1887

Microbiology Society logo

Volume 151, Issue 1

analyses of constitutive and regulated promoters in halophilic archaea Free

Abstract

The two promoters and of , and the promoter of were investigated with respect to growth-phase-dependent expression and regulation in transformants using the reading frame encoding BgaH, an enzyme with -galactosidase activity, as reporter. For comparison, the promoter of the ferredoxin gene of and the promoter of (formerly ) were analysed. , driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas and the three promoters yielded the largest activities during the stationary growth phase. Compared to , the basal promoter activities of and were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE. The promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE. To investigate whether the -TATA box and the BRE element were the reason for the lack of the basal activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger promoter and investigated in transformants. All these promoter chimera did not yield a detectable basal promoter activity. However, whenever the -BRE element was substituted for the -BRE, an enhanced cGvpE-mediated activation was observed. The promoter chimeras harbouring -BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins. The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of -BRE. None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of .

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): TBP, TATA box-binding protein
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27541-0
2005-01-01
2024-04-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/1/mic1510025.html?itemId=/content/journal/micro/10.1099/mic.0.27541-0&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 Biology vol 1 New York: Greene Publishing Associates & Wiley-Interscience;
     [Google Scholar]
  2. Baliga 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 Microbiol 36:1184–1185 [CrossRef]
     [Google Scholar]
  3. Bell S. D., Jackson S. 1998; Transcription and translation in archaea: a mosaic of eukaryal and bacterial features. Trends Microbiol 6:222–228 [CrossRef]
     [Google Scholar]
  4. Bell S. D., Cairns S. S., Robson R. L., Jackson S. P. 1999a; Transcriptional regulation of an archaeal operon in vivo and in vitro . Mol Cell 4:971–982 [CrossRef]
     [Google Scholar]
  5. Bell S. D., Kosa P., Sigler P., Jackson S. 1999b; Orientation of the transcription preinitiation complex in archaea. Proc Natl Acad Sci U S A 96:13662–13667 [CrossRef]
     [Google Scholar]
  6. Brinkman A. B., Dahlke I., Tuininga J. 7 other authors 2000; An Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus is negatively autoregulated. J Biol Chem 272:38160–38169
     [Google Scholar]
  7. Danner S., Soppa J. 1996; Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection. Mol Microbiol 19:1265–1276 [CrossRef]
     [Google Scholar]
  8. Englert C., Pfeifer F, Krüger K., Offner S. 1992a; Three different but related gene clusters encoding gas vesicles in halophilic archaea. J Mol Biol 227:586–592 [CrossRef]
     [Google Scholar]
  9. Enoru-Eta J., Gigot D., Thia-Toong T., Glansdorf N., Charlier D. 2000; Purification and characterization of Sa-Lrp, a DNA-binding protein from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius homologous to the bacterial global transcription regulator Lrp. J Bacteriol 182:3661–3672 [CrossRef]
     [Google Scholar]
  10. Gregor D., Pfeifer F. 2001; The use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea. Microbiology 147:1745–1754
     [Google Scholar]
  11. Hochheimer A., Hedderich R., Thauer R. K. 1999; The DNA binding protein Tfx from Methanobacterium thermoautotrophicum : structure, DNA binding properties and transcriptional regulation. Mol Microbiol 31:641–650 [CrossRef]
     [Google Scholar]
  12. Hofacker A., Schmitz K. M., Cichonczyk A., Sartorius-Neef S., Pfeifer F. 2004; GvpE- and GvpD-mediated transcription regulation of the p- gvp genes encoding gas vesicles in Halobacterium salinarum . Microbiology 150:1829–1838 [CrossRef]
     [Google Scholar]
  13. Holmes M. L., Dyall-Smith M. 2000; Sequence and expression of a halobacterial β -galactosidase gene. Mol Microbiol 36:114–122 [CrossRef]
     [Google Scholar]
  14. 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; 1337276–286 [CrossRef]
