The obligate aerobe Streptomyces coelicolor A3(2) synthesizes three active respiratory nitrate reductases

Abstract

Streptomyces coelicolorA3(2) synthesizes three membrane-associated respiratory nitrate reductases (Nars). During aerobic growth in liquid medium the bacterium was able to reduce 50 mM nitrate stoichiometrically to nitrite. Construction and analysis of a mutant in which all threenarGHJIoperons were deleted showed that it failed to reduce nitrate. Deletion of the gene encoding MoaA, which catalyses the first step in molybdenum cofactor biosynthesis, also prevented nitrate reduction, consistent with the Nars being molybdoenzymes. In contrast to the triplenarGHJImutant, themoaAmutant was also unable to use nitrate as sole nitrogen source, which indicates that the assimilatory nitrate reductases inS. coelicolorare also molybdenum-dependent. Analysis ofS. coelicolorgrowth on solid medium demonstrated that Nar activity is present in both spores and mycelium (hypha). Development of a survival assay with the nitrate analogue chlorate revealed that wild-typeS. coelicolorspores and mycelium were sensitive to chlorate after anaerobic incubation, independent of the presence of nitrate, while both themoaAand triplenarmutants were chlorate-resistant. Complementation of the triplenarmutant with the individualnarGHJIoperons delivered on cosmids revealed that each operon encoded an enzyme that was synthesized and active in nitrate or chlorate reduction. The data obtained from these studies allow a tentative assignment of Nar1 activity to spores, Nar2 to spores and mycelium, and Nar3 exclusively to mycelium.