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840800.9984The defensome of prokaryotes in aquifers. Groundwater harbors a pristine biosphere where microbes co-evolve with less human interference, yet the ancient and ongoing arms race between prokaryotes and viruses remains largely unknown in such ecosystems. Based on our recent nationwide groundwater monitoring campaign across China, we construct a metagenomic groundwater prokaryotic defensome catalogue (GPDC), encompassing 190,810 defense genes, 90,824 defense systems, 139 defense families, and 669 defense islands from 141 prokaryotic phyla. Over 94% of the defense genes in GPDC are novel and contribute vast microbial immune resources in groundwater. We find that candidate phyla radiation (CPR) bacteria possess higher defense system density and diversity against intense phage infection, while microbes as a whole exhibit an inverse relationship between defense systems and adaptive traits like resistance genes in groundwater. We further identify five first-line defense families covering 69.2% of the total defense systems, and high-turnover accessory immune genes are mostly conveyed to defense islands by mobile genetic elements. Our study also reveals viral resistance to microbial defense through co-localized anti-defense genes and interactions between CRISPR-Cas9 and anti-CRISPR protein. These findings expand our understanding of microbial immunity in pristine ecosystems and offer valuable immune resources for potential biotechnological applications.202540659683
840910.9981Comparative genomics reveals key adaptive mechanisms in pathogen host-niche specialization. INTRODUCTION: Understanding the key factors that enable bacterial pathogens to adapt to new hosts is crucial, as host-microbe interactions not only influence host health but also drive bacterial genome diversification, thereby enhancing pathogen survival in various ecological niches. METHODS: We conducted a comparative genomic analysis of 4,366 high-quality bacterial genomes isolated from various hosts and environments. Bioinformatics databases and machine learning approaches were used to identify genomic differences in functional categories, virulence factors, and antibiotic resistance genes across different ecological niches. RESULTS: Significant variability in bacterial adaptive strategies was observed. Human-associated bacteria, particularly from the phylum Pseudomonadota, exhibited higher detection rates of carbohydrate-active enzyme genes and virulence factors related to immune modulation and adhesion, indicating co-evolution with the human host. In contrast, bacteria from environmental sources, particularly those from the phyla Bacillota and Actinomycetota, showed greater enrichment in genes related to metabolism and transcriptional regulation, highlighting their high adaptability to diverse environments. Bacteria from clinical settings had higher detection rates of antibiotic resistance genes, particularly those related to fluoroquinolone resistance. Animal hosts were identified as important reservoirs of resistance genes. Key host-specific bacterial genes, such as hypB, were found to potentially play crucial roles in regulating metabolism and immune adaptation in human-associated bacteria. DISCUSSION: These findings highlight niche-specific genomic features and adaptive mechanisms of bacterial pathogens. This study provides valuable insights into the genetic basis of host-pathogen interactions and offers evidence to inform pathogen transmission control, infection management, and antibiotic stewardship.202540547794
966520.9981Time-calibrated genomic evolution of a monomorphic bacterium during its establishment as an endemic crop pathogen. Horizontal gene transfer is of major evolutionary importance as it allows for the redistribution of phenotypically important genes among lineages. Such genes with essential functions include those involved in resistance to antimicrobial compounds and virulence factors in pathogenic bacteria. Understanding gene turnover at microevolutionary scales is critical to assess the pace of this evolutionary process. Here, we characterized and quantified gene turnover for the epidemic lineage of a bacterial plant pathogen of major agricultural importance worldwide. Relying on a dense geographic sampling spanning 39 years of evolution, we estimated both the dynamics of single nucleotide polymorphism accumulation and gene content turnover. We identified extensive gene content variation among lineages even at the smallest phylogenetic and geographic scales. Gene turnover rate exceeded nucleotide substitution rate by three orders of magnitude. Accessory genes were found preferentially located on plasmids, but we identified a highly plastic chromosomal region hosting ecologically important genes such as transcription activator-like effectors. Whereas most changes in the gene content are probably transient, the rapid spread of a mobile element conferring resistance to copper compounds widely used for the management of plant bacterial pathogens illustrates how some accessory genes can become ubiquitous within a population over short timeframes.202133305421
