Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its <i>oriT</i> Region and Relaxase

<jats:title>ABSTRACT</jats:title> <jats:p> NAH7 and pWW0 from gammaproteobacterial <jats:named-content content-type="genus-species">Pseudomonas putida</jats:named-content> strains are IncP-9 conjugative plasmids that carry the genes for degradation of naphthalene and toluene, respectively. Although such genes on these plasmids are well-characterized, 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 ( <jats:italic>oriT</jats:italic> )-containing region and its relaxase, which specifically nicks within the <jats:italic>oriT</jats:italic> region for initiation of transfer, and (ii) comparing the conjugation systems between NAH7 and pWW0. The NAH7 <jats:italic>oriT</jats:italic> ( <jats:italic>oriT</jats:italic> <jats:sub>N</jats:sub> ) region was located within a 430-bp fragment, and the strand-specific nicking ( <jats:italic>nic</jats:italic> ) site and its upstream sequences that were important for efficient conjugation in the <jats:italic>oriT</jats:italic> <jats:sub>N</jats:sub> 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 <jats:italic>oriT</jats:italic> <jats:sub>N</jats:sub> region, between two copies of the pWW0 <jats:italic>oriT</jats:italic> ( <jats:italic>oriT</jats:italic> <jats:sub>W</jats:sub> ) region (which is clearly different from the <jats:italic>oriT</jats:italic> <jats:sub>N</jats:sub> region), and between the <jats:italic>oriT</jats:italic> <jats:sub>N</jats:sub> and <jats:italic>oriT</jats:italic> <jats:sub>W</jats:sub> 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 <jats:italic>oriT</jats:italic> <jats:sub>N</jats:sub> -containing plasmid. Such a plasmid was, in the presence of the NAH7Δ <jats:italic>nic</jats:italic> 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. </jats:p> <jats:p> <jats:bold>IMPORTANCE</jats:bold> Various studies have strongly suggested an important contribution of conjugative transfer of catabolic plasmids to the rapid and wide dissemination of the plasmid-loaded degradation genes to microbial populations. Degradation genes on such plasmids are often loaded on transposons, which can be inserted into the genomes of the recipient bacterial strains where the transferred plasmids cannot replicate. The aim was to advance detailed molecular knowledge of the determinants of host range for plasmids. This aim is expected to be easily and comprehensively achieved using an experimental strategy in which the <jats:italic>oriT</jats:italic> region is connected with a plasmid that has a broad host range of replication. Using such a strategy in this study, we showed that (i) the NAH7 <jats:italic>oriT</jats:italic> -relaxase system has unique properties that are significantly different from other well-studied systems and (ii) the host range of the NAH7 conjugation system is broader than previously thought. </jats:p>