REQUIREMENTS FOR STRAND-SPECIFIC AND SITE-SPECIFIC CLEAVAGE WITHIN THE ORIT REGION OF TN4399, A MOBILIZING TRANSPOSON FROM BACTEROIDES-FRAGILIS

Replicons that contain Tn4399, a conjugal mobilizing transposon isolated from Bacteroides fragilis, can be mobilized in the presence of broad-host-range IncP plasmids RP4 and R751 in Escherichia coli to B. fragilis or E. coli recipients (C. G. Murphy and M. H. Malamy, J. Bacteriol. 175:5814-5823. 1993). To identify the initial DNA processing events involved in Tn4399-mediated mobilization in E. coli, plasmid DNA from pCGM328 (a pUC7 vector that contains the mobilization region of Tn4399) was isolated from donor cells following the release of plasmid DNA from the relaxation complex. Site- and strand-specific cleavage within the oriT region of Tn4399 was detected by denaturing gel electrophoresis and Southern hybridization analysis of this DNA in the presence or absence of IncP plasmids. Mutations in either moc4 or mocB, two genes which are encoded by Tn4399 and are required for mobilization, significantly decrease the amount of specifically nicked DNA detected. These results suggest roles for the MocA and MocB gene products in specific processing of Tn4399-containing plasmid DNA prior to mobilization. By isolation of the nicked strand and primer extension of this template. we mapped the precise 5' end of the single-stranded cleavage reaction. The nucleotide position of nic(Tn4399) is adjacent to two sets of inverted repeats, a genetic arrangement similar to those of pre,iously characterized oriT region., Two site-directed mutations which remote nic(Tn4399) (oriT Delta) and oriT Delta 2)) cannot be mobilized to recipients when they are present in trans along with functional MocA and MocB proteins and an IncP mobilizing plasmid; they are cis-dominant loss-of-function mutations.