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Introns were traditionally thought to be rare in bacteria, yet their occurrence and diversity may have been underestimated. Here, we present the first comprehensive overview of group I and group II introns in Patescibacteria. While most introns are readily identified, group I introns inserted at position 35/36 within the anticodon loop often escape detection by standard annotation tools; through experimental verification, we demonstrate that these introns are accurately spliced despite their unusual insertion site. Notably, approximately 40% of genomes in both Patescibacteria and Cyanobacteriota harbor group I introns; however, while around 20% of Cyanobacteriota genomes also contain group II introns, none were detected in Patescibacteria. These results illustrate a previously overlooked phylogenetic distribution of group I and group II introns across the bacterial domain.

Mobile genetic elements are important contributors to horizontal gene transfer, including of antimicrobial resistance genes. Understanding which microbes carry these mobile elements is vital to assess the spread of resistance. Here, we use a nanopore adaptive sampling approach to increase detection of low-abundance bacteria and mobile elements and use DNA methylation detection and Hi-C sequencing to determine mobile element hosts. By introducing a known bacterium and isolating a native strain, we could evaluate the performance of these methods, indicating that although powerful, they require careful experimental design, interpretation, and validation. However, when combined, these approaches enable a comprehensive investigation of mobile elements and gene transfer dynamics in complex environments.

Mobile genetic elements (MGEs) or mobilomes promote the mobilization and dissemination of antimicrobial resistance genes (ARGs), serving as critical drivers for antimicrobial resistance (AMR) accumulation, interaction, and persistence. However, systematic and quantitative evaluations of the role of mobilome in spreading resistome in a bacterial pathogen remain unaddressed, partially due to the lack of closed genomes. Here, we examined MGEs across 1,817 Salmonella isolates with complete genomic sequences from 58 countries between 1911 and 2022. We found the plasmid harboring 69.8% ARGs to be the largest ARG reservoir, correlated with serovar-based evolution in most Salmonella lineages. Prophages, specifically RCS47 and SJ46, play a crucial role in the plasmids’ plasticity and the acquisition of ARGs. Furthermore, distinct ARG accumulation, including resistance toward last-resort antibiotics, exhibited an MGE-favored manner. Certain socioeconomic and ecological factors, as additional layers of mediators, are associated with the preferential distribution of MGE-mediated ARGs in Salmonella . Collectively, this study demonstrated an uncharted knowledge of the segmentation of Salmonella resistome driven by mobilome, elucidating dynamic drivers and distinct mediators for resistome development that are of immediate relevance for targeted interventions. Antimicrobial resistance (AMR) has become a significant global challenge, with an estimated 10 million deaths annually by 2050. The emergence of AMR is mainly attributed to mobile genetic elements (MGEs or mobilomes), which accelerate wide dissemination among pathogens. The interaction between mobilomes and AMR genes (or resistomes) in Salmonella , a primary cause of diarrheal diseases that results in over 90 million cases annually, remains poorly understood. The available fragmented or incomplete genomes remain a significant limitation in investigating the relationship between AMR and MGEs. Here, we collected the most extensive closed Salmonella genomes ( n = 1,817) from various sources across 58 countries. Notably, our results demonstrate that resistome transmission between Salmonella lineages follows a specific pattern of MGEs and is influenced by external drivers, including certain socioeconomic factors. Therefore, targeted interventions are urgently needed to mitigate the catastrophic consequences of Salmonella AMR.

Integrative and conjugative elements (ICEs) are mobile DNA elements that drive bacterial conjugation, a major process by which bacteria exchange genes. Although conjugation has been studied for decades, the focus has been almost exclusively on donor cells and the ICE itself, leaving the role of recipient cells largely overlooked. Using the wall-less ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis as minimal models, we discovered that a single recipient lipoprotein is required for efficient ICE uptake. Our data show that nucleoside recognition by P48, but not transport, is critical for conjugation, revealing an unexpected mechanistic link between nutrient sensing and gene acquisition. These findings shift the paradigm of conjugation from a donor-driven process to one jointly determined by donor and recipient functions. By identifying a recipient-encoded determinant of ICE transfer, this work opens new avenues to control horizontal gene flow in both pathogenic and engineered bacteria.

