1887

Abstract

High-level, inducible expression of heterologous genes in the cyanobacterium sp. strain PCC 7942 was obtained using the promoter and repressor. The gene of sp. strain PCC 7937 encoding plastocyanin precursor protein and the gene encoding β-glucuronidase were initially placed under the control of the promoter and repressor by cloning into the pTrc99C expression vector and were introduced into the chromosomal platform for integration in (PIM) of the R2-PIM9 recipient strain. These pTrc99C-derived constructs often gave rise to transformants that did not contain a complete insert gene, probably because of gene conversion events. Selection of the desired R2-PIM9 transformants was vastly improved using the new pTrcIS vector that contains the gene encoding streptomycin resistance as an extra antibiotic resistance marker. The influence of IPTG concentration and induction time on gene expression with the system in was determined using β-glucuronidase as a reporter. The PCC 7937 gene in was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis. The general usability of pTrcIS as a cloning vector for inducible heterologous gene expression in was confirmed by the introduction of several more genes.

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1995-04-01
2024-12-14
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References

  1. Amann E., Ochs B., Abel K.-J. 1988; Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli . Gene 69:301–315
    [Google Scholar]
  2. Arfman N., Worrell V., Ingram L. O. 1992; Use of the tac promoter and lacIq for the controlled expression of Zymomonas mobilis fermentation genes in Escherichia coli and Zymomonas mobilis . J Bacteriol 174:7370–7378
    [Google Scholar]
  3. Berry J., Bjorkman O. 1980; Photosynthetic response and adaptation to temperature in higher plants. Annu Rep Plant Physiol 31:491–543
    [Google Scholar]
  4. Borbély G., Surdnyi G., Korcz A., Pcilfi Z. 1985; Effect of heat shock on protein synthesis in the cyanobacterium Synechococcus sp. strain PCC 6301. J Bacteriol 161:1125–1130
    [Google Scholar]
  5. Bovy A., de Vrieze G., Borrias M., Weisbeek P. 1992; Transcriptional regulation of the plastocyanin and cytochrome c553 genes from the cyanobacterium Anabaena species PCC 7937. Mol Microbiol 6:1507–1513
    [Google Scholar]
  6. Bovy A., de Vrieze G., Lugones L., van Horssen P., van den Bert C., Borrias M., Weisbeek P. 1993; Iron-dependent stability of the ferredoxin I transcripts from the cyanobacterial strains Synechococcus PCC 7942 and Anabaena PCC 7937. Mol Microbiol 7:429–439
    [Google Scholar]
  7. Brosius J. 1987 Expression vectors employing lambda-, trp-, lac-, and lpp-derived promoters.. In Vectors: a Survey of Molecular Cloning Vectors and Their Use pp 205–225 Edited by R. J. Rodriguez; D. T. Denhardt; Boston: Butterworths.:
    [Google Scholar]
  8. Bryant D. A. 1992 . Molecular biology of photosystem I. In Topics in Photosynthesis,vol 11 The Photosystems: Structure, Function and Molecular Biology pp 501–549 Edited by J. Barber; Amsterdam : Elsevier:
    [Google Scholar]
  9. Cai Y., Wolk C. P. 1990; Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J Bacteriol1723138–3145
    [Google Scholar]
  10. Carter P., Bedouelle H., Winter G. 1985; Improved oligonucleotide site-directed mutagenesis using Ml3 vectors. Nucleic Acids Res 13:4431–4443
    [Google Scholar]
  11. Dente L., Cortese R. 1987; . pEMBL: a new family of singlestranded plasmids for sequencing DNA. Methods Ensymol 155:111–118
    [Google Scholar]
  12. Elhai J. 1993; Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120. FEMS Microbiol Lett 114:179–184
    [Google Scholar]
  13. Feinberg A. P., Vogelstein B. 1983; A technique for radio-labeling DNA restriction endonuclease fragments to high specificity. Anal Biochem 132:6–13
    [Google Scholar]
  14. Ferino F., Chauvat F. 1989; . A promoter-probe vector-host system for the cyanobacterium, Synechocystis PCC6803. Gene 84:257–266
    [Google Scholar]
  15. Friedberg D., Seijffers J. 1986; Controlled gene expression utilising lambda phage regulatory signals in a cyanobacterial host. Mol Gen Genet 203:505–510
    [Google Scholar]
  16. Geerts D., Schubert H., de Vrieze G., Borrias M., Matthijs H. C.P., Weisbeek P. J. 1994; Expression of Anabaena PCC 7937 plastocyanin in Synechococcus PCC 7942 enhances photosynthetic electron transfer and alters the electron distribution between PS I and cytochrome c oxidase. J Biol Chem 269:28068–28075
    [Google Scholar]
  17. Gray M. W. 1989; The evolutionary origins of organelles. Trends Genet 5:294–299
    [Google Scholar]
