In Situ Detection of High Levels of Horizontal Plasmid Transfer in Marine Bacterial Communities

Gene transfer of the conjugative plasmid pBF1 fromPseudomonas putida to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) (C. Dahlberg et al., Mol. Biol. Evol. 15:385–390, 1998). pBF1 was tagged with thegfp gene controlled by alac promoter which is down regulated in the donor cell by a chromosomal repressor (lacI q ). The plasmid donor cells (Pseudomonas putida KT2442) subsequently do not expressgfp . Transfer to recipient strains lacking the repressor results in expression ofgfp . The transconjugant can subsequently be detected by epifluorescence microscopy on a single-cell level. By using this method, transfer of pBF1::gfp and expression of thegfp gene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater. Second, we measured transfer of pBF1 fromP. putida to the marine bacterial community directly in seawater samples, on a single-cell level, without limiting the detection of gene transfer to the culturable fraction of bacteria. Plasmid transfer was detected on surfaces and in bulk seawater. Seawater bacteria with different morphologies were shown to receive the plasmid. Gene transfer frequencies of 2.3 × 10−6 to 2.2 × 10−4 transconjugants per recipient were recorded after 3 days of incubation.