Plasmid Detection, Characterization, and Ecology

Abstract

Plasmids are important vehicles for rapid adaptation of bacterial populations to changing environmental conditions. It is thought that to reduce the cost of plasmid carriage, only a fraction of a local population carries plasmids or is permissive to plasmid uptake. Plasmids provide various accessory traits which might be beneficial under particular conditions. The genetic variation generated by plasmid carriage within populations ensures the robustness toward environmental changes. Plasmid-mediated gene transfer plays an important role not only in the mobilization and dissemination of antibiotic resistance genes but also in the spread of degradative pathways and pathogenicity determinants of pathogens. Here we summarize the state-of-the-art methods to study the occurrence, abundance, and diversity of plasmids in environmental bacteria. Increasingly, cultivation-independent total-community DNA-based methods are being used to characterize and quantify the diversity and abundance of plasmids in relation to various biotic and abiotic factors. An improved understanding of the ecology of plasmids and their hosts is crucial in the development of intervention strategies for antibiotic-resistance-gene spread. We discuss the potentials and limitations of methods used to determine the host range of plasmids, as the ecology of plasmids is tightly linked to their hosts. The recent advances in sequencing technologies provide an enormous potential for plasmid classification, diversity, and evolution studies, but numerous challenges still exist.

Figure 1

Overview of applying Hi-C technology to a mixed bacterial population to reliably associate plasmids with the chromosomes of their hosts (modified from Burton et al. [41]). (A) Rectangles indicate different cells carrying plasmids or not. Plasmids are cross-linked with bacterial chromosomes in close proximity (red circles). (B) The DNA in the cross-linked protein complexes is digested with HindIII endonuclease following cell lysis and free DNA ends are tagged with biotin. After ligation of blunt-ended DNA fragments under highly dilute conditions, which preferentially ligates fragments that are within the same cross-linked DNA/protein complex, cross-links are removed, DNA is purified, biotin is eliminated from un-ligated ends, DNA is size-selected, and ligation products are selected for through a biotin pull-down. The resulting Hi-C library is further analyzed by sequencing. (C) Workflow to create individual species/plasmid assemblies from a metagenome sample by combining shotgun, Hi-C, and (optionally) mate-pair libraries.

Figure 2

Exogenous capturing of plasmids by means of (A) biparental and (B) triparental mating. For biparental mating environmental bacteria are mixed with recipient cells and after a filter mating the cells are being re-suspended and plated on media containing rifampicin (Rif), kanamycin (Kan) (to select for the recipient) and antibiotics or heavy metals to which the recipient is sensitive. Triparental matings involve a second donor carrying a small mobilizable IncQ plasmid and the plasmid capturing is exclusively based on their plasmid-mobilizing capacity. To facilitate the identification of transconjugants the recipient is labeled with the green fluorescent protein (gfp).