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Appl Environ Microbiol, June 1998, p. 2247-2255, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Establishment of New Genetic Traits in a Microbial Biofilm Community

Bjarke B. Christensen,1 Claus Sternberg,1 Jens Bo Andersen,1 Leo Eberl,2 Søren Møller,3 Michael Givskov,1 and Søren Molin1,*

Department of Microbiology, The Technical University of Denmark, DK-2800 Lyngby,1 and NovoNordisk A/S, DK-2880 Bagsværd,3 Denmark, and Lehrstuhl für Mikrobiologie, Technische Universität München, D-80290 Munich, Germany2

Received 6 February 1998/Accepted 19 March 1998

Conjugational transfer of the TOL plasmid (pWWO) was analyzed in a flow chamber biofilm community engaged in benzyl alcohol degradation. The community consisted of three species, Pseudomonas putida RI, Acinetobacter sp. strain C6, and an unidentified isolate, D8. Only P. putida RI could act as a recipient for the TOL plasmid. Cells carrying a chromosomally integrated lacIq gene and a lacp-gfp-tagged version of the TOL plasmid were introduced as donor strains in the biofilm community after its formation. The occurrence of plasmid-carrying cells was analyzed by viable-count-based enumeration of donors and transconjugants. Upon transfer of the plasmids to the recipient cells, expression of green fluorescence was activated as a result of zygotic induction of the gfp gene. This allowed a direct in situ identification of cells receiving the gfp-tagged version of the TOL plasmid. Our data suggest that the frequency of horizontal plasmid transfer was low, and growth (vertical transfer) of the recipient strain was the major cause of plasmid establishment in the biofilm community. Employment of scanning confocal laser microscopy on fixed biofilms, combined with simultaneous identification of P. putida cells and transconjugants by 16S rRNA hybridization and expression of green fluorescence, showed that transconjugants were always associated with noninfected P. putida RI recipient microcolonies. Pure colonies of transconjugants were never observed, indicating that proliferation of transconjugant cells preferentially took place on preexisting P. putida RI microcolonies in the biofilm.

* Corresponding author. Mailing address: Department of Microbiology, Building 301, The Technical University of Denmark, DK-2800 Lyngby, Denmark. Phone: 45 45 25 25 13. Fax: 45 45 88 73 28. E-mail: sm{at}im.dtu.dk.

Appl Environ Microbiol, June 1998, p. 2247-2255, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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