ErmK leader peptide : amino acid sequence critical for induction by erythromycin.
The ermK gene from Bacillus lichenformis encodes an inducible rRNA methylase that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino acid leader peptide together with its ribosome binding site. The secondary structure of ermK leader mRNA and a leader peptide sequence have been reported as the elements that control expression. In this study, the contribution of specific leader peptide amino acid residues to induction of ermK was studied using the PCR-based megaprimer mutation method. ermK methylases with altered leader peptide codons were translationally fused to E. coil beta-galactosidase reporter gene. The deletion of the codons for Thr-2 through Ser-4 reduced inducibility by erythromycin, whereas that for Thr-2 and His-3 was not. The replacement of the individual codons for Ser-4, Met-5 and Arg-6 with termination codon led to loss of inducibility, but stop mutation of codon Phe-9 restored inducibility by erythromycin. Collectively, these findings suggest that the codons for residue 4, 5 and 6 comprise the critical region for induction. The stop mutation at Leu-7 expressed constitutively ermK gene. Thus, ribosome stalling at codon 7 appears to be important for ermK induction.
Kwon
Ae-Ran
AR
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea.
Min
Yu-Hong
YH
Yoon
Eun-Jeong
EJ
Kim
Jung-A
JA
Shim
Mi-Ja
MJ
Choi
Eung-Chil
EC
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001407:Bacillus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D001412:Bacillus subtilis / Q000254:growth & development; D004790:Enzyme Induction; D004917:Erythromycin / Q000494:pharmacology*; D007763:Lac Operon / Q000235:genetics; D008780:Methyltransferases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects
D000900:Anti-Bacterial Agents; D010455:Peptides; D004917:Erythromycin; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase