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Phosphoribosylamine (PRA) is the first intermediate in the common purine/thiamine biosynthetic pathway and is primarily synthesized by the product of the purF gene, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14). Past genetic and biochemical studies have shown that multiple mechanisms for the synthesis of PRA independent of PurF are present in Salmonella enterica. Here, we describe mutant alleles of the essential prsA gene, which encodes PRPP synthetase (E.C. 2.7.6.1), that allow PurF-independent thiamine synthesis. The mutant alleles resulted in reduced PrsA activity in extracts, caused nutritional requirements indicative of PRPP limitation and allowed non-enzymic formation of PRA due to a build-up of ribose 5-phosphate (R5P). These results emphasize the balance that must be reached between pathways competing for the same substrate to maintain robustness of the metabolic network. DOI 10.1099/mic.0.033050-0

The response of a cell to integrated stresses was investigated using environmental and/or genetic perturbations that disrupted labile iron homeostasis and increased oxidative stress. The effects of the perturbations were monitored as nutritional requirements, and were traced to specific enzymic targets. A yggX gshA cyaY mutant strain required exogenous thiamine and methionine for growth. The thiamine requirement, which had previously been linked to the Fe-S cluster proteins ThiH and ThiC, was responsive to oxidative stress and was not directly affected by manipulation of the iron pool. The methionine requirement was associated with the activity of sulfite reductase, an enzyme that appeared responsive to disruption of labile iron homeostasis. The results are incorporated in a model to suggest how the activity of iron-containing enzymes not directly sensitive to oxygen can be decreased by oxidation of the labile iron pool.

Several cellular pathways have been identified which affect the efficiency of thiamine biosynthesis in Salmonella enterica. Mutants defective in iron-sulfur (Fe-S) cluster metabolism are less efficient at synthesis of the pyrimidine moiety of thiamine. These mutants are compromised for the conversion of aminoimidazole ribotide (AIR) to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P), not the synthesis of AIR. The gene product ThiC contains potential ligands for an Fe-S cluster that are required for function in vivo. The conversion of AIR to HMP-P is sensitive to oxidative stress, and variants of ThiC have been identified that have increased sensitivity to oxidative growth conditions. The data are consistent with ThiC or an as-yet-unidentified protein involved in HMP-P synthesis containing an Fe-S cluster required for its physiological function.