1887

Abstract

The reading frame encoding a β-galactosidase of ‘’ was used as a reporter gene to investigate three different promoter regions derived from genes of (mc-) and (c- and p-) in transformants. The fusion of at the start codon of each reading frame (A1– fusion genes) caused translational problems in some cases. Transformants containing constructs with fusions further downstream in the reading frame (A–) produced β-galactosidase, and colonies on agar plates turned blue when sprayed with X-Gal. The β-galactosidase activities quantified by standard ONPG assays correlated well with the mRNA data determined with transformants containing the respective genes: the cA– fusion gene was completely inactive, the mcA– transformants showed low amounts of products, whereas the pA– fusion gene was constitutively expressed in the respective transformants. The transcription of each A– gene was activated by the homologous transcriptional activator protein GvpE. The cGvpE, pGvpE and mcGvpE proteins were able to activate the promoter of pA– and mcA–, whereas the promoter of cA– was only activated by cGvpE. Among the three GvpE proteins tested, cGvpE appeared to be the strongest transcriptional activator.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-7-1745
2001-07-01
2020-04-02
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/7/1471745a.html?itemId=/content/journal/micro/10.1099/00221287-147-7-1745&mimeType=html&fmt=ahah

References

  1. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1988; Current Protocols in Molecular. Biologyvol. 1: New York: Greene Publishing Associates and Wiley-Interscience;
    [Google Scholar]
  2. Baglia N., Goo Y. A., Ng W. V., Hood L., Daniels C., DaSarma S. 2000; Is gene expression in Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors?. Mol Microbiol36:1184–1185[CrossRef]
    [Google Scholar]
  3. Bell S., Jaxel C., Nadal M., Kosa P., Jackson S. 1998; Temperature, template topology, and factor requirements of archaeal transcription. Proc Natl Acad Sci USA95:15218–15222[CrossRef]
    [Google Scholar]
  4. Bell S., Kosa P., Sigler P., Jackson S. 1999; Orientation of the transcription preinitiation complex in Archaea. Proc Natl Acad Sci USA96:13662–13667[CrossRef]
    [Google Scholar]
  5. Chomczynski P., Sacchi N. 1987; Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem162:156–159
    [Google Scholar]
  6. Cline S. W., Schalkwyk L. C., Doolittle W. F. 1989; Transformation of the archaebacterium Halobacterium volcanii with genomic DNA. J Bacteriol171:4987–4991
    [Google Scholar]
  7. DasSarma S., Arora P., Lin F., Molinari E., Yin L. 1994; Wild-type gas vesicle formation requires at least ten genes in the gvp gene cluster of Halobacterium halobium plasmid pNRC100. J Bacteriol176:7646–7652
    [Google Scholar]
  8. Ellenberger T. E., Brandl C. J., Struhl K., Harrison S. C. 1992; The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted α helices: crystal structure of the protein–DNA complex. Cell71:1223–1237[CrossRef]
    [Google Scholar]
  9. Englert C., Horne M., Pfeifer F. 1990; Expression of the major gas vesicle protein in the halophilic archaebacterium Haloferax mediterranei is modulated by salt. Mol Gen Genet222:225–232[CrossRef]
    [Google Scholar]
  10. Englert C., Pfeifer F, Krüger K., Offner S.. 1992a; Three different but related gene clusters encoding gas vesicles in halophilic archaea. J Mol Biol227:586–592[CrossRef]
    [Google Scholar]
  11. Englert C., Wanner G., Pfeifer F. 1992b; Functional analysis of the gas-vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac-region boundary and suggests a regulatory role for the gvpD gene or its product. Mol Microbiol6:3543–3550[CrossRef]
    [Google Scholar]
  12. Hausner W., Wettach J., Hethke C., Thomm M. 1996; Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase. J Biol Chem271:30144–30148[CrossRef]
    [Google Scholar]
  13. Holmes M. L., Dyall-Smith M. 2000; Sequence and expression of a halobacterial β-galactosidase gene. Mol Microbiol36:114–122[CrossRef]
    [Google Scholar]
  14. Holmes M. L., Nuttall S. D., Dyall-Smith M. 1991; Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. J Bacteriol12:3807–3813
    [Google Scholar]
  15. Holmes M. L., Scopes R., Moritz R., Simpson R., Englert C., Pfeifer F., Dyall-Smith M. 1997; Purification and analysis of an extremely halophilic β-galactosidase from Haloferax alicantei Biochim Biophys Acta; 1337;276–286[CrossRef]
    [Google Scholar]
  16. Horne M., Pfeifer F. 1989; Expression of two gas vacuole protein genes in Halobacterium halobium and other related species. Mol Gen Genet218:437–444[CrossRef]
