Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis.
A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harboring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3'-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tumefaciens.
Kang
H W
HW
Institute for Biochemical Regulation, Faculty of Agriculture, Nagoya University, Japan.
Wirawan
I G
IG
Kojima
M
M
D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000472:pathogenicity; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D010957:Plasmids; D011487:Protein Conformation; D014774:Virulence / Q000235:genetics
D004251:DNA Transposable Elements; D004269:DNA, Bacterial