Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo insertion mutagenesis of the cloned gene

Clones containing the pbpB gene which encodes penicillin-binding protein (PBP) 3 of Pseudomonas aerugi-nosa were detected by hybridization by PCR amplification with primers based on the conserved sequences of high-molecular-weight PBPs. The translated amino acid sequence demonstrated 45 % identity and had a total of 66 % conserved amino acids relative to the Escherichia coli PBP3. The pbpB gene was located upstream of a gene homologous to the E. coli murE gene, which encodes uridine diphosphate-N-acetyl muramic acid-tripep-tide synthetase. The overexpressed pbpB gene product reacted with 3H-penicillin G and had an apparent molecular weight of 60,000. Pseudomonas aeruginosa is an opportunistic pathogen that is intrinsically resistant to a wide range of antibiotics. Infections by this bacterium are often treated with b-lactam antibiotics. b-Lactams exert their effects by acting as the substrate analogs of the peptidoglycan biosynthetic enzymes transpeptidase and D-alanine carboxypeptidase (21), which are located within the cytoplasmic membrane and are commonly named penicillin-binding proteins (PBPs) because of their abilities to covalentl