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Appl Environ Microbiol, May 1998, p. 1902-1909, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effect of Bacterial Distribution and Activity on
Conjugal Gene Transfer on the Phylloplane of the Bush Bean
(Phaseolus vulgaris)
Bo
Normander,1
Bjarke B.
Christensen,2
Søren
Molin,2 and
Niels
Kroer1,*
National Environmental Research Institute,
Department of Marine Ecology and Microbiology, DK-4000
Roskilde,1 and
Department of
Microbiology, Technical University of Denmark, DK-2800
Lyngby,2 Denmark
Received 18 September 1997/Accepted 22 February 1998
Conjugal plasmid transfer was examined on the phylloplane of bean
(Phaseolus vulgaris) and related to the spatial
distribution pattern and metabolic activity of the bacteria. The donor
(Pseudomonas putida KT2442) harbored a derivative of the
TOL plasmid, which conferred kanamycin resistance and had the
gfp gene inserted downstream of a lac promoter.
A chromosomal insertion of lacIq prevented
expression of the gfp gene. The recipient (P. putida KT2440) had a chromosomal tetracycline resistance marker.
Thus, transconjugants could be enumerated by plating and visualized in
situ as green fluorescent cells. Sterile bean seedlings were inoculated
with donors and recipients at densities of approximately 105 cells per cm2. To manipulate the density
and metabolic activity (measured by incorporation of
[3H]leucine) of the inoculated bacteria, plants were
grown at various relative humidities (RH). At 100% RH, the
transconjugants reached a density of 3 × 103
CFU/cm2, corresponding to about one-third of the recipient
population. At 25% RH, numbers of transconjugants were below the
detection limit. Immediately after inoculation onto the leaves, the
per-cell metabolic activity of the inocula increased by up to eight
times (100% RH), followed by a decrease to the initial level after
96 h. The metabolic activity of the bacteria was not rate limiting for conjugation, and no correlation between the two parameters was
observed. Apparently, leaf exudates insured that the activity of the
bacteria was above a threshold value for transfer to occur. Transconjugants were primarily observed in junctures between epidermal cells and in substomatal cavities. The distribution of the
transconjugants was similar to the distribution of indigenous bacteria
on nonsterile leaves. Compared to polycarbonate filters, with cell
densities equal to the overall density on the leaves, transfer ratios
on leaves were up to 30 times higher. Thus, aggregation of the bacteria into microhabitats on the phylloplane had a great stimulatory effect on
transfer.
*
Corresponding author. Mailing address: Dept. of Marine
Ecology and Microbiology, National Environmental Research Institute, Frederiksborgvej 399, DK-4000 Roskilde, Denmark. Phone: 45 46 30 13 88. Fax: 45 46 30 12 16. E-mail: nk{at}dmu.dk.
Appl Environ Microbiol, May 1998, p. 1902-1909, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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