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. 1978 Jun;75(6):2631–2635. doi: 10.1073/pnas.75.6.2631

Isolation of a mutant of Escherichia coli lacking penicillin-sensitive D-alanine carboxypeptidase IA.

M Matsuhashi, I N Maruyama, Y Takagaki, S Tamaki, Y Nishimura, Y Hirota
PMCID: PMC392616  PMID: 351612

Abstract

A mutant of Escherichia coli that is deficient in D-alanine carboxypeptidase IA has been isolated. The enzyme is membrane bound and moderately sensitive to penicillin. It catalyzes in vitro both D-alanine carboxypeptidase and transpeptidase reactions. Being able to synthesize crosslinked peptidoglycan both in vivo and in vitro despite the absence of enzyme activity, the newly isolated mutant grew normally under a wide range of growth conditions. Therefore, this enzyme, like D-alanine carboxypeptidase IB, is not required for normal peptidoglycan synthesis in E. coli. The defect in the activity of D-alanine carboxypeptidase IA in the mutant however was not associated with disappearance of penicillin-binding proteins 5 and 6 (which have been shown to be D-alanine carboxypeptidase IA) or any of the other protein bands that bind [14C]penicillin G. Genetic mapping studies showed that the mutation (dacA) is located close to leuS(13.7 min) on the E. coli chromosome map. Double mutants (dacA dacB) that are deficient in both D-alanine carboxypeptidases IA and IB were obtained. These double mutants also were found to grow normally and to catalyze normal formation of crosslinked peptidoglycan.

