Genetically engineered mutant of the cyanobacterium Synechococcus PCC 7942 defective in nitrate transport

Abstract

Nitrate-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) contain a 45-kDa protein as a major protein in the cytoplasmic membrane but ammonium-grown cells lack it. A mutant (M45) was constructed by inactivating the gene encoding the 45-kDa protein. M45 did not grow under low concentrations of nitrate but high concentrations of nitrate could support its growth, with the optimal concentration being 40-70 mM. The growth rate of M45 was as high as that of the wild-type cells when ammonium was the nitrogen source. The 45-kDa protein was absent in M45 irrespective of the growth conditions. The activities of nitrate and nitrite reductases were higher in M45 than in wild type. The rate of nitrate-dependent O2 evolution in wild type measured in the presence of L-methionine D,L-sulfoximine and D,L-glyceraldehyde showed saturation kinetics with respect to nitrate concentration in the external medium. The nitrate concentration required to produce half the maximal rate was 1 μM. In M45, the rate of nitrate-dependent O2 evolution was nearly zero at nitrate concentrations <1 mM and was linearly increased as the concentration increased. The presumed absence of nitrate transport in M45 demonstrated by these results suggested that the 45-kDa protein is a nitrate transporter.