     [Google Scholar]
  15. Horne M., Englert C., Wimmer C., Pfeifer F. 1991; A DNA region of 9 kbp contains all genes necessary for gas vesicle synthesis in halophilic archaebacteria. Mol Microbiol 5:1159–1174 [CrossRef]
     [Google Scholar]
  16. Kosa P., Ghosh G., DeDecker B., Sigler P. 1997; The 2·1-Å crystal structure of an archaeal preinitiation complex: TATA box-binding protein/transcription factor (II)B core/TATA box. Proc Natl Acad Sci U S A 94:6042–6047 [CrossRef]
     [Google Scholar]
  17. 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 salinarum PHH4. J Bacteriol 178:4012–4019
     [Google Scholar]
  18. 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 Biol 279:761–771 [CrossRef]
     [Google Scholar]
  19. Leonard P. M., Smiths S. H., Sedelnikova S. E., Brinkman A. B., Rice D. W., Rafferty J. B, de Vos W. M., van der Oost J. 2001; Crystal structure of the Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus . EMBO J 20:990–997 [CrossRef]
     [Google Scholar]
  20. Littlefield O., Korkhin Y., Sigler P. 1999; The structural basis for the oriented assembly of a TBP/TFB/promoter complex. Proc Natl Acad Sci U S A 96:13668–13673 [CrossRef]
     [Google Scholar]
  21. Ng W. V., Kennedy S. P., Mahairas G. 40 other authors 2000; Genome sequence of Halobacterium species NRC-1. Proc Natl Acad Sci U S A 97:12176–12181 [CrossRef]
     [Google Scholar]
  22. Offner S., Pfeifer F. 1995; Complementation studies with the gas vesicle-encoding p-vac region of Halobacterium salinarum PHH1 reveal a regulatory role for thep- gvpDE genes. Mol Microbiol 16:9–19 [CrossRef]
     [Google Scholar]
  23. Offner S., Wanner G., Pfeifer F. 1996; Functional studies of the gvpACNO operon of Halobacterium salinarum reveal that the GvpC protein shapes gas vesicles. J Bacteriol 178:2071–2078
     [Google Scholar]
  24. Ouhammouch M., Dewhurst R., Hausner W., Thomm M., Geiduschek E. P. 2003; Activation of archaeal transcription by recruitment of the TATA-binding protein. Proc Natl Acad Sci U S A 100:5097–5102 [CrossRef]
     [Google Scholar]
  25. 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 Microbiol 36:102–113
     [Google Scholar]
  26. Pfeifer F., Ghahraman P. 1993; Plasmid pHH1 of Halobacterium salinarum : characterization of the replicon region, the gas vesicle gene cluster and insertion elements. Mol Gen Genet 238:193–200
     [Google Scholar]
  27. Pfeifer F., Griffig J., Oesterhelt D. 1993; The fdx gene encoding the 2Fe-2S ferredoxin of Halobacterium salinarum ( H. halobium . Mol Gen Genet 239:66–71
     [Google Scholar]
  28. Pfeifer F., Ziesche S., Offner S, Krüger K., Röder R., Mayr M. 1997; Gas vesicle formation in halophilic archaea. Arch Microbiol 167:259–268 [CrossRef]
     [Google Scholar]
  29. 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 . Microbiology 147:63–73
     [Google Scholar]
  30. Plößer P., Pfeifer F. 2002; A bZIP protein from halophilic archaea: structural features and dimer formation of cGvpE from Halobacterium salinarum . Mol Microbiol 45:511–520 [CrossRef]
     [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 Cell 1:389–400 [CrossRef]
     [Google Scholar]
  32. 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. Microbiol 142:1715–1723 [CrossRef]
     [Google Scholar]
  33. Shukla H. D., DasSarma S. 2004; Complexitiy of gas vesicle biogenesis in Halobacterium sp. strain NRC-1: identification of five new proteins. J Bacteriol 186:3182–3186 [CrossRef]
     [Google Scholar]
  34. Soppa J. 1999; Normalized nucleotide frequencies allow the definition of archaeal promoter elements for different archaeal groups and reveal base-specific TFB contacts upstream of the TATA box. Mol Microbiol 31:1589–1601 [CrossRef]
     [Google Scholar]
  35. Zimmermann P., Pfeifer F. 2003; Regulation of gas vesicle formation in Haloferax mediterranei : the two regulatory proteins GvpD and GvpE interact. Mol Microbiol 49:783–794
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27541-0
Loading
In vivo analyses of constitutive and regulated promoters in halophilic archaea
Microbiology 151, 25 (2005); https://doi.org/10.1099/mic.0.27541-0
/content/journal/micro/10.1099/mic.0.27541-0
/content/journal/micro/10.1099/mic.0.27541-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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