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Salmonella typhimurium Mutants with Alternate Requirements for Vitamin B₆ or Isoleucine

Department of Biochemistry, University of California, Berkeley, California 94720

ABSTRACT

Several mutants of Salmonella typhimurium LT-2, isolated as auxotrophs for vitamin B₆, grew without the added vitamin when supplied with either isoleucine, -ketobutyrate, or -keto-ß-methylvalerate, but not with threonine or with other -keto acids. When grown on minimal medium supplemented with isoleucine, these mutants synthesized vitamin B₆ in amounts comparable to wild-type cells; they thus appeared to contain a modified L-threonine dehydratase and to belong to genotype ilvA (threonine dehydratase) instead of pdx (pyridoxine). Direct assays confirmed this hypothesis. Wild-type cells (toluene-treated) showed approximately the same threonine dehydratase activity whether grown in the presence or absence of added pyridoxal-P; mutant cells approached the activity of wild-type cells only when they were grown with added vitamin B₆ and were assayed in the presence of pyridoxal-P. In cell-free extracts, the threonine dehydratase from mutant cells was cold labile and more labile to oxidative inactivation than the wild-type enzyme; furthermore, activation of the mutant apoenzyme required a 10- to 20-fold higher concentration of pyridoxal-P than was required for the wild-type apoenzyme. These results show that cultures which appear auxotrophic for a given vitamin may synthesize that vitamin in normal amounts, the exogenous requirement arising from impaired binding of the vitamin-derived coenzyme to a genetically altered apoenzyme dependent on that coenzyme. Inadequate nutritional data to support the genetic findings can lead to erroneous genotype classification for such mutants.

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