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. 1994 May;176(10):3013–3020. doi: 10.1128/jb.176.10.3013-3020.1994

Escherichia coli signal peptides direct inefficient secretion of an outer membrane protein (OmpA) and periplasmic proteins (maltose-binding protein, ribose-binding protein, and alkaline phosphatase) in Bacillus subtilis.

D N Collier 1
PMCID: PMC205459  PMID: 8188602

Abstract

Signal peptides of gram-positive exoproteins generally carry a higher net positive charge at their amino termini (N regions) and have longer hydrophobic cores (h regions) and carboxy termini (C regions) than do signal peptides of Escherichia coli envelope proteins. To determine if these differences are functionally significant, the ability of Bacillus subtilis to secrete four different E. coli envelope proteins was tested. A pulse-chase analysis demonstrated that the periplasmic maltose-binding protein (MBP), ribose-binding protein (RBP), alkaline phosphatase (PhoA), and outer membrane protein OmpA were only inefficiently secreted. Inefficient secretion could be ascribed largely to properties of the homologous signal peptides, since replacing them with the B. amyloliquefaciens alkaline protease signal peptide resulted in significant increases in both the rate and extent of export. The relative efficiency with which the native precursors were secreted (OmpA >> RBP > MBP > PhoA) was most closely correlated with the overall hydrophobicity of their h regions. This correlation was strengthened by the observation that the B. amyloliquefaciens levansucrase signal peptide, whose h region has an overall hydrophobicity similar to that of E. coli signal peptides, was able to direct secretion of only modest levels of MBP and OmpA. These results imply that there are differences between the secretion machineries of B. subtilis and E. coli and demonstrate that the outer membrane protein OmpA can be translocated across the cytoplasmic membrane of B. subtilis.

