Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium |
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Authors: | Paul A. Wiersma John E. Hachey William L. Crosby Maurice M. Moloney |
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Affiliation: | (1) Department of Biological Sciences, University of Calgary, 2500 University Drive NW, T2N 1N4 Calgary, Alberta, Canada;(2) Plant Biotechnology Institute, National Research Council Canada, 110 Gymnasium Road, S7N 0W9 Saskatoon, Saskatchewan, Canada |
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Abstract: | Summary The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5 end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed. |
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Keywords: | Acetolactate synthase Bacterial expression Brassica napus Herbicide resistance Salmonella typhimurium |
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