Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Feb;174(4):1314–1323. doi: 10.1128/jb.174.4.1314-1323.1992

Role of the two-component leader sequence and mature amino acid sequences in extracellular export of endoglucanase EGL from Pseudomonas solanacearum.

J Z Huang 1, M A Schell 1
PMCID: PMC206427  PMID: 1735723

Abstract

The egl gene of Pseudomonas solanacearum encodes a 43-kDa extracellular endoglucanase (mEGL) involved in wilt disease caused by this phytopathogen. Egl is initially translated with a 45-residue, two-part leader sequence. The first 19 residues are apparently removed by signal peptidase II during export of Egl across the inner membrane (IM); the remaining residues of the leader sequence (modified with palmitate) are removed during export across the outer membrane (OM). Localization of Egl-PhoA fusion proteins showed that the first 26 residues of the Egl leader sequence are required and sufficient to direct lipid modification, processing, and export of Egl or PhoA across the IM but not the OM. Fusions of the complete 45-residue leader sequence or of the leader and increasing portions of mEgl sequences to PhoA did not cause its export across the OM. In-frame deletion of portions of mEGL-coding sequences blocked export of the truncated polypeptides across the OM without affecting export across the IM. These results indicate that the first part of the leader sequence functions independently to direct export of Egl across the IM while the second part and sequences and structures in mEGL are involved in export across the OM. Computer analysis of the mEgl amino acid sequence obtained from its nucleotide sequence identified a region of mEGL similar in amino acid sequence to regions in other prokaryotic endoglucanases.

