Protocol for Evaluating the Permissiveness of Bacterial Communities Toward Conjugal Plasmids by Quantification and Isolation of Transconjugants

Abstract

Transfer of conjugal plasmids is the main bacterial process of horizontal gene transfer to potentially distantly related bacteria. These extra-chromosomal, circular DNA molecules host genes that code for their own replication and transfer to other organisms. Because additional accessory genes may encode catabolic pathways, virulence factors and antibiotic or metal resistances it is of environmental, evolutionary and medical relevance to track and monitor the fate of plasmids in mixed microbial community. When assessing the short-term and long-term implications of conjugal plasmid transfer the ability of a plasmid to invade a mixed community is crucial. The main parameter that controls the possible extent of horizontal plasmid transfer (HGT) in a bacterial community is the in-situ community permissiveness for the considered plasmid. Permissiveness describes the fraction of a microbial community able to receive an introduced plasmid at both quantitative and phylogenetic levels. In this chapter we describe a protocol for simultaneous quantification of plasmid transfer frequency to and high-throughput isolation of transconjugants from a mixed bacterial community after introducing a gfp-tagged plasmid in a mCherry red fluorescently tagged donor strain repressing gfp expression. We take advantage of fluorescent marker genes to microscopically detect plasmid transfer events and use subsequent high throughput fluorescent activated cell sorting (FACS) to isolate these transconjugants from the complex community.