Metabolic Defects Caused by Mutations in the isc Gene Cluster in Salmonella enterica Serovar Typhimurium: Implications for Thiamine Synthesis

Historically, biosynthetic genes have been identified by mutations resulting in a predicted nutritional requirement. When the biosynthetic genes of several pathways were identified, it became clear that nutritional requirements were generated by lesions in genes that do not encode biosynthetic enzymes but rather have indirect roles in the respective pathways. The characterization of mutations that indirectly affect thiamine biosynthesis has provided insight into the synthesis of this essential vitamin and uncovered new features of metabolism in Salmonella enterica (1, 7, 8, 18, 19, 23, 25, 40). Thiamine pyrophosphate is an essential cofactor for several well-characterized enzymes, including pyruvate dehydrogenase, pyruvate decarboxylase, α-ketoglutarate dehydrogenase, and transketolase (57). Thiamine pyrophosphate is formed by the condensation and subsequent phosphorylation of 4-amino-5-hydroxymethyl pyrimidine pyrophosphate (HMP-PP) and 4-methyl-5-(β-hydroxyethyl)-thiazole monophosphate (THZ-P) (2). HMP-PP is derived from aminoimidazole ribotide, which is a shared intermediate with the purine pathway (20, 21, 36–38). Extensive labeling studies determined that cysteine, tyrosine, and 1-deoxy-d-xylulose contribute to the THZ-P moiety (10, 11, 15, 22, 52). Our present understanding of the thiamine biosynthetic pathway is depicted in Fig. ​Fig.1.1. FIG. 1 Biosynthetic pathway for thiamine synthesis. Enzymes for the catalysis of each reaction are indicated above the relevant arrow. Enzymes that have not been demonstrated but are predicted to function at certain steps in thiamine biosynthesis are in parentheses. ... The strB genetic locus was defined genetically by mutations that resulted in low-level streptomycin resistance, affected histidine biosynthesis, and generated a thiamine and/or nicotinic acid auxotrophy (14, 44, 45). Mutations in this locus were reported to be resistant to 50 μg of streptomycin/ml in nutrient broth and 500 μg/ml in minimal salts medium, distinguishing them from the classical strA alleles of ribosomal protein S12 (rpsL), which are typically resistant to 2 mg of streptomycin/ml (12). Work by Zhu and Roth determined that the nicotinic acid (NA) requirement was due to a reduced activity of quinolinic acid synthetase in these strains (61). Here we mapped two independently isolated insertions in the strB locus and determined that it was allelic with the isc gene cluster in S. enterica. The isc locus was originally described in Azotobacter vinlandii (59) and contained homologs of nif genes that are involved in formation of the iron-sulfur ([Fe-S]) center of nitrogenase (29, 58). Specifically, in a strain with nifS deleted (NifS is involved in the mobilization of sulfur for nitrogenase) (60), an enzyme with l-cysteine desulfurase activity was isolated and designated IscS. The complex locus containing the iscS gene was sequenced and found to be conserved in many non-nitrogen-fixing bacteria (59). The prevalence of this isc gene cluster and the presence of genes similar to those with a demonstrated role in [Fe-S] cluster assembly in nitrogenase suggested that genes in this locus had a general cellular role in [Fe-S] cluster assembly (59). Consistent with this proposal, Nakamura et al. recently demonstrated that coexpression of the isc gene cluster increased the production of five reporter ferredoxins (35). The authors attributed this increase in production to an increased ability to form stable [Fe-S] centers. A second study also showed that the specific isc gene products required for overproduction varied with each reporter [Fe-S] protein tested (50). Recently, Kambampati and Lauhon (31) isolated IscS based on its ability to transfer sulfur to the 4-thiouridine modification of tRNA via a ThiI intermediate. ThiI has been shown to be required for the synthesis of both the thiazole (THZ) moiety of thiamine (55) and the 4-thiouridine modification of tRNA (34), causing the authors to suggest that IscS also has a role in the sulfur donation to THZ. The results presented in this study support this hypothesis. We have identified insertion mutations in two genes in the isc locus, iscR and iscA (Fig. ​(Fig.2).2). The insertions were polar on downstream genes, and complementation analyses showed that the iscR mutation affected two distinct steps in the synthesis of the THZ moiety of thiamine. Both insertion mutants in the isc gene cluster had decreased activities of at least two enzymes containing [Fe-S] centers and displayed novel metabolic phenotypes. In particular, spent medium from these mutants had increased levels of pyruvate when high levels of iron were present in the growth medium, and this increase was eliminated by a null mutation in fur, the gene that encodes the ferric uptake regulator (48). FIG. 2 Organization of the isc gene cluster in S. enterica serovar Typhi. The relative locations of two MudJ insertions are indicated by solid triangles, and the assigned allele numbers are shown. The proposed functions of the products are listed below each ...