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. 1986 Jun;166(3):1061–1066. doi: 10.1128/jb.166.3.1061-1066.1986

Cloning and expression of the phospho-beta-galactosidase gene of Staphylococcus aureus in Escherichia coli.

F Breidt Jr, G C Stewart
PMCID: PMC215232  PMID: 3011732

Abstract

The phospho-beta-galactosidase gene of Staphylococcus aureus was cloned in Escherichia coli. This was done by first isolating a staphylococcal transposon Tn551-induced mutant which rendered phospho-beta-galactosidase synthesis partially constitutive because of an insertion nearby this lac structural gene. This allowed selection in E. coli of chimeric plasmids which expressed the erythromycin resistance determinant of Tn551. A 26-kilobase (kb) BamHI insert in plasmid pBR322 was isolated which encoded phospho-beta-galactosidase, as determined by phospho-beta-galactosidase activity measurements. Maxicell experiments showed the presence of 56-, 13.5-, and 31-kilodalton proteins encoded by the staphylococcal DNA. The presence of the 56-kilodalton protein correlated with phospho-beta-galactosidase activity and corresponded in molecular weight to the reported value for the purified enzyme. The nature of the other proteins is unknown. Phospho-beta-galactosidase was apparently expressed in E. coli by a promoter contained within a 2.1-kb EcoRI chromosomal DNA fragment. This fragment, when inserted into a chloramphenicol acetyl transferase promoter detection plasmid, was transcriptionally active in both E. coli and Bacillus subtilis but was much more active in the latter host.

