Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1986 Mar;29(3):432–439. doi: 10.1128/aac.29.3.432

In vivo target of benzylpenicillin in Gaffkya homari.

P W Wrezel, L F Ellis, F C Neuhaus
PMCID: PMC180409  PMID: 3717943

Abstract

It has been established that the DD-carboxypeptidase is the primary in vitro target of benzylpenicillin in Gaffkya homari (W. P. Hammes, Eur. J. Biochem. 70:107-113, 1976). To determine whether this enzyme is also the primary target of benzylpenicillin in vivo, we compared the effects of this beta-lactam, cefmenoxime, cephalothin, and cefoxitin on growth with their acylation of penicillin-binding protein (PBP) 9, the DD-carboxypeptidase. Results of three types of experiments with membrane-walls indicated that PBP 9 is this enzyme and that it is the primary in vitro target of these beta-lactams in the synthesis of sodium dodecyl sulfate (SDS)-insoluble peptidoglycan. First, the acylation of PBP 9 by these beta-lactams paralleled the inhibition of DD-carboxypeptidase and the inhibition of SDS-insoluble peptidoglycan synthesis. Second, the rate of benzylpenicillin release from PBP 9 correlated with the recovery of DD-carboxypeptidase. Third, DD-carboxypeptidase activity was detected in a protein with the same apparent molecular weight as PBP 9 after elution from an SDS-polyacrylamide gel. When intact cells were treated with benzylpenicillin, the minimum growth inhibitory concentration (MGIC) correlated with the concentration of [35S]benzylpenicillin required to acylate PBPs 6 and 9 by 50%. When intact cells were treated with cefmenoxime, cephalothin, or cefoxitin, the MGICs correlated with the concentration of unlabeled beta-lactam required to reduce the subsequent binding of [35S]benzylpenicillin by 50% (ED50) for PBP 6. In contrast, the MGICs of these beta-lactams did not correlate with the ED50s for PBP 9. PBP 9 was not acylated by cefmenoxime or cephalothin at their MGICs, whereas this PBP was fully acylated by cefoxitin at one-tenth of its MGIC. It is suggested that PBP 6 may be a primary target of growth inhibition by benzylpenicillin, cefmenoxime, cephalothin, and cefoxitin; PBP 9, the DD-carboxypeptidase, is dispensable for growth under laboratory conditions; and PBP 9 does not appear to be a primary in vivo target of these beta-lactams, even though this PBP is their primary target in vitro.

