Regulation of NAD biosynthesis was examined through the construction of nad-lac fusions in Salmonella typhimurium. The nadA (17 unit map position) and nadB (55 units) genetic loci involved with quinolinic acid biosynthesis were both found to be regulated by the product of a nadR locus (99 units) in a repression/derepression manner while nadC (3 units) expression appeared constitutive at the transcriptional level. Increases in nadAB transcription directly correlated with decreases in intracellular NAD(P) levels, and kinetic studies indicated that the NAD analogue 6-amino NAD was ineffective in repressing either nadA or nadB. The presence of cAMP + cAMP receptor protein was essential for the complete derepression of nadA while no effect was evident upon nadB. Transfer of cultures from aerobic to anaerobic conditions, however, resulted in the partial derepression of both nadA and nadB. Thus, there appears to be a very complex set of controls regulating NAD biosynthesis.
Two classes of pyridine nucleotide uptake mutants isolated previously in a strain of Salmonella typhimurium defective in both de novo NAD biosynthesis (nad) and pyridine nucleotide recycling (pncA) were analysed in terms of their genetic relationship to each other and their roles in the transport of nicotinamide mononucleotide as a precursor to NAD. The first class of uptake mutants, pnuA (99 units), failed to grow on nicotinamide mononucleotide (NMN) as a precursor for NAD. The second class, pnuB, grew on lower than normal levels of NMN and suppressed pnuA mutations. A third class of uptake mutant, pnuC, isolated in a nadB pncA pnuB background, also failed to grow on NMN. Transport studies and enzyme analyses confirmed these strains as defective in NMN uptake. A fourth locus, designated pnuD, was found to diminish NMN utilization in a nad pncA+ background. Tn10 insertions near pnuA, pnuC and pnuD were isolated and utilized in mapping studies. pnuA was found to map between thr and serB near trpR. The pnuC locus was cotransducible with nadA at 17 units while pnuD mapped at approximately 60 units. The biochemical and genetic data suggest that the pnuA and pnuC gene products cooperate in the utilization of NMN under normal conditions. A pnuB mutant, however, does not require the pnuA gene product for NMN uptake but does rely on the pnuC product. Fusion studies indicate that pnuC is regulated by internal NAD concentrations.
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