The transfer of plasmids between bacterial cells by conjugation is the result of two processes: (i) mating-pair formation between the donor and recipient, and (ii) a DNA processing reaction that prepares the plasmid for transfer (Willetts and Wilkins, 1984; Pansegrau and Lanka, 1996) . Conjugation can proceed only after the formation of a protein-DNA complex (relaxosome) at the oriT site, which is often located in the intergenic region upstream of its cognate relaxase gene (Francia et al., 2004) . The oriT of the 89K PAI was predicted to be a sequence of~40 nt positioned within the 05SSU0915 coding sequence, which is predicted to encode a polypeptide of unknown function with 62% aa identity to a hypothetical protein (SMSK597-1197, accession number ZP_07642092.1) of S. mitis SK597. This segment contains a perfect 9 nt inverted repeat sequence (predicted by the DNA Strider program) that may be necessary to form a stable nucleoprotein complex during conjugation (Bhattacharjee and Meyer, 1993 ). The precise nic (nickase cleavage) site was predicted to reside at position 3′-(887140)CTCG/ CAAA(887147) on the negative strand, which is located 8 nt downstream of the inverted repeats within the oriT region based on an extensive sequence alignment with other oriT sites that have been experimentally determined previously. Figure 4 shows the overall gene organization of the DNA processing region of 89K. We noticed that MobA89K has an N-terminal relaxase domain but lacks a T4SS-mediated transfer of 89K PAI in S. suis 2 1675 C-terminal helicase domain, which usually possesses a processive 5′ to 3′ unwinding activity that provides the motive force for strand transfer (Llosa et al., 1996; Matson et al., 2001) . Instead, we found that ORF 05SSU0934 within 89K encodes a match to the DNA helicase (accession number ACF56805.1) of S. pneumoniae G54, with 93% aa identity. In vivo mutational analysis demonstrated that this gene (designated hlc, helicase) is not required for 89K excision and circularization (Fig. 3B ) but is essential for 89K transfer (Table 1) .