Interaction of native and mutant MecI repressors with sequences that regulate mecA, the gene encoding penicillin binding protein 2a in methicillin-resistant staphylococci.
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.
Sharma
V K
VK
Department of Medicine, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond 23298-0049, USA.
Hackbarth
C J
CJ
Dickinson
T M
TM
Archer
G L
GL
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D003850:Deoxyribonuclease I; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006602:Hexosyltransferases; D016106:Methicillin Resistance / Q000235:genetics*; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000096:biosynthesis* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; C101640:MecI protein, Staphylococcus; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D003850:Deoxyribonuclease I; D002267:Muramoylpentapeptide Carboxypeptidase