Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate.
The mecA gene from methicillin-resistant Staphylococcus aureus 27r, which encodes the membrane-bound penicillin-binding protein 2a (PBP 2a), was cloned, sequenced, and expressed in Escherichia coli. PBP 2a is the major factor that mediates methicillin resistance in staphylococci. The DNA sequence of the mecA gene from strain 27r was greater than 99% identical to the DNA sequence of other S. aureus mecA genes and the mecA gene from Staphylococcus epidermidis. Analysis of the deduced amino acid sequence of PBP 2a from strain 27r revealed a hydrophobic region at the amino terminus that possessed characteristics of an uncleaved signal peptide such as those found in type II integral membrane proteins. Site-specific mutagenesis was used to modify the strain 27r mecA gene to permit removal of the region encoding the putative transmembrane region (amino acids 2 to 22). When it was expressed in E. coli, the modified mecA gene from strain 27r encoded a water-soluble form of PBP 2a that was detectable in the cytoplasm of transformants. The water-soluble form of PBP 2a protein from S. aureus 27r retained the same binding efficiency for beta-lactam antibiotics as the unmodified membrane-bound PBP 2a from S. aureus 27r.
Wu
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Infectious Disease Research, Eli Lilly and Company, Indianapolis, Indiana 46285.
Hoskins
J
J
Blaszczak
L C
LC
Preston
D A
DA
Skatrud
P L
PL
D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins; D004926:Escherichia coli; D005822:Genetic Vectors; D006602:Hexosyltransferases / Q000235:genetics* / Q000302:isolation & purification; D016106:Methicillin Resistance; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification; D002267:Muramoylpentapeptide Carboxypeptidase; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase