Natural transformation is assumed to be the most likely mechanism by which
DNA from transgenic plants could be horizontally transferred to bacteria. In
order to determine the occurrence of naturally transformable bacteria
amongst bulk and rhizosphere soil bacteria, different transformation
strategies were employed using either plasmid DNA (IncQ plasmids pSM1890 and
pSM1885, conferring GFP, Smr, Gmr and GFP, Smr, Tcr,
respectively) or genomic DNA from rhizosphere isolates, which were
chromosomally tagged with mini-Tn5~(GFP, Tcr, as transforming DNA.
Transformation assays were done in microtiter plates (262 isolates and
pSM1890 or pSM1885), on filters (i) with rhizosphere bacterial community
mixed with pSM1890 or pSM1885, (ii) with 24 rhizosphere or soil bacterial
isolates mixed with genomic DNA of the corresponding mini-Tn5-tagged strains,
and in the rhizosphere of tobacco plants inoculated with
rifampicin-resistant bacterial isolates and genomic DNA of the corresponding
mini-Tn5-tagged strains added. One transformant colony was obtained when
Brevundimonas vesicularis was transformed with genomic DNA of the corresponding mini-Tn5-tagged
strain. Attempts to reproduce this result were unsuccessful. With this
single exception, transformants were neither detected in the collection of
isolates nor in the rhizosphere bacterial community. Acinetobacter baylyi BD413 used as a
positive control showed drastically reduced transformation frequencies with
plasmid pSM1890 as transforming DNA when mixed with the rhizosphere pellet.
All transformants were characterized by BOX-PCR fingerprints, and three
different BOX patterns were revealed. Sequencing the 16S rRNA gene showed
that all transformants could be assigned to Acinetobacter sp. Since transformants were only
observed in the positive control, the introduced BD413 either underwent
genomic rearrangements, or competence of the Acinetobacter population present in the
rhizosphere was stimulated by the introduction of BD413. The various
transformation assays performed indicate that the proportion of rhizosphere
or bulk soil bacteria which are naturally transformable is negligibly low.