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Selected References

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

  1. Bissett D. L., Anderson R. L. Lactose and D0galactose metabolism in Staphylococcus aureus: pathway of D-galactose 6-phosphate degradation. Biochem Biophys Res Commun. 1973 May 15;52(2):641–647. doi: 10.1016/0006-291x(73)90761-4. [DOI] [PubMed] [Google Scholar]
  2. Botsford J. L. Cyclic nucleotides in procaryotes. Microbiol Rev. 1981 Dec;45(4):620–642. doi: 10.1128/mr.45.4.620-642.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  4. Deutscher J., Beyreuther K., Sobek H. M., Stüber K., Hengstenberg W. Phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus: factor IIIlac, a trimeric phospho-carrier protein that also acts as a phase transfer catalyst. Biochemistry. 1982 Sep 28;21(20):4867–4873. doi: 10.1021/bi00263a006. [DOI] [PubMed] [Google Scholar]
  5. Dyer D. W., Iandolo J. J. Rapid isolation of DNA from Staphylococcus aureus. Appl Environ Microbiol. 1983 Jul;46(1):283–285. doi: 10.1128/aem.46.1.283-285.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hays J. B., Simoni R. D., Roseman S. Sugar transport. V. A trimeric lactose-specific phosphocarrier protein of the Staphylococcus aureus phosphotransferase system. J Biol Chem. 1973 Feb 10;248(3):941–956. [PubMed] [Google Scholar]
  7. Hengstenberg W., Penberthy W. K., Hill K. L., Morse M. L. Metabolism of lactose by Staphylococcus aureus. J Bacteriol. 1968 Dec;96(6):2187–2188. doi: 10.1128/jb.96.6.2187-2188.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hengstenberg W., Penberthy W. K., Morse M. L. Purification of the staphylococcal 6-phospho-beta-D-- galactosidase. Eur J Biochem. 1970 May 1;14(1):27–32. doi: 10.1111/j.1432-1033.1970.tb00256.x. [DOI] [PubMed] [Google Scholar]
  9. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  10. Kreiswirth B. N., Löfdahl S., Betley M. J., O'Reilly M., Schlievert P. M., Bergdoll M. S., Novick R. P. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature. 1983 Oct 20;305(5936):709–712. doi: 10.1038/305709a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Lee L. J., Hansen J. B., Jagusztyn-Krynicka E. K., Chassy B. M. Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12. J Bacteriol. 1982 Dec;152(3):1138–1146. doi: 10.1128/jb.152.3.1138-1146.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. MCCLATCHY J. K., ROSENBLUM E. D. INDUCTION OF LACTOSE UTILIZATION IN STAPHYLOCOCCUS AUREUS. J Bacteriol. 1963 Dec;86:1211–1215. doi: 10.1128/jb.86.6.1211-1215.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. MORSE M. L., ALIRE M. L. An agar medium indicating acid production. J Bacteriol. 1958 Sep;76(3):270–271. doi: 10.1128/jb.76.3.270-271.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maeda S., Gasson M. J. Cloning, expression and location of the Streptococcus lactis gene for phospho-beta-D-galactosidase. J Gen Microbiol. 1986 Feb;132(2):331–340. doi: 10.1099/00221287-132-2-331. [DOI] [PubMed] [Google Scholar]
  16. Morse M. L., Hill K. L., Egan J. B., Hengstenberg W. Metabolism of lactose by Staphylococcus aureus and its genetic basis. J Bacteriol. 1968 Jun;95(6):2270–2274. doi: 10.1128/jb.95.6.2270-2274.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Murphy E., Phillips S., Edelman I., Novick R. P. Tn554: isolation and characterization of plasmid insertions. Plasmid. 1981 May;5(3):292–305. doi: 10.1016/0147-619x(81)90006-8. [DOI] [PubMed] [Google Scholar]
  18. NOVICK R. P. ANALYSIS BY TRANSDUCTION OF MUTATIONS AFFECTING PENICILLINASE FORMATION IN STAPHYLOCOCCUS AUREUS. J Gen Microbiol. 1963 Oct;33:121–136. doi: 10.1099/00221287-33-1-121. [DOI] [PubMed] [Google Scholar]
  19. NOVICK R. P., RICHMOND M. H. NATURE AND INTERACTIONS OF THE GENETIC ELEMENTS GOVERNING PENICILLINASE SYNTHESIS IN STAPHYLOCOCCUS AUREUS. J Bacteriol. 1965 Aug;90:467–480. doi: 10.1128/jb.90.2.467-480.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Norgard M. V. Rapid and simple removal of contaminating RNA from plasmid DNA without the use of RNase. Anal Biochem. 1981 May 1;113(1):34–42. doi: 10.1016/0003-2697(81)90040-3. [DOI] [PubMed] [Google Scholar]
  21. Novick R. P., Edelman I., Schwesinger M. D., Gruss A. D., Swanson E. C., Pattee P. A. Genetic translocation in Staphylococcus aureus. Proc Natl Acad Sci U S A. 1979 Jan;76(1):400–404. doi: 10.1073/pnas.76.1.400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Novick R. P., Murphy E., Gryczan T. J., Baron E., Edelman I. Penicillinase plasmids of Staphylococcus aureus: restriction-deletion maps. Plasmid. 1979 Jan;2(1):109–129. doi: 10.1016/0147-619x(79)90010-6. [DOI] [PubMed] [Google Scholar]
  23. Rubin S. J., Rosenblum E. D. Effects of the recipient strain and ultraviolet irradiation on transduction kinetics of the penicillinase plasmid of Staphylococcus aureus. J Bacteriol. 1971 Dec;108(3):1192–1199. doi: 10.1128/jb.108.3.1192-1199.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Scheer-Abramowitz J., Gryczan T. J., Dubnau D. Origin and mode of replication of plasmids pE194 and pUB110. Plasmid. 1981 Jul;6(1):67–77. doi: 10.1016/0147-619x(81)90054-8. [DOI] [PubMed] [Google Scholar]
  26. Schäfer A., Schrecker O., Hengstenberg W. The staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Purification and characterisation of the galactoside-specific membrane-component enzyme II. Eur J Biochem. 1981 Jan;113(2):289–294. doi: 10.1111/j.1432-1033.1981.tb05065.x. [DOI] [PubMed] [Google Scholar]
  27. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  28. Simoni R. D., Nakazawa T., Hays J. B., Roseman S. Sugar transport. IV. Isolation and characterization of the lactose phosphotransferase system in Staphylococcus aureus. J Biol Chem. 1973 Feb 10;248(3):932–940. [PubMed] [Google Scholar]
  29. Simoni R. D., Smith M. F., Roseman S. Resolution of a staphylococcal phosphotransferase system into four protein components and its relation to sugar transport. Biochem Biophys Res Commun. 1968 Jun 10;31(5):804–811. doi: 10.1016/0006-291x(68)90634-7. [DOI] [PubMed] [Google Scholar]
  30. Snook R. J., McKay L. L. Conjugal Transfer of Lactose-Fermenting Ability Among Streptococcus cremoris and Streptococcus lactis Strains. Appl Environ Microbiol. 1981 Nov;42(5):904–911. doi: 10.1128/aem.42.5.904-911.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stahl M. L., Pattee P. A. Computer-assisted chromosome mapping by protoplast fusion in Staphylococcus aureus. J Bacteriol. 1983 Apr;154(1):395–405. doi: 10.1128/jb.154.1.395-405.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stüber D., Bujard H. Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci U S A. 1981 Jan;78(1):167–171. doi: 10.1073/pnas.78.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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