Penicillin-binding Protein 1Bs Research Articles

Mutation causing overproduction of the short peptide component gamma of penicillin-binding protein 1Bs of Escherichia coli.

An Escherichia coli mutant was isolated which overproduces component γ of penicillin-binding protein (PBP)-1Bs. The mutant formed normal amounts of components α and its proteolytic product β. The mutation was located in mrcB, the structural gene coding for PBP-1Bα and γ. The overproduction of the mutant-type PBP-1Bγ was caused by an alteration of one nucleotide which changed the GGC coding for glycine at residue 8 from the N-terminal of PBP-1Bγ to GAC coding for aspartate.

Reactivation of peptidoglycan synthesis in ether-permeabilized Escherichia coli after inhibition by beta-lactam antibiotics

The recovery of peptidoglycan-synthesizing activity after inhibition by beta-lactam antibiotics was investigated in ether-permeabilized cells of Escherichia coli B. Such cells synthesize sodium dodecyl sulfate-insoluble peptidoglycan when provided with UDP-linked precursors and Mg2+. The ability of beta-lactam antibiotics to inhibit the synthesis of peptidoglycan was correlated with their affinity for penicillin-binding proteins 1A and 1Bs. Penicillin-binding protein 1Bs is thought to be the major peptidoglycan synthetase in E. coli and is a major lethal target for beta-lactam antibiotics. Ether-treated bacteria were preincubated with concentrations of beta-lactams sufficient to completely inhibit peptidoglycan synthesis and then treated with beta-lactamases to inactivate free antibiotic prior to measurement of peptidoglycan synthesis. At 40 min after beta-lactamase treatment, the rate of peptidoglycan synthesis was about 74% of the control rate in cells pretreated with ampicillin, but only 15% of the control in cells pretreated with penicillin G or azlocillin. Reversal of inhibition by several other antibiotics fell between these extremes. When cross-linking of peptidoglycan was measured specifically, reversal of inhibition by ampicillin also occurred more readily than that by penicillin G. Reactivation of peptidoglycan synthesis was not due to de novo synthesis of penicillin-binding proteins since it occurred under conditions that did not allow incorporation of [14C]leucine. We conclude that there is considerable variation in the stability of the inactive acyl enzymes formed between various beta-lactams and penicillin-binding protein 1Bs, with those formed by penicillin G being relatively long-lived.

Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli.

A chromosomal region of Escherichia coli contiguous to the fabE gene at 71 min on the chromosomal map contains multiple genes that are responsible for determination of the rod shape and sensitivity to the amidinopenicillin mecillinam. The so-called mre region was cloned and analyzed by complementation of two closely related but distinct E. coli mutants characterized, respectively, by the mutations mre-129 and mre-678, that showed a rounded to irregular cell shape and altered sensitivities to mecillinam; the mre-129 mutant was supersensitive to mecillinam at 30 degrees C, but the mre-678 mutant was resistant. The mre-678 mutation also caused simultaneous overproduction of penicillin-binding proteins 1Bs and 3. A chromosomal region of the wild-type DNA containing the total mre region and the fabE gene was first cloned on a lambda phage; a 7-kilobase (kb) fragment containing the whole mre region, but not the fabE gene, was then recloned on a mini F plasmid, pLG339; and finally, a 2.8-kb fragment complementing only mre-129 was also cloned on this low-copy-number plasmid. The whole 7-kb fragment was required for complementing the mre-678 mutant phenotypes. Fragments containing fabE but not the mre-129 region could be cloned on a high-copy-number plasmid. Southern blot hybridization indicated that the mre-678 mutant had a large deletion of 5.25 kb in its DNA, covering at least part of the mre-129 gene.

Functional biosynthesis of cell wall peptidoglycan by polymorphic bifunctional polypeptides. Penicillin-binding protein 1Bs of Escherichia coli with activities of transglycosylase and transpeptidase.