Full text

PDF
432

Images in this article

434Image
on p.434
436Image
on p.436
437Image
on p.437

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bardin C., Sinha R. K., Kalomiris E., Neuhaus F. C. Biosynthesis of peptidoglycan in Gaffkya homari: processing of nascent glycan by reactivated membranes. J Bacteriol. 1984 Feb;157(2):398–404. doi: 10.1128/jb.157.2.398-404.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blumberg P. M., Strominger J. L. Inactivation of D-alanine carboxypeptidase by penicillins and cephalosporins is not lethal in Bacillus subtilis. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2814–2817. doi: 10.1073/pnas.68.11.2814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blumberg P. M., Strominger J. L. Interaction of penicillin with the bacterial cell: penicillin-binding proteins and penicillin-sensitive enzymes. Bacteriol Rev. 1974 Sep;38(3):291–335. doi: 10.1128/br.38.3.291-335.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carpenter C. V., Goyer S., Neuhaus F. C. Steric effects on penicillin-sensitive peptidoglycan synthesis in a membrane-wall system Gaffkya homari. Biochemistry. 1976 Jul 13;15(14):3146–3152. doi: 10.1021/bi00659a031. [DOI] [PubMed] [Google Scholar]
  5. Chase H. A., Reynolds P. E., Ward J. B. Purification and characterization of the penicillin-binding protein that is the lethal target of penicillin in Bacillus megaterium and Bacillus licheniformis. Protein exchange and complex stability. Eur J Biochem. 1978 Jul 17;88(1):275–285. doi: 10.1111/j.1432-1033.1978.tb12448.x. [DOI] [PubMed] [Google Scholar]
  6. Coyette J., Ghuysen J. M., Fontana R. The penicillin-binding proteins in Streptococcus faecalis ATCC 9790. Eur J Biochem. 1980 Sep;110(2):445–456. doi: 10.1111/j.1432-1033.1980.tb04886.x. [DOI] [PubMed] [Google Scholar]
  7. Frère J. M., Joris B. Penicillin-sensitive enzymes in peptidoglycan biosynthesis. Crit Rev Microbiol. 1985;11(4):299–396. doi: 10.3109/10408418409105906. [DOI] [PubMed] [Google Scholar]
  8. Georgopapadakou N. H., Liu F. Y. Binding of beta-lactam antibiotics to penicillin-binding proteins of Staphylococcus aureus and Streptococcus faecalis: relation to antibacterial activity. Antimicrob Agents Chemother. 1980 Nov;18(5):834–836. doi: 10.1128/aac.18.5.834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  10. Hammes W. P. Biosynthesis of peptidoglycan in Gaffkya homari. The mode of action of penicillin G and mecillinam. Eur J Biochem. 1976 Nov 1;70(1):107–113. doi: 10.1111/j.1432-1033.1976.tb10961.x. [DOI] [PubMed] [Google Scholar]
  11. Hammes W. P., Kandler O. Biosynthesis of peptidoglycan in Gaffkya homari. The incorporation of peptidoglycan into the cell wall and the direction of transpeptidation. Eur J Biochem. 1976 Nov 1;70(1):97–106. doi: 10.1111/j.1432-1033.1976.tb10960.x. [DOI] [PubMed] [Google Scholar]
  12. Hammes W. P., Neuhaus F. C. Biosynthesis of peptidoglycan in Gaffkya homari: role of the peptide subunit of uridine diphosphate-N-acetylmuramyl-pentapeptide. J Bacteriol. 1974 Oct;120(1):210–218. doi: 10.1128/jb.120.1.210-218.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hammes W. P., Neuhaus F. C. On the specificity of phospho-N-acetylmuramyl-pentapeptide translocase. The peptide subunit of uridine diphosphate-N-actylmuramyl-pentapeptide. J Biol Chem. 1974 May 25;249(10):3140–3150. [PubMed] [Google Scholar]
  14. Hammes W. P., Seidel H. The activities in vitro of DD-carboxypeptidase and LD-carboxypeptidase of Gaffkya homari during biosynthesis of peptidoglycan. Eur J Biochem. 1978 Mar;84(1):141–147. doi: 10.1111/j.1432-1033.1978.tb12150.x. [DOI] [PubMed] [Google Scholar]
  15. Hammes W. P. The LD-carboxypeptidase activity in Gaffkya homari. The target of the action of D-amino acids or glycine on the formation of wall-bound peptidoglycan. Eur J Biochem. 1978 Nov 15;91(2):501–507. doi: 10.1111/j.1432-1033.1978.tb12703.x. [DOI] [PubMed] [Google Scholar]
  16. Izaki K., Matsuhashi M., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. 8. Peptidoglycan transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reaction in strains of Escherichia coli. J Biol Chem. 1968 Jun 10;243(11):3180–3192. [PubMed] [Google Scholar]
  17. Kelly K. F., Evans J. B. Deoxyribonucleic acid homology among strains of the lobster pathogen 'Gaffkya homari' and Aerococcus viridans. J Gen Microbiol. 1974 Mar;81(1):257–260. doi: 10.1099/00221287-81-1-257. [DOI] [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Mirelman D., Yashouv-Gan Y., Schwarz U. Peptidoglycan biosynthesis in a thermosensitive division mutant of Escherichia coli. Biochemistry. 1976 May 4;15(9):1781–1790. doi: 10.1021/bi00654a001. [DOI] [PubMed] [Google Scholar]
  21. Nakagawa J., Matsuhashi M. Molecular divergence of a major peptidoglycan synthetase with transglycosylase-transpeptidase activities in Escherichia coli --- penicillin-binding protein 1Bs. Biochem Biophys Res Commun. 1982 Apr 29;105(4):1546–1553. doi: 10.1016/0006-291x(82)90964-0. [DOI] [PubMed] [Google Scholar]
  22. Neuhaus F. C., Tobin C. E., Ahlgren J. A. Membrane-wall interrelationship in Gaffkya homari: sulfhydryl sensitivity and heat lability of nascent peptidoglycan incorporation into walls. J Bacteriol. 1980 Jul;143(1):112–119. doi: 10.1128/jb.143.1.112-119.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reynolds P. E., Shepherd S. T., Chase H. A. Identification of the binding protein which may be the target of penicillin action in Bacillus megaterium. Nature. 1978 Feb 9;271(5645):568–570. doi: 10.1038/271568a0. [DOI] [PubMed] [Google Scholar]
  24. Spratt B. G. Properties of the penicillin-binding proteins of Escherichia coli K12,. Eur J Biochem. 1977 Jan;72(2):341–352. doi: 10.1111/j.1432-1033.1977.tb11258.x. [DOI] [PubMed] [Google Scholar]
  25. Waxman D. J., Strominger J. L. Penicillin-binding proteins and the mechanism of action of beta-lactam antibiotics. Annu Rev Biochem. 1983;52:825–869. doi: 10.1146/annurev.bi.52.070183.004141. [DOI] [PubMed] [Google Scholar]
  26. Wyke A. W., Ward J. B., Hayes M. V., Curtis N. A. A role in vivo for penicillin-binding protein-4 of Staphylococcus aureus. Eur J Biochem. 1981 Oct;119(2):389–393. doi: 10.1111/j.1432-1033.1981.tb05620.x. [DOI] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES