Streptomyces spp. contain class Ia and class II ribonucleotide reductases: expression analysis of the genes in vegetative growth.
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 approximately 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 (3-5x10(5) 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 (2.8x10(4) copies), and decreased markedly at the mid-exponential (4x10(3) copies) and late-exponential phases (1.1x10(3) copies) of growth. A possible role for the involvement of two RNRs during vegetative growth is discussed.
Borovok
Ilya
I
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel1.
Kreisberg-Zakarin
Rachel
R
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel1.
Yanko
Michaela
M
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel1.
Schreiber
Rachel
R
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel1.
Myslovati
Margarita
M
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel1.
Aslund
Fredrik
F
Department of Biochemistry 1, Medical Nobel Institute, MBB, Karolinska Institutet, S-17177 Stockholm, Sweden2.
Holmgren
Arne
A
Department of Biochemistry 1, Medical Nobel Institute, MBB, Karolinska Institutet, S-17177 Stockholm, Sweden2.
Cohen
Gerald
G
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel1.
Aharonowitz
Yair
Y
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel1.
D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D004269:DNA, Bacterial / Q000235:genetics; D019143:Evolution, Molecular; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000145:classification* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012264:Ribonucleotide Reductases