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Selected References

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

  1. Araki Y., Shimada A., Ito E. Effect of penicillin on cell wall mucopeptide synthesis in a Escherichia coli particulate system. Biochem Biophys Res Commun. 1966 May 25;23(4):518–525. doi: 10.1016/0006-291x(66)90760-1. [DOI] [PubMed] [Google Scholar]
  2. Bachmann B. J., Low K. B., Taylor A. L. Recalibrated linkage map of Escherichia coli K-12. Bacteriol Rev. 1976 Mar;40(1):116–167. doi: 10.1128/br.40.1.116-167.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barnett H. J. D-alanine carboxypeptidases of Bacillus stearothermophilus: solubilisation of particulate enzymes and mechanism of action of penicillin. Biochim Biophys Acta. 1973 Apr 28;304(2):332–352. doi: 10.1016/0304-4165(73)90252-3. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Iwaya M., Strominger J. L. Simultaneous deletion of D-alanine carboxypeptidase IB-C and penicillin-binding component IV in a mutant of Escherichia coli K12. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2980–2984. doi: 10.1073/pnas.74.7.2980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Izaki K., Matsuhashi M., Strominger J. L. Glycopeptide transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reactions. Proc Natl Acad Sci U S A. 1966 Mar;55(3):656–663. doi: 10.1073/pnas.55.3.656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kamiryo T., Strominger J. L. Penicillin-resistant temperature-sensitive mutants of Escherichia coli which synthesize hypo- or hyper-cross-linked peptidoglycan. J Bacteriol. 1974 Feb;117(2):568–577. doi: 10.1128/jb.117.2.568-577.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. LEDERBERG J. Mechanism of action of penicillin. J Bacteriol. 1957 Jan;73(1):144–144. doi: 10.1128/jb.73.1.144-144.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  11. Matsuhashi M., Takagaki Y., Maruyama I. N., Tamaki S., Nishimura Y., Suzuki H., Ogino U., Hirota Y. Mutants of Escherichia coli lacking in highly penicillin-sensitive D-alanine carboxypeptidase activity. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2976–2979. doi: 10.1073/pnas.74.7.2976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Miyakawa T., Matsuzawa H., Matsuhashi M., Sugino Y. Cell wall peptidoglycan mutants of Escherichia coli K-12: existence of two clusters of genes, mra and mrb, for cell wall peptidoglycan biosynthesis. J Bacteriol. 1972 Nov;112(2):950–958. doi: 10.1128/jb.112.2.950-958.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nguyen-Distèche M., Ghuysen J. M., Pollock J. J., Reynolds P., Perkins H. R., Coyette J., Salton M. R. Enzymes involved in wall peptide crosslinking in Escherichia coli K12, strain 44. Eur J Biochem. 1974 Feb 1;41(3):447–455. doi: 10.1111/j.1432-1033.1974.tb03286.x. [DOI] [PubMed] [Google Scholar]
  14. Nguyen-Distèche M., Pollock J. J., Ghuysen J. M., Puig J., Reynolds P., Perkins H. R., Coyette J., Salton M. R. Sensitivity to ampicillin and cephalothin of enzymes involved in wall peptide crosslinking in Escherichia coli K12, strain 44. Eur J Biochem. 1974 Feb 1;41(3):457–463. doi: 10.1111/j.1432-1033.1974.tb03287.x. [DOI] [PubMed] [Google Scholar]
  15. Pollock J. J., Nguyen-Distèche M., Ghuysen J. M., Coyette J., Linder R., Salton M. R., Kim K. S., Perkins H. R., Reynolds P. Fractionation of the DD-carboxypeptidase-transpeptidase activities solubilized from membranes of Escherichia coli K12, strain 44. Eur J Biochem. 1974 Feb 1;41(3):439–446. doi: 10.1111/j.1432-1033.1974.tb03285.x. [DOI] [PubMed] [Google Scholar]
  16. Spratt B. G. Distinct penicillin binding proteins involved in the division, elongation, and shape of Escherichia coli K12. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2999–3003. doi: 10.1073/pnas.72.8.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Spratt B. G., Pardee A. B. Penicillin-binding proteins and cell shape in E. coli. Nature. 1975 Apr 10;254(5500):516–517. doi: 10.1038/254516a0. [DOI] [PubMed] [Google Scholar]
  18. Spratt B. G., Strominger J. L. Identification of the major penicillin-binding proteins of Escherichia coli as D-alanine carboxypeptidase IA. J Bacteriol. 1976 Jul;127(1):660–663. doi: 10.1128/jb.127.1.660-663.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Strominger J. L., Izaki K., Matsuhashi M., Tipper D. J. Peptidoglycan transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reactions. Fed Proc. 1967 Jan-Feb;26(1):9–22. [PubMed] [Google Scholar]
  20. Strominger J. L., Willoughby E., Kamiryo T., Blumberg P. M., Yocum R. R. Penicillin-sensitive enzymes and penicillin-binding components in bacterial cells. Ann N Y Acad Sci. 1974 May 10;235(0):210–224. doi: 10.1111/j.1749-6632.1974.tb43267.x. [DOI] [PubMed] [Google Scholar]
  21. Suzuki H., Nishimura Y., Hirota Y. On the process of cellular division in Escherichia coli: a series of mutants of E. coli altered in the penicillin-binding proteins. Proc Natl Acad Sci U S A. 1978 Feb;75(2):664–668. doi: 10.1073/pnas.75.2.664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tamaki S., Nakajima S., Matsuhashi M. Thermosensitive mutation in Escherichia coli simultaneously causing defects in penicillin-binding protein-1Bs and in enzyme activity for peptidoglycan synthesis in vitro. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5472–5476. doi: 10.1073/pnas.74.12.5472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tamura T., Imae Y., Strominger J. L. Purification to homogeneity and properties of two D-alanine carboxypeptidases I From Escherichia coli. J Biol Chem. 1976 Jan 25;251(2):414–423. [PubMed] [Google Scholar]
  24. Tipper D. J., Strominger J. L. Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-D-alanyl-D-alanine. Proc Natl Acad Sci U S A. 1965 Oct;54(4):1133–1141. doi: 10.1073/pnas.54.4.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wise E. M., Jr, Park J. T. Penicillin: its basic site of action as an inhibitor of a peptide cross-linking reaction in cell wall mucopeptide synthesis. Proc Natl Acad Sci U S A. 1965 Jul;54(1):75–81. doi: 10.1073/pnas.54.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]

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