Penicillin Binding Components of Bacterial Cells and Their Relationship to the Mechanism of Penicillin Action

A new technique has been devised to investigate the penetration of antibiotics through the gram-negative outer membrane; the application here was to study intrinsic resistance of Escherichia coli K-12. Exponential cells in broth were briefly treated with 2.5 mM ethylenediaminetetraacetic acid at 5°C to disrupt the outer membrane penetration barrier, and the response of treated and untreated cells to antibiotics was compared by turbidimetry. A barrier index was derived to describe the ability of 7 beta-lactam and 10 other antibiotics to penetrate the outer membrane of strain Y10. There was correlation between the molecular weight and logio barrier index (r = 0.59, P = 0.01). The envelope mutant D22 (envA) had low barrier indexes for erythromycin, rifampin, ampicillin, and cloxacillin. For the beta-lactams, outer membrane penetration and affinity for inner membrane target site(s) triggering cell lysis were measured as independent components of the overall activity; although penetration and overall activity varied greatly, the affinities of most were within a narrow range.The limiting layers of the gram-negative cell envelope are the cytoplasmic or inner membrane (IM) and the outer membrane (OM) (29). Between the IM and OM is a murein sacculus within a tenuous space known as the periplasm, which contains a number of wall-associated enzymes (28).The OM is a barrier to molecules above a critical size; in Escherichia coli and Salmonella typhimurium, the penetration of oligopeptides (25) and oligosaccharides (8) declines sharply as the molecular weight exceeds 600, and they are essentially excluded at 1,000. Similarly, there is at least partial exclusion of some antibiotics by the OM contributing to "intrinsic" resistance (12), although in most cases the effect has not been well quantified. According to a popular model, solutes may penetrate the OM through narrow aqueous pores which restrict entry nonspecifically by size, and so provide a molecular sieving function (8). This function involves certain OM proteins which have been called "porins" (14,17,18,19,22).In this paper, we describe a technique which was used to measure the penetration of 17 antibiotics through the OM of E. coli K-12. A preliminary report has been made (30). The OM penetrability barrier was disrupted by brief treatment with ethylenediaminetetraacetic acid (EDTA), and the turbidimetric response of treated and normal cells to antibiotics was then compared over a short period of growth. (There was no adjustment of the external osmotic pressure). In support of the pore model, we found that antibiotic penetration through the OM was limited by molecular weight, and that the limits agreed well with those for oligosaccharides (8). However, it was clear that in some cases other variables must also control penetration.Beta-lactam antibiotics were differentiated with respect to penetration ofthe OM and affinity for the IM target site(s) promoting cell lysis. This gave an insight into the basis for the wide range of activities against E. coli which are encount...

Section: Discussionsupporting

confidence: 67%

“…y = 375.6 + 132.4x (3) According to equation 3, the BI reaches unity at 375 daltons, meaning that molecules below this weight should have unhindered access through the OM. At 1,000 daltons, the predicted BI is 52,000, which is tantamount to exclusion.…”

Section: Discussionmentioning

confidence: 99%

Outer-Membrane Penetration Barriers as Components of Intrinsic Resistance to Beta-Lactam and Other Antibiotics in Escherichia coli K-12

Scudamore

Beveridge

Goldner

1979

“…P. aeruginosa PA0503 was treated with ethyl methane sulfonate (EMS; Eastman Kodak Co.). Between 0.03 and 0.05 ml of EMS was added to 1 ml of a washed exponential-phase culture and suspended to approximately 109 cells per ml in fresh prewarmed TSB. The treated culture was incubated at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Penicillins, cephalosporins, and cephamycins collectively described as 3-lactam antibiotics inhibit bacteria by interfering with steps in the synthesis of the bacterial peptidoglycan layer (1,2). This layer comprises the major structural support for the cell, and inhibition of it results in malformation of the cell and eventual lysis (19).…”

mentioning

confidence: 99%

Mutation of Pseudomonas aeruginosa specifying reduced affinity for penicillin G

Godfrey¹,

Bryan²

1982

A mutant of Pseudomonas aeruginosa strain PAO503 was isolated after ethane-methane-sulfonate mutagenesis and selection of ticarcillin. The mutant, PCC17, displayed reduced affinity for [14C] penicillin G at all of its penicillin-binding proteins as well as a general increase in resistance to all the beta-lactam antibiotics tested. The mutation designated pbpA has been mapped by FP-2-mediated conjugation and was located distal to the proA locus and 33% linked to it. The two loci were not cotransducible with phage F116L. PCC17 and exconjugants produced from it had similar phenotypes, displayed the reduced affinity for [14C] penicillin G, had similar resistance profiles, and had an increased amount of protein corresponding to penicillin-binding protein 6. On back mutation the pbpA locus reverted to the PAO503 phenotype.

“…6,13). Several penicillin-sensitive enzymes are known (1,3,13,17), and direct labeling experiments or affinity chromatography procedures have shown several penicillin-binding proteins (PBP) in Bacillus subtilis, Bacillus stearothermophilus, Bacillus cereus, Staphylococcus aureus, and Escherichia coli (3,5,15,16). Of these PBP, only the D-alanine carboxypeptidase has been identified (3).…”

mentioning

confidence: 99%

Effect of Cephapirin on Formation of d -Alanine Carboxypeptidase in Growing Bacillus subtilis Cells

White

Astill

Lawrence

1979

Cephapirin was utilized to examine the interaction of β-lactam antibiotics with growing Bacillus subtilis cells and the biological effects simultaneously produced. Saturation binding and quantitative cell death were observed at the cephapirin concentration of 0.1 μg/ml. Cephapirin bound to all penicillin-binding proteins except the d -alanine carboxypeptidase. A specific [ 14 C]benzylpenicillin-binding assay was developed for the d -alanine carboxypeptidase. At the lowest saturating concentration of antibiotic (0.1 μg/ml), cephapirin inhibited formation of the d -alanine carboxypeptidase. Upon incubation with cephapirin, 18% of the membranous d -alanine carboxypeptidase was released into the media. The data suggest that β-lactam antibiotics may affect the formation of bacterial cytoplasmic membranes in addition to their effect on cell wall synthesis.