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

Ilya Borovok, Rachel Kreisberg-Zakarin, Michaela Yanko, Rachel Schreiber, Margarita Myslovati, Fredrik Åslund, Arne Holmgren, Gerald Cohen, Yair Aharonowitz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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 (3-5 × 105 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 (2.8 × 104 copies), and decreased markedly at the mid-exponential (4 × 103 copies) and late-exponential phases (1.1 × 103 copies) of growth. A possible role for the involvement of two RNRs during vegetative growth is discussed.

Original languageEnglish
Pages (from-to)391-404
Number of pages14
JournalMicrobiology
Volume148
Issue number2
DOIs
StatePublished - 2002

Keywords

  • 5′-deoxyadenosylcobalamine
  • Expression
  • Phylogeny
  • Ribonucleotide reductase genes
  • Streptomyces

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