Search

Methicillin-resistant clinical isolates of Staphylococcus aureus are intrinsically resistant to beta-lactam antibiotics in that the resistance mechanism is unrelated to the possession of beta-lactamases. We have demonstrated that a new, high-molecular-mass penicillin-binding protein (PBP) is present in these strains with a low affinity for beta-lactams and that its amount is regulated by the growth conditions. The new PBP from all strains that have been examined has an identical mobility on SDS gel electrophoresis and is the only PBP still present in an uncomplexed state with beta-lactams (and therefore the only functional PBP when these strains are grown in media containing concentrations of beta-lactam antibiotics sufficient to kill sensitive strains.

The differences in the outer membrane permeation between two major subgroups of β-lactam antibiotics were studied. The permeation of cephalosporins was closely related to porin channels in the outer membrane. In contrast, the outer membrane permeation of penicillins did not decrease in porin-deficient mutants and, in Rd-type mutants, their permeability became proportional to the hydrophobicity of the molecules. The activation enthalpy of the penicillin permeation was significantly larger than that of cephalosporins. These observations indicate that penicillins can use the hydrophobic region for the major route of outer membrane passage whereas the cephalosporin permeation is restricted to the pathway via the porin pore.

A polyclonal antibody (anti-PepT1/C) was raised against the rabbit intestinal H+-coupled oligopeptide transporter, PepT1. Anti-PepT1/C detected 70–80-kDa protein in crude membranes obtained from rabbit duodenum, jejunum and ileum. PepT1 was localized in the brush-border of the absorptive epithelial cells by subcellular fractionation of membranes on a sucrose density gradient and by immunohistochemistry using light and electron microscopy. Transport activity for cephalosporins and dipeptide expressed in Xenopus laevis oocytes injected with total mRNA obtained from rabbit small intestine was eliminated completely by prehybridization of the mRNA with antisense oligonucleotide against the 5′-coding region of rabbit PepT1 cDNA.

A gene fusion that links the COOH-terminal 349 amino acids of penicillin-binding protein 3 (60 kDa) of E.coli to the NH2-terminus of β-galactosidase has been constructed. The fusion protein (38.5 kDa) retains the ability to bind benzylpenicillin with high affinity, establishing that the penicillin-binding domain (and presumably the penicillin-sensitive transpeptidase activity) of this high molecular mass penicillin-binding protein is located on a COOH-terminal functional domain.

Penicillin-G acylase (EC 3.5.1.11) from Escherichia coli catalyzed the synthesis of various beta-lactam antibiotics in ice at -20 degrees C with higher yields than obtained in solution at 20 degrees C. The initial ratio between aminolysis and hydrolysis of the acyl-enzyme complex in the synthesis of cephalexin increased from 1.3 at 20 degrees C to 25 at -20 degrees C. The effect on the other antibiotics studied was less, leading us to conclude that freezing of the reaction medium influences the hydrolysis of each nucleophile-acyl-enzyme complex to a different extent. Only free penicillin-G acylase could perform transformations in frozen media: immobilized preparations showed a low, predominantly hydrolytic activity under these conditions.