A novel insertion mutation in Streptomyces coelicolor ribosomal S12 protein results in paromomycin resistance and antibiotic overproduction.
We identified a novel paromomycin resistance-associated mutation in rpsL, caused by the insertion of a glycine residue at position 92, in Streptomyces coelicolor ribosomal protein S12. This insertion mutation (GI92) resulted in a 20-fold increase in the paromomycin resistance level. In combination with another S12 mutation, K88E, the GI92 mutation markedly enhanced the production of the blue-colored polyketide antibiotic actinorhodin and the red-colored antibiotic undecylprodigiosin. The gene replacement experiments demonstrated that the K88E-GI92 double mutation in the rpsL gene was responsible for the marked enhancement of antibiotic production observed. Ribosomes with the K88E-GI92 double mutation were characterized by error restrictiveness (i.e., hyperaccuracy). Using a cell-free translation system, we found that mutant ribosomes harboring the K88E-GI92 double mutation but not ribosomes harboring the GI92 mutation alone displayed sixfold greater translation activity relative to that of the wild-type ribosomes at late growth phase. This resulted in the overproduction of actinorhodin, caused by the transcriptional activation of the pathway-specific regulatory gene actII-orf4, possibly due to the increased translation of transcripts encoding activators of actII-orf4. The mutant with the K88E-GI92 double mutation accumulated a high level of ribosome recycling factor at late stationary phase, underlying the high level of protein synthesis activity observed.
Wang
Guojun
G
National Food Research Institute, Tsukuba, Ibaraki, Japan.
Inaoka
Takashi
T
Okamoto
Susumu
S
Ochi
Kozo
K
D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002474:Cell-Free System; D004269:DNA, Bacterial / Q000737:chemistry; D024881:Drug Resistance, Bacterial; D015964:Gene Expression Regulation, Bacterial; D005998:Glycine / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D008826:Microbial Sensitivity Tests; D015394:Molecular Structure; D016254:Mutagenesis, Insertional; D010303:Paromomycin / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012269:Ribosomal Proteins; C034212:ribosomal protein S12; D049452:Green Fluorescent Proteins; D010303:Paromomycin; D005998:Glycine