Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis

Biosci Biotechnol Biochem. 1992 Dec;56(12):1924-8. doi: 10.1271/bbb.56.1924.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens / genetics*
  • Agrobacterium tumefaciens / pathogenicity
  • Amino Acid Sequence
  • Base Sequence
  • Blotting, Southern
  • Chromosomes, Bacterial*
  • Cloning, Molecular
  • Conjugation, Genetic
  • DNA Transposable Elements*
  • DNA, Bacterial / genetics
  • DNA, Bacterial / isolation & purification
  • Escherichia coli / genetics
  • Molecular Sequence Data
  • Mutagenesis, Insertional*
  • Open Reading Frames
  • Plasmids
  • Protein Conformation
  • Virulence / genetics

Substances

  • DNA Transposable Elements
  • DNA, Bacterial