Full text

PDF
1317

Images in this article

1320Image
on p.1320
1320Image
on p.1320
1321Image
on p.1321
1321Image
on p.1321

Selected References

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

  1. Aldea M., Garrido T., Hernández-Chico C., Vicente M., Kushner S. R. Induction of a growth-phase-dependent promoter triggers transcription of bolA, an Escherichia coli morphogene. EMBO J. 1989 Dec 1;8(12):3923–3931. doi: 10.1002/j.1460-2075.1989.tb08573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andro T., Chambost J. P., Kotoujansky A., Cattaneo J., Bertheau Y., Barras F., Van Gijsegem F., Coleno A. Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase. J Bacteriol. 1984 Dec;160(3):1199–1203. doi: 10.1128/jb.160.3.1199-1203.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  5. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dröge W., Lehmann V., Lüderitz O., Westphal O. Structural investigations on the 2-keto-3-deoxyoctonate region of lipopolysaccharides. Eur J Biochem. 1970 May 1;14(1):175–184. doi: 10.1111/j.1432-1033.1970.tb00276.x. [DOI] [PubMed] [Google Scholar]
  7. Filloux A., Bally M., Ball G., Akrim M., Tommassen J., Lazdunski A. Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria. EMBO J. 1990 Dec;9(13):4323–4329. doi: 10.1002/j.1460-2075.1990.tb07881.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Freudl R., Schwarz H., Klose M., Movva N. R., Henning U. The nature of information, required for export and sorting, present within the outer membrane protein OmpA of Escherichia coli K-12. EMBO J. 1985 Dec 16;4(13A):3593–3598. doi: 10.1002/j.1460-2075.1985.tb04122.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fukumori F., Kudo T., Narahashi Y., Horikoshi K. Molecular cloning and nucleotide sequence of the alkaline cellulase gene from the alkalophilic Bacillus sp. strain 1139. J Gen Microbiol. 1986 Aug;132(8):2329–2335. doi: 10.1099/00221287-132-8-2329. [DOI] [PubMed] [Google Scholar]
  10. GAREN A., LEVINTHAL C. A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim Biophys Acta. 1960 Mar 11;38:470–483. doi: 10.1016/0006-3002(60)91282-8. [DOI] [PubMed] [Google Scholar]
  11. Gilkes N. R., Henrissat B., Kilburn D. G., Miller R. C., Jr, Warren R. A. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303–315. doi: 10.1128/mr.55.2.303-315.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. He S. Y., Lindeberg M., Chatterjee A. K., Collmer A. Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1079–1083. doi: 10.1073/pnas.88.3.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Henrissat B., Claeyssens M., Tomme P., Lemesle L., Mornon J. P. Cellulase families revealed by hydrophobic cluster analysis. Gene. 1989 Sep 1;81(1):83–95. doi: 10.1016/0378-1119(89)90339-9. [DOI] [PubMed] [Google Scholar]
  14. Hirst T. R., Welch R. A. Mechanisms for secretion of extracellular proteins by gram-negative bacteria. Trends Biochem Sci. 1988 Jul;13(7):265–269. doi: 10.1016/0968-0004(88)90160-0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Huang J. H., Schell M. A. DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum. J Bacteriol. 1990 Jul;172(7):3879–3887. doi: 10.1128/jb.172.7.3879-3887.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang J. Z., Schell M. A. Evidence that extracellular export of the endoglucanase encoded by egl of Pseudomonas solanacearum occurs by a two-step process involving a lipoprotein intermediate. J Biol Chem. 1990 Jul 15;265(20):11628–11632. [PubMed] [Google Scholar]
  18. Huang J. Z., Sukordhaman M., Schell M. A. Excretion of the egl gene product of Pseudomonas solanacearum. J Bacteriol. 1989 Jul;171(7):3767–3774. doi: 10.1128/jb.171.7.3767-3774.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Inukai M., Takeuchi M., Shimizu K., Arai M. Mechanism of action of globomycin. J Antibiot (Tokyo) 1978 Nov;31(11):1203–1205. doi: 10.7164/antibiotics.31.1203. [DOI] [PubMed] [Google Scholar]
  20. Kasahara M., Anraku Y. Succinate dehydrogenase of Escherichia coli membrane vesicles. Activation and properties of the enzyme. J Biochem. 1974 Nov;76(5):959–966. [PubMed] [Google Scholar]
  21. Kranz R. G., Foster-Hartnett D. Transcriptional regulatory cascade of nitrogen-fixation genes in anoxygenic photosynthetic bacteria: oxygen- and nitrogen-responsive factors. Mol Microbiol. 1990 Nov;4(11):1793–1800. doi: 10.1111/j.1365-2958.1990.tb02027.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Manoil C., Mekalanos J. J., Beckwith J. Alkaline phosphatase fusions: sensors of subcellular location. J Bacteriol. 1990 Feb;172(2):515–518. doi: 10.1128/jb.172.2.515-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McGavin M. J., Forsberg C. W., Crosby B., Bell A. W., Dignard D., Thomas D. Y. Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3. J Bacteriol. 1989 Oct;171(10):5587–5595. doi: 10.1128/jb.171.10.5587-5595.