Project description:Plasmids are autonomous, self-replicating, extrachromosomal genetic elements that are typically not essential for growth of their host. They may encode metabolic capabilities, which promote the maintenance of these genetic elements, and may allow adaption to specific ecological niches and consequently enhance survival. Genome sequencing of 16 Lactococcus lactis strains revealed the presence of 83 plasmids, including two megaplasmids. The limitations of Pacific Biosciences SMRT sequencing in detecting the total plasmid complement of lactococcal strains is examined, while a combined Illumina/SMRT sequencing approach is proposed to combat these issues. Comparative genome analysis of these plasmid sequences combined with other publicly available plasmid sequence data allowed the definition of the lactococcal plasmidome, and facilitated an investigation into (bio) technologically important plasmid-encoded traits such as conjugation, bacteriocin production, exopolysaccharide (EPS) production, and (bacterio) phage resistance.
Project description:Plasmids are self-replicating genetic elements capable of mobilization between different hosts. Plasmids often serve as mediators of lateral gene transfer, a process considered to be a strong and sculpting evolutionary force in microbial environments. Our aim was to characterize the overall plasmid population in the environment of the bovine rumen, which houses a complex and dense microbiota that holds enormous significance for humans. We developed a procedure for the isolation of total rumen plasmid DNA, termed rumen plasmidome, and subjected it to deep sequencing using the Illumina paired-end protocol and analysis using public and custom-made bioinformatics tools. A large number of plasmidome contigs aligned with plasmids of rumen bacteria isolated from different locations and at various time points, suggesting that not only the bacterial taxa, but also their plasmids, are defined by the ecological niche. The bacterial phylum distribution of the plasmidome was different from that of the rumen bacterial taxa. Nevertheless, both shared a dominance of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. Evidently, the rumen plasmidome is of a highly mosaic nature that can cross phyla. Interestingly, when we compared the functional profile of the rumen plasmidome to two plasmid databases and two recently published rumen metagenomes, it became apparent that the rumen plasmidome codes for functions, which are enriched in the rumen ecological niche and could confer advantages to their hosts, suggesting that the functional profiles of mobile genetic elements are associated with their environment, as has been previously implied for viruses.
Project description:The rapid spread of antibiotic resistance among bacterial pathogens is a serious human health threat. While a range of environments have been identified as reservoirs of antibiotic resistance genes (ARGs), we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to our inability to identify the natural bacterial hosts of ARGs and the mobile genetic elements that mediate this spread, such as plasmids and integrons. Here we demonstrate that the in vivo proximity-ligation method Hi-C can reconstruct a known plasmid-host association from a wastewater community, and identify the in situ host range of ARGs, plasmids, and integrons by physically linking them to their host chromosomes. Hi-C detected both previously known and novel associations between ARGs, mobile genetic elements and host genomes, thus validating this method. We showed that IncQ plasmids and class 1 integrons had the broadest host range in this wastewater, and identified bacteria belonging to Moraxellaceae, Bacteroides, and Prevotella, and especially Aeromonadaceae as the most likely reservoirs of ARGs in this community. A better identification of the natural carriers of ARGs will aid the development of strategies to limit resistance spread to pathogens.
Project description:Plasmids can provide a selective advantage for microorganisms to survive and adapt to new environmental conditions. Plasmid-encoded traits, such as antimicrobial resistance (AMR) or virulence, impact the ecology and evolution of bacteria and can significantly influence the burden of infectious diseases. Insight about the identity and functions encoded on plasmids on the global scale are largely lacking. Here, we investigate the plasmidome of 24 samples (22 countries, 5 continents) from the global sewage surveillance project. We obtained 105-Gbp Oxford Nanopore and 167-Gbp Illumina NextSeq DNA sequences from plasmid DNA preparations and assembled 165,302 contigs (159,322 circular). Of these, 58,429 carried genes encoding for plasmid-related and 11,222 for virus/phage-related proteins. About 90% of the circular DNA elements did not have any similarity to known plasmids. Those that exhibited similarity had similarity to plasmids whose hosts were previously detected in these sewage samples (e.g., Acinetobacter, Escherichia, Moraxella, Enterobacter, Bacteroides, and Klebsiella). Some AMR classes were detected at a higher abundance in plasmidomes (e.g., macrolide-lincosamide-streptogramin B, macrolide, and quinolone) compared to the respective complex sewage samples. In addition to AMR genes, a range of functions were encoded on the candidate plasmids, including plasmid replication and maintenance, mobilization, and conjugation. In summary, we describe a laboratory and bioinformatics workflow for the recovery of plasmids and other potential extrachromosomal DNA elements from complex microbiomes. Moreover, the obtained data could provide further valuable insight into the ecology and evolution of microbiomes, knowledge about AMR transmission, and the discovery of novel functions. IMPORTANCE This is, to the best of our knowledge, the first study to investigate plasmidomes at a global scale using long read sequencing from complex untreated domestic sewage. Previous metagenomic surveys have detected AMR genes in a variety of environments, including sewage. However, it is unknown whether the AMR genes were present on the microbial chromosome or located on extrachromosomal elements, such as plasmids. Using our approach, we recovered a large number of plasmids, of which most appear novel. We identified distinct AMR genes that were preferentially located on plasmids, potentially contributing to their transmissibility. Overall, plasmids are of great importance for the biology of microorganisms in their natural environments (free-living and host-associated), as well as for molecular biology and biotechnology. Plasmidome collections may therefore be valuable resources for the discovery of fundamental biological mechanisms and novel functions useful in a variety of contexts.
Project description:Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments. However, while the number of marine microbiome studies is constantly increasing, very little is known about marine plasmids, and they are very poorly represented in public databases. To extend the repertoire of environmental marine plasmids, we established a pipeline for the de novo assembly of plasmids in the marine environment by analyzing available microbiome metagenomic sequencing data. By applying the pipeline to data from the Red Sea, we identified 362 plasmid candidates. We showed that the distribution of plasmids corresponds to environmental conditions, particularly, depth, temperature, and physical location. At least 7 of the 362 candidates are most probably real plasmids, based on a functional analysis of their open reading frames (ORFs). Only one of the seven has been described previously. Three plasmids were identified in other public marine metagenomic data from different locations all over the world; these plasmids contained different cassettes of functional genes at each location. Analysis of antibiotic and metal resistance genes revealed that the same positions that were enriched with genes encoding resistance to antibiotics were also enriched with resistance to metals, suggesting that plasmids contribute site-dependent phenotypic modules to their ecological niches. Finally, half of the ORFs (50.8%) could not be assigned to a function, emphasizing the untapped potential of the unique marine plasmids to provide proteins with multiple novel functions. IMPORTANCE Marine plasmids are understudied and hence underrepresented in databases. Plasmid functional annotation and characterization is complicated but, if successful, may provide a pool of novel genes and unknown functions. Newly discovered plasmids and their functional repertoire are potentially valuable tools for predicting the dissemination of antimicrobial resistance, providing vectors for molecular cloning and an understanding of plasmid-bacterial interactions in various environments.