Isolation and characterization of a chromosomal gene that affectsvir gene expression in Agrobacterium tumefaciens

John Gray, Purdue University

Abstract

vir regulon expression in Agrobacterium tumefaciens involves both chromosome- and Ti-plasmid-encoded gene products. We have isolated and characterized a chromosomal gene that when mutated results in a 2 to 10 fold reduction in the induced expression of vir genes by acetosyringone. This reduced expression occurs in AB minimal medium containing either sucrose or glucose, at a pH of 5.5, and in the presence of high or low phosphate concentrations. A slight reduction in virulence was observed on red potato, but not on tobacco, tomato, Kalanchoe or sunflower by these mutant A. tumefaciens strains. The locus was cloned and used to complement A. tumefaciens strains harboring Tn5 insertions in the gene. Sequence analysis of this locus revealed an ORF with strong homology to genes that encode tRNA:isopentenyltransferase enzymes responsible for the specific modification of the A-37 residue in UNN codon tRNA species. The function of the homologous gene in A. tumefaciens was proven by genetic complementation of E. coli miaA mutant strains and by tRNA analysis. tRNA undermodification in Agrobacterium tumefaciens miaA mutant strains may reduce vir gene expression by causing a reduced translation efficiency. The effect of a miaA mutation in A. tumefaciens on the expression of lacZ gene fusions in five non-vir genes varied from no effect to as much as a 44% reduction in gene expression. Of all single copy gene fusions examined vir gene expression remained most severely affected by the miaA mutation. The total protein translation rate in an A. tumefaciens miaA strain not carrying any vir genes differed only slightly from that of a wild-type strain, suggesting a possible indirect effect of the miaA mutation on vir gene expression. The expression of individual proteins in an A. tumefaciens miaA strain carrying pTiR10 both in the presence and absence of acetosyringone was examined by two-dimensional separation of $\sp{14}$C-labelled proteins. We did not observe any large changes in the quantitative patterns of protein expression in this strain compared to a wild-type strain but we did observe apparent repression and derepression of three non-vir genes. These results suggest that a miaA mutation may have slight effects on protein translation but leaves open the possibility that the expression of some genes including vir genes are affected in their expression by specific mechanisms mediated by undermodified tRNA.

Degree

Ph.D.

Advisors

Gelvin, Purdue University.

Subject Area

Molecular biology|Genetics

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