No abstract

No abstract

SummaryThe mobilization region of plasmid CloDF13 was localized to a 3.6 kb DNA segment that was analysed by transposon mutagenesis and DNA sequencing. Analysis of the DNA sequence allowed us to identify two mobilization genes and the CloDF13 origin of conjugative transfer (oriT), which was localized to a 661 bp segment at one end of the mobilization (Mob) region. Thus, the overall organization was oriT±mobB±mobC. Plasmid CloDF13 DNA was isolated mainly as a relaxed form that contained a unique strand and site-specific cleavage site (nic). The position of nic was mapped to the sequence 5 H -GGGTG/GTCGGG-3 H by primer extension and sequencing reactions. Analysis of Mob 2 insertion mutants showed that mobC was essential for CloDF13 relaxation in vivo. The sequence of mobC predicts a protein (MobC) of 243 amino acids without significant similarity to previously reported relaxases. In addition to MobC, the product of mobB was also required for CloDF13 mobilization and for oriT relaxation in vivo. mobB codes for a protein (MobB) of 653 amino acids with three predicted transmembrane segments at the N-terminus and the NTP-binding motifs characteristic of the TraG family of conjugative coupling proteins. Membership of the TraG family was confirmed by the fact that CloDF13 mobilization by plasmid R388 was independent of TrwB and only required PIL W . However, contrary to the activities found for other coupling proteins, MobB was required for efficient oriT cleavage in vivo, suggesting an additional role for this particular protein during oriT processing for mobilization. Additionally, the cleavage site produced by the joint activities of MobB and MobC was shown to contain unblocked ends, suggesting that no stable covalent intermediates between relaxase and DNA were formed during the nic cleavage reaction. This is the first report of a conjugative transfer system in which nic cleavage results in a free nicked-DNA intermediate.

SummaryThe conjugative transfer region of the IncX plasmid R6K (TRA X ) was analysed by transposon mutagenesis and DNA sequencing. Tn5 tac1 insertional mutations localized TRA X to a 14.8 kb segment containing the ␣ origin of transfer (oriT␣), genes involved in conjugative DNA-processing (Dtr X ) and genes involved in pilus synthesis and assembly (Mpf X ). A second functional oriT, oriT ␤, was located at a distance of 5.3 kb from oriT␣ and was outside TRA X . Mpf X occupied a segment of 10 kb, as judged by the location of insertions conferring resistance to infection by the X pilusspecific phage X-2. At both sides of Mpf X there were insertions that were Tra ¹ but X-2 sensitive, suggesting that the mutations were in Dtr X . This region was sequenced and three genes were identified: taxA, taxB, and taxC. The overall organization was oriT␣-taxA-taxC-Mpf X -taxB. taxC coded for a oriT-relaxase that belongs to the VirD2 family. taxA coded for a protein of 181 amino acids that showed similarity to TraY of F-like plasmids and to the Arc-repressor superfamily. TaxB showed similarity to TraG-like proteins, a protein superfamily probably involved in coupling the relaxosome to the DNA-transport apparatus. TaxA and TaxC are required for oriT nicking in vivo. The nicking reaction was mistakenly assumed by Flashner et al. (1996) to represent a feature of the vegetative replication origins. However, insertions or deletions disrupting taxA and taxC affected conjugation but not replication of R6K. Conversely, protein , which is absolutely required for replication of R6K, was not required for conjugative transfer. In addition, protein DDP3, which is also assumed to have a role in replication, was found to be a positive modulator of bacterial conjugation. Taken together, these results rule out a direct and essential involvement of conjugation proteins in R6K vegetative replication, and also rule out the requirement of replication protein for conjugation.