Chapter 23 : Plasmid Detection, Characterization, and Ecology

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Plasmid-mediated horizontal gene transfer is recognized as a major driving force for bacterial adaptation and diversification. Different environmental settings have distinct bacterial community compositions, which determine—possibly with the exception of broad host range plasmids—the type of dominant plasmids that can be found. It is assumed that only a fraction of a population carries plasmids, which ensures a rapid adaptation of the population to changing environmental conditions ( ). Without a doubt, plasmid-mediated spread of antibiotic resistance genes among bacteria of different taxa is one of the most impressive examples of bacterial plasticity in response to various selective pressures ( ). While the molecular biology of the plasmid-encoded replication, maintenance, and transfer processes of some plasmids has been studied for decades, little attention has been paid to their dissemination in the environment, their ecology, and the factors that drive their spread and diversification. In an overwhelming number of studies, the investigated plasmid-carrying strains originate from clinical specimens or diseased plant material, mostly human or plant pathogens. The reason for the lack of studies of the ecology of plasmids in natural settings was mainly the lack of tools to detect and quantify plasmids and to successfully culture their hosts.

Citation: Smalla K, Jechalke S, Top E. 2015. Plasmid Detection, Characterization, and Ecology, p 445-458. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0038-2014
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Figure 1

Overview of applying Hi-C technology to a mixed bacterial community to reliably associate plasmids with the chromosomes of their hosts (modified from Burton et al. [ ]). (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 III 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 unligated 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.

Citation: Smalla K, Jechalke S, Top E. 2015. Plasmid Detection, Characterization, and Ecology, p 445-458. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0038-2014
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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 resuspended 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 the plasmid-mobilizing capacity. To facilitate the identification of transconjugants the recipient is labeled with the green fluorescent protein ().

Citation: Smalla K, Jechalke S, Top E. 2015. Plasmid Detection, Characterization, and Ecology, p 445-458. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0038-2014
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