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Selected References

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  1. Bachmair A., Finley D., Varshavsky A. In vivo half-life of a protein is a function of its amino-terminal residue. Science. 1986 Oct 10;234(4773):179–186. doi: 10.1126/science.3018930. [DOI] [PubMed] [Google Scholar]
  2. Bankaitis V. A., Rasmussen B. A., Bassford P. J., Jr Intragenic suppressor mutations that restore export of maltose binding protein with a truncated signal peptide. Cell. 1984 May;37(1):243–252. doi: 10.1016/0092-8674(84)90320-9. [DOI] [PubMed] [Google Scholar]
  3. Bedouelle H., Bassford P. J., Jr, Fowler A. V., Zabin I., Beckwith J., Hofnung M. Mutations which alter the function of the signal sequence of the maltose binding protein of Escherichia coli. Nature. 1980 May 8;285(5760):78–81. doi: 10.1038/285078a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Briggs M. S., Gierasch L. M. Exploring the conformational roles of signal sequences: synthesis and conformational analysis of lambda receptor protein wild-type and mutant signal peptides. Biochemistry. 1984 Jul 3;23(14):3111–3114. doi: 10.1021/bi00309a001. [DOI] [PubMed] [Google Scholar]
  6. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  7. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  8. Chen M. W., Nagarajan V. Chloramphenicol acetyltransferase, a cytoplasmic protein is incompatible for export from Bacillus subtilis. J Bacteriol. 1993 Sep;175(17):5697–5700. doi: 10.1128/jb.175.17.5697-5700.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  10. Collier D. N., Bankaitis V. A., Weiss J. B., Bassford P. J., Jr The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell. 1988 Apr 22;53(2):273–283. doi: 10.1016/0092-8674(88)90389-3. [DOI] [PubMed] [Google Scholar]
  11. Collier D. N., Bassford P. J., Jr Mutations that improve export of maltose-binding protein in SecB- cells of Escherichia coli. J Bacteriol. 1989 Sep;171(9):4640–4647. doi: 10.1128/jb.171.9.4640-4647.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Collier D. N. SecB: a molecular chaperone of Escherichia coli protein secretion pathway. Adv Protein Chem. 1993;44:151–193. doi: 10.1016/s0065-3233(08)60567-7. [DOI] [PubMed] [Google Scholar]
  13. Collier D. N., Strobel S. M., Bassford P. J., Jr SecB-independent export of Escherichia coli ribose-binding protein (RBP): some comparisons with export of maltose-binding protein (MBP) and studies with RBP-MBP hybrid proteins. J Bacteriol. 1990 Dec;172(12):6875–6884. doi: 10.1128/jb.172.12.6875-6884.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cunningham K., Wickner W. Specific recognition of the leader region of precursor proteins is required for the activation of translocation ATPase of Escherichia coli. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8630–8634. doi: 10.1073/pnas.86.22.8630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Doud S. K., Chou M. M., Kendall D. A. Titration of protein transport activity by incremental changes in signal peptide hydrophobicity. Biochemistry. 1993 Feb 9;32(5):1251–1256. doi: 10.1021/bi00056a008. [DOI] [PubMed] [Google Scholar]
  17. Duplay P., Bedouelle H., Fowler A., Zabin I., Saurin W., Hofnung M. Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12. J Biol Chem. 1984 Aug 25;259(16):10606–10613. [PubMed] [Google Scholar]
  18. Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
  19. Emr S. D., Silhavy T. J. Importance of secondary structure in the signal sequence for protein secretion. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4599–4603. doi: 10.1073/pnas.80.15.4599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fikes J. D., Bassford P. J., Jr Export of unprocessed precursor maltose-binding protein to the periplasm of Escherichia coli cells. J Bacteriol. 1987 Jun;169(6):2352–2359. doi: 10.1128/jb.169.6.2352-2359.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Freudl R., Schwarz H., Stierhof Y. D., Gamon K., Hindennach I., Henning U. An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export. J Biol Chem. 1986 Aug 25;261(24):11355–11361. [PubMed] [Google Scholar]
  22. Gennity J., Goldstein J., Inouye M. Signal peptide mutants of Escherichia coli. J Bioenerg Biomembr. 1990 Jun;22(3):233–269. doi: 10.1007/BF00763167. [DOI] [PubMed] [Google Scholar]
  23. Iida A., Groarke J. M., Park S., Thom J., Zabicky J. H., Hazelbauer G. L., Randall L. L. A signal sequence mutant defective in export of ribose-binding protein and a corresponding pseudorevertant isolated without imposed selection. EMBO J. 1985 Jul;4(7):1875–1880. doi: 10.1002/j.1460-2075.1985.tb03863.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jeong S. M., Yoshikawa H., Takahashi H. Isolation and characterization of the secE homologue gene of Bacillus subtilis. Mol Microbiol. 1993 Oct;10(1):133–142. doi: 10.1111/j.1365-2958.1993.tb00910.x. [DOI] [PubMed] [Google Scholar]
  25. Kallio P., Simonen M., Palva I., Sarvas M. Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis. J Gen Microbiol. 1986 Mar;132(3):677–687. doi: 10.1099/00221287-132-3-677. [DOI] [PubMed] [Google Scholar]
  26. Kikuchi Y., Yoda K., Yamasaki M., Tamura G. The nucleotide sequence of the promoter and the amino-terminal region of alkaline phosphatase structural gene (phoA) of Escherichia coli. Nucleic Acids Res. 1981 Nov 11;9(21):5671–5678. doi: 10.1093/nar/9.21.5671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kumamoto C. A., Gannon P. M. Effects of Escherichia coli secB mutations on pre-maltose binding protein conformation and export kinetics. J Biol Chem. 1988 Aug 15;263(23):11554–11558. [PubMed] [Google Scholar]
