Randomly selected suppressor mutations in genes for NADH : quinone oxidoreductase-1, which rescue motility of a Salmonella ubiquinone-biosynthesis mutant strain

Abstract

The primary mobile electron-carrier in the aerobic respiratory chain of Salmonella is ubiquinone. Demethylmenaquinone and menaquinone are alternative electron-carriers involved in anaerobic respiration. Ubiquinone biosynthesis was disrupted in strains bearing deletions of the ubiA or ubiE genes. In soft tryptone agar both mutant strains swam poorly. However, the ubiA deletion mutant strain produced suppressor mutant strains with somewhat rescued motility and growth. Six independent suppressor mutants were purified and comparative genome sequence analysis revealed that they each bore a single new missense mutation, which localized to genes for subunits of NADH : quinone oxidoreductase-1. Four mutants bore an identical nuoG(Q297K) mutation, one mutant bore a nuoM(A254S) mutation and one mutant bore a nuoN(A444E) mutation. The NuoG subunit is part of the hydrophilic domain of NADH : quinone oxidoreductase-1 and the NuoM and NuoN subunits are part of the hydrophobic membrane-embedded domain. Respiration was rescued and the suppressed mutant strains grew better in Luria–Bertani broth medium and could use l-malate as a sole carbon source. The quinone pool of the cytoplasmic membrane was characterized by reversed-phase HPLC. Wild-type cells made ubiquinone and menaquinone. Strains with a ubiA deletion mutation made demethylmenaquinone and menaquinone and the ubiE deletion mutant strain made demethylmenaquinone and 2-octaprenyl-6-methoxy-1,4-benzoquinone; the total quinone pool was reduced. Immunoblotting found increased NADH : quinone oxidoreductase-1 levels for ubiquinone-biosynthesis mutant strains and enzyme assays measured electron transfer from NADH to demethylmenaquinone or menaquinone. Under certain growth conditions the suppressor mutations improved electron flow activity of NADH : quinone oxidoreductase-1 for cells bearing a ubiA deletion mutation.