Molecular analysis of the ribosomal L11 protein gene (rplK = relC) of Streptomyces griseus and identification of a deletion allele.
The rplK (= relC) gene, which codes for ribosomal protein L11, was cloned from Streptomyces griseus IFO13189 using a screening procedure based on polymerase chain reaction amplification of a gene segment that subsequently allowed the isolation of the complete gene from a gene library. rplK lies between the nusG gene, encoding a protein involved in antitermination of transcription, and the rplA gene, which encodes the ribosomal protein L1. Comparison of the rplK gene sequences of the wild-type strain and the presumed relC mutant strain 3-3 (originally isolated as a thiopeptin-resistant isolate) revealed a 12-bp deletion within the rplK gene from the mutant, flanked by a 4-bp repeat sequence in the corresponding region in the wild type. When the wild-type rplK gene was propagated on a low-copy-number vector in the relC mutant 3-3, the ability to produce guanosine 5'-diphosphate 3'-diphosphate, streptomycin, and submerged spores was completely restored to parental levels. The impaired ability to form aerial mycelium was, however, unaffected. Western blotting analysis showed that the ribosomes from the relC mutant 3-3 incorporate the mutant L11 protein normally, although the level of incorporation is approximately one-third that of the wild-type L11 protein in ribosomes of the parent strain. Propagation of the mutant rplK gene in the wild-type strain resulted in marked defects in growth, streptomycin production, and aerial mycelium formation, indicating that the mutant L11 protein exerts certain negative effects in the cells.
Kawamoto
S
S
National Food Research Institute, Ibaraki, Japan.
Zhang
D
D
Ochi
K
K
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012269:Ribosomal Proteins / Q000235:genetics* / Q000302:isolation & purification; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013305:Streptomyces griseus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D013307:Streptomycin / Q000096:biosynthesis
D017931:DNA Primers; D004269:DNA, Bacterial; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; D006159:Guanosine Tetraphosphate; D013307:Streptomycin