Lesions in gshA (Encoding γ-l-Glutamyl-l-Cysteine Synthetase) Prevent Aerobic Synthesis of Thiamine in Salmonella enterica Serovar Typhimurium LT2

Thiamine pyrophosphate (TPP) is an essential cofactor for several enzymes, including pyruvate dehydrogenase, transketolase, and acetolactate synthase, for which it functions as a carbon unit carrier and electron sink. TPP is composed of two independently synthesized moieties, 4-methyl-5-β-hydroxyethyl-thiazole monophosphate (THZ-P) and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP). Although several recent studies have increased our understanding of the synthesis of TPP in Escherichia coli and Salmonella enterica (5, 31, 39, 46, 51, 52, 53), many central issues have yet to be resolved. The current understanding of the substrates and enzymes in TPP synthesis are outlined in Fig. ​Fig.1A1A (6, 15, 20–23, 43, 54, 55). Significantly, l-Tyr donates a single carbon and the nitrogen atom to the thiazole (THZ) ring. FIG. 1 Biosynthetic pathways for thiamine and GSH. (A) Our current understanding of thiamine biosynthesis in S. enterica. Enzymes for each of the reactions are indicated above the relevant arrows. Enzymes that have not been demonstrated but are predicted to ... Of the five genes implicated in the synthesis of THZ-P in S. enterica and E. coli (5, 49, 51), the products of thiFSGI have been assigned functions based on in vitro activity assays (31, 46, 47). However, the order of the steps and the specific metabolites in this biosynthesis have not been rigorously defined. The precursor for the pyrimidine (HMP) moiety was shown to be the purine biosynthetic intermediate, aminoimidazole ribotide (AIR) (22). The steps involved in the conversion of AIR to HMP are unknown, and to date, mutations in only one gene, thiC, have resulted in an unconditional requirement for HMP or thiamine. Past work showed that mutations in several loci distinct from the thi genes could result in a thiamine auxotrophy. Characterization of several mutations that indirectly affected thiamine synthesis has increased our understanding of thiamine synthesis and its integration with other metabolic processes (12, 13, 17, 18, 24, 25, 40, 42; J. Zilles, J. Kappock, J. Stubbe, and D. M. Downs, unpublished data). Mutations that affected thiamine synthesis often resulted in phenotypes dependent on growth conditions and strain backgrounds. Functional characterization of two of these loci in particular, apbE and apbC, has been difficult since each contains a novel open reading frame (ORF) and no significant functional information has been uncovered by sequence analyses. ApbE is a periplasmic lipoprotein (3, 4), and apbC (mrp) encodes a protein of unknown function (38) that has been implicated in a variety of metabolic processes (J. Escalante-Semerena, J. R. Roth, and R. LaRossa, personal communication). Both apbE and apbC were required for thiamine synthesis under some conditions, and lesions in either of these genes resulted in a requirement for both the HMP and THZ moieties of thiamine for growth (4). Here we show that organisms of a laboratory strain of S. enterica with mutations in the gshA gene, encoding γ-l-glutamyl-l-cysteine synthetase (EC 6.3.2.2), are thiamine auxotrophs. The gshA mutants displayed phenotypes similar to apbC and apbE mutants, including (i) a requirement for both HMP and THZ, (ii) the ability of l-Tyr to satisfy the THZ requirement, (iii) the suppression of the thiamine requirement by anaerobic growth, and (iv) the suppression by second-site mutations at a single locus. GshA is one of two gene products that are required for the synthesis of glutathione (GSH), the major free thiol in the cell (Fig. ​(Fig.1B).1B). The extensive literature on GshA and the role of GSH in metabolism, the demonstration that mutant strains defective in [Fe-S] cluster formation or repair have similar THZ phenotypes (i to iv above) (42), and the work described here led to a model to explain the THZ requirement in these strains (27, 37). Results from genetic experiments here are consistent with the THZ requirement in this group of strains (gshA, apbE, and apbC) being caused by inhibition of ThiH activity. We suggest that ThiH contains an [Fe-S] cluster, and in these mutants, synthesis and/or repair of this cluster is defective. This defect results in a loss of ThiH activity and compromises the ability of the cell to synthesize THZ. The specific defect resulting in a requirement for HMP in these strains has not yet been addressed.