Cultivation-independent analyses were carried out to compare the bacterial
community structure found in the rhizospheres of a transplastomic tobacco
plant carrying the antibiotic resistance marker-gene aadA and its non-engineered
parental line. PCR- and reverse transcriptase PCR-amplifications of 16S rRNA
and their corresponding genes were carried out with primers targeting the
domain Bacteria. The diversity of PCR-products amplified from total nucleic acids
extracted from rhizospheres of 10-week-old plants, which had been grown in
potting soil in the greenhouse, was visualized by genetic profiling using
the single-strand conformation polymorphism (SSCP) technique. The SSCP
profiles generated from DNA extracted with two different protocols, one
including total RNA and the other only DNA, did not show any differences.
The SSCP profiles amplified from RNA and DNA were also highly similar to
each other, indicating that the dominant bacteria detected were
metabolically active. High similarities were seen between the SSCP profiles
from the transplastomic and the non-engineered plants, except for a single
band that consistently occurred with samples from the non-engineered plants
(six replicates), but not, or only weakly, with their engineered
counterparts. DNA sequencing and database analysis revealed that the partial
rRNA gene matched to a Flavobacterium sp. Other bands of the SSCP-profiles, related to
Burkholderia and Bordetella were variable between individual plants but not affected by the
transplastomic modification. Thus, the transplastomic modification caused a
relative decline of a specific Flavobacterium population but not of other bacteria.
Further studies including additional tobacco cultivars, soils and conditions
of cultivation would be desirable, to elucidate the ecological importance of
this difference.