435730.9980Comparative genomic analysis of 255 Oenococcus oeni isolates from China: unveiling strain diversity and genotype-phenotype associations of acid resistance. Oenococcus oeni, the only species of lactic acid bacteria capable of fully completing malolactic fermentation under challenging wine conditions, continues to intrigue researchers owing to its remarkable adaptability, particularly in combating acid stress. However, the mechanism underlying its superior adaptation to wine stresses still remains elusive due to the lack of viable genetic manipulation tools for this species. In this study, we conducted genomic sequencing and acid resistance phenotype analysis of 255 O. oeni isolates derived from diverse wine regions across China, aiming to elucidate their strain diversity and genotype-phenotype associations of acid resistance through comparative genomics. A significant correlation between phenotypes and evolutionary relationships was observed. Notably, phylogroup B predominantly consisted of acid-resistant isolates, primarily originating from Shandong and Shaanxi wine regions. Furthermore, we uncovered a noteworthy linkage between prophage genomic islands and acid resistance phenotype. Using genome-wide association studies, we identified key genes correlated with acid resistance, primarily involved in carbohydrates and amino acid metabolism processes. This study offers profound insights into the genetic diversity and genetic basis underlying adaptation mechanisms to acid stress in O. oeni.IMPORTANCEThis study provides valuable insights into the genetic basis of acid resistance in Oenococcus oeni, a key lactic acid bacterium in winemaking. By analyzing 255 isolates from diverse wine regions in China, we identified significant correlations between strain diversity, genomic islands, and acid resistance phenotypes. Our findings reveal that certain prophage-related genomic islands and specific genes are closely linked to acid resistance, offering a deeper understanding of how O. oeni adapts to acidic environments. These discoveries not only advance our knowledge of microbial stress responses but also pave the way for selecting and engineering acid-resistant strains, enhancing malolactic fermentation efficiency and wine quality. This research underscores the importance of genomics in improving winemaking practices and addressing challenges posed by high-acidity wines.202540261018
935040.9980Genome DNA Sequence Variation, Evolution, and Function in Bacteria and Archaea. Comparative genomics has revealed that variations in bacterial and archaeal genome DNA sequences cannot be explained by only neutral mutations. Virus resistance and plasmid distribution systems have resulted in changes in bacterial and archaeal genome sequences during evolution. The restriction-modification system, a virus resistance system, leads to avoidance of palindromic DNA sequences in genomes. Clustered, regularly interspaced, short palindromic repeats (CRISPRs) found in genomes represent yet another virus resistance system. Comparative genomics has shown that bacteria and archaea have failed to gain any DNA with GC content higher than the GC content of their chromosomes. Thus, horizontally transferred DNA regions have lower GC content than the host chromosomal DNA does. Some nucleoid-associated proteins bind DNA regions with low GC content and inhibit the expression of genes contained in those regions. This form of gene repression is another type of virus resistance system. On the other hand, bacteria and archaea have used plasmids to gain additional genes. Virus resistance systems influence plasmid distribution. Interestingly, the restriction-modification system and nucleoid-associated protein genes have been distributed via plasmids. Thus, GC content and genomic signatures do not reflect bacterial and archaeal evolutionary relationships.201322772895
838350.9980Novel insights into carbohydrate utilisation, antimicrobial resistance, and sporulation potential in Roseburia intestinalis isolates across diverse geographical locations. Roseburia intestinalis is one of the most abundant and important butyrate-producing human gut anaerobic bacteria that plays an important role in maintaining health and is a potential next-generation probiotic. We investigated the pangenome of 16 distinct strains, isolated over several decades, identifying local and time-specific adaptations. More than 50% of the genes in each individual strain were assigned to the core genome, and 77% of the cloud genes were unique to individual strains, revealing the high level of genome conservation. Co-carriage of the same enzymes involved in carbohydrate binding and degradation in all strains highlighted major pathways in carbohydrate utilization and reveal the importance of xylan, starch and mannose as key growth substrates. A single strain had adapted to use rhamnose as a sole growth substrate, the first time this has been reported. The ubiquitous presence of motility and sporulation gene clusters demonstrates the importance of these phenotypes for gut survival and acquisition of this bacterium. More than half the strains contained functional, potentially transferable, tetracycline resistance genes. This study advances our understanding of the importance of R. intestinalis within the gut ecosystem by elucidating conserved metabolic characteristics among different strains, isolated from different locations. This information will help to devise dietary strategies to increase the abundance of this species providing health benefits.202540089923
921460.9980Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi. The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans(1-6). However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.201930617347
