Multiple pathways of plasmid DNA transfer in Helicobacter pylori

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of
different size and gene content, the function of which is not well
understood. A subgroup of these plasmids (e.g. pHel4, pHel12),
contain a mobilisation region, but no cognate type IV secretion
system (T4SS) for conjugative transfer. Instead, certain H. pylori
strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to
four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we
show that such indigenous plasmids can be efficiently transferred
between H. pylori strains, even in the presence of extracellular
DNaseI eliminating natural transformation. Knockout of a
plasmid-encoded mobA relaxase gene significantly reduced plasmid
DNA transfer in the presence of DNaseI, suggesting a DNA
conjugation or mobilisation process. To identify the T4SS involved
in this conjugative DNA transfer, each individual T4SS was
consecutively deleted from the bacterial chromosome. Using a
marker-free counterselectable gene deletion procedure (rpsL
counterselection method), a P12 mutant strain was finally obtained
with no single T4SS (P12ΔT4SS). Mating experiments using these
mutants identified the comB T4SS in the recipient strain as the
major mediator of plasmid DNA transfer between H. pylori strains,
both in a DNaseI-sensitive (natural transformation) as well as a
DNaseI-resistant manner (conjugative transfer). However, transfer
of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a
P12ΔT4SS recipient strain provided evidence for the existence of a
third, T4SS-independent mechanism of DNA transfer. This novel type
of plasmid DNA transfer, designated as alternate DNaseI-Resistant
(ADR) mechanism, is observed at a rather low frequency under in
vitro conditions. Taken together, our study describes for the first
time the existence of three distinct pathways of plasmid DNA
transfer between H. pylori underscoring the importance of
horizontal gene transfer for this species.