  18. Houmard J., Tandeau de Marsac N. 1988; Cyanobacterial genetic tools: current status. Methods Ensymol 167:808–847
    [Google Scholar]
  19. Jefferson R. A., Burgess S. M.c, Hirsh D. 1986; β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83:8447–8451
    [Google Scholar]
  20. Kuritz T., Ernst A., Black T., Wolk C. P. 1993; .High-resolution mapping of genetic loci of Anabaena PCC 7120 required for photosynthesis and nitrogen fixation. Mol Microbiol 8:101–110
    [Google Scholar]
  21. Labes M., PUhler A., Simon R. 1990; .A new family of RSFl0l0-derived expression and lac-fusion broad-host-range vectors for Gram-negative bacteria. Gene 89:37–46
    [Google Scholar]
  22. Laemmli U. K. 1970; .Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  23. Marraccini P., Bulteau S., Cassier-Chauvat C., Mermet-Bouvier P., Chauvat F. 1993; . A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis. . Plant Mol Biol 23:905–909
    [Google Scholar]
  24. Mermet-Bouvier P., Chauvat F. 1994; . A conditional expression vector for the cyanobacteria Synechocystis sp. strains PCC 6803 and PCC 6714 and Synechococcus sp. strains PCC 7942 and PCC 6301. Curr Microbiol 28:145–148
    [Google Scholar]
  25. Pilon M., de Boer A. D., Knols S. L., Koppelman M. H.G. M.,, van der Graaff R. M., de Kruijff B., Weisbeek P. J. 1990; .Expression in E. coli and purification of a translocation-competent precursor of the chloroplast protein ferredoxin. J Biol Chem 265:3358–3361
    [Google Scholar]
  26. van der Plas J., Bovy A., Kruyt F., de Vrieze G., Dassne E., Klein B., Weisbeek P. 1989; . The gene for the precursor of plastocyanin from the cyanobacterium Anabaena sp. PCC7937: isolation, sequence and regulation. Mol Microbiol 3:275–284
    [Google Scholar]
  27. van der Plas J., Hegeman H., de Vrieze G., Tyul M., Borrias M., Weisbeek P. 1990; . Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin. Gene. 95:39–48
    [Google Scholar]
  28. Porter R. D. 1986; . Transformation in cyanobacteria. Crit Rev Microbiol 13:111–132
    [Google Scholar]
  29. Prentki P., Binda A., Epstein A. 1991; . Plasmid vectors for selecting IS1-promoted deletions in cloned DNA : sequence analysis of the omega interposon. Gene 103:17–23
    [Google Scholar]
  30. Rippka R., Deruelles J., Waterbury J. B., Herdman M., Stanier R. Y. 1979; . Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61
    [Google Scholar]
  31. Rose R. E. 1988; . The nucleotide sequence of pACYC184. Nucleic AcidsRes 16, 355.:
    [Google Scholar]
  32. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn Spring Harbor, NY:: Cold Spring Harbor Laboratory.;
    [Google Scholar]
  33. Scanlan D. J., Bloye S. A., Mann N. H., Hodgson D. A., Carr N. G. 1990; . Construction of lacZ promoter probe vectors for use in Synechococcus: application to the identification of CO2-regulated promoters. Gene 90:43–49
    [Google Scholar]
  34. Sleat D. E., Gallie D. R., Jefferson R. A., Bevan M. W., Turner P.C, Wilson T. M.A. 1987; . Characterization of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro. Gene 60:217–225
    [Google Scholar]
  35. Stanier R. Y., Cohen-Bazire G. 1977; . Phototrophic prokaryotes : the cyanobacteria. Annu Rev Microbiol 31:225–274
    [Google Scholar]
  36. Tandeau de Marsac N., Houmard J. 1993; . Adaptation of cyanobacteria to environmental stimuli : new steps towards molecular mechanisms. FEMS Microbiol Rev 104:119–190
    [Google Scholar]
  37. Thiel T., Poo H. 1989; . Transformation of a filamentous cyanobacterium by electroporation. J Bacteriol 171:5743–5746
    [Google Scholar]
  38. Vermaas W. 1993; . Molecular biology approaches to analyze photosystem II structure and function. Annu Rev Plant Physiol Plant Mol Biol 44:457–481
    [Google Scholar]
  39. Vorst O., van Dam F., Oosterhoff-Teertstra R., Smeekens S., Weisbeek P. 1990; . Tissue-specific expression directed by an Arabidopsis thaliana pre-ferredoxin promoter in transgenic tobacco plants. Plant Mol Biol 14:491–499
    [Google Scholar]
  40. Wada H., Murata N. 1990; . Temperature-induced changes in the fatty acid composition of the cyanobacterium, Synechocystis PCC 6803. Plant Physiol 92:1062–1069
    [Google Scholar]
  41. Williams J. G.K., Szalay A. A. 1983; . Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2. Gene 24:37–51
    [Google Scholar]
  42. Wolk C. P., Vonshak A., Kehoe P., Elhai J. 1984; . Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc Natl Acad Sci USA 81:1561–1565
    [Google Scholar]
  43. Yansura D. G., Henner D. J. 1984; .Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis . Proc Natl Acad Sci USA 81:439–443
    [Google Scholar]
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