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Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7: Characterization of Its oriT Region and Relaxase and Host Range of Conjugative System.

Research paper by Kouhei K Kishida, Kei K Inoue, Yoshiyuki Y Ohtsubo, Yuji Y Nagata, Masataka M Tsuda

Indexed on: 16 Oct '16Published on: 16 Oct '16Published in: Applied and environmental microbiology



Abstract

NAH7 and pWW0 from γ-proteobacterial 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 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 (oriT)-containing region and its relaxase, which specifically nicks within the oriT region for initiation of transfer, and by (ii) comparing the conjugation systems between NAH7 and pWW0. The NAH7 oriT (oriTN) region was located within a 430-bp fragment, and the strand-specific nicking (nic) site and its upstream sequences important for efficient conjugation in the oriTN 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 oriTN region, between two copies of the pWW0 oriT (oriTW) region (which is clearly different from the oriTN region) and between the oriTN and oriTW regions. The pWW0 relaxase, which is also clearly different from the NAH7 one, was strongly suggested to have the ability to conjugatively and efficiently mobilize the oriTN -containing plasmid. Such a plasmid was, in the presence of the NAH7Δnic derivative, conjugatively transferable to α- and β-proteobacterial strains in which the NAH7 replication machinery is non-functional, indicating that the NAH7 conjugation system has a broader host range than its replication system.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 oriT region is connected with a plasmid having a broad host range of replication. Using such a strategy in this study, we showed that (i) the NAH7 oriT-relaxase system has unique properties significantly different from other well-studied systems, and (ii) the host range of the NAH7 conjugation system is broader than previously thought.