Establishment of New Genetic Traits in a Microbial Biofilm Community

Abstract

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 lacI q gene and alacp-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 thegfp-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.

The ecology of transfer of mobile genetic elements

“…By contrast, another suite of techniques has been developed that allow direct in situ observations to be made on HGT and selection in a non-disruptive manner. These approaches have been made possible by exploiting the potential of £uorescent markers, such as the gfp gene, for studying the self-transfer or mobilization of plasmids [17,20,21]. The studies by Molin et al [20^23] have been pivotal in this respect.…”

“…[16]), when employed in DNA microarrays, allows the study of the expression of speci¢c plasmid and/or host genes in relation to HGT in the natural habitat. In addition, the resolving power o¡ered by reporter gene technology, in particular on the basis of the green £uorescent protein (GFP), has allowed detailed analyses of the patterns of gene transfer in microcolonies or bio¢lms to be made [17]. The impetus of these developments in fostering our understanding of environmental HGT and its impact on natural microbial communities is only beginning to emerge.…”

The transfer dynamics of Pseudomonas sp. plasmid pQBR11 in biofilms

“…All assays were initiated with V5U10 6 cells with a donor:recipient ratio of 1:10 and incubated at 20 ‡C for 24 h. The resultant ratios of transconjugants to donors (TC/D) and to recipients (TC/R) were estimated by plate count methods and compared with controls. Twelve marked plasmids were transferred into P. putida SM1443 (KT2440 lacI q ) [6] where gfp expression was repressed by the lacI q repressor. Transfer of marked plasmids to recipients was detected by epi£uorescence microscopy.…”

Spatially resolved abundances of antibiotic resistance genes and intI1 in wastewater treatment biofilms

“…One relevant factor is oxygen availability, which is greatest at biofilm upper surfaces and may contribute significantly to transfer rates of MGEs (Król et al, 2011). Furthermore, plasmid invasion from external donors into biofilms has been found to occur primarily at upper surfaces where organisms are metabolically active and consistently replicating (Christensen et al, 1998;Haagensen, Hansen, Johansen, & Molin, 2002;Stalder & Top, 2016).…”