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

Abstract

Streptomyces coelicolor A3(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 three narGHJI operons 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 triple narGHJI mutant, the moaA mutant was also unable to use nitrate as sole nitrogen source, which indicates that the assimilatory nitrate reductases in S. coelicolor are also molybdenumdependent. Analysis of S. coelicolor growth 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-type S. coelicolor spores and mycelium were sensitive to chlorate after anaerobic incubation, independent of the presence of nitrate, while both the moaA and triple nar mutants were chlorate-resistant. Complementation of the triple nar mutant with the individual narGHJI operons 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.

RNA-Seq and RNA Immunoprecipitation Analyses of the Transcriptome of Streptomyces coelicolor Identify Substrates for RNase III

“…4-fold higher in JSE1880 than in M145. S. coelicolor contains operons for three nitrate reductases (14), and transcript levels for other subunits of reductase 3 and for several subunits of reductase 2 also increased in JSE1880, by 2.0-to 3.4-fold (see Table S2 in the supplemental material).…”

Oxygen-Dependent Control of Respiratory Nitrate Reduction in Mycelium of Streptomyces coelicolor A3(2)

“…In liquid medium, spore suspensions (20 ml of a suspension with an OD 450 of 5) were anaerobically incubated with 50 mM MOPS-NaOH buffer and 5 mM nitrate in serum flasks. Excreted nitrite was determined colorimetrically as described below, and the presence of nitrite was revealed as a dark violet staining of the well (12).…”

“…Nar1 has recently been characterized as the first known spore-specific Nar enzyme (13) and is always present in a ready-to-use mode in mature spores, but it is not found in mycelium. The in vivo activity of Nar1-dependent nitrate reduction is initiated only in the absence of ambient oxygen and results in the stoichiometric release of nitrite when exogenous nitrate is available (12,13). Less is known about the Nar2 and Nar3 enzymes, other than the fact that they are primarily active in mycelium and not in spores (12).…”

“…In contrast to M. tuberculosis, the genome of S. coelicolor A3(2) encodes three nonredundant, active Nar enzymes (12). Nar1 has recently been characterized as the first known spore-specific Nar enzyme (13) and is always present in a ready-to-use mode in mature spores, but it is not found in mycelium.…”

“…The in vivo activity of Nar1-dependent nitrate reduction is initiated only in the absence of ambient oxygen and results in the stoichiometric release of nitrite when exogenous nitrate is available (12,13). Less is known about the Nar2 and Nar3 enzymes, other than the fact that they are primarily active in mycelium and not in spores (12). It is conceivable that, like for Nar1, the regulation of the synthesis of Nar2 and Nar3 differs from the regulatory mechanisms controlling Nar enzyme synthesis in other microorganisms.…”