1887

Abstract

Summary: High-level synthesis of exportable β-galactosidase (LacZ) fusion proteins in results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible fusion gene system for this purpose, but frequent mutations . which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional secretion mutant after nitrosoguanidine mutagenesis. At 42°C, but not at 30°C, this mutant displayed extreme growth retardation when the LacZ fusion protein was produced, and was also defective in the secretion of subtilisin Carlsberg. The processing kinetics and secretion of a subtilisin Carlsberg-alkaline phosphatase fusion derivative were found to be defective specifically at the non-permissive temperature. The secretion defect was not linked to the locus.

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1995-07-01
2021-10-25
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References

  1. Altman E., Kumamoto C.A., Emr S. 1991; Heat-shock proteins can substitute for SecB function during protein export in Escherichia coli.. EMBO J 10:239–245
    [Google Scholar]
  2. Bassford P.J., Silhavy T.J., Beckwith J.R. 1979; Use of a gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.. J Bacteriol 139:19–31
    [Google Scholar]
  3. Boylan R.J., Mendelson N.H., Brooks D., Young F.E. 1972; Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid.. J Bacteriol 110:281–290
    [Google Scholar]
  4. van Dijl J.M., de Jong A., Vehmaanpera J., Venema G., Bron S. 1992; Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases.. EMBO J 11:2819–2828
    [Google Scholar]
  5. Errington J., Vogt C.H. 1990; Isolation and characterization of mutations in the gene encoding endogenous Bacillus subtilis -galactosidase and its regulator.. J Bacteriol 172:488–490
    [Google Scholar]
  6. Gardel C., Benson S., Hunt J., Michaelis S., Beckwith J. 1987; secD, a new gene involved in protein export in Escherichia coli.. J Bacteriol 169:1286–1290
    [Google Scholar]
  7. Gruss A., Ehrlich S.D. 1989; The family of highly inter-related ssDNA plasmids.. Microbiol Rev 53:231–241
    [Google Scholar]
  8. Hartl F.-U., Lecker S., Schiebel E., Hendrick J.P., Wickner W. 1990; The binding cascade of SecB to Sec A to SecY /E mediates preprotein targeting to the E. coli plasma membrane.. Cell 63:269–279
    [Google Scholar]
  9. Hastrup S. 1988; Analysis of the xylose regulon.. In Genetics and Biotechnology of Bacilli 2 pp. 79–81 Ganesan A.T., Hoch J.A. Edited by San Diego:: Academic Press.;
    [Google Scholar]
  10. Hastrup S. 1993; Gene expression system.. European Patent 0242220B1 21July 1993
    [Google Scholar]
  11. Hastrup S., Jacobs M.F. 1990; Lethal phenotype conferred by xylose-induced overproduction of an apr-lacZ fusion protein.. In Genetics and Biotechnology of Bacilli 3 pp. 33–41 Zukowski M.M., Ganesan A.T., Hoch J.A. Edited by San Diego:: Academic Press.;
    [Google Scholar]
  12. Honda K., Nakamura K., Nishiguchi M., Yamane K. 1993; Cloning and characterization of a Bacillus subtilis gene encoding a homolog of the 54-kilodalton subunit of mammalian signal recognition particle and Escherichia coli Ffh.. J Bacteriol 175:4885–4894
    [Google Scholar]
  13. Jacobs M.F. 1995; Expression of the subtilisin Carlsberg-encoding gene in Bacillus licheniformis and Bacillus subtilis.. Gene 152:69–74
    [Google Scholar]
  14. Jacobs M.F., Andersen J.B., Kontinen V., Sarvas M. 1993; Bacillus subtilis PrsA is required in vivo as an extracytoplasmic chaperone for secretion of active enzymes synthesized either with or without pro-sequences.. Mol Microbiol 8:957–966
    [Google Scholar]
  15. Jannière L., Bruand C., Ehrlich S.D. 1990; Structurally stable Bacillus subtilis cloning vectors.. Gene 87:53–61
    [Google Scholar]
  16. Jeong S.M., Yoshikawa H., Takahashi H. 1993; Isolation and characterization of the secE homologue gene of Bacillus subtilis.. Mol Microbiol 10:133–142
    [Google Scholar]
  17. Kontinen V.P., Sarvas M. 1988; Mutants of Bacillus subtilisdefective in protein transport.. J Gen Microbiol 134:2333–2344
    [Google Scholar]
  18. Kumamoto C.A., Beckwith J. 1983; Mutations in a new gene, secB, cause defective protein localization in Escherichia coli.. J Bacteriol 154:253–260
    [Google Scholar]
  19. Kumamoto C.A., Francetic O. 1993; Highly selective binding of nascent polypeptides by an Escherichia coli chaperone protein in vivo.. J Bacteriol 175:2184–2188
