Fig. 4.
Point mutations in nadD restore nitrate growth to an mpa mutant and increase NAD abundance in bacteria. (A) Amino acid substitutions in NadD partially rescue growth of an mpa mutant in PB-nitrate. Strains WT, MHD149, MHD1294, MHD1300, MHD1301, and MHD1311 are represented. Note that strains MHD1294, MHD1300, MHD1301, and MHD1311 each have transposon insertions in unrelated genes (see SI Appendix, Table S1 for full genotypes). (B) nadD mutations restore nitrite secretion by the mpa strain to WT levels during growth in PB-nitrate. (C) Ectopic expression of WT nadD or nadDV62A partially rescues growth of an mpa mutant in PB-nitrate (Left) and lowers nitrite secretion by the mpa mutant (Right); statistical significance was determined using one-way ANOVA. Strains MHD1350, MHD1352, MHD1440, and MHD1456 are represented. (D) An mpa mutant contains less NAD than a WT strain, a defect that is rescued both by mutations in nadD and by disruption of hrcA (MHD1297). Total NAD [oxidized (NADH) and reduced (NAD+) forms] was quantified in lysates of bacteria grown in PB-nitrate; statistical significance is indicated by comparison with the mpa single mutant (one-way ANOVA). Experiments in A–D each contain data from three replicate cultures. **P < 0.01; ***P < 0.001.