840270.9980Exploring phage-host interactions in Burkholderia cepacia complex bacterium to reveal host factors and phage resistance genes using CRISPRi functional genomics and transcriptomics. Complex interactions of bacteriophages with their bacterial hosts determine phage host range and infectivity. While phage defense systems and host factors have been identified in model bacteria, they remain challenging to predict in non-model bacteria. In this paper, we integrate functional genomics and transcriptomics to investigate phage-host interactions, revealing active phage resistance and host factor genes in Burkholderia cenocepacia K56-2. Burkholderia cepacia complex species are commonly found in soil and are opportunistic pathogens in immunocompromised patients. We studied infection of B. cenocepacia K56-2 with Bcep176, a temperate phage isolated from Burkholderia multivorans. A genome-wide dCas9 knockdown library targeting B. cenocepacia K56-2 was constructed, and a pooled infection experiment identified 63 novel genes or operons coding for candidate host factors or phage resistance genes. The activities of a subset of candidate host factor and resistance genes were validated via single-gene knockdowns. Transcriptomics of B. cenocepacia K56-2 during Bcep176 infection revealed that expression of genes coding for host factor and resistance candidates identified in this screen was significantly altered during infection by 4 h post-infection. Identifying which bacterial genes are involved in phage infection is important to understand the ecological niches of B. cenocepacia and its phages, and for designing phage therapies.IMPORTANCEBurkholderia cepacia complex bacteria are opportunistic pathogens inherently resistant to antibiotics, and phage therapy is a promising alternative treatment for chronically infected patients. Burkholderia bacteria are also ubiquitous in soil microbiomes. To develop improved phage therapies for pathogenic Burkholderia bacteria, or engineer phages for applications, such as microbiome editing, it's essential to know the bacterial host factors required by the phage to kill bacteria, as well as how the bacteria prevent phage infection. This work identified 65 genes involved in phage-host interactions in Burkholderia cenocepacia K56-2 and tracked their expression during infection. These findings establish a knowledge base to select and engineer phages infecting or transducing Burkholderia bacteria.202541036840
986180.9980Comparative genomics of native plasmids from plant pathogenic Gammaproteobacteria. Plasmids are key in the evolution and adaptation of plant pathogenic Gammaproteobacteria (PPG), yet their diversity and functional contributions remain underexplored. Here, comparative genomics revealed extensive variation in plasmid size, replicon types, mobility, and genetic content across PPG. Most plasmids are small (< 200 kb), except in Pantoea, exhibiting high coding densities (76% to 78%). Five ancestral replicon types were identified across multiple orders, indicating vertical descent yet efficient horizontal transfer across taxa, although with limited genetic conservation. Virulence plasmids are widespread (56% to 68%) but differ in virulence gene content across orders: type III effector (T3E) genes are common in Pseudomonas and Xanthomonas, but rare in Enterobacterales and Xylella, aligning with their smaller effector repertoires. Plasmids frequently carry regulatory genes, highlighting their role in bacterial phenotype modulation. Distinct patterns were observed among orders: Enterobacterales plasmids often harbor thiamin biosynthesis operons and transcriptional regulators but lack post-transcriptional regulators, while most Pseudomonas and Xanthomonas plasmids are mobile, enriched in T3E genes, and exhibit high insertion sequence densities, fostering DNA mobility. Resistance to ultraviolet light is common, but not to antimicrobial compounds. These findings highlight the dynamic role of plasmids in spreading adaptive traits, shaping virulence, and driving the evolution of plant pathogenic bacteria.202540273218
838490.9980In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors. Symbiosis is often characterized by co-evolutionary changes in the genomes of the partners involved. An understanding of these changes can provide insight into the nature of the relationship, including the mechanisms that initiate and maintain an association between organisms. In this study we examined the genome sequences of bacteria isolated from the Drosophila melanogaster gut with the objective of identifying genes that are important for function in the host. We compared microbiota isolates with con-specific or closely related bacterial species isolated from non-fly environments. First the phenotype of germ-free Drosophila (axenic flies) was compared to that of flies colonized with specific bacteria (gnotobiotic flies) as a measure of symbiotic function. Non-fly isolates were functionally distinct from bacteria isolated from flies, conferring slower development and an altered nutrient profile in the host, traits known to be microbiota-dependent. Comparative genomic methods were next employed to identify putative symbiosis factors: genes found in bacteria that restore microbiota-dependent traits to gnotobiotic flies, but absent from those that do not. Factors identified include riboflavin synthesis and stress resistance. We also used a phylogenomic approach to identify protein coding genes for which fly-isolate sequences were more similar to each other than to other sequences, reasoning that these genes may have a shared function unique to the fly environment. This method identified genes in Acetobacter species that cluster in two distinct genomic loci: one predicted to be involved in oxidative stress detoxification and another encoding an efflux pump. In summary, we leveraged genomic and in vivo functional comparisons to identify candidate traits that distinguish symbiotic bacteria. These candidates can serve as the basis for further work investigating the genetic requirements of bacteria for function and persistence in the Drosophila gut.201425408687
