Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1992 Mar;36(3):533–539. doi: 10.1128/aac.36.3.533

Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate.

C Y Wu 1, J Hoskins 1, L C Blaszczak 1, D A Preston 1, P L Skatrud 1
PMCID: PMC190552  PMID: 1622161

Abstract

The mecA gene from methicillin-resistant Staphylococcus aureus 27r, which encodes the membrane-bound penicillin-binding protein 2a (PBP 2a), was cloned, sequenced, and expressed in Escherichia coli. PBP 2a is the major factor that mediates methicillin resistance in staphylococci. The DNA sequence of the mecA gene from strain 27r was greater than 99% identical to the DNA sequence of other S. aureus mecA genes and the mecA gene from Staphylococcus epidermidis. Analysis of the deduced amino acid sequence of PBP 2a from strain 27r revealed a hydrophobic region at the amino terminus that possessed characteristics of an uncleaved signal peptide such as those found in type II integral membrane proteins. Site-specific mutagenesis was used to modify the strain 27r mecA gene to permit removal of the region encoding the putative transmembrane region (amino acids 2 to 22). When it was expressed in E. coli, the modified mecA gene from strain 27r encoded a water-soluble form of PBP 2a that was detectable in the cytoplasm of transformants. The water-soluble form of PBP 2a protein from S. aureus 27r retained the same binding efficiency for beta-lactam antibiotics as the unmodified membrane-bound PBP 2a from S. aureus 27r.

Full text

PDF
533

Images in this article

535Image
on p.535
536Image
on p.536
537Image
on p.537
537Image
on p.537

Selected References

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

  1. Adachi H., Ohta T., Matsuzawa H. A water-soluble form of penicillin-binding protein 2 of Escherichia coli constructed by site-directed mutagenesis. FEBS Lett. 1987 Dec 21;226(1):150–154. doi: 10.1016/0014-5793(87)80569-0. [DOI] [PubMed] [Google Scholar]
  2. Asoh S., Matsuzawa H., Ishino F., Strominger J. L., Matsuhashi M., Ohta T. Nucleotide sequence of the pbpA gene and characteristics of the deduced amino acid sequence of penicillin-binding protein 2 of Escherichia coli K12. Eur J Biochem. 1986 Oct 15;160(2):231–238. doi: 10.1111/j.1432-1033.1986.tb09961.x. [DOI] [PubMed] [Google Scholar]
  3. Chambers H. F., Hackbarth C. J. Effect of NaCl and nafcillin on penicillin-binding protein 2a and heterogeneous expression of methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1987 Dec;31(12):1982–1988. doi: 10.1128/aac.31.12.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hackbarth C. J., Chambers H. F. Methicillin-resistant staphylococci: detection methods and treatment of infections. Antimicrob Agents Chemother. 1989 Jul;33(7):995–999. doi: 10.1128/aac.33.7.995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hartman B. J., Tomasz A. Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus. J Bacteriol. 1984 May;158(2):513–516. doi: 10.1128/jb.158.2.513-516.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Inglis B., Matthews P. R., Stewart P. R. Induced deletions within a cluster of resistance genes in the mec region of the chromosome of Staphylococcus aureus. J Gen Microbiol. 1990 Nov;136(11):2231–2239. doi: 10.1099/00221287-136-11-2231. [DOI] [PubMed] [Google Scholar]
  7. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  9. Matsuhashi M., Song M. D., Ishino F., Wachi M., Doi M., Inoue M., Ubukata K., Yamashita N., Konno M. Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus. J Bacteriol. 1986 Sep;167(3):975–980. doi: 10.1128/jb.167.3.975-980.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mottl H., Terpstra P., Keck W. Penicillin-binding protein 4 of Escherichia coli shows a novel type of primary structure among penicillin-interacting proteins. FEMS Microbiol Lett. 1991 Mar 1;62(2-3):213–220. doi: 10.1016/0378-1097(91)90160-c. [DOI] [PubMed] [Google Scholar]
  11. Murakami K., Nomura K., Doi M., Yoshida T. Production of low-affinity penicillin-binding protein by low- and high-resistance groups of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1987 Sep;31(9):1307–1311. doi: 10.1128/aac.31.9.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  13. Parks G. D., Lamb R. A. Topology of eukaryotic type II membrane proteins: importance of N-terminal positively charged residues flanking the hydrophobic domain. Cell. 1991 Feb 22;64(4):777–787. doi: 10.1016/0092-8674(91)90507-u. [DOI] [PubMed] [Google Scholar]
  14. Pierre J., Williamson R., Bornet M., Gutmann L. Presence of an additional penicillin-binding protein in methicillin-resistant Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus simulans with a low affinity for methicillin, cephalothin, and cefamandole. Antimicrob Agents Chemother. 1990 Sep;34(9):1691–1694. doi: 10.1128/aac.34.9.1691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Preston D. A., Wu C. Y., Blaszczak L. C., Seitz D. E., Halligan N. G. Biological characterization of a new radioactive labeling reagent for bacterial penicillin-binding proteins. Antimicrob Agents Chemother. 1990 May;34(5):718–721. doi: 10.1128/aac.34.5.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ryffel C., Tesch W., Birch-Machin I., Reynolds P. E., Barberis-Maino L., Kayser F. H., Berger-Bächi B. Sequence comparison of mecA genes isolated from methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Gene. 1990 Sep 28;94(1):137–138. doi: 10.1016/0378-1119(90)90481-6. [DOI] [PubMed] [Google Scholar]
  17. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  18. Tesch W., Strässle A., Berger-Bächi B., O'Hara D., Reynolds P., Kayser F. H. Cloning and expression of methicillin resistance from Staphylococcus epidermidis in Staphylococcus carnosus. Antimicrob Agents Chemother. 1988 Oct;32(10):1494–1499. doi: 10.1128/aac.32.10.1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tonin E., Tomasz A. Beta-lactam-specific resistant mutants of Staphylococcus aureus. Antimicrob Agents Chemother. 1986 Oct;30(4):577–583. doi: 10.1128/aac.30.4.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ubukata K., Yamashita N., Konno M. Occurrence of a beta-lactam-inducible penicillin-binding protein in methicillin-resistant staphylococci. Antimicrob Agents Chemother. 1985 May;27(5):851–857. doi: 10.1128/aac.27.5.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. von Heijne G. The signal peptide. J Membr Biol. 1990 May;115(3):195–201. doi: 10.1007/BF01868635. [DOI] [PubMed] [Google Scholar]

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

RESOURCES