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Abstract

Antibiotic resistance is a serious global problem. Antibiotic resistance
genes (ARG), which are widespread in environmental bacteria, can be
transferred to pathogenic bacteria via horizontal gene transfer (HGT).
Gut microbiomes are especially apt for the emergence and dissemination
of ARG. Conjugation is the HGT route that is predominantly responsible
for the spread of ARG. Little is known about conjugative elements of
Gram-positive bacteria, including those of the phylum Firmicutes, which
are abundantly present in gut microbiomes. A critical step in the
conjugation process is the relaxase-mediated site-and strand-specific
nick in the oriT region of the conjugative element. This generates a
single-stranded DNA molecule that is transferred from the donor to the
recipient cell via a connecting channel. Here we identified and
characterized the relaxosome components oriT and the relaxase of the
conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show
that the relaxase gene, named rel(LS20), is essential for conjugation,
that it can function in trans and provide evidence that Tyr26
constitutes the active site residue. In vivo and in vitro analyses
revealed that the oriT is located far upstream of the relaxase gene and
that the nick site within oriT is located on the template strand of the
conjugation genes. Surprisingly, the Rel(LS20) shows very limited
similarity to known relaxases. However, more than 800 genes to which no
function had been attributed so far are predicted to encode proteins
showing significant similarity to RelLS20. Interestingly, these putative
relaxases are encoded almost exclusively in Firmicutes bacteria. Thus,
RelLS20 constitutes the prototype of a new family of relaxases. The
identification of this novel relaxase family will have an important
impact in different aspects of future research in the field of HGT in
Gram-positive bacteria in general, and specifically in the phylum of
Firmicutes, and in gut microbiome research.