Vancomycin is an antibiotic used to treat infections caused by multidrug-resistant Gram-positive bacteria. Vancomycin resistance is common in clinical isolates of the Gram-positive pathogen Enterococcus faecium . Vancomycin-resistant E. faecium (VREfm) is a significant public health concern because of limited treatment options. Genomic surveillance can be used to monitor VREfm transmission and evolution. Genomic analysis of VREfm has not been reported for the Dallas/Fort Worth/Arlington, TX, area, which is currently the 4th largest metropolitan area in the United States. Our study aimed to address this gap in knowledge by analyzing the genomes of 46 VREfm strains and 1 vancomycin-sensitive comparator collected during routine fecal surveillance of high-risk patients upon admission to a Dallas, TX, hospital system (August to October 2015). Thirty-one complete and 16 draft genome sequences were generated. The closed VREfm genomes possessed up to 12 extrachromosomal elements each. Overall, 251 closed putative plasmid sequences assigned to previously described and newly defined rep family types were obtained. Phylogenetic analysis identified 10 different sequence types (STs) among the isolates, with the most prevalent being ST17 and ST18. Strikingly, all but three of the VREfm isolates encoded vanA -type vancomycin resistance within Tn 1546 -like elements on a pRUM-like ( rep17 ) plasmid backbone. Relative to a previously reported typing scheme for the vanA -carrying Tn 1546 , new variants of the Tn 1546 were identified that harbored a combination of 7 insertion sequences (IS), including 3 novel IS elements reported here (IS Efa16 , IS Efa17 , and IS Efa18 ). We conclude that pRUM-like plasmids are important vectors for vancomycin resistance in the Dallas, TX, area and should be a focus of plasmid surveillance efforts. IMPORTANCE Vancomycin is an antibiotic used to treat infections caused by multidrug-resistant Gram-positive bacteria. Vancomycin resistance is common in clinical isolates of the Gram-positive pathogen Enterococcus faecium . Among E. faecium strains, vancomycin resistance genes can be disseminated by plasmids with different host ranges and transfer efficiencies. Surveillance of resistance plasmids is critical to understanding antibiotic resistance transmission. This study analyzed the genome sequences of VREfm isolates collected from the Dallas, TX, area, with particular focus on the mobile elements associated with vancomycin resistance genes. We found that a single plasmid family, the pRUM-like family, was associated with vancomycin resistance in the majority of isolates sampled. Our work suggests that the pRUM-like plasmids should continue to be studied to understand their mechanisms of maintenance, transmission, and evolution in VREfm.

Background Symbioses between chemoautotrophic bacteria and marine invertebrates are rare examples of living systems that are virtually independent of photosynthetic primary production. These associations have evolved multiple times in marine habitats, such as deep-sea hydrothermal vents and reducing sediments, characterized by steep gradients of oxygen and reduced chemicals. Due to difficulties associated with maintaining these symbioses in the laboratory and culturing the symbiotic bacteria, studies of chemosynthetic symbioses rely heavily on culture independent methods. The symbiosis between the coastal bivalve, Solemya velum , and its intracellular symbiont is a model for chemosynthetic symbioses given its accessibility in intertidal environments and the ability to maintain it under laboratory conditions . To better understand this symbiosis, the genome of the S. velum endosymbiont was sequenced. Results Relative to the genomes of obligate symbiotic bacteria, which commonly undergo erosion and reduction, the S. velum symbiont genome was large (2.7 Mb), GC-rich (51%), and contained a large number (78) of mobile genetic elements. Comparative genomics identified sets of genes specific to the chemosynthetic lifestyle and necessary to sustain the symbiosis. In addition, a number of inferred metabolic pathways and cellular processes, including heterotrophy, branched electron transport, and motility, suggested that besides the ability to function as an endosymbiont, the bacterium may have the capacity to live outside the host. Conclusions The physiological dexterity indicated by the genome substantially improves our understanding of the genetic and metabolic capabilities of the S. velum symbiont and the breadth of niches the partners may inhabit during their lifecycle.

