In situ detection of horizontal transfer of mobile genetic elements.
Plasmid transfer was investigated in microbial populations associated with different types of surfaces. The general strategy behind these investigations was to label the transferable plasmid with a gene encoding a fluorescent protein in order to make it a transfer reporter. This was achieved by fusing the reporter gene with a lac promoter expression cassette and combining this with a donor cell-associated lacI repressor cassette. After construction of a range of strains and plasmids with combinations of genes expressing fluorescent proteins from constitutive (cell tagging) or regulated promoters (transfer reporters) it was thus possible to detect transfer events in situ and correlate these with either the location of donor and recipient cells or with the growth activity of the cells. In some cases, expression of unstable Gfp from a growth-controlled promoter, rrnB from Escherichia coli, was used to monitor bacterial growth activity in situ. Differential tagging of mobilizing and mobilizable plasmids with different genes encoding fluorescent proteins with varying emission wavelengths allowed in situ detection of plasmid mobilization and detection of retro-transfer on agar surfaces. The obtained data show that the several different types of fluorescent reporters, which are now available, allow more informative in situ investigations of horizontal gene transfer to be carried out, and by combining these genes with various expression systems it is possible to simultaneously monitor donor/recipient positioning, cellular activity and appearance of transconjugants.
Haagensen
Janus A J
JA
BioCentrum-DTU, Molecular Microbial Ecology Group, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark.
Hansen
S K
SK
Johansen
T
T
Molin
S
S