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J Bacteriol, April 1998, p. 2160-2166, Vol. 180, No. 8
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Interaction of Native and Mutant MecI Repressors with Sequences That Regulate mecA, the Gene Encoding Penicillin Binding Protein 2a in Methicillin-Resistant Staphylococci

Vijay K. Sharma,1,dagger Corinne J. Hackbarth,2,Dagger Tanja M. Dickinson,3 and Gordon L. Archer1,3,*

Departments of Medicine1 and Microbiology/Immunology,3 Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia, and Department of Medicine, University of California, San Francisco, California2

Received 2 December 1997/Accepted 30 January 1998

Methicillin resistance in staphylococci is mediated by PBP2a, a penicillin binding protein with low affinity for beta -lactam antibiotics. The gene encoding PBP2a, mecA, is transcriptionally regulated in some clinical isolates by mecR1 and mecI, genes divergently transcribed from mecA that encode a signal transducer and repressor, respectively. The biochemical basis of MecI-mediated mecA transcriptional repression was investigated by using purified MecI. In DNase I protection studies, MecI protected a 30-bp palindrome encompassing the predicted mecA -10 and the mecR1 -35 promoter sequences. The larger palindrome contained 15 bp of dyad symmetry within which was a smaller 6-bp palindrome. Electrophoretic mobility shift assays established a requirement for the entire 15-bp half-site for initial repressor binding. Fragments containing the 30-bp palindrome and the entire mecA-mecR1 intergenic region were retarded in gels as multiple discrete bands varying in molecular size, characteristic of cooperative DNA binding. Glutaraldehyde cross-linking confirmed oligomerization of repressor in solution. A naturally occurring MecI mutant (MecI*; D39G) repressed mecA transcription sixfold less well than the wild type in vivo. Although MecI* protected the same target sequences and exhibited similar gel shift patterns to MecI, 5- to 10-fold more protein was required. MecI* exhibited defective oligomerization in solution, suggesting that the MecI amino terminus is important in protein-protein interactions and that protein oligomerization is necessary for optimum repression.

* Corresponding author. Mailing address: Medical College of Virginia Campus of Virginia Commonwealth University, Department of Medicine, Division of Infectious Diseases, Sanger Hall, Room 7-082, 1101 E. Marshall St., Richmond, VA 23298-0049. Phone: (804) 828-9711. Fax: (804) 828-3097. E-mail: GARCHER{at}GEMS.VCU.EDU.

dagger Present address: National Animal Disease Center, U.S. Department of Agriculture, Ames, IA.

Dagger Present address: Versicor, Inc., Fremont, Calif.




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