9606100.9980Rapid identification of key antibiotic resistance genes in E. coli using high-resolution genome-scale CRISPRi screening. Bacteria possess a vast repertoire of genes to adapt to environmental challenges. Understanding the gene fitness landscape under antibiotic stress is crucial for elucidating bacterial resistance mechanisms and antibiotic action. To explore this, we conducted a genome-scale CRISPRi screen using a high-density sgRNA library in Escherichia coli exposed to various antibiotics. This screen identified essential genes under antibiotic-induced stress and offered insights into the molecular mechanisms underlying bacterial responses. We uncovered previously unrecognized genes involved in antibiotic resistance, including essential membrane proteins. The screen also underscored the importance of transcriptional modulation of essential genes in antibiotic tolerance. Our findings emphasize the utility of genome-wide CRISPRi screening in mapping the genetic landscape of antibiotic resistance. This study provides a valuable resource for identifying potential targets for antibiotics or antimicrobial strategies. Moreover, it offers a framework for exploring transcriptional regulatory networks and resistance mechanisms in E. coli and other bacterial pathogens.202540352728
9345110.9980Replacement of the arginine biosynthesis operon in Xanthomonadales by lateral gene transfer. The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the gamma-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution.200818305979
9663120.9979The structure of temperate phage-bacteria infection networks changes with the phylogenetic distance of the host bacteria. With their ability to integrate into the bacterial chromosome and thereby transfer virulence or drug-resistance genes across bacterial species, temperate phage play a key role in bacterial evolution. Thus, it is paramount to understand who infects whom to be able to predict the movement of DNA across the prokaryotic world and ultimately the emergence of novel (drug-resistant) pathogens. We empirically investigated lytic infection patterns among Vibrio spp. from distinct phylogenetic clades and their derived temperate phage. We found that across distantly related clades, infections occur preferentially within modules of the same clade. However, when the genetic distance of the host bacteria decreases, these clade-specific infections disappear. This indicates that the structure of temperate phage-bacteria infection networks changes with the phylogenetic distance of the host bacteria.201830429242
9661130.9979Pangenomes of human gut microbiota uncover links between genetic diversity and stress response. The genetic diversity of the gut microbiota has a central role in host health. Here, we created pangenomes for 728 human gut prokaryotic species, quadrupling the genes of strain-specific genomes. Each of these species has a core set of a thousand genes, differing even between closely related species, and an accessory set of genes unique to the different strains. Functional analysis shows high strain variability associates with sporulation, whereas low variability is linked with antibiotic resistance. We further map the antibiotic resistome across the human gut population and find 237 cases of extreme resistance even to last-resort antibiotics, with a predominance among Enterobacteriaceae. Lastly, the presence of specific genes in the microbiota relates to host age and sex. Our study underscores the genetic complexity of the human gut microbiota, emphasizing its significant implications for host health. The pangenomes and antibiotic resistance map constitute a valuable resource for further research.202439353429
8711140.9979Novel soil bacteria possess diverse genes for secondary metabolite biosynthesis. In soil ecosystems, microorganisms produce diverse secondary metabolites such as antibiotics, antifungals and siderophores that mediate communication, competition and interactions with other organisms and the environment(1,2). Most known antibiotics are derived from a few culturable microbial taxa (3) , and the biosynthetic potential of the vast majority of bacteria in soil has rarely been investigated (4) . Here we reconstruct hundreds of near-complete genomes from grassland soil metagenomes and identify microorganisms from previously understudied phyla that encode diverse polyketide and nonribosomal peptide biosynthetic gene clusters that are divergent from well-studied clusters. These biosynthetic loci are encoded by newly identified members of the Acidobacteria, Verrucomicobia and Gemmatimonadetes, and the candidate phylum Rokubacteria. Bacteria from these groups are highly abundant in soils(5-7), but have not previously been genomically linked to secondary metabolite production with confidence. In particular, large numbers of biosynthetic