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. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129

TnphoA: a transposon probe for protein export signals.

C Manoil, J Beckwith
PMCID: PMC391456  PMID: 2999794

Abstract

We constructed a derivative of transposon Tn5 that permits the generation of hybrid proteins composed of alkaline phosphatase (EC 3.1.3.1) lacking its signal peptide fused to amino-terminal sequences of other proteins. Such a hybrid gives alkaline phosphatase activity if the protein fused to alkaline phosphatase contributes sequences that promote export and thus compensate for the missing alkaline phosphatase signal peptide. Fusions to both a secreted periplasmic protein and a complex cytoplasmic membrane protein led to alkaline phosphatase activity. TnphoA fusions should help localize export signals within the structure of a protein, such as a transmembrane protein, as well as identify new chromosomal genes for secreted and transmembrane proteins.

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Selected References

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  1. Achtman M. Mating aggregates in Escherichia coli conjugation. J Bacteriol. 1975 Aug;123(2):505–515. doi: 10.1128/jb.123.2.505-515.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auerswald E. A., Ludwig G., Schaller H. Structural analysis of Tn5. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):107–113. doi: 10.1101/sqb.1981.045.01.019. [DOI] [PubMed] [Google Scholar]
  3. Bassford P. J., Jr, Silhavy T. J., Beckwith J. R. Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol. 1979 Jul;139(1):19–31. doi: 10.1128/jb.139.1.19-31.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beckwith J. R., Signer E. R., Epstein W. Transposition of the Lac region of E. coli. Cold Spring Harb Symp Quant Biol. 1966;31:393–401. doi: 10.1101/sqb.1966.031.01.051. [DOI] [PubMed] [Google Scholar]
  5. Beckwith J., Silhavy T. J. Genetic analysis of protein export in Escherichia coli. Methods Enzymol. 1983;97:3–11. doi: 10.1016/0076-6879(83)97114-8. [DOI] [PubMed] [Google Scholar]
  6. Bellofatto V., Shapiro L., Hodgson D. A. Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1035–1039. doi: 10.1073/pnas.81.4.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Berg D. E., Schmandt M. A., Lowe J. B. Specificity of transposon Tn5 insertion. Genetics. 1983 Dec;105(4):813–828. doi: 10.1093/genetics/105.4.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brosius J., Cate R. L., Perlmutter A. P. Precise location of two promoters for the beta-lactamase gene of pBR322. S1 mapping of ribonucleic acid isolated from Escherichia coli or synthesized in vitro. J Biol Chem. 1982 Aug 10;257(15):9205–9210. [PubMed] [Google Scholar]
  9. Copeland B. R., Richter R. J., Furlong C. E. Renaturation and identification of periplasmic proteins in two-dimensional gels of Escherichia coli. J Biol Chem. 1982 Dec 25;257(24):15065–15071. [PubMed] [Google Scholar]
  10. Hoffman C. S., Wright A. Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5107–5111. doi: 10.1073/pnas.82.15.5107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Inouye H., Barnes W., Beckwith J. Signal sequence of alkaline phosphatase of Escherichia coli. J Bacteriol. 1982 Feb;149(2):434–439. doi: 10.1128/jb.149.2.434-439.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Isberg R. R., Lazaar A. L., Syvanen M. Regulation of Tn5 by the right-repeat proteins: control at the level of the transposition reaction? Cell. 1982 Oct;30(3):883–892. doi: 10.1016/0092-8674(82)90293-8. [DOI] [PubMed] [Google Scholar]
  13. Isberg R. R., Syvanen M. Replicon fusions promoted by the inverted repeats of Tn5. The right repeat is an insertion sequence. J Mol Biol. 1981 Jul 25;150(1):15–32. doi: 10.1016/0022-2836(81)90322-3. [DOI] [PubMed] [Google Scholar]
  14. Ito K., Bassford P. J., Jr, Beckwith J. Protein localization in E. coli: is there a common step in the secretion of periplasmic and outer-membrane proteins? Cell. 1981 Jun;24(3):707–717. doi: 10.1016/0092-8674(81)90097-0. [DOI] [PubMed] [Google Scholar]
  15. Kadonaga J. T., Gautier A. E., Straus D. R., Charles A. D., Edge M. D., Knowles J. R. The role of the beta-lactamase signal sequence in the secretion of proteins by Escherichia coli. J Biol Chem. 1984 Feb 25;259(4):2149–2154. [PubMed] [Google Scholar]
  16. Koshland D., Botstein D. Evidence for posttranslational translocation of beta-lactamase across the bacterial inner membrane. Cell. 1982 Oct;30(3):893–902. doi: 10.1016/0092-8674(82)90294-x. [DOI] [PubMed] [Google Scholar]
  17. Koshland D., Botstein D. Secretion of beta-lactamase requires the carboxy end of the protein. Cell. 1980 Jul;20(3):749–760. doi: 10.1016/0092-8674(80)90321-9. [DOI] [PubMed] [Google Scholar]
  18. Kroos L., Kaiser D. Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5816–5820. doi: 10.1073/pnas.81.18.5816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  21. Michaelis S., Beckwith J. Mechanism of incorporation of cell envelope proteins in Escherichia coli. Annu Rev Microbiol. 1982;36:435–465. doi: 10.1146/annurev.mi.36.100182.002251. [DOI] [PubMed] [Google Scholar]
  22. Michaelis S., Inouye H., Oliver D., Beckwith J. Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. J Bacteriol. 1983 Apr;154(1):366–374. doi: 10.1128/jb.154.1.366-374.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nguyen T. T., Postle K., Bertrand K. P. Sequence homology between the tetracycline-resistance determinants of Tn10 and pBR322. Gene. 1983 Nov;25(1):83–92. doi: 10.1016/0378-1119(83)90170-1. [DOI] [PubMed] [Google Scholar]
  24. Prentki P., Karch F., Iida S., Meyer J. The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication. Gene. 1981 Sep;14(4):289–299. doi: 10.1016/0378-1119(81)90161-x. [DOI] [PubMed] [Google Scholar]
  25. Sarthy A., Michaelis S., Beckwith J. Deletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA. J Bacteriol. 1981 Jan;145(1):288–292. doi: 10.1128/jb.145.1.288-292.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  27. Talmadge K., Gilbert W. Cellular location affects protein stability in Escherichia coli. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1830–1833. doi: 10.1073/pnas.79.6.1830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. de Bruijn F. J., Lupski J. R. The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids--a review. Gene. 1984 Feb;27(2):131–149. doi: 10.1016/0378-1119(84)90135-5. [DOI] [PubMed] [Google Scholar]

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