Characterization of transposon-generated protease mutant of Xanthomonas campestris pathovar glycine 8ra. Enzyme activity, cloning, and mapping of flanking DNA.
Protease negative mutant of Xanthomonas campestris pathovar glycine 8ra (prt-mutant) was constructed by mutagenesis employing artificial transposon Omegon-Km. Transposon delivery was conducted through diparental conjugation using X. campestris pathovar glycine 8ra as recipient and Escherichia coli S17-1 carrying pJFF 3500 plasmid as the donor. The frequency of transconjugation was found 1.9 x 10(-7) per recipient. Enzyme analysis indicated the presence of mutant with lower protease activity than that of the wild-type. Genetic analysis employing pulsed-field gel electrophoresis (PFGE) of the genomic DNA digested with AseI or SpeI restriction endonuclease could significantly differentiate X. campestris pathovar glycine 8ra prt from the wild-type parent. The 9.85 kb pLR omega 6 plasmid was constructed from the genomic DNA of the prt mutant after being digested with KpnI restriction endonuclease and ligated with T4 DNA ligase.
Rosana
L
L
Laboratory for Biochemistry and Microbiology IUC Biotechnology IPB, Bogor Agricultural University, Indonesia.
Suhartono
M T
MT
Suwanto
A
A
D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics*; D005998:Glycine / Q000378:metabolism; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D020161:Physical Chromosome Mapping; D010957:Plasmids; D016959:Xanthomonas campestris / Q000235:genetics*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D010450:Endopeptidases; D005998:Glycine