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. 1997 Oct;63(10):3965–3971. doi: 10.1128/aem.63.10.3965-3971.1997

Use of alkaline phosphatase as a reporter polypeptide to study the role of the subtilin leader segment and the SpaT transporter in the posttranslational modifications and secretion of subtilin in Bacillus subtilis 168.

G Izaguirre 1, J N Hansen 1
PMCID: PMC168708  PMID: 9327561

Abstract

The subtilin leader segment of presubtilin was fused to alkaline phosphatase (AP), which was used as a reporter polypeptide to study the role of the subtilin leader segment in posttranslational modifications during the conversion of presubtilin to subtilin and in the translocation of presubtilin from the cytoplasm of Bacillus subtilis 168 to the extracellular medium. It was observed that the subtilin leader segment could be utilized by a wild-type transporter, but secretion was enhanced if the subtilin transporter was available. The subtilin leader was not cleaved away from the AP component of the precursor until the precursor had been transported to the cell wall, and none of the AP was released into the medium until after cleavage had occurred. The role of SpaT, which is an ABC transporter that has been implicated in subtilin secretion, was explored by making a large in-frame deletion from the central region of SpaT and observing the effect on translocation of the AP reporter. Instead of having an effect on translocation, the deletion disrupted proteolytic cleavage of the subtilin leader segment and release of the AP reporter into the medium. The AP that was secreted by means of the subtilin leader segment had not undergone any posttranslational modifications, as assessed by amino acid composition analysis and enzymatic activity analysis.

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Selected References

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  1. Banerjee S., Hansen J. N. Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J Biol Chem. 1988 Jul 5;263(19):9508–9514. [PubMed] [Google Scholar]
  2. Breitling R., Dubnau D. A membrane protein with similarity to N-methylphenylalanine pilins is essential for DNA binding by competent Bacillus subtilis. J Bacteriol. 1990 Mar;172(3):1499–1508. doi: 10.1128/jb.172.3.1499-1508.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chakicherla A., Hansen J. N. Role of the leader and structural regions of prelantibiotic peptides as assessed by expressing nisin-subtilin chimeras in Bacillus subtilis 168, and characterization of their physical, chemical, and antimicrobial properties. J Biol Chem. 1995 Oct 6;270(40):23533–23539. doi: 10.1074/jbc.270.40.23533. [DOI] [PubMed] [Google Scholar]
  4. Chung Y. J., Hansen J. N. Determination of the sequence of spaE and identification of a promoter in the subtilin (spa) operon in Bacillus subtilis. J Bacteriol. 1992 Oct;174(20):6699–6702. doi: 10.1128/jb.174.20.6699-6702.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chung Y. J., Steen M. T., Hansen J. N. The subtilin gene of Bacillus subtilis ATCC 6633 is encoded in an operon that contains a homolog of the hemolysin B transport protein. J Bacteriol. 1992 Feb;174(4):1417–1422. doi: 10.1128/jb.174.4.1417-1422.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fath M. J., Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. doi: 10.1128/mr.57.4.995-1017.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hall B. G., Sharp P. M. Molecular population genetics of Escherichia coli: DNA sequence diversity at the celC, crr, and gutB loci of natural isolates. Mol Biol Evol. 1992 Jul;9(4):654–665. doi: 10.1093/oxfordjournals.molbev.a040751. [DOI] [PubMed] [Google Scholar]
  8. Hansen J. N. Antibiotics synthesized by posttranslational modification. Annu Rev Microbiol. 1993;47:535–564. doi: 10.1146/annurev.mi.47.100193.002535. [DOI] [PubMed] [Google Scholar]
  9. Håvarstein L. S., Diep D. B., Nes I. F. A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export. Mol Microbiol. 1995 Apr;16(2):229–240. doi: 10.1111/j.1365-2958.1995.tb02295.x. [DOI] [PubMed] [Google Scholar]
  10. Klein C., Entian K. D. Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633. Appl Environ Microbiol. 1994 Aug;60(8):2793–2801. doi: 10.1128/aem.60.8.2793-2801.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Klein C., Kaletta C., Schnell N., Entian K. D. Analysis of genes involved in biosynthesis of the lantibiotic subtilin. Appl Environ Microbiol. 1992 Jan;58(1):132–142. doi: 10.1128/aem.58.1.132-142.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Liu W., Hansen J. N. Conversion of Bacillus subtilis 168 to a subtilin producer by competence transformation. J Bacteriol. 1991 Nov;173(22):7387–7390. doi: 10.1128/jb.173.22.7387-7390.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Liu W., Hansen J. N. Enhancement of the chemical and antimicrobial properties of subtilin by site-directed mutagenesis. J Biol Chem. 1992 Dec 15;267(35):25078–25085. [PubMed] [Google Scholar]
  15. Meyer C., Bierbaum G., Heidrich C., Reis M., Süling J., Iglesias-Wind M. I., Kempter C., Molitor E., Sahl H. G. Nucleotide sequence of the lantibiotic Pep5 biosynthetic gene cluster and functional analysis of PepP and PepC. Evidence for a role of PepC in thioether formation. Eur J Biochem. 1995 Sep 1;232(2):478–489. doi: 10.1111/j.1432-1033.1995.tb20834.x. [DOI] [PubMed] [Google Scholar]
  16. Nes I. F., Tagg J. R. Novel lantibiotics and their pre-peptides. Antonie Van Leeuwenhoek. 1996 Feb;69(2):89–97. doi: 10.1007/BF00399414. [DOI] [PubMed] [Google Scholar]
  17. Nishio C., Komura S., Kurahashi K. Peptide antibiotic subtilin is synthesized via precursor proteins. Biochem Biophys Res Commun. 1983 Oct 31;116(2):751–758. doi: 10.1016/0006-291x(83)90588-0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Sahl H. G. Gene-encoded antibiotics made in bacteria. Ciba Found Symp. 1994;186:27–53. doi: 10.1002/9780470514658.ch3. [DOI] [PubMed] [Google Scholar]
  20. Sahl H. G., Jack R. W., Bierbaum G. Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. Eur J Biochem. 1995 Jun 15;230(3):827–853. doi: 10.1111/j.1432-1033.1995.tb20627.x. [DOI] [PubMed] [Google Scholar]
  21. Siezen R. J., Kuipers O. P., de Vos W. M. Comparison of lantibiotic gene clusters and encoded proteins. Antonie Van Leeuwenhoek. 1996 Feb;69(2):171–184. doi: 10.1007/BF00399422. [DOI] [PubMed] [Google Scholar]
  22. Sloma A., Ally A., Ally D., Pero J. Gene encoding a minor extracellular protease in Bacillus subtilis. J Bacteriol. 1988 Dec;170(12):5557–5563. doi: 10.1128/jb.170.12.5557-5563.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sullivan M. A., Yasbin R. E., Young F. E. New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments. Gene. 1984 Jul-Aug;29(1-2):21–26. doi: 10.1016/0378-1119(84)90161-6. [DOI] [PubMed] [Google Scholar]
  24. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wickner W., Driessen A. J., Hartl F. U. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem. 1991;60:101–124. doi: 10.1146/annurev.bi.60.070191.000533. [DOI] [PubMed] [Google Scholar]
  26. de Vos W. M., Kuipers O. P., van der Meer J. R., Siezen R. J. Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria. Mol Microbiol. 1995 Aug;17(3):427–437. doi: 10.1111/j.1365-2958.1995.mmi_17030427.x. [DOI] [PubMed] [Google Scholar]

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