Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad- host-range plasmid pBBR1, which was isolated from the gram-negative bacteri um Bordetella bronchiseptica (R, Antoine and C, Locht, Mel. Microbiol, 6:17 85-1799, 1992), Plasmid pBBR1 consists of two functional cassettes and pres ents sequence similarities with the transfer origins of several plasmids an d mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in additi on to the transfer origin, the promoter of the mob gene. We demonstrate tha t this gene is autoregulated. The binding of the Mob protein to the 52-bp s equence could thus allow the formation of a protein-DNA complex with a doub le function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4 555 and the streptococcal plasmid pMV158, The oriT of the latter is charact eristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in g ram-negative bacteria and does not replicate via a rolling-circle mechanism . In addition, we identified two amino acids of the Mob protein necessary f or its activity, and we discuss their involvement in the mobilization mecha nism.