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Plasmid
transfer from Pseudomonas putida to the indigenous bacteria on alfalfa sprouts:
characterization, direct quantification, and in situ location of transconjugant
cells.
Appl Environ Microbiol. 2003 Sep;69(9):5536-42.
Molbak L, Licht TR, Kvist T, Kroer N, Andersen SR.
Department of Environmental Chemistry and Microbiology, National Environmental
Research Institute, DK-4000 Roskilde, Denmark.
The transfer of the plasmids pJKJ5 and TOL (pWWO) from Pseudomonas putida to the
indigenous bacterial community on alfalfa sprouts was studied. Tagging with
fluorescent protein markers allowed direct quantification of the introduced
donor bacteria and of indigenous bacteria that had received the plasmids. The
sprouts were observed for 9 days; during this time alfalfa seeds, inoculated
with donor bacteria, developed to edible and subsequently decaying sprouts. The
first transconjugants were detected on day 6 after donor inoculation and
occurred at frequencies of 3.4 x 10(-4) and 2.0 x 10(-6) transconjugant cells
per donor cell for pKJK5::gfp and TOL::gfp, respectively. Confocal laser
scanning microscopy revealed that the sprouts were heavily colonized with donors
and that most transconjugants were located around the hypocotyl and root areas.
Randomly selected members of the indigenous bacterial community from both
inoculated and uninoculated sprouts, as well as a representative part of the
community that had received the plasmids, were characterized by polymorphisms of
PCR-amplified ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes,
followed by partial 16S rDNA sequencing. This showed that the initially
dominating genera Erwinia and Paenibacillus were gradually replaced by
Pseudomonas on the fully developed sprouts. Transconjugants carrying either of
the investigated plasmids mainly belonged to the genera Pseudomonas and ERWINIA:
The numbers of transconjugant cells did not reach detectable levels until 6 days
after the onset of germination, at which point these species constituted the
majority of the indigenous bacteria. In conclusion, the alfalfa sprouts provided
an environment that allowed noteworthy frequencies of plasmid transfer from P.
putida in the absence of selective pressure that could favor the presence of the
investigated plasmids.
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