    [Google Scholar]
  20. Kusukawa N., Yura T., Ueguchi C., Akiyama Y., Ito K. 1989; Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli.. EMBO J 8:3517–3521
    [Google Scholar]
  21. Marusyk R., Sargeant A. 1980; A simple method for dialysis of small-volume samples.. Anal Biochem 105:403–404
    [Google Scholar]
  22. McKenzie T., Hoshino T., Tanaka T., Sueoka N. 1987; A revision of the nucleotide sequence and functional map of pUB110.. Plasmid 17:83–85
    [Google Scholar]
  23. Nagarajan V., Borchert T.V. 1991; Levansucrase: a tool to study protein secretion in Bacillus subtilis. . Res Microbiol 142:787–792
    [Google Scholar]
  24. Nakamura K., Takamatsu H., Akiyama Y., Ito K., Yamane K. 1990; Complementation of the protein transport defect of an Escherichia coli secY mutant (secY24) by Bacillus subtilis secYhomologue.. FEBS Lett 273:75–78
    [Google Scholar]
  25. Oliver D.B., Beckwith J. 1981; Escherichia coli mutant pleiotropically defective in the export of secreted proteins.. Cell 25:765–772
    [Google Scholar]
  26. Overhoff B., Klein M., Spies M., Freudl R. 1991; Identification of a gene fragment which codes for the amino-terminal 364 amino-acid residues of a SecA homologue from Bacillus subtilis: further evidence for the conservation of the protein export apparatus in gram-positive and gram-negative bacteria.. Mol & Gen Genet 228:417–423
    [Google Scholar]
  27. Phillips G.J., Silhavy T.J. 1990; Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli.. Nature 344:882–884
    [Google Scholar]
  28. Sadaie Y., Kada T. 1983; Effect of septum-initiation mutations on sporulation and competent cell formation in Bacillus subtilis.. Mol & Gen Genet 190:176–178
    [Google Scholar]
  29. Sadaie Y., Takamatsu H., Nakamura K., Yamane K. 1991; Sequencing reveals similarity of the wild-type div + gene of Bacillus subtilis to the Escherichia coli sec A gene.. Gene 98:101–105
    [Google Scholar]
  30. Sambrook J., Fritsch E.F., Maniatis T. 1989 In Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory.;
    [Google Scholar]
  31. Schaeffer P., Millet J., Aubert J.-P. 1965; Catabolic repression of bacterial sporulation.. Proc Natl Acad Sci USA 54704–711
    [Google Scholar]
  32. Schatz P.J., Beckwith J. 1990; Genetic analysis of protein export in Escherichia coli.. Annu Rev Genet 24:215–248
    [Google Scholar]
  33. Simon D., Chopin A. 1988; Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis.. Biochimie 70:559–566
    [Google Scholar]
  34. Simons K., Sarvas M., Garoff H., Helenius A. 1978; Membrane bound and secreted forms of penicillinase from Bacillus licheniformis.. J Mol Biol 126:673–690
    [Google Scholar]
  35. Snyder W.B., Silhavy T.J. 1995; Galactosidase is inactivated by intermolecular disulfide bonds and is toxic when secreted to the periplasm of Escherichia coli.. J Bacteriol 177:953–963
    [Google Scholar]
  36. Suh J.-W., Boylan A., Thomas S.M., Dolan K.M., Oliver D.B., Price C.W. 1990; Isolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria.. Mol Microbiol 4:305–314
    [Google Scholar]
  37. Takamatsu H., Fuma S., Nakamura K., Sadaie Y., Shinkai A., Matsuyama S., Mizushima S., Yamane K. 1992; In vivo and in vitro characterization of the sec A gene product of Bacillus subtilis.. J Bacteriol 174:4308–4316
    [Google Scholar]
  38. Wild J., Altman E., Yura T., Gross C.A. 1992; DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli.. Genes & Dev 6:1165–1172
    [Google Scholar]
  39. Yoshikawa H., Jeong S.M., Hirata A., Kawamura F., Doi R.H., Takahashi H. 1993; Temperature-sensitive sporulation caused by a mutation in the Bacillus subtilis secY gene.. J Bacteriol 175:3656–3660
    [Google Scholar]
  40. Zagorec M., Steinmetz M. 1990; Expression of levansucrase--galactosidase hybrids inhibits secretion and is lethal in Bacillus subtilis.. J Gen Microbiol 136:1137–1143
    [Google Scholar]
  41. Zagorec M., Steinmetz M. 1991; Construction of a derivative of Tn917 containing an outward-directed promoter and its use in Bacillus subtilis.. J Gen Microbiol 137:107–112
    [Google Scholar]
  42. Zuber U., Schumann W. 1994; CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis.. J Bacteriol 176:1359–1363
    [Google Scholar]
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