Localization of hup determinants in Rhizobium sp. sesbania

Rhizobium sp. sesbania strain SRS 78, a Hup wild type carrying three plasmids was mutagenised using Tn5-sac BR cassette and clones carrying Tn5 insertion in plasmids were identified on the basis of transfer frequency of Tn5-sac BR labelled plasmids from Rhizobium to A. tumefaciens. Neomycin sensitive (Nms) clones were isolated from clones containing Tn5 labelled plasmids by plating on sucrose (5%). Nms clones did not show loss of hup activity. Preliminary results on curing studies indicated that hup genes are located on chromosome. These results were later confirmed by southern hybridization using plasmid pKHT30 containing hup genes of Azotobacter chroococcum. None of the three plasmids showed hybridization with hup gene probe while chromosomal DNA left in the wells showed positive signal.