Sarunyou Chusri, Division of Infectious Diseases, Department of Internal Medicine, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand, Tel +66 8 973 40446, Email sarunyouchusri@hotmail.comPurpose: The spread of New Delhi metallo-β-lactamase (NDM) encoded by the blaNDM gene has been a global health crisis for many years. Most of blaNDM-harboring bacteria commonly carry various antimicrobial resistance (AMR) genes on their chromosomes or plasmids, leading to limited treatment options. Thus, we aimed to evaluate the synergistic effects of fosfomycin in combination with other antimicrobial agents against blaNDM-harboring carbapenem-resistant Escherichia coli (CREC) and to characterize the whole-genome and plasmid sequences of these pathogens.Methods: Thirty-eight CREC isolates were collected from patients in the Medicine Ward, Songklanagarind Hospital, Thailand. The activity of fosfomycin in combination with other antimicrobial agents against CREC isolates harboring blaNDM on the plasmid was evaluated using the checkerboard method. In this method, the serial dilutions of two antibiotics were mixed with the cultured CREC, the mixtures were incubated, and FICI was calculated to interpret the synergistic activity of the combination. The whole-genome and particular plasmids of these pathogens were sequenced using next-generation sequencing. Sequence analysis, especially on antimicrobial resistance (AMR) genes, mobile-genetic elements (MGEs), and virulence genes was performed using many bioinformatics tools.Results: Of the E. coli 38 isolates, only 3 isolates contained the blaNDM-1 gene, which is located on the IncN2 plasmid. The combinations of fosfomycin with aminoglycosides, colistin, tigecycline, sitafloxacin, and ciprofloxacin were synergies against blaNDM-1-harboring CREC isolates. Genomic analysis revealed that these isolates harbored many β-lactam resistance genes and other AMR genes that may confer resistance to aminoglycoside, fluoroquinolone, rifampicin, trimethoprim, sulfonamide, tetracycline, and macrolide. Also, various MGEs, especially the blaNDM-1-bearing IncN2 plasmid, were present in these isolates.Conclusion: Our study demonstrated some synergistic effects of antimicrobial combination against CREC isolates harboring blaNDM-1 on the IncN2 plasmid. Also, our data on the whole-genome and plasmid sequences might be beneficial in the control of the spread of blaNDM-1-harboring CREC isolates. The linkages between blaNDM-1-carrying plasmid, patient information, and time of collection will be elucidated to track the horizontal gene transfer in the future.

SignificanceGenome sequences are being produced for more and more eukaryotic species. The bulk of these genomes are composed of parasitic, self-mobilizing transposable elements (TEs) that play important roles in organismal evolution. Thus there is a pressing need for developing software that can accurately identify the diverse set of TEs dispersed in genome sequences. Here we introduce RepeatModeler2, an easy-to-use package for the production of reference TE libraries which can be applied to any eukaryotic species. Through several major improvements over the previous version, RepeatModeler2 is able to produce libraries that recapitulate the known composition of three model species with some of the most complex TE landscapes. Thus RepeatModeler2 will greatly enhance the discovery and annotation of TEs in genome sequences. The accelerating pace of genome sequencing throughout the tree of life is driving the need for improved unsupervised annotation of genome components such as transposable elements (TEs). Because the types and sequences of TEs are highly variable across species, automated TE discovery and annotation are challenging and time-consuming tasks. A critical first step is the de novo identification and accurate compilation of sequence models representing all of the unique TE families dispersed in the genome. Here we introduce RepeatModeler2, a pipeline that greatly facilitates this process. This program brings substantial improvements over the original version of RepeatModeler, one of the most widely used tools for TE discovery. In particular, this version incorporates a module for structural discovery of complete long terminal repeat (LTR) retroelements, which are widespread in eukaryotic genomes but recalcitrant to automated identification because of their size and sequence complexity. We benchmarked RepeatModeler2 on three model species with diverse TE landscapes and high-quality, manually curated TE libraries: Drosophila melanogaster (fruit fly), Danio rerio (zebrafish), and Oryza sativa (rice). In these three species, RepeatModeler2 identified approximately 3 times more consensus sequences matching with >95% sequence identity and sequence coverage to the manually curated sequences than the original RepeatModeler. As expected, the greatest improvement is for LTR retroelements. Thus, RepeatModeler2 represents a valuable addition to the genome annotation toolkit that will enhance the identification and study of TEs in eukaryotic genome sequences. RepeatModeler2 is available as source code or a containerized package under an open license (https://github.com/Dfam-consortium/RepeatModeler, http://www.repeatmasker.org/RepeatModeler/).

It is well established that plasmids play an important role in the dissemination of antimicrobial resistance (AMR) genes; however, little is known about the role of the underlying interactions between different plasmid categories and other mobile genetic elements (MGEs) in shaping the promiscuous spread of AMR genes. Here, we developed a tool designed for plasmid classification, AMR gene annotation, and plasmid visualization and found that most plasmid-borne AMR genes, including those localized on class 1 integrons, are enriched in conjugative plasmids. Notably, we report the discovery and characterization of a massive insertion sequence (IS)-associated AMR gene transfer network (245 combinations covering 59 AMR gene subtypes and 53 ISs) linking conjugative plasmids and phylogenetically distant pathogens, suggesting a general evolutionary mechanism for the horizontal transfer of AMR genes mediated by the interaction between conjugative plasmids and ISs. Moreover, our experimental results confirmed the importance of the observed interactions in aiding the horizontal transfer and expanding the genetic range of AMR genes within complex microbial communities.

CRISPR-Cas, the bacterial and archaeal adaptive immunity systems, encompass a complex machinery that integrates fragments of foreign nucleic acids, mostly from mobile genetic elements (MGE), into CRISPR arrays embedded in microbial genomes. Transcripts of the inserted segments (spacers) are employed by CRISPR-Cas systems as guide (g)RNAs for recognition and inactivation of the cognate targets. The CRISPR-Cas systems consist of distinct adaptation and effector modules whose evolutionary trajectories appear to be at least partially independent. Comparative genome analysis reveals the origin of the adaptation module from casposons, a distinct type of transposons, which employ a homologue of Cas1 protein, the integrase responsible for the spacer incorporation into CRISPR arrays, as the transposase. The origin of the effector module(s) is far less clear. The CRISPR-Cas systems are partitioned into two classes, class 1 with multisubunit effectors, and class 2 in which the effector consists of a single, large protein. The class 2 effectors originate from nucleases encoded by different MGE, whereas the origin of the class 1 effector complexes remains murky. However, the recent discovery of a signalling pathway built into the type III systems of class 1 might offer a clue, suggesting that type III effector modules could have evolved from a signal transduction system involved in stress-induced programmed cell death. The subsequent evolution of the class 1 effector complexes through serial gene duplication and displacement, primarily of genes for proteins containing RNA recognition motif domains, can be hypothetically reconstructed. In addition to the multiple contributions of MGE to the evolution of CRISPR-Cas, the reverse flow of information is notable, namely, recruitment of minimalist variants of CRISPR-Cas systems by MGE for functions that remain to be elucidated. Here, we attempt a synthesis of the diverse threads that shed light on CRISPR-Cas origins and evolution. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.