EVIDENCE FOR 2 NAD KINASES IN SALMONELLA-TYPHIMURIUM

The electron-carrying cofactor NADP is formed by phosphorylation of NA D. A strategy for the isolation of NAD kinase mutants revealed two cla sses of temperature-sensitive mutations, nadF and nadG, mapping at min 13 and 72 of the Salmonella chromosome. Both mutant types grew on nut rient broth at both 30 and 42 degrees C but on minimal medium showed a temperature sensitive growth defect which was not corrected by any of the single nutritional supplements tested. A nadF deletion mutant gre w on nutrient broth but not on minimal medium. A double mutant with th e nadF deletion and a nadG(Ts) mutation showed temperature-sensitive g rowth on all media. We propose that Salmonella typhimurium has two NAD kinases, one encoded by the nadF and one by the nadG gene. This is su pported by the fact that temperature-sensitive mutants of both genes p roduce kinase activity with altered heat stability. Results suggest th at either one of two NAD kinases is sufficient for growth on rich medi um, but that both are needed for growth on minimal media. Enzyme assay s show that the nadF gene is responsible for about 70% of total NAD ki nase activity, and that the nadG gene dictates the remaining 30%. Whil e testing nutritional phenotypes of nadF and nadG mutants, we found th at the biosynthetic intermediate, quinolinic acid (QA) inhibited growt h of nadF mutants on nutrient broth. This suggested that the NadG enzy me might be inhibited by QA. Enzyme assays demonstrated that QA inhibi ts the NadG but not the NadF enzyme. This suggests the existence of a regulatory mechanism which controls NADP levels.