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mead D. A., Skorupa E. S., Kemper B. Single stranded DNA SP6 promoter plasmids for engineering mutant RNAs and proteins: synthesis of a 'stretched' preproparathyroid hormone. Nucleic Acids Res. 1985 Feb 25;13(4):1103–1118. doi: 10.1093/nar/13.4.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Murata H., Fons M., Chatterjee A., Collmer A., Chatterjee A. K. Characterization of transposon insertion out- mutants of Erwinia carotovora subsp. carotovora defective in enzyme export and of a DNA segment that complements out mutations in E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi. J Bacteriol. 1990 Jun;172(6):2970–2978. doi: 10.1128/jb.172.6.2970-2978.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Oliver D. Protein secretion in Escherichia coli. Annu Rev Microbiol. 1985;39:615–648. doi: 10.1146/annurev.mi.39.100185.003151. [DOI] [PubMed] [Google Scholar]
  30. Pugsley A. P., Kornacker M. G. Secretion of the cell surface lipoprotein pullulanase in Escherichia coli. Cooperation or competition between the specific secretion pathway and the lipoprotein sorting pathway. J Biol Chem. 1991 Jul 25;266(21):13640–13645. [PubMed] [Google Scholar]
  31. Pugsley A. P., Reyss I. Five genes at the 3' end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion. Mol Microbiol. 1990 Mar;4(3):365–379. doi: 10.1111/j.1365-2958.1990.tb00604.x. [DOI] [PubMed] [Google Scholar]
  32. Randall L. L., Hardy S. J., Thom J. R. Export of protein: a biochemical view. Annu Rev Microbiol. 1987;41:507–541. doi: 10.1146/annurev.mi.41.100187.002451. [DOI] [PubMed] [Google Scholar]
  33. Randall L. L., Hardy S. J. Unity in function in the absence of consensus in sequence: role of leader peptides in export. Science. 1989 Mar 3;243(4895):1156–1159. doi: 10.1126/science.2646712. [DOI] [PubMed] [Google Scholar]
  34. Roberts D. P., Denny T. P., Schell M. A. Cloning of the egl gene of Pseudomonas solanacearum and analysis of its role in phytopathogenicity. J Bacteriol. 1988 Apr;170(4):1445–1451. doi: 10.1128/jb.170.4.1445-1451.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Saloheimo M., Lehtovaara P., Penttilä M., Teeri T. T., Ståhlberg J., Johansson G., Pettersson G., Claeyssens M., Tomme P., Knowles J. K. EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. Gene. 1988;63(1):11–22. doi: 10.1016/0378-1119(88)90541-0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schell M. A. Purification and Characterization of an Endoglucanase from Pseudomonas solanacearum. Appl Environ Microbiol. 1987 Sep;53(9):2237–2241. doi: 10.1128/aem.53.9.2237-2241.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schell M. A., Roberts D. P., Denny T. P. Analysis of the Pseudomonas solanacearum polygalacturonase encoded by pglA and its involvement in phytopathogenicity. J Bacteriol. 1988 Oct;170(10):4501–4508. doi: 10.1128/jb.170.10.4501-4508.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schnaitman C. A. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol. 1971 Oct;108(1):545–552. doi: 10.1128/jb.108.1.545-552.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Spök A., Stubenrauch G., Schörgendorfer K., Schwab H. Molecular cloning and sequencing of a pectinesterase gene from Pseudomonas solanacearum. J Gen Microbiol. 1991 Jan;137(1):131–140. doi: 10.1099/00221287-137-1-131. [DOI] [PubMed] [Google Scholar]
  42. Thom J. R., Randall L. L. Role of the leader peptide of maltose-binding protein in two steps of the export process. J Bacteriol. 1988 Dec;170(12):5654–5661. doi: 10.1128/jb.170.12.5654-5661.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Thurn K. K., Chatterjee A. K. Single-site chromosomal Tn5 insertions affect the export of pectolytic and cellulolytic enzymes in Erwinia chrysanthemi EC16. Appl Environ Microbiol. 1985 Oct;50(4):894–898. doi: 10.1128/aem.50.4.894-898.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wickner W. Secretion and membrane assembly. Trends Biochem Sci. 1989 Jul;14(7):280–283. doi: 10.1016/0968-0004(89)90064-9. [DOI] [PubMed] [Google Scholar]
  45. Wretlind B., Pavlovskis O. R. Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins. J Bacteriol. 1984 Jun;158(3):801–808. doi: 10.1128/jb.158.3.801-808.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yamaguchi K., Yu F., Inouye M. A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell. 1988 May 6;53(3):423–432. doi: 10.1016/0092-8674(88)90162-6. [DOI] [PubMed] [Google Scholar]
  47. Yanagida N., Uozumi T., Beppu T. Specific excretion of Serratia marcescens protease through the outer membrane of Escherichia coli. J Bacteriol. 1986 Jun;166(3):937–944. doi: 10.1128/jb.166.3.937-944.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. d'Enfert C., Ryter A., Pugsley A. P. Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase. EMBO J. 1987 Nov;6(11):3531–3538. doi: 10.1002/j.1460-2075.1987.tb02679.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES