Mobile Genetic Elements Drive Antimicrobial Resistance Gene Spread in Pasteurellaceae Species

Mobile genetic elements (MGEs) and antimicrobial resistance (AMR) drive important ecological relationships in microbial communities and pathogen-host interaction. In this study, we investigated the resistome-associated mobilome in 345 publicly available Pasteurellaceae genomes, a large family of Gram-negative bacteria including major human and animal pathogens. We generated a comprehensive dataset of the mobilome integrated into genomes, including 10,820 insertion sequences, 2,939 prophages, and 43 integrative and conjugative elements. Also, we assessed plasmid sequences of Pasteurellaceae. Our findings greatly expand the diversity of MGEs for the family, including a description of novel elements. We discovered that MGEs are comparable and dispersed across species and that they also co-occur in genomes, contributing to the family’s ecology via gene transfer. In addition, we investigated the impact of these elements in the dissemination and shaping of AMR genes. A total of 55 different AMR genes were mapped to 721 locations in the dataset. MGEs are linked with 77.6% of AMR genes discovered, indicating their important involvement in the acquisition and transmission of such genes. This study provides an uncharted view of the Pasteurellaceae by demonstrating the global distribution of resistance genes linked with MGEs.

Complete Genome Sequencing and Comparative Phenotypic Analysis Reveal the Discrepancy Between Clostridioides difficile ST81 and ST37 Isolates

Toxin A-negative, toxin B-positive Clostridioides difficile strains, which primarily include the ST81 and ST37 genotypes, are predominant in C. difficile infections leading to antibiotic-associated diarrhea in China. Recently, ST81 has been reported as the most prevalent genotype rather than ST37, although the genetic and functional characteristics of the two genotypes remain ambiguous. In this study, we conducted comprehensive comparative analysis of these two genotypes through complete genome sequencing and phenotypic profiling. The whole genome sequencing revealed that the ST81 and ST37 isolates were closely related genetically with similar gene compositions, and high rate of the core genome shared. The integrative and conjugative elements identified in ST81 were similar to those in ST37, albeit with more diverse and insertion regions. By characterizing the phenotypes related to colonization or survival in the host, we found that the ST81 isolates exhibited robust colonization ability and survival both in vitro and in vivo, enhanced spore production, and slightly increased motility, which may be attributable to the discrepancy in non-synonymous single-nucleotide polymorphisms in the relevant functional genes. Furthermore, the ST81 isolates displayed a significantly higher rate of resistance to fluoroquinolones compared with the ST37 isolates (94.12% vs. 62.5%) and mostly carried the amino acid substitution Asp426Val in GyrB. In summary, the results of our study indicate that ST81 isolates exhibit enhanced ability to transmit between hosts and survive in harsh environments, providing key genetic insights for further epidemiological investigations and surveillance of C. difficile infection.

Biology and engineering of integrative and conjugative elements: Construction and analyses of hybrid ICEs reveal element functions that affect species-specific efficiencies

Integrative and conjugative elements (ICEs) are mobile genetic elements that reside in a bacterial host chromosome and are prominent drivers of bacterial evolution. They are also powerful tools for genetic analyses and engineering. Transfer of an ICE to a new host involves many steps, including excision from the chromosome, DNA processing and replication, transfer across the envelope of the donor and recipient, processing of the DNA, and eventual integration into the chromosome of the new host (now a stable transconjugant). Interactions between an ICE and its hosts throughout the life cycle likely influence the efficiencies of acquisition by new hosts. Here, we investigated how different functional modules of two ICEs, Tn916 and ICEBs1, affect the transfer efficiencies into different host bacteria. We constructed hybrid elements that utilize the high-efficiency regulatory and excision modules of ICEBs1 and the conjugation genes of Tn916. These elements produced more transconjugants than Tn916, likely due to increased excision frequencies. We also found that several Tn916 and ICEBs1 components can substitute for one other. Using B. subtilis donors and three Enterococcus species as recipients, we found that different hybrid elements were more readily acquired by some species than others, demonstrating species-specific interactions in steps of the ICE life cycle. This work demonstrates that hybrid elements utilizing the efficient regulatory functions of ICEBs1 can be built to enable efficient transfer into and engineering of a variety of other species.

Giant Starship elements mobilize accessory genes in fungal genomes

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a novel group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27-393 kb and last shared a common ancestor ca. 400 mya. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize 4 additional Starships whose past and ongoing activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first known agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.

A New ICEclc Subfamily Integrative and Conjugative Element Responsible for Horizontal Transfer of Biphenyl and Salicylic Acid Catabolic Pathway in the PCB-Degrading Strain Pseudomonas stutzeri KF716

Integrative and conjugative elements (ICEs) are chromosomally integrated self-transmissible mobile genetic elements. Although some ICEs are known to carry genes for the degradation o-f aromatic compounds, information on their genetic features is limited. We identified a new member of the ICEclc family carrying biphenyl catabolic bph genes and salicylic acid catabolic sal genes from the PCB-degrading strain Pseudomonas stutzeri KF716. The 117-kb ICEbph-salKF716 contains common core regions exhibiting homology with those of degradative ICEclc from P. knackmussii B13 and ICEXTD from Azoarcus sp. CIB. A comparison of the gene loci collected from the public database revealed that several putative ICEs from P. putida B6-2, P, alcaliphila JAB1, P. stutzeri AN10, and P. stutzeri 2A20 had highly conserved core regions with those of ICEbph-salKF716, along with the variable region that encodes the catabolic genes for biphenyl, naphthalene, toluene, or phenol. These data indicate that this type of ICE subfamily is ubiquitously distributed within aromatic compound-degrading bacteria. ICEbph-salKF716 was transferred from P. stutzeri KF716 to P. aeruginosa PAO1 via a circular extrachromosomal intermediate form. In this study, we describe the structure and genetic features of ICEbph-salKF716 compared to other catabolic ICEs.

Why are rhizobial symbiosis genes mobile?

Rhizobia are one of the most important and best studied groups of bacterial symbionts. They are defined by their ability to establish nitrogen-fixing intracellular infections within plant hosts. One surprising feature of this symbiosis is that the bacterial genes required for this complex trait are not fixed within the chromosome, but are encoded on mobile genetic elements (MGEs), namely plasmids or integrative and conjugative elements. Evidence suggests that many of these elements are actively mobilizing within rhizobial populations, suggesting that regular symbiosis gene transfer is part of the ecology of rhizobial symbionts. At first glance, this is counterintuitive. The symbiosis trait is highly complex, multipartite and tightly coevolved with the legume hosts, while transfer of genes can be costly and disrupt coadaptation between the chromosome and the symbiosis genes. However, horizontal gene transfer is a process driven not only by the interests of the host bacterium, but also, and perhaps predominantly, by the interests of the MGEs that facilitate it. Thus understanding the role of horizontal gene transfer in the rhizobium–legume symbiosis requires a ‘mobile genetic element's-eye view' on the ecology and evolution of this important symbiosis. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

GntR-like SCO3932 Protein Provides a Link between Actinomycete Integrative and Conjugative Elements and Secondary Metabolism

Streptomyces bacteria produce a plethora of secondary metabolites including the majority of medically important antibiotics. The onset of secondary metabolism is correlated with morphological differentiation and controlled by a complex regulatory network involving numerous regulatory proteins. Control over these pathways at the molecular level has a medical and industrial importance. Here we describe a GntR-like DNA binding transcription factor SCO3932, encoded within an actinomycete integrative and conjugative element, which is involved in the secondary metabolite biosynthesis regulation. Affinity chromatography, electrophoresis mobility shift assay, footprinting and chromatin immunoprecipitation experiments revealed, both in vitro and in vivo, SCO3932 binding capability to its own promoter region shared with the neighboring gene SCO3933, as well as promoters of polyketide metabolite genes, such as cpkD, a coelimycin biosynthetic gene, and actII-orf4—an activator of actinorhodin biosynthesis. Increased activity of SCO3932 target promoters, as a result of SCO3932 overproduction, indicates an activatory role of this protein in Streptomyces coelicolor A3(2) metabolite synthesis pathways.

