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As our knowledge of discrete biochemical pathways increases, it has become important to understand how these pathways are integrated to result in an effective physiology. We have examined thiamine synthesis in Salmonella enterica serovar Typhimurium as a model system for detecting metabolic pathway integration (8, 13, 15, 23). Thiamine monophosphate is synthesized by the condensation of 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) pyrophosphate and 4-methyl-5-(-hydroxyethyl)-thiazole phosphate. Thiamine monophosphate phosphorylation produces thiamine pyrophosphate, the coenzymic form of the vitamin (5, 6). As depicted in Fig. 1A, HMP pyrophosphate and 4-methyl-5-(-hydroxyethyl)-thiazole phosphate are synthesized by independent pathways. Despite rigorous in vivo labeling experiments, the biochemical reactions that result in the synthesis of these moieties are not defined.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Relevant metabolic pathways. (A) Illustrated are the biosynthetic pathways involved in purine and thiamine synthesis. Names of genes whose products are known are indicated. The reaction(s) involved in the formation of PRA independent of PurF has not been defined biochemically or genetically. (B) Glycolysis, the pentose phosphate pathway, and the Entner-Doudoroff pathway are schematically represented. Names of genes whose products are required are indicated. Abbreviations: P, phosphate; PRPP, 5-phosphoribosyl-1-pyrophosphate AIR, aminoimidazole ribotide; PP, pyrophosphate; THZ-P, 4-methyl-5-(-hydroxyethyl)-thiazole phosphate; TMP, thiamine monophosphate; G-3-P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; TCA, tricarboxylic acid.

The HMP moiety of thiamine is synthesized from the de novo purine pathway intermediate aminoimidazole ribotide, the last intermediate common to the two pathways (16, 20, 21). Work in our laboratory demonstrated that thiamine synthesis could occur independently of the purF gene product, and this synthesis was attributed to the alternative pyrimidine biosynthetic (APB) pathway (10, 12). Recent work has demonstrated that the four purine biosynthetic reactions after that catalyzed by PurF are needed for HMP synthesis via the APB pathway, making it synonymous with PurF-independent thiamine synthesis (13). However, the proposed step(s) forming phosphoribosylamine (PRA) in the absence of PurF has not been defined genetically or biochemically. To date, mutations preventing PurF-independent thiamine synthesis (Apb) have been involved in the pantothenate biosynthetic pathway (e.g., panE) (11, 17), in loci thought to affect the conversion of aminoimidazole ribotide to HMP (i.e., apbC and apbE) (4, 22), or in genes encoding enzymes of the oxidative pentose phosphate (OPP) pathway (e.g., gnd and zwf) (15, 23). Based on analyses of the last class of mutants, it was proposed that the OPP pathway contributed ribose-5-phosphate for the formation of PRA via the APB pathway (14, 24). Work presented here was initiated to identify genes encoding functions needed to synthesize PRA in the absence of PurF.

Herein we describe a new class of mutations that block PurF-independent thiamine synthesis. These lesions were located in the nuo operon, which encodes NADH dehydrogenase I (NADH dHI). NADH dHI transfers electrons to ubiquinone in the electron transport chain and, unlike NADH dHII (encoded by ndh), produces a proton motive force (18). Two general classes of metabolic phenotypes have been reported for nuo mutants: those due to lack of energy generation (2, 27) and those due to the resulting imbalance in the ratio of reduced to oxidized pyridine nucleotide pools (25). We report here that, in addition to having a defect in thiamine synthesis, nuo mutants have a reduced capacity for flux through the OPP pathway and suggest that this is a consequence of the imbalance in pyridine nucleotide pools in these mutants.

Rationale for mutant isolation. In the majority of mutants isolated for their defect in PurF-independent thiamine synthesis, a purE block was able to restore thiamine-independent growth to the levels of wild-type strains (4, 22, 24). To eliminate this predominant class of mutants, we screened MudJ(Km) insertion mutations for those that prevented strain DM2353 (purF purE) (Table 1) from growing on gluconate-adenine medium lacking thiamine. Of the approximately 35,000 Km^r transductants screened, 11 mutants carried insertions that blocked thiamine synthesis in a purF purE mutant but that had no effect on the ability of a wild-type strain to grow on minimal medium. Three of these mutants had lesions in gnd (encoding gluconate-6-phosphate dehydrogenase), and one had a lesion in panE (which encodes ketopantoate reductase), both of which were expected based on previously described phenotypes (11, 15, 17). The remaining seven mutants had insertions that were unlinked to loci previously shown to prevent PurF-independent thiamine synthesis and thus represented a new class of mutants.

