Assessing the permissiveness of complex bacterial communities towards conjugal plasmids ? Development of a novel method

KLÜMPER, ULI; RIBER, LEISE; SANNAZZARO, ANALIA INÉS; DECHESNE, ARNAUD; MUSOVIC, SANIN ; HANSEN, LARS; SØRENSEN, SØREN; SMETS, BERTH

Ljubljana

Congreso; 12th Symposium on Bacterial Genetics and Ecology; 2013

A crucial parameter governing the significance of horizontal gene transfer (HGT) in a bacterial community is the community?s permissiveness. Permissiveness describes the fraction of a microbial community able to receive an introduced plasmid at quantitative and phylogenetic levels. Permissiveness in complex, natural communities has not been studied intensively, because no suitable methods are available. The purpose of this work was to develop a method for estimating permissiveness of a complex, natural, microbial community towards plasmids that allows to simultaneously: ? Estimate plasmid transfer frequency to the recipient community ? Isolate and taxonomically identify the transconjugants Three mCherry-tagged donor strains (P. putida, E. coli & Kluyvera spp.) carrying one of four gfp-tagged plasmids (RP4, pKJK5, pIPO2Tet, pRO101) were mixed with a soil bacterial community in a filter mating assay mimicking natural nutrient conditions. Plasmid transfer frequency was quantified by detecting green fluorescent transconjugant microcolonies using stereomicroscopy and image analysis. Transconjugants were isolated using fluorescent activated cell sorting (FACS) with triple gating for bacterial size, gfp-based green fluorescence and exclusion of mCherry based red fluorescence. Sorted transconjugants were subsequently analyzed by 454 amplicon pyrosequencing. Transfer frequency differed depending on plasmid and donor strain. For all combinations with visually detectable transfer, sorting of transconjugants by FACS was possible. Plating of more than 200 sorted transconjugants showed no recovery of red fluorescent donor cells, whereas more than 99.5% of the colonies were gfp-positive. The number of sorted cells obtained after FACS was considered high enough for subsequent pyrosequencing for taxonomic analysis of the transconjugal communities. The combination of filter mating assays, stereomicroscopy and FACS enables us to isolate transconjugants from a complex donor-recipient mixture, and subsequently identify these at a phylogenetic level by pyrosequencing. We can therefore relate the transfer frequency of a plasmid to the fraction and identity of bacteria that actually take part in HGT, thereby determining the permissiveness of the community towards plasmids.