Comparative analysis of integrative and conjugative mobile genetic elements in the genus Mesorhizobium

Members of the Mesorhizobium genus are soil bacteria that often form nitrogen-fixing symbioses with legumes. Most characterised Mesorhizobium spp. genomes are ~8 Mb in size and harbour extensive pangenomes including large integrative and conjugative elements (ICEs) carrying genes required for symbiosis (ICESyms). Here, we document and compare the conjugative mobilome of 41 complete Mesorhizobium genomes. We delineated 56 ICEs and 24 integrative and mobilizable elements (IMEs) collectively occupying 16 distinct integration sites, along with 24 plasmids. We also demonstrated horizontal transfer of the largest (853,775 bp) documented ICE, the tripartite ICEMspSymAA22. The conjugation systems of all identified ICEs and several plasmids were related to those of the paradigm ICESym ICEMlSymR7A, with each carrying conserved genes for conjugative pilus formation (trb), excision (rdfS), DNA transfer (rlxS) and regulation (fseA). ICESyms have likely evolved from a common ancestor, despite occupying a variety of distinct integration sites and specifying symbiosis with diverse legumes. We found extensive evidence for recombination between ICEs and particularly ICESyms, which all uniquely lack the conjugation entry-exclusion factor gene trbK. Frequent duplication, replacement and pseudogenization of genes for quorum-sensing-mediated activation and antiactivation of ICE transfer suggests ICE transfer regulation is constantly evolving. Pangenome-wide association analysis of the ICE identified genes potentially involved in symbiosis, rhizosphere colonisation and/or adaptation to distinct legume hosts. In summary, the Mesorhizobium genus has accumulated a large and dynamic pangenome that evolves through ongoing horizontal gene transfer of large conjugative elements related to ICEMlSymR7A.

A Mating Procedure for Genetic Transfer of Integrative and Conjugative Elements (ICEs) of Streptococci and Enterococci

DNA sequencing of whole bacterial genomes has revealed that the entire set of mobile genes (mobilome) represents as much as 25% of the bacterial genome. Despite the huge availability of sequence data, the functional analysis of the mobile genetic elements (MGEs) is rarely reported. Therefore, established laboratory protocols are needed to investigate the biology of this important part of the bacterial genome. Conjugation is a mechanism of horizontal gene transfer which allows the exchange of MGEs among strains of the same or different bacterial species. In streptococci and enterococci, integrative and conjugative elements (ICEs) represent a large part of the mobilome. Here, we describe an efficient and easy-to-perform plate mating protocol for in vitro conjugative transfer of ICEs in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis. Conjugative transfer is carried out on solid media and selection of transconjugants is performed with a multilayer plating. This protocol allows the transfer of large genetic elements with a size up to 81 kb, and a transfer frequency up to 6.7 × 10−3 transconjugants/donor cells.

EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a recent single-cell genomic method based on a fusion-PCR allowing us to link a functional sequence of interest to a 16S rRNA gene fragment and use the mass sequencing of the resulting amplicons for taxonomic assignment of the functional sequence-carrying bacteria. Although it is interesting because it presents the highest efficiency for assigning a bacterial host to a marker, epicPCR remains a complex multistage procedure with technical difficulties that may easily impair the approach depth and quality. Here, we described how to adapt epicPCR to new gene targets and environmental matrices while identifying the natural host range of SXT/R391 integrative and conjugative elements in water microbial communities from the Meurthe River (France). We notably show that adding a supplementary PCR step allowed us to increase the amplicon yield and thus the number of reads obtained after sequencing. A comparison of operational taxonomic unit (OTU) identification approaches when using biological and technical replicates demonstrated that, although OTUs can be validated when obtained from three out of three technical replicates, up to now, results obtained from two or three biological replicates give a similar and even a better confidence level in OTU identification, while allowing us to detect poorly represented SXT/R391 hosts in microbial communities.