ErmK leader peptide : amino acid sequence critical for induction by erythromycin
Ae-Ran Kwon1, Yu-Hong Min, Eun-Jeong Yoon
1College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea.
Abstract:
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.
Insights
Investigating the ermK gene in Bacillus lichenformis revealed specific leader peptide amino acid residues crucial for antibiotic resistance induction. Mutations altering codons 4-6 significantly impacted ermK gene expression, highlighting their regulatory role.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The ermK gene from Bacillus lichenformis confers resistance to macrolide-lincosamide-streptogramin B antibiotics.
- ermK expression is regulated by its mRNA leader sequence, including a 14-amino acid leader peptide and its secondary structure.
Purpose of the Study:
- To investigate the contribution of specific leader peptide amino acid residues to the induction of the ermK gene.
- To identify critical residues within the ermK leader peptide involved in regulating antibiotic resistance gene expression.
Main Methods:
- Utilized the PCR-based megaprimer mutation method to create alterations in ermK leader peptide codons.
- Constructed translational fusions of ermK methylase with an E. coli beta-galactosidase reporter gene to assess inducibility.
Main Results:
- Deletion of codons for Thr-2 through Ser-4 reduced erythromycin inducibility.
- Replacing codons for Ser-4, Met-5, and Arg-6 with stop codons resulted in loss of inducibility.
- A stop mutation at Phe-9 restored inducibility, while a stop mutation at Leu-7 led to constitutive ermK gene expression.
Conclusions:
- Codons for residues 4, 5, and 6 in the ermK leader peptide form a critical region for gene induction.
- Ribosome stalling at codon 7 is essential for ermK induction, suggesting a mechanism of translational control.
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