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Streptomyces spp. contain class Ia and class II ribonucleotide reductases: expression analysis of the genes in vegetative growth

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel¹
Department of Biochemistry 1, Medical Nobel Institute, MBB, Karolinska Institutet, S-17177 Stockholm, Sweden²

Author for correspondence: Yair Aharonowitz. Tel: +972 3 6409411. Fax: +972 3 6422245. e-mail: yaira{at}post.tau.ac.il

Genes encoding two ribonucleotide reductases (RNRs) were identified in members of the genus Streptomyces. 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. The Streptomyces clavuligerus nrdJ gene 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 the S. clavuligerus class II RNR shares significant similarity with most other bacterial and archaeal class II RNRs. Two other genes, nrdA and nrdB, were initially identified in the Streptomyces coelicolor genome database in unannotated ORFs as encoding a class Ia RNR. Southern analysis demonstrated that the nrdAB genes were present in different Streptomyces spp. The S. coelicolor nrdAB genes were cloned and expressed in Escherichia coli, and the recombinant proteins were shown to represent a class I RNR. It was shown, using quantitative real-time PCR, that the S. clavuligerus class Ia and class II RNR genes were differentially transcribed during vegetative growth. The copy number of the class II nrdJ transcripts was approximately constant throughout the exponential phase of vegetative growth (3–5x10⁵ copies per 400 ng total RNA after reverse transcription). In contrast, the copy number of the class Ia nrdAB transcripts was some 10- to 20-fold less than that of nrdJ in the early-exponential growth phase (2·8x10⁴ copies), and decreased markedly at the mid-exponential (4x10³ copies) and late-exponential phases (1·1x10³ copies) of growth. A possible role for the involvement of two RNRs during vegetative growth is discussed.

Keywords: Streptomyces, 5'-deoxyadenosylcobalamine, expression, phylogeny, ribonucleotide reductase genes

Abbreviations: coenzyme B₁₂, 5'-deoxyadenosylcobalamine; RNR, ribonucleotide reductase

The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this paper are AJ224870, AJ276618, AJ277778, AJ295338 and AJ295339.

^a These two authors contributed equally to this study.

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