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. 1993 Sep;175(17):5697–5700. doi: 10.1128/jb.175.17.5697-5700.1993

Chloramphenicol acetyltransferase, a cytoplasmic protein is incompatible for export from Bacillus subtilis.

M W Chen 1, V Nagarajan 1
PMCID: PMC206629  PMID: 8366055

Abstract

Bacillus subtilis cells expressing a hybrid protein (Lvsss-Cat) consisting of the B. amyloliquefaciens levansucrase signal peptide fused to B. pumilus chloramphenicol acetyltransferase (Cat) are unable to export Cat protein into the growth medium. A series of tripartite protein fusions was constructed by inserting various lengths of the Cat sequences between the levansucrase signal peptide and staphylococcal protein A or Escherichia coli alkaline phosphatase. Biochemical characterization of the various Cat protein fusions revealed that multiple regions in the Cat protein were causing the export defect.

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Selected References

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  1. Borchert T. V., Nagarajan V. Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis. J Bacteriol. 1991 Jan;173(1):276–282. doi: 10.1128/jb.173.1.276-282.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Davis N. G., Model P. An artificial anchor domain: hydrophobicity suffices to stop transfer. Cell. 1985 Jun;41(2):607–614. doi: 10.1016/s0092-8674(85)80033-7. [DOI] [PubMed] [Google Scholar]
  3. Freudl R., Schwarz H., Kramps S., Hindennach I., Henning U. Dihydrofolate reductase (mouse) and beta-galactosidase (Escherichia coli) can be translocated across the plasma membrane of E. coli. J Biol Chem. 1988 Nov 15;263(32):17084–17091. [PubMed] [Google Scholar]
  4. Harwood C. R., Williams D. M., Lovett P. S. Nucleotide sequence of a Bacillus pumilus gene specifying chloramphenicol acetyltransferase. Gene. 1983 Oct;24(2-3):163–169. doi: 10.1016/0378-1119(83)90076-8. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Lee C., Li P., Inouye H., Brickman E. R., Beckwith J. Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane. J Bacteriol. 1989 Sep;171(9):4609–4616. doi: 10.1128/jb.171.9.4609-4616.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Leslie A. G., Moody P. C., Shaw W. V. Structure of chloramphenicol acetyltransferase at 1.75-A resolution. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4133–4137. doi: 10.1073/pnas.85.12.4133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Li P., Beckwith J., Inouye H. Alteration of the amino terminus of the mature sequence of a periplasmic protein can severely affect protein export in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7685–7689. doi: 10.1073/pnas.85.20.7685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Manoil C., Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 1986 Sep 26;233(4771):1403–1408. doi: 10.1126/science.3529391. [DOI] [PubMed] [Google Scholar]
  10. Nagarajan V., Albertson H., Chen M., Ribbe J. Modular expression and secretion vectors for Bacillus subtilis. Gene. 1992 May 1;114(1):121–126. doi: 10.1016/0378-1119(92)90717-4. [DOI] [PubMed] [Google Scholar]
  11. Nagarajan V., Borchert T. V. Levansucrase: a tool to study protein secretion in Bacillus subtilis. Res Microbiol. 1991 Sep-Oct;142(7-8):787–792. doi: 10.1016/0923-2508(91)90056-g. [DOI] [PubMed] [Google Scholar]
  12. Payne M. S., Jackson E. N. Use of alkaline phosphatase fusions to study protein secretion in Bacillus subtilis. J Bacteriol. 1991 Apr;173(7):2278–2282. doi: 10.1128/jb.173.7.2278-2282.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Randall L. L., Hardy S. J. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. doi: 10.1016/0092-8674(86)90074-7. [DOI] [PubMed] [Google Scholar]
  14. Rasmussen B. A., Bankaitis V. A., Bassford P. J., Jr Export and processing of MalE-LacZ hybrid proteins in Escherichia coli. J Bacteriol. 1984 Nov;160(2):612–617. doi: 10.1128/jb.160.2.612-617.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Silhavy T. J., Beckwith J. R. Uses of lac fusions for the study of biological problems. Microbiol Rev. 1985 Dec;49(4):398–418. doi: 10.1128/mr.49.4.398-418.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Summers R. G., Harris C. R., Knowles J. R. A conservative amino acid substitution, arginine for lysine, abolishes export of a hybrid protein in Escherichia coli. Implications for the mechanism of protein secretion. J Biol Chem. 1989 Nov 25;264(33):20082–20088. [PubMed] [Google Scholar]
  17. Summers R. G., Knowles J. R. Illicit secretion of a cytoplasmic protein into the periplasm of Escherichia coli requires a signal peptide plus a portion of the cognate secreted protein. Demarcation of the critical region of the mature protein. J Biol Chem. 1989 Nov 25;264(33):20074–20081. [PubMed] [Google Scholar]
  18. Tang L. B., Lenstra R., Borchert T. V., Nagarajan V. Isolation and characterization of levansucrase-encoding gene from Bacillus amyloliquefaciens. Gene. 1990 Nov 30;96(1):89–93. doi: 10.1016/0378-1119(90)90345-r. [DOI] [PubMed] [Google Scholar]
  19. Zagorec M., Steinmetz M. Expression of levansucrase-beta-galactosidase hybrids inhibits secretion and is lethal in Bacillus subtilis. J Gen Microbiol. 1990 Jun;136(6):1137–1143. doi: 10.1099/00221287-136-6-1137. [DOI] [PubMed] [Google Scholar]

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