genes were characterized in newly identified members of the Acidobacteria, which is the most abundant bacterial phylum across soil biomes (5) . We identify two acidobacterial genomes from divergent lineages, each of which encodes an unusually large repertoire of biosynthetic genes with up to fifteen large polyketide and nonribosomal peptide biosynthetic loci per genome. To track gene expression of genes encoding polyketide synthases and nonribosomal peptide synthetases in the soil ecosystem that we studied, we sampled 120 time points in a microcosm manipulation experiment and, using metatranscriptomics, found that gene clusters were differentially co-expressed in response to environmental perturbations. Transcriptional co-expression networks for specific organisms associated biosynthetic genes with two-component systems, transcriptional activation, putative antimicrobial resistance and iron regulation, linking metabolite biosynthesis to processes of environmental sensing and ecological competition. We conclude that the biosynthetic potential of abundant and phylogenetically diverse soil microorganisms has previously been underestimated. These organisms may represent a source of natural products that can address needs for new antibiotics and other pharmaceutical compounds.201829899444
8377150.9979Genome-Wide Association Analyses in the Model Rhizobium Ensifer meliloti. Genome-wide association studies (GWAS) can identify genetic variants responsible for naturally occurring and quantitative phenotypic variation. Association studies therefore provide a powerful complement to approaches that rely on de novo mutations for characterizing gene function. Although bacteria should be amenable to GWAS, few GWAS have been conducted on bacteria, and the extent to which nonindependence among genomic variants (e.g., linkage disequilibrium [LD]) and the genetic architecture of phenotypic traits will affect GWAS performance is unclear. We apply association analyses to identify candidate genes underlying variation in 20 biochemical, growth, and symbiotic phenotypes among 153 strains of Ensifer meliloti For 11 traits, we find genotype-phenotype associations that are stronger than expected by chance, with the candidates in relatively small linkage groups, indicating that LD does not preclude resolving association candidates to relatively small genomic regions. The significant candidates show an enrichment for nucleotide polymorphisms (SNPs) over gene presence-absence variation (PAV), and for five traits, candidates are enriched in large linkage groups, a possible signature of epistasis. Many of the variants most strongly associated with symbiosis phenotypes were in genes previously identified as being involved in nitrogen fixation or nodulation. For other traits, apparently strong associations were not stronger than the range of associations detected in permuted data. In sum, our data show that GWAS in bacteria may be a powerful tool for characterizing genetic architecture and identifying genes responsible for phenotypic variation. However, careful evaluation of candidates is necessary to avoid false signals of association.IMPORTANCE Genome-wide association analyses are a powerful approach for identifying gene function. These analyses are becoming commonplace in studies of humans, domesticated animals, and crop plants but have rarely been conducted in bacteria. We applied association analyses to 20 traits measured in Ensifer meliloti, an agriculturally and ecologically important bacterium because it fixes nitrogen when in symbiosis with leguminous plants. We identified candidate alleles and gene presence-absence variants underlying variation in symbiosis traits, antibiotic resistance, and use of various carbon sources; some of these candidates are in genes previously known to affect these traits whereas others were in genes that have not been well characterized. Our results point to the potential power of association analyses in bacteria, but also to the need to carefully evaluate the potential for false associations.201830355664
9608160.9979A genome-wide atlas of antibiotic susceptibility targets and pathways to tolerance. Detailed knowledge on how bacteria evade antibiotics and eventually develop resistance could open avenues for novel therapeutics and diagnostics. It is thereby key to develop a comprehensive genome-wide understanding of how bacteria process antibiotic stress, and how modulation of the involved processes affects their ability to overcome said stress. Here we undertake a comprehensive genetic analysis of how the human pathogen Streptococcus pneumoniae responds to 20 antibiotics. We build a genome-wide atlas of drug susceptibility determinants and generated a genetic interaction network that connects cellular processes and genes of unknown function, which we show can be used as therapeutic targets. Pathway analysis reveals a genome-wide atlas of cellular processes that can make a bacterium less susceptible, and often tolerant, in an antibiotic specific manner. Importantly, modulation of these processes confers fitness benefits during active infections under antibiotic selection. Moreover, screening of sequenced clinical isolates demonstrates that mutations in genes that decrease antibiotic sensitivity and increase tolerance readily evolve and are frequently associated with resistant strains, indicating such mutations could be harbingers for the emergence of antibiotic resistance.202235672367