Dual enzyme activities for the biosynthesis of peptidoglycan of the cell wall are located in major higher molecular weight penicillin-binding proteins (PBP) of Escherichia coli. Each of these proteins catalyzes the two successive final reactions in the synthesis of cross-linked peptidoglycan from the precursor N-acetylglucosaminyl-N-acetylmuramyl peptide linked to undecaprenol diphosphate; namely, the transglycosylation that extends the glycan chain and the penicillin-sensitive DD-transpeptidation that cross-links the glycan chains through two peptide side chains. Both transglycosylation and transpeptidation catalyzed by PBP-1Bs represent de novo synthesis of cross-linked peptidoglycan. Under appropriate conditions, about 25% cross-linkage was observed during the reaction, the main reaction product supposedly being a regularly cross-linked network of peptidoglycan. The two domains for the transglycosylase and transpeptidase activities were found to be located on a 50-kDa portion of the PBP-1Bs, which are about 90 kDa. Gene recombination experiments indicated that the transglycosylase domain is located upstream, i.e. on the N-terminal side of the transpeptidase domain, suggesting that the gene for these bifunctional peptides may have been formed by fusion of the genes for transglycosylase and transpeptidase that were previously located separately on the chromosome in this order.

Penicillin-binding proteins and role of amdinocillin in causing bacterial cell death

The activity of penicillins against bacteria is in large part related to binding to specific receptor proteins involved in cell wall biosynthesis. These proteins have been designated penicillin-binding proteins. They can be separated into distinct entities through the use of acrylamide gel electrophoresis and binding of radioactive 14C-labeled penicillin G. Six major proteins have been defined in the Enterobacteriaceae, penicillin-binding proteins 1 to 6. Selection of mutants has shown that there are three essential proteins: penicillin-binding protein 1, which is divided into penicillin-binding protein 1Bs, a peptidoglycan transpeptidase, and penicillin-binding protein 1A, which acts as a replacement for penicillin-binding protein 1Bs. Penicillin-binding protein 2 is a murein-elongation initiating enzyme and penicillin-binding protein 3 is a septal murein-synthesizing enzyme. Penicillin-binding proteins 4, 5, and 6 are not essential for bacterial survival. Binding of penicillins to penicillin-binding protein 1Bs produces lysis, binding to penicillin-binding protein 2 produces round cells, and binding to penicillin-binding protein 3 produces long filaments. Amdinocillin is a beta-amidino penicillanic acid derivative that binds specifically to penicillin-binding protein 2. The compound is more beta-lactamase stable than ampicillin and has no major delay in entry into the periplasmic space as do some penicillins. Amdinocillin inhibits most of the Enterobacteriaceae, with the exception of some indole-positive Proteus species, but it does not inhibit gram-positive cocci or Pseudomonas aeruginosa. Amdinocillin produces spherical bacterial cells that eventually lyse. Its activity in vitro is markedly affected by ionic content of media. This agent acts synergistically with many penicillins, such as ampicillin, carbenicillin, and the like, and with cephalosporins, cefazolin, cefamandole, or cefoxitin to inhibit gram-negative bacilli, probably on the basis of binding to different proteins needed for the production of the peptidoglycan of the bacterial cell wall. Amdinocillin possesses a number of the essentials for effective antimicrobial activity and, by virtue of its enhancement of the activity of other beta-lactams, may prove to be a useful agent in the chemotherapy of certain infections.

Molecular divergence of a major peptidoglycan synthetase with transglycosylase - transpeptidase activities in Escherichia coli — Penicillin-binding protein 1Bs

Penicillin-binding protein 1Bs of Escherichia coli (Mr ca. 9 × 10 4) gave three protein bands with slightly different mobilities on sodium dodecylsulfate — polyacrylamide gel electrophoresis. The enzymatic activities of each of these proteins were identified after renaturation of the proteins separated by electrophoresis. Each of them had two enzymatic activities of the last steps of synthesis of peptidoglycan from lipid-linked precursor, i. e., activity of transglycosylase, which extends the glycan chain, and activity of penicillin-sensitive transpeptidase, which crosslinks glycan chains with peptide cross-bridges. Trypsin treatment of each of the three proteins resulted in formation of a doublet of penicillin-binding proteins (Mr ca. 5 × 10 4). The results strongly indicate that penicillin-binding protein 1Bs are bifunctional peptidoglycan synthetase proteins differing slightly in molecular structure.

