Host range of the conjugative transfer system of IncP-9 naphthalene-catabolic plasmid NAH7 and characterization of its oriT region and relaxase

NAH7 and pWW0 from gammaproteobacterial Pseudomonas putida strains are IncP-9 conjugative plasmids that carry the genes for degradation of naphthalene and toluene, respectively. Although such genes on these plasmids are wellcharacterized, experimental investigation of their conjugation systems remains at a primitive level. To clarify these conjugation systems, in this study, we investigated the NAH7-encoded conjugation system by (i) analyzing the origin of its conjugative transfer (oriT)-containing region and its relaxase, which specifically nicks within the oriT region for initiation of transfer, and (ii) comparing the conjugation systems between NAH7 and pWW0. The NAH7 oriT (oriT_N) region was located within a 430-bp fragment, and the strand-specific nicking (nic) site and its upstream sequences that were important for efficient conjugation in the oriT_N region were identified. Unlike many other relaxases, the NAH7 relaxase exhibited unique features in its ability to catalyze, in a conjugation-independent manner, the site-specific intramolecular recombination between two copies of the oriT_N region, between two copies of the pWW0 oriT (oriT_W) region (which is clearly different from the oriT_N region), and between the oriT_N and oriT_W regions. The pWW0 relaxase, which is also clearly different from the NAH7 relaxase, was strongly suggested to have the ability to conjugatively and efficiently mobilize the oriT_Ncontaining plasmid. Such a plasmid was, in the presence of the NAH7?nic derivative, conjugatively transferable to alphaproteobacterial and betaproteobacterial strains in which the NAH7 replication machinery is nonfunctional, indicating that the NAH7 conjugation system has a broader host range than its replication system.