9664170.9979Distribution of Genetic Determinants Associated with CRISPR-Cas Systems and Resistance to Antibiotics in the Genomes of Archaea and Bacteria. The CRISPR-Cas system represents an adaptive immune mechanism found across diverse Archaea and Bacteria, allowing them to defend against invading genetic elements such as viruses and plasmids. Despite its broad distribution, the prevalence and complexity of CRISPR-Cas systems differ significantly between these domains. This study aimed to characterize and compare the genomic distribution, structural features, and functional implications of CRISPR-Cas systems and associated antibiotic resistance genes in 30 archaeal and 30 bacterial genomes. Through bioinformatic analyses of CRISPR arrays, cas gene architectures, direct repeats (DRs), and thermodynamic properties, we observed that Archaea exhibit a higher number and greater complexity of CRISPR loci, with more diverse cas gene subtypes exclusively of Class 1. Bacteria, in contrast, showed fewer CRISPR loci, comprising a mix of Class 1 and Class 2 systems, with Class 1 representing the majority (~75%) of the detected systems. Notably, Bacteria lacking CRISPR-Cas systems displayed a higher prevalence of antibiotic resistance genes, suggesting a possible inverse correlation between the presence of these immune systems and the acquisition of such genes. Phylogenetic and thermodynamic analyses further highlighted domain-specific adaptations and conservation patterns. These findings support the hypothesis that CRISPR-Cas systems play a dual role: first, as a defense mechanism preventing the integration of foreign genetic material-reflected in the higher complexity and diversity of CRISPR loci in Archaea-and second, as a regulator of horizontal gene transfer, evidenced by the lower frequency of antibiotic resistance genes in organisms with active CRISPR-Cas systems. Together, these results underscore the evolutionary and functional diversification of CRISPR-Cas systems in response to environmental and selective pressures.202540572209
3777180.9979A Bioinformatic Analysis of Integrative Mobile Genetic Elements Highlights Their Role in Bacterial Adaptation. Mobile genetic elements (MGEs) contribute to bacterial adaptation and evolution; however, high-throughput, unbiased MGE detection remains challenging. We describe MGEfinder, a bioinformatic toolbox that identifies integrative MGEs and their insertion sites by using short-read sequencing data. MGEfinder identifies the genomic site of each MGE insertion and infers the identity of the inserted sequence. We apply MGEfinder to 12,374 sequenced isolates of 9 prevalent bacterial pathogens, including Mycobacterium tuberculosis, Staphylococcus aureus, and Escherichia coli, and identify thousands of MGEs, including candidate insertion sequences, conjugative transposons, and prophage elements. The MGE repertoire and insertion rates vary across species, and integration sites often cluster near genes related to antibiotic resistance, virulence, and pathogenicity. MGE insertions likely contribute to antibiotic resistance in laboratory experiments and clinical isolates. Additionally, we identified thousands of mobility genes, a subset of which have unknown function opening avenues for exploration. Future application of MGEfinder to commensal bacteria will further illuminate bacterial adaptation and evolution.202031862382
8664190.9979Genome-centric metagenomics reveals the host-driven dynamics and ecological role of CPR bacteria in an activated sludge system. BACKGROUND: Candidate phyla radiation (CPR) constitutes highly diverse bacteria with small cell sizes and are likely obligate intracellular symbionts. Given their distribution and complex associations with bacterial hosts, genetic and biological features of CPR bacteria in low-nutrient environments have received increasing attention. However, CPR bacteria in wastewater treatment systems remain poorly understood. We utilized genome-centric metagenomics to answer how CPR communities shift over 11 years and what kind of ecological roles they act in an activated sludge system. RESULTS: We found that approximately 9% (135) of the 1,526 non-redundant bacterial and archaeal metagenome-assembled genomes were affiliated with CPR. CPR bacteria were consistently abundant with a relative abundance of up to 7.5% in the studied activated sludge system. The observed striking fluctuations in CPR community compositions and the limited metabolic and biosynthetic capabilities in CPR bacteria collectively revealed the nature that CPR dynamics may be directly determined by the available hosts. Similarity-based network analysis further confirmed the broad bacterial hosts of CPR lineages. The proteome contents of activated sludge-associated CPR had a higher similarity to those of environmental-associated CPR than to those of human-associated ones. Comparative genomic analysis observed significant enrichment of genes for oxygen stress resistance in activated sludge-associated CPR bacteria. Furthermore, genes for carbon cycling and horizontal gene transfer were extensively identified in activated sludge-associated CPR genomes. CONCLUSIONS: These findings highlight the presence of specific host interactions among CPR lineages in activated sludge systems. Despite the lack of key metabolic pathways, these small, yet abundant bacteria may have significant involvements in biogeochemical cycling and bacterial evolution in activated sludge systems. Video Abstract.202336945052