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- Borovok, I, et al.
(författare)
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Streptomyces spp. contain class Ia and class II ribonucleotide reductases: expression analysis of the genes in vegetative growth
- 2002
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Ingår i: Microbiology (Reading, England). - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 148:Pt 2, s. 391-404
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Tidskriftsartikel (refereegranskat)abstract
- Genes encoding two ribonucleotide reductases (RNRs) were identified in members of the genusStreptomyces. One gene,nrdJ, encoded an oligomeric protein comprising four identical subunits each with a molecular mass of ∼108 kDa. The activity of this protein depended on the presence of 5′-deoxyadenosylcobalamine (coenzyme B12), establishing it as a class II RNR. TheStreptomyces clavuligerus nrdJgene was cloned, using internal peptide sequences from the purified protein, and was found to encode a polypeptide of 961 aa. Molecular phylogenetic analysis showed that theS. clavuligerusclass II RNR shares significant similarity with most other bacterial and archaeal class II RNRs. Two other genes,nrdAandnrdB, were initially identified in theStreptomyces coelicolorgenome database in unannotated ORFs as encoding a class Ia RNR. Southern analysis demonstrated that thenrdABgenes were present in differentStreptomycesspp. TheS. coelicolor nrdABgenes were cloned and expressed inEscherichia coli, and the recombinant proteins were shown to represent a class I RNR. It was shown, using quantitative real-time PCR, that theS. clavuligerusclass Ia and class II RNR genes were differentially transcribed during vegetative growth. The copy number of the class IInrdJtranscripts was approximately constant throughout the exponential phase of vegetative growth (3–5×105copies per 400 ng total RNA after reverse transcription). In contrast, the copy number of the class IanrdABtranscripts was some 10- to 20-fold less than that ofnrdJin the early-exponential growth phase (2·8×104copies), and decreased markedly at the mid-exponential (4×103copies) and late-exponential phases (1·1×103copies) of growth. A possible role for the involvement of two RNRs during vegetative growth is discussed.
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