    [Google Scholar]
  17. Horne M., Englert C., Wimmer C., Pfeifer F. 1991; A DNA region of 9 kb contains all genes necessary for gas vesicle synthesis in halophilic archaebacteria. Mol Microbiol5:1159–1174[CrossRef]
    [Google Scholar]
  18. Krüger K., Pfeifer F. 1996; Transcript analysis of the c-vac region, and differential synthesis of the two regulatory gas-vesicle proteins GvpD and GvpE in Halobacterium salinarium PHH4. J Bacteriol178:4012–4019
    [Google Scholar]
  19. Krüger K.. Hermann T., Armbruster V., Pfeifer F. 1998; The transcriptional activator GvpE for the halobacterial gas vesicle genes resembles a basic region leucine-zipper regulatory protein. J Mol Biol279:761–771[CrossRef]
    [Google Scholar]
  20. Lam W. L., Doolittle W. F. 1989; Shuttle vectors for the archaebacterium Halobacterium volcanii . Proc Natl Acad Sci USA86:5478–5482[CrossRef]
    [Google Scholar]
  21. Ng W. L., Ciufo S., Smith T. 9 other authors 1998; Snapshot of a large dynamic replicon in a halophilic archaeon: megaplasmid or minichromosome?. Genome Res8:1131–1141
    [Google Scholar]
  22. Ng W. L., Kenney S., Mahairas G. 14 other authors 2000; Genome sequence of Halobacterium species NRC-1. Proc Natl Acad Sci USA97:12176–12181[CrossRef]
    [Google Scholar]
  23. Offner S., Wanner G., Pfeifer F. 1996; Functional studies of the gvpACNO operon of Halobacterium salinarium reveal that the GvpC protein shapes gas vesicles. J Bacteriol178:2071–2078
    [Google Scholar]
  24. Offner S., Ziese U., Wanner G., Typke D., Pfeifer F. 1998; Structural characteristics of halobacterial gas vesicles. Microbiology144:1331–1342[CrossRef]
    [Google Scholar]
  25. Palmer B., Marinus M. 1994; The dam and dcm strains of Escherichia coli – a review. Gene143:1–12[CrossRef]
    [Google Scholar]
  26. Patenge N., Haase A., Bolhuis H., Oesterhelt D. 2000; The gene for a halophilic β-galactosidase ( bgaH ) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum . Mol Microbiol36:102–113
    [Google Scholar]
  27. Pfeifer F., Blaseio U. 1989; Insertion elements and deletion formation in a halophilic archaebacterium. J Bacteriol171:5135–5140
    [Google Scholar]
  28. Pfeifer F., Ghahraman P. 1993; Plasmid pHH1 of Halobacterium salinarium : characterization of the replicon region, the gas-vesicle gene cluster and insertion elements. Mol Gen Genet238:193–200
    [Google Scholar]
  29. Pfeifer F., Offner S., Englert C, Krüger K., Ghahraman P.. 1994; Transformation of halophilic archaea and investigation of gas-vesicle synthesis. Syst Appl Microbiol16:569–577
    [Google Scholar]
  30. Pfeifer F., Zotzel J., Kurenbach B., Röder R., Zimmermann P. 2001; A p-loop motif and two basic regions in the regulatory protein GvpD are important for the repression of gas vesicle formation in the archaeon Haloferax mediterranei . Microbiology147:63–73
    [Google Scholar]
  31. Qureshi S., Jackson S. 1998; Sequence-specific DNA binding by the S. shibatae TFIIB homolog, TFB, and its effect on promoter strength. Mol Cell1:389–400[CrossRef]
    [Google Scholar]
  32. Qureshi S., Baumann P., Rowlands T., Khoo B., Jackson S. 1995; Cloning and functional analysis of the TATA binding protein from Sulfolobus shibatae. Nucleic Acids Res23:1775–1781[CrossRef]
    [Google Scholar]
  33. Reeve J. N., Sandman K., Daniels C. 1997; Archaeal histones, nucleosomes, and transcription initiation. Cell89:999–1002[CrossRef]
    [Google Scholar]
  34. Röder R., Pfeifer F. 1996; Influence of salt on the transcription of the gas-vesicle genes of Haloferax mediterranei and identification of the endogenous transcriptional activator gene. Microbiology142:1715–1723[CrossRef]
    [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T. 1989; Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. Thomm M. 1996; Archaeal transcription factors and their role in transcription initiation. FEMS Microbiol Rev18:159–171[CrossRef]
    [Google Scholar]
  37. Thompson D. K., Palmer J. R., Daniels C. J. 1999; Expression and heat-responsive regulation of a TFIIB homologue from the archaeon Haloferax volcanii. Mol Microbiol33:1081–1092[CrossRef]
    [Google Scholar]
  38. Zillig W., Palm P., Klenk H.-P., Langer D., Hüdepohl U., Hain J., Lanzendörfer M., Holz I.. 1993; Transcription in archaea. In The Biochemistry of Archaea pp367–391 Edited by Kates M.. Amsterdam: Elsevier Science;
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-7-1745
Loading
/content/journal/micro/10.1099/00221287-147-7-1745
Loading

Data & Media loading...

Most cited this month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error