Plasmid transfer from Pseudomonas putida to the indigenous bacteria on alfalfa sprouts: characterization, direct quantification, and in situ location of transconjugant cells

Abstract

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.

FIG. 1.

Numbers of CFU of the total bacterial population (○), non-lactose-fermenting gram-negative bacteria (▵), Pseudomonas including the donor strain (□), and the donor strain (▪) measured on sprouts inoculated with donor bacteria containing either pKJK5::gfp (A), TOL::gfp (B), or without donor inoculation (C). The error bars represent the standard errors.

FIG. 1.

Numbers of CFU of the total bacterial population (○), non-lactose-fermenting gram-negative bacteria (▵), Pseudomonas including the donor strain (□), and the donor strain (▪) measured on sprouts inoculated with donor bacteria containing either pKJK5::gfp (A), TOL::gfp (B), or without donor inoculation (C). The error bars represent the standard errors.

FIG. 2.

Relative densities of major bacterial populations present on uninoculated sprouts at selected time points as identified by polymorphisms of PCR-amplified rDNA spacer regions and subsequent partial 16S rDNA sequencing. The results for Pseudomonas (), Erwinia (□), and Paenibacillus (▪) spp. and for species either not identified or present in proportions of <5% (▨) are presented.

FIG. 3.

Direct counts of total bacteria (□), donor cells (▪), and transconjugant cells () counted in sprout samples at days 1, 6, and 9 after inoculation with donor cells containing either pKJK5::gfp (A) or TOL::gfp (B). The error bars represent the standard errors.

FIG. 4.

Confocal laser scanning micrographs of alfalfa sprouts 6 days after the onset of germination and inoculation with red fluorescent donor bacteria P. putida LM50/pKJK5::gfp. Bacteria belonging to the indigenous microbial population on the sprouts, which have received the pKJK5::gfp plasmid, appear green. (A) Root area heavily colonized by donor cells. Several transconjugant bacteria are visible. Scale bar, 20 μm. (B) Xyz scan of hypocotyl area with microcolony of donor and transconjugant bacteria. Scale bar, 5 μm. (C and D) Micrographs of transconjugant bacteria with different cell morphologies. Scale bar, 5 μm. Observations of sprouts inoculated with P. putida LM50/TOL::gfp were similar to those of sprouts colonized with P. putida LM50/pKJK5::gfp (data not shown).