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

Abstract

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 loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in gshA, encoding gamma-L-glutamyl-L-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and 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 warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of L-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that 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.

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 function at certain steps in thiamine biosynthesis are in parentheses. The nitrogen and carbon atoms donated by tyrosine are boxed in the THZ structure. The position of ThiH with respect to ThiG, -F, and -S is based partially on work reported here. (B) The GSH biosynthetic pathway. Abbreviations: PRPP, phosphoribosylpyrophosphate; Gln, glutamine; AIR, 5-aminoimidazole ribotide; THZ-P, 4-methyl-5-β-hydroxyethyl-thiazole monophosphate; HMP-PP, 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate; TMP, thiamine monophosphate; TPP, thiamine pyrophosphate.

FIG. 2

Growth requirements of gshA mutations in S. enterica. Soft agar containing strain DM4620 (gshA) was overlaid on minimal glucose medium. Compounds were spotted in 2-μl aliquots on the solidified agar as indicated, at the following concentrations: thiamine (B₁), HMP, and THZ, 100 μM; GSH and γ-GC, 10 mM.

FIG. 3

Mutations in gshB and gshA have opposite effects on sensitivity of the strain to paraquat. Cultures were grown as described in Materials and Methods. (A) Cultures were grown in Luria broth supplemented with 4 μM paraquat (methyl viologen). (B) Strains were grown in Luria broth supplemented with 0.4 μM paraquat. In each case, the strains were LT2 (wild type, □), DM4496 (gshB mutant, ▴), and DM4620 (gshA mutant, ●).

FIG. 4

TMP synthesis in resting cells. (A) Titration of TMP synthesis in DM3012 (thiL mutant, ■) and DM5490 (thiL apbE mutant, ●). Equal amounts of cells were resuspended in minimal media supplemented with HMP and THZ as described in Materials and Methods. (B) Measurement of TMP formation over time in resting cells from strains DM3012 (thiL mutant, ■), DM5490 (thiL apbE mutant, ●), and DM5537 (thiL thiE mutant, ▴). Equal amounts of cells were resuspended in minimal media, and samples were removed for TMP determination. The time of addition of HMP plus THZ (25 nM each) is indicated by the arrow.

FIG. 5

Representative growth curves of Thz* and thiH null mutants. (A) Representative growth of Thz* mutant DM4106 (thiH1106). (B) Growth response of a thiH null mutant, DM460. Strains were grown in minimal medium (○), minimal medium supplemented with 100 μM l-tyrosine (□), or 1 μM thiamine (●).

FIG. 6

Plasmid construction. Plasmid pthiCH was isolated as described in the text. BamHI (B) digestion allowed individual subcloning of thiH on a 2.4-kb fragment, generating plasmid pthiH. The insert of pthiH was ligated into BamHI-cut pGEM7 (Promega), generating plasmids pthiH-7(+) and pthiH-7(−), with thiH in the correct orientation for expression from the T₇ promoter and from the lac promoter, respectively. Plasmid pthiES1 was generated via BamHI and EcoRI (E) digestion of pthiCH and ligation into pSU18. Black arrows represent complete ORFs, and boxes represent vector DNA.

FIG. 7

Activity of ThiH follows that of ThiG-, -F, and -S in THZ synthesis. Cells of strain DM4797 (thiH1106 thiG1113) were overlaid in soft agar on a minimal glucose plate and incubated at 30°C for the indicated time. At the appropriate times, 2 μmol of l-tyrosine in 20 μl was spotted in the middle of duplicate plates. One of the plates was then returned to 30°C and one was incubated at 42°C. Growth was assessed after 18 h. Pluses and minuses represent our interpretation of growth on the relevant plates.