Lesions in gshA (encoding gamma-L-glutamyl-L-cysteine synthetase) prevent aerobic synthesis of thiamine in Salmonella enterica serovar Typhimurium LT2

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella enterica serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter lo ci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is cha llenged. Mutations in gshA, encoding gamma-L-glutamyl-L-cysteine synthetase , prevent the synthesis of glutathione, the major free thiol in the cell, a nd are shown here to result in a thiamine auxotrophy in some of the strains tested, including S. enterica LT2. Phenotypic characterization of the gshA mutants indicated they were similar enough to apbC and apbE mutants to war rant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiami ne, (ii) the ability of L-tryosine to satisfy the THZ requirement, (iii) su ppression of the thiamine requirement by anaerobic growth, and (iv) suppres sion by a second-site mutation at a single locus. Genetic data indicated th at a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.