  28. Kusukawa N., Yura T., Ueguchi C., Akiyama Y., Ito K. Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli. EMBO J. 1989 Nov;8(11):3517–3521. doi: 10.1002/j.1460-2075.1989.tb08517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Liu G. P., Topping T. B., Cover W. H., Randall L. L. Retardation of folding as a possible means of suppression of a mutation in the leader sequence of an exported protein. J Biol Chem. 1988 Oct 15;263(29):14790–14793. [PubMed] [Google Scholar]
  32. McKnight C. J., Briggs M. S., Gierasch L. M. Functional and nonfunctional LamB signal sequences can be distinguished by their biophysical properties. J Biol Chem. 1989 Oct 15;264(29):17293–17297. [PubMed] [Google Scholar]
  33. Meens J., Frings E., Klose M., Freudl R. An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtilis. Mol Microbiol. 1993 Aug;9(4):847–855. doi: 10.1111/j.1365-2958.1993.tb01743.x. [DOI] [PubMed] [Google Scholar]
  34. Movva N. R., Nakamura K., Inouye M. Amino acid sequence of the signal peptide of ompA protein, a major outer membrane protein of Escherichia coli. J Biol Chem. 1980 Jan 10;255(1):27–29. [PubMed] [Google Scholar]
  35. 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]
  36. Nagarajan V. System for secretion of heterologous proteins in Bacillus subtilis. Methods Enzymol. 1990;185:214–223. doi: 10.1016/0076-6879(90)85021-f. [DOI] [PubMed] [Google Scholar]
  37. Nakamura K., Fujita Y., Itoh Y., Yamane K. Modification of length, hydrophobic properties and electric charge of Bacillus subtilis alpha-amylase signal peptide and their different effects on the production of secretory proteins in B. subtilis and Escherichia coli cells. Mol Gen Genet. 1989 Mar;216(1):1–9. doi: 10.1007/BF00332223. [DOI] [PubMed] [Google Scholar]
  38. Palva I., Sarvas M., Lehtovaara P., Sibakov M., Käriäinen L. Secretion of Escherichia coli beta-lactamase from Bacillus subtilis by the aid of alpha-amylase signal sequence. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5582–5586. doi: 10.1073/pnas.79.18.5582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Park S., Liu G., Topping T. B., Cover W. H., Randall L. L. Modulation of folding pathways of exported proteins by the leader sequence. Science. 1988 Feb 26;239(4843):1033–1035. doi: 10.1126/science.3278378. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Puohiniemi R., Butcher S., Tarkka E., Sarvas M. High level production of Escherichia coli outer membrane proteins OmpA and OmpF intracellularly in Bacillus subtilis. FEMS Microbiol Lett. 1991 Sep 15;67(1):29–33. doi: 10.1016/0378-1097(91)90438-g. [DOI] [PubMed] [Google Scholar]
  42. Puohiniemi R., Simonen M., Muttilainen S., Himanen J. P., Sarvas M. Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step. Mol Microbiol. 1992 Apr;6(8):981–990. doi: 10.1111/j.1365-2958.1992.tb02164.x. [DOI] [PubMed] [Google Scholar]
  43. Puziss J. W., Fikes J. D., Bassford P. J., Jr Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide. J Bacteriol. 1989 May;171(5):2303–2311. doi: 10.1128/jb.171.5.2303-2311.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Puziss J. W., Strobel S. M., Bassford P. J., Jr Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations. J Bacteriol. 1992 Jan;174(1):92–101. doi: 10.1128/jb.174.1.92-101.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Ribbe J., Nagarajan V. Characterization of the secretion efficiency of a plant signal peptide in Bacillus subtilis. Mol Gen Genet. 1992 Nov;235(2-3):333–339. doi: 10.1007/BF00279378. [DOI] [PubMed] [Google Scholar]
  47. Ryan J. P., Duncan M. C., Bankaitis V. A., Bassford P. J., Jr Intragenic reversion mutations that improve export of maltose-binding protein in Escherichia coli malE signal sequence mutants. J Biol Chem. 1986 Mar 5;261(7):3389–3395. [PubMed] [Google Scholar]
  48. Sadaie Y., Takamatsu H., Nakamura K., Yamane K. Sequencing reveals similarity of the wild-type div+ gene of Bacillus subtilis to the Escherichia coli secA gene. Gene. 1991 Feb 1;98(1):101–105. doi: 10.1016/0378-1119(91)90110-w. [DOI] [PubMed] [Google Scholar]
  49. Schatz P. J., Beckwith J. Genetic analysis of protein export in Escherichia coli. Annu Rev Genet. 1990;24:215–248. doi: 10.1146/annurev.ge.24.120190.001243. [DOI] [PubMed] [Google Scholar]
  50. Suh J. W., Boylan S. A., Thomas S. M., Dolan K. M., Oliver D. B., Price C. W. Isolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria. Mol Microbiol. 1990 Feb;4(2):305–314. doi: 10.1111/j.1365-2958.1990.tb00597.x. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Vasantha N., Thompson L. D., Rhodes C., Banner C., Nagle J., Filpula D. Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. J Bacteriol. 1984 Sep;159(3):811–819. doi: 10.1128/jb.159.3.811-819.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Watson M. E. Compilation of published signal sequences. Nucleic Acids Res. 1984 Jul 11;12(13):5145–5164. doi: 10.1093/nar/12.13.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wild J., Altman E., Yura T., Gross C. A. DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli. Genes Dev. 1992 Jul;6(7):1165–1172. doi: 10.1101/gad.6.7.1165. [DOI] [PubMed] [Google Scholar]
  55. van Dijl J. M., de Jong A., Vehmaanperä J., Venema G., Bron S. Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases. EMBO J. 1992 Aug;11(8):2819–2828. doi: 10.1002/j.1460-2075.1992.tb05349.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. von Heijne G., Abrahmsén L. Species-specific variation in signal peptide design. Implications for protein secretion in foreign hosts. FEBS Lett. 1989 Feb 27;244(2):439–446. doi: 10.1016/0014-5793(89)80579-4. [DOI] [PubMed] [Google Scholar]
  57. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

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