No abstract

Penicillin-binding proteins 1A and 1 B of Escherichia coli are the major peptidoglycan transglycosylasetranspeptidases that catalyse the polymerisation and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. The nucleotide sequence of a 2764-base-pair fragment of DNA that contained the ponA gene, encoding penicillin-binding protein 1 A, was determined. The sequence predicted that penicillin-binding protein 1A had a relative molecular mass of 93500 (850 amino acids). The amino-terminus of the protein had the features of a signal peptide but it is not known if this peptide is removed during insertion of the protein into the cytoplasmic membrane. The nucleotide sequence of a 2758-base-pair fragment of DNA that contained the ponB gene, encoding penicillin-binding protein 1 B, was also determined. Penicillin-binding protein 1 B consists of two major components which were shown to result from the use of alternative sites for the initiation of translation. The large and small forms of penicillin-binding protein 1B were predicted to have relative molecular masses of 94 100 and 88 800 (844 and 799 amino acids). The amino acid sequences of penicillin-binding proteins 1 A and 1 B could be aligned if two large gaps were introduced into the latter sequence and the two proteins then showed about 30% identitiy. The amino acid sequences of the proteins showed no extensive similarity to the sequences of penicillin-binding proteins 3 or 5, or to the class A or class C p-lactamases. Two short regions of amino acid similarity were, however, found between penicillin-binding proteins 1 A and 1 B and the other penicillin-binding proteins and fi-lactamases. One of these included the predicted active-site serine residue which was located towards the middle of the sequences of penicillin-binding proteins 1 A, 1B and 3, within the conserved sequence Gly-SerXaa-Xaa-Lys-Pro. The other region was 19 -40 residues to the amino-terminal side of the active-site serine and may be part of a conserved penicillin-binding site in these proteins.fi-Lactam antibiotics kill Escherichia coli by inactivating a series of penicillin-binding proteins (PBPs) that are located in the cytoplasmic membrane and which catalyse the final stages of peptidoglycan synthesis [l, 21. In E. coli there are seven well characterised PBPs with relative molecular masses (Mr) between 40000 and 91 000 [3]. Genetic analysis of the role of each of the E. coli PBPs in the lethal effects of p-lactam antibiotics has shown that the lower-Mr PBPs 4, 5 and 6 are non-essential for bacterial growth and are therefore not of primary importance in the killing action of p-lactams [l, 4-71. The higher-M, PBPs 1A/lB, 2 and 3 have been shown to be essential enzymes and p-lactams exert their lethal effects by the inactivation of one or more of these killing targets [l, 81. Inactivation of PBP 3 results in the inhibition of cell division and the formation of long filamentous cells (EC 3.1.23.10), EcoRI (EC 3.1.23.13), EcoRV (EC 3.1.23.-)), H@I Subsequently PB...

The coding region for the mature form of TEM beta-lactamase was fused to random positions within the coding region of the penicillin-binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in-frame fusions were determined. All fusion proteins that contained at least the NH2-terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the beta-lactamase moiety had been translocated to the periplasm. Fusion proteins that contained less than or equal to 63 residues of PBP 1B possessed beta-lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the beta-lactamase moiety of these fusion proteins remained in the cytoplasm. The beta-lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic transmembrane segment (residues 64-87), with a short NH2-terminal domain (residues 1-63), and the remainder of the polypeptide (residues 88-844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.

Accession nos. dacA=X06479 and dacC=X06480 Penicillin-binding proteins (PBPs) 5 and 6 are the major D-alanine carboxypeptidases of Escherichia coli. The PBP 5 and PBP 6 genes (dacA and dacC respectively) have been cloned 1T2and the nucleotide sequence of a 1597