The affinities of a range of penicillins and cephalosporins for ther penicillinbinding proteins of Escherichia coli K-12 have been studied, and the results were compared with the antibacterial activity of the compounds against E. coli K-12 and an isogenic permeability mutant. Different penicillins and cephalosporins exhibited different affinities for the "essential" penicillin-binding proteins of E. coli K-12, in a manner which directly correlated with their observed effects upon bacterial morphology. Furthermore, the affinities of the compounds for their "primary" lethal penicillin-binding protein targets showed close agreement with their antibacterial activities against the permeability mutant.

The degree of cross-linking of the peptidoglycan of Staphylococcus aureus H and mutants lacking penicillinbinding proteins 1 and 4 was studied. No major changes were observed in organisms lacking protein 1 whereas loss of protein 4 was accompanied by a marked reduction in the degree of cross-linking and the absence of a membrane-bound 'model' transpeptidase activity. A similar effect was achieved when cultures of the staphylococci were treated with the P-lactam antibiotic cefoxitin. At low concentrations (0.05 pg m1-l) cefoxitin shows highest affinity for protein 4 to which it appears to bind irreversibly. Treatment of the mutant lacking protein 4 with this concentration of the antibiotic did not affect the degree of cross-linkage.The possibility that the decrease in cross-linkage was a consequence of DD-carboxypeptidase activity on peptidoglycan precursors was investigated. Although both S. uureus H and the mutants possessed such activity it was insensitive to benzylpenicillin and cefoxitin and the role of this enzyme(s) in peptidoglycan biosynthesis remains unknown.We conclude that in vivo protein 4 acts as a transpeptidase involved in the secondary cross-linking of peptido-,glycan and this activity is necessary to achieve the high degree of cross-linkage observed in the peptidoglycan of staphylococci.The penicillin-binding proteins of both gram-positive and gram-negative bacteria have received considerable attention in recent years [I -31. These investigations have resulted in the general conclusion that the interaction of B-lactams with one or more of the binding proteins is responsible for the lethal effects of these antibiotics. However, only in Escherichia coli has the role of individual penicillin-binding proteins been investigated in detail. In this organism distinct penicillinbinding proteins have been implicated in the determination of cell shape [4,5] ; cell division [6] and elongation [4,5,7,8]. With the exception of penicillin-binding protein 2, all have now been isolated and purified [9-141 and in the case of protein 1A [I21 and 1B [9,10,13] shown to catalyse in vitro transglycosylation and transpeptidation reactions using lipid intermediates as the peptidoglycan precursors. The lower molecular weight proteins (4, 5 and 6) catalyse DD-carboxypeptidase and in the case of proteins 5 and 6 'model' transpeptidase activity [I 1,141. Mutants lacking these proteins (4 = ducB 5 = ducA) grow normally under various conditions and it was concluded these proteins were 'non-essential' Similar studies on gram-positive bacteria have tended to concentrate on Staphylococcus aureus and various Bacillus spp. (For review, see [2].) In the bacilli, four to six distinct penicillin-binding proteins have been described in which the one of lowest molecular weight (protein 5 in Bacillus subtilis) has DD-carboxypeptidase and 'model' transpeptidase activity [I 9,201 Enzyme. DD-Carboxypeptidase or muramoyl-pentapeptide carboxypeptidase (EC 3.4.17.8).H. A. Chase, unpublished observations). A similar situation exists in S. uur...

Selected aminothiazolyl-oxime cephalosporin congeners substituted at C-3' with a catechol moiety were used to probe the basis of the enhanced antibacterial activity against Escherichia coli K-12 often associated with chemical modifications of this type. Evidence is presented for a tonB-dependent illicit transport of the compounds across the outer membrane of E. coli K-12, the process involving jointly and specifically the Fiu and Cir iron-regulated outer membrane proteins. Thus, both tonB and flu cir mutants showed a comparably reduced susceptibility to the probe compounds, whereas mutants singularly lacking any one of the six iron-regulated outer membrane proteins (Fiu, FepA, FecA, FhuA, FhuE, and Cir) or lacking any combination of any two of these proteins (except Fiu plus Cir) did not show this resistance. Mutants devoid of all six iron-regulated outer membrane proteins were no more resistant to the probe compounds than fiu cir or tonB strains. In addition to the latter genes, the products of the exbB and possibly the exbC loci were necessary for maximal antibacterial potency. A dependence of antibacterial activity on the level of expression of the uptake system components was noted. Comparison of penicillin-binding protein target affinity with antibacterial activity suggested a possible periplasmic accumulation of active compounds by E. coli K-12. Free vicinal hydroxyl groups of the catechol residue were a primary chemical requirement for recognition by the uptake pathway and thus for high antibacterial activity.Recent reports have described the enhanced antibacterial potency against members of the family Enterobacteriaceae and pseudomonads of ,B-lactam antibiotics variously substituted with catechol, derivatized catechol, and related moieties (2, 6, 29-31, 33, 34, 45). Using cephalosporin E-0702 (featuring a catechol derivative at C-7), Watanabe et al. (45) reported the isolation of spontaneous Escherichia coli K-12 mutants specifically resistant to this compound owing to a defect at the tonB locus. From the known functionality of the tonB gene product in the transport of iron-chelated siderophores by E. coli K-12, it was hypothesized that the potency of E-0702 resulted from the incorporation of the compound by a tonB-dependent siderophore transport route (45). Unpublished studies in this laboratory have shown that such (tonB) mutants of E. coli K-12 can be isolated irrespective of the chemical diversity of the catechol-substituted cephalosporin (catechol-cephalosporin) used. These mutants, like tonB mutants independently isolated as multiply colicintolerant variants of sensitive strains (3,24,25), are specifically cross resistant to catechol-cephalosporins as a chemical class (unpublished observations and this report).In view of the pivotal importance of the tonB gene product in determining the sensitivity of E. coli K-12 to these agents, we attempted to identify whether any of the known tonBdependent uptake processes of E. coli K-12 could be implicated in catechol-cephalosporin activity.The tonB gene pro...

The competition of a number of beta-lactam morphogenic probes for the penicillin-binding proteins (PBPs) of Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella aerogenes, Proteus rettgeri, and Escherichia coli has been studied. The results indicate that the various gram-negative bacteria have similar, but not identical, PBP patterns and that the individual proteins probably perform similar morphogenic functions as in E. coli K-12. Comparison of the 50% binding concentrations of the compounds for the various PBPs of the five strains with their antibacterial activity indicates that the different antibiotics are excluded to a greater or lesser degree by the outer membrane permeability barrier and that the exclusion is most pronounced in P. aeruginosa.

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