Mutations in the nuo operon prevent PurF-independent thiamine synthesis. A Tn10d(Tc) insertion [zeg-8661::Tn10d(Tc)] was isolated 50% linked to MudJ(Km) in strain DM4134 (purF2085 purE3041 nuo-354::MudJ), and found to be linked to the remaining six MudJ insertion mutations, suggesting that the seven mutants were defective in the same locus.

Mutations in nuo reduce flux through the OPP pathway in vivo. Previously we showed that flux through the OPP pathway was required for PurF-independent thiamine synthesis (14). Therefore, we tested whether nuo mutations resulted in reduced flux through the OPP pathway and, if so, whether the reduction was sufficient to cause the observed thiamine requirement. We reasoned that eliminating the major NADH dehydrogenase activity would result in a higher cellular NADH/NAD pool ratio, an idea previously proposed by others (25). If this assumption was correct, the cell might compensate by decreasing NADPH pools in an attempt to restore internal redox balance. Since the OPP pathway provides a source of NADPH, this pathway may be a site for such regulation. Results from the following experiments indicated that the flux capacity of the OPP pathway was reduced in a nuo mutant but that this reduction was not solely responsible for the resulting thiamine requirement.

Gnd is the limiting step of the OPP pathway in nuo mutants. Based on the described routes for gluconate catabolism, the above results indicated that a nuo mutation resulted in reduced flux through the OPP pathway. The rationale for the model presented above suggested that Gnd, the enzyme directly responsible for production of NADPH, may be the limiting step in gluconate utilization in the nuo edd mutant. To address this possibility, pMN6 (Gnd⁺) (19) was introduced into DM5399 (nuo edd) and growth of the resulting strain (DM5400) was assessed with gluconate as the sole carbon source. Representative data from these experiments, shown in Table 3, demonstrated that pMN6 restored a wild-type growth rate to the double mutant. Assays in cell-free extracts determined that strains carrying the pMN6 plasmid had ~10-fold more gluconate-6-phosphate dehydrogenase activity than those with only chromosomal gnd (data not shown). These results were consistent with a reduction of Gnd activity preventing a nuo edd double mutant from utilizing gluconate.

Reduced flux through the OPP pathway does not cause the Thi phenotype of nuo mutants. Having determined that nuo mutants had reduced flux through the OPP pathway, we asked whether this reduction was responsible for the thiamine requirement observed in a purF nuo mutant. Two results indicated that the defect in the OPP pathway was not sufficient to generate the thiamine requirement of a purF nuo mutant. First, the presence of pMN6(Gnd⁺) in a purF nuo double mutant failed to restore thiamine-independent growth. The specific growth rates of the strain with and without this plasmid were not significantly different in medium with gluconate as the sole carbon and energy source and containing adenine (µ = 0.134 and 0.198, respectively). That the pMN6 plasmid restored flux through the OPP pathway suggested that an additional effect of the nuo mutation was involved in causing the thiamine requirement. Second, when exogenous ribose is spotted on a top agar lawn of purF gnd cells on gluconate adenine medium, thiamine-independent growth is restored (14). Exogenous ribose was unable to restore thiamine synthesis in either DM4615 (purF nuo) or DM4484 (purF nuo purE) when the strains were tested in the same way (data not shown). This result also supported the conclusion that a nuo mutation did not cause a thiamine requirement solely by reducing flux through the OPP pathway.

Summary. The studies described here made two contributions to our understanding of metabolism in Salmonella serovar Typhimurium. First, we have shown that mutations in the nuo operon are unable to perform PurF-independent thiamine synthesis. Second, we showed that strains defective in the major energy-generating NADH dehydrogenase complex are impaired in the OPP pathway. Our data are consistent with the model that lesions in this complex result in an increased NADH/NAD ratio that inhibits the activity of Gnd. Such an inhibition may be designed to reduce the NADPH/NADP ratio and thus restore the balance of reduced to oxidized pyridine nucleotide pools in the cell.