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Site-directed mutagenesis of the mecA gene from a methicillin-resistant strain of Staphylococcus aureus
C Y Wu1, W E Alborn, J E Flokowitsch
1Infectious Disease Research, Eli Lilly and Company, Indianapolis, Indiana 46285.
Abstract:
The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.
Insights
The mecA-27r gene in Staphylococcus aureus produces penicillin-binding protein 2a (PBP2a-27r), conferring methicillin resistance. Key motifs within the transpeptidase region are crucial for penicillin binding.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Staphylococcus aureus is a common pathogen.
- Methicillin resistance in S. aureus is a significant public health concern.
- The mecA gene encodes penicillin-binding protein 2a (PBP2a), a key determinant of methicillin resistance.
Purpose of the Study:
- To investigate the functional domains of the mecA-27r gene product, PBP2a-27r.
- To identify the specific regions and motifs responsible for penicillin binding activity.
Main Methods:
- Site-directed mutagenesis was used to alter specific domains and motifs of PBP2a-27r.
- Penicillin-binding assays were performed to assess the activity of modified PBP2a-27r variants.
Main Results:
- Removal of the N-terminal transmembrane domain did not affect penicillin binding.
- Deletion of portions of the putative transglycosylase domain abolished penicillin-binding activity.
- Alterations in the SXXK, SXN, and KSG motifs within the transpeptidase region eliminated penicillin-binding activity.
Conclusions:
- The transglycosylase domain and specific motifs (SXXK, SXN, KSG) within the transpeptidase region of PBP2a-27r are essential for penicillin binding.
- These findings provide insights into the mechanism of methicillin resistance in Staphylococcus aureus.