to antibiotic resistance. METHODS: Large-scale comparative genomics was performed using publicly available bacterial genomes carrying mfmAB homologs. Phylogenomic reconstruction, average nucleotide identity analysis, genomic context comparison, plasmid characterization, and insertion sequence mapping were used to infer evolutionary history and identify mechanisms of horizontal gene transfer. RESULTS: mfmAB homologs were identified in twelve Aminobacter and three Pseudomonas genomes within a conserved ∼8.2 kb gene cluster. Phylogenomic analyses showed that metformin-degrading capacity emerged independently in multiple Aminobacter lineages across distinct continents, consistent with convergent evolution under anthropogenic selective pressure. Genomic comparisons indicated a chromosomal origin of mfmAB, followed by mobilization onto conjugative plasmids through IS1182-mediated transposition. In Pseudomonas, additional IS3/IS6-mediated transposition events integrated mfmAB into diverse plasmid backbones, frequently within composite transposons also encoding guanylurea and biguanide degradation pathways (guuH, bguH). These findings reveal a dynamic modular assembly of metabolic functions facilitating adaptation to pharmaceutical pollutants. CONCLUSIONS: Metformin pollution appears to promote the emergence and mobilization of pharmaceutical-degrading genes through mechanisms paralleling antibiotic resistance evolution. Although no clinical impact has yet been demonstrated, the potential spread of such genes into human-associated microbiomes and their possible co-selection with antibiotic resistance determinants represent an emerging One Health concern. Environmental surveillance of pharmaceutical-degrading genes is warranted to anticipate future threats to drug efficacy.

Convergent evolution↗