Differences between ceftizoxime and its stereoisomer in antibacterial activity and affinity for penicillin-binding proteins

A new cephalosporin derivative, ceftizoxime (syn FK 749), and its anti isomer, FR 14060, were compared in antibacterial activity, outer membrane permeability, stability to beta-lactamases, and affinity for penicillin-binding proteins (PBPs), using Escherichia coli NIHJ JC-2 and Enterobacter cloacae 58-5 as the test organisms. Although ceftizoxime was superior in antibacterial activity to FR 14060, no marked differences between the two agents were found in outer membrane permeability and stability to cephalosporinase. However, the affinity for PBPs and stability to penicillinase of ceftizoxime and FR 14060 differed significantly. Concentrations of ceftizoxime required to reduce [14C]penicillin G binding by 50% were below 1 microgram/ml for PBPs 1a and 1bs of E. cloacae 58-5 and below 3.2 microgram/ml for PBPs 1a and 1bs of E. coli NIHJ JC-2. A more than 10-fold-higher concentration of FR 14060 was required for 50% reduction of (14C]penicillin G binding to PBP 1bs of strains tested. Ceftizoxime was severalfold more stable than FR 14060 to penicillinase, but the antibacterial activity of both drugs against penicillinase-producing E. coli was as strong as against non-penicillinase-producing E. coli. These results indicate that the difference between the two compounds in antibacterial activity is likely to be due to differences in their abilities to inhibit peptidoglycan polymerization.

Fragmentation of penicillin-binding protein-1Bs of Escherichia coli by proteolytic enzymes: A preliminary study on the location of the penicillin-binding site on the bifunctional peptidoglycan synthetase protein.

Fragmentation of penicillin-binding protein-1Bs of Escherichia coli by proteolytic enzymes: A preliminary study on the location of the penicillin-binding site on the bifunctional peptidoglycan synthetase protein.

Purified penicillin binding proteins 1Bs from Escherichia coli membrane showing activities of both peptidoglycan polymerase and peptidoglycan crosslinking enzyme.

(1979). Purified Penicillin Binding Proteins 1Bs from Escherichia coli Membrane Showing Activities of Both Peptidoglycan Polymerase and Peptidoglycan Crosslinking Enzyme. Agricultural and Biological Chemistry: Vol. 43, No. 6, pp. 1379-1380.

Thermosensitive mutation in Escherichia coli simultaneously causing defects in penicillin-binding protein-1Bs and in enzyme activity for peptidoglycan synthesis in vitro.

A thermosensitive mutant of Escherichia coli K-12 was isolated in which the membrane fractions were deficient both in penicillin-binding protein-1Bs, the major components of protein 1 [Spratt, B.G. & Pardee, A.B. (1975) Nature 254, 516-517] and in activity for in vitro peptidoglycan synthesis. The mutant was also supersensitive to many kinds of beta-lactam antibiotics. All these phenotypic changes were found to be caused by a single mutation (mrc). Genetic mapping studies show that the mrc mutation was located at about 3.3 min on the E. coli chromosome linkage map [Bachmann, B.J., Low, K.B. & Taylor, A.L. (1976) Bacteriol. Rev. 40, 116-167]. Penicillin-binding protein-1Bs seemed to be identical to one of the essential enzymes involved in crosslinking of peptidoglycan and the target of cell-lytic action of penicillins. Possible functions of some other penicillin-binding proteins in compensating for lack of protein-1Bs were also proposed.