ΔD transformants containing all 14 genes of required for gas vesicle formation except for are gas vesicle overproducers (Vac), whereas ΔD/D transformants containing the reading frame under ferredoxin promoter control on a second construct in addition to ΔD did not form gas vesicles (Vac). The amino acid sequence of GvpD indicates three interesting regions (a putative nucleotide-binding site called the p-loop motif, and two basic regions); these were altered by mutation, and the resulting GvpD proteins tested in ΔD/D transformants for their ability to repress gas vesicle formation. The exchange of amino acids at conserved positions in the p-loop motif resulted in Vac ΔD/D transformants, indicating that these GvpD proteins were unable to repress gas vesicle formation. In contrast, a GvpD protein with an alteration of a non-conserved proline in the p-loop region (P41A) was still able to repress. The repressing function of the various GvpD proteins was also investigated at the promoter level of the gene. This promoter is only activated during the stationary phase, depending on the transcriptional activator protein GvpE. Whereas the Vac ΔD transformants contained very high amounts of mRNA predominantly in the stationary growth phase, the amount of this transcript was significantly reduced in the Vac transformants ΔD/D and ΔD/D. In contrast, the Vac ΔD/D transformants harbouring GvpD with mutations at conserved positions in the p-loop motif contained large amounts of mRNA already during exponential growth, suggesting that this motif is important for the GvpD repressor function during this growth phase. The GvpD mutants containing mutations in the two basic regions were mostly defective in the repressing function. The GvpD protein containing an exchange of the three arginine residues 494RRR496 to alanine residues was able to repress gas vesicle formation. No mRNA was detectable in this transformant, demonstrating that this GvpD protein was acting as a strong repressor. All these results imply that the GvpD protein is able to prevent the GvpE-mediated promoter activation, and that the p-loop motif as well as the two basic regions are important for this function.


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  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 and Wiley-Interscience.
  2. Chomczynski, P. & Sacchi, N.(1987). Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156-159. [Google Scholar]
  3. Cohen-Kupiec, R., Blank, C. & Leigh, J. A.(1997). Transcriptional regulation in Archaea: in vivo demonstration of a repressor binding site in a methanogen. Proc Natl Acad Sci USA 94, 1316-1320.[CrossRef] [Google Scholar]
  4. 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 Bacteriol 176, 7646-7652. [Google Scholar]
  5. Deyrup, A., Krishnan, S., Cockburn, B. N. & Schwatz, N. B.(1998). Deletion and site-directed mutagenesis of the ATP-binding motif (p-loop) in the bifunctional murine ATP-sulfurylase/adenosine 5′-phosphosulfate kinase enzyme. J Bacteriol 273, 9450-9456. [Google Scholar]
  6. 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 a helices: crystal structure of the protein–DNA complex. Cell 71, 1223-1237.[CrossRef] [Google Scholar]
  7. Englert, C. & Pfeifer, F.(1993). Analysis of gas vesicle gene expression in Haloferax mediterranei reveals that GvpA and GvpC are both gas vesicle structural proteins. J Biol Chem 268, 9329-9336. [Google Scholar]
  8. 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 Genet 222, 225-232.[CrossRef] [Google Scholar]
  9. Englert, C., Krüger, K., Offner, S. & Pfeifer, F.(1992a). Three different but related gene clusters encoding gas vesicles in halophilic archaea. J Mol Biol 227, 586-592.[CrossRef] [Google Scholar]
  10. 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 Microbiol 6, 3543-3550.[CrossRef] [Google Scholar]
  11. Halladay, J., Jones, J., Lin, F., MacDonald, F. & DasSarma, S.(1993). The rightward gas-vesicle operon in Halobacterium plasmid pNRC100: identification of the gvpA and gvpC gene products by use of antibody probes and genetic analysis of the region downstream of gvpC. J Bacteriol 175, 684-692. [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 Chem 27, 30144-30148. [Google Scholar]
  13. Hayes, P. K., Buchholz, B. & Walsby, A. E.(1992). Gas vesicles are strengthened by the outer-surface protein, GvpC. Arch Microbiol 157, 229-234.[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 Bacteriol 12, 3807-3813. [Google Scholar]
  15. Jones, J. G., Young, D. C. & DasSarma, S.(1991). Structure and organization of the gas-vesicle gene cluster on the Halobacterium halobium plasmid pNRC100. Gene 102, 117-122.[CrossRef] [Google Scholar]
  16. Kinsman, R., Walsby, A. E. & Hayes, P. K.(1995). GvpCs with reduced numbers of repeating sequence elements bind to and strengthen cyanobacterial gas vesicles. Mol Microbiol 17, 147-154.[CrossRef] [Google Scholar]
  17. Konola, J. T., Logan, K. M. & Knight, K. L.(1994). Functional characterization of residues in the p-loop motif of the RecA protein ATP binding site. J Mol Biol 237, 20-34.[CrossRef] [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. Lagrange, T., Kapanidis, A., Tang, H., Reinberg, D. & Ebright, R.(1998). New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB. Genes Dev 12, 34-44.[CrossRef] [Google Scholar]
  20. Lam, W. L. & Doolittle, W. F.(1989). Shuttle vectors for the archaebacterium Halobacterium volcanii. Proc Natl Acad Sci USA 86, 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 Res 8, 1131–1141. [Google Scholar]
  22. Offner, S. & Pfeifer, F.(1995). Complementation studies with the gas vesicle-encoding p-vac region of Halobacterium salinarium PHH1 reveal a regulatory role for the p-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 salinarium reveal that the GvpC protein shapes gas vesicles. J Bacteriol 178, 2071-2078. [Google Scholar]
  24. Offner, S., Hofacker, A., Wanner, G. & Pfeifer, F.(2000). Eight of fourteen gvp genes are sufficient for the formation of gas vesicles in halophilic archaea. J Bacteriol 182, 4328-4336.[CrossRef] [Google Scholar]
  25. Palmer, B. & Marinus, M.(1994). The dam and dcm strains of Escherichia coli – a review. Gene 143, 1-12.[CrossRef] [Google Scholar]
  26. Pfeifer, F. & Englert, C.(1992). Function and biosynthesis of gas vesicles in halophilic archaea. J Bioenerg Biomembr 24, 577-585.[CrossRef] [Google Scholar]
  27. 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 Genet 238, 193-200. [Google Scholar]
  28. Pfeifer, F., Offner, S., Krüger, K., Ghahraman, P. & Englert, C.(1994). Transformation of halophilic archaea and investigation of gas-vesicle synthesis. Syst Appl Microbiol 16, 569-577. [Google Scholar]
  29. 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]
  30. 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 Res 23, 1775-1781.[CrossRef] [Google Scholar]
  31. 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. Microbiology 142, 1715-1723.[CrossRef] [Google Scholar]
  32. Rodriguez-Valera, F., Juez, G. & Kushner, D.(1983).Halobacterium mediterranei spec. nov., a new carbohydrate-utilizing extreme halophile. Syst Appl Microbiol 4, 369-381.[CrossRef] [Google Scholar]
  33. Ruepp, A. & Soppa, J.(1996). Fermentative arginine degradation in Halobacterium salinarum: genes, gene products and transcripts of the arcRACB gene cluster. J Bacteriol 178, 4942-4947. [Google Scholar]
  34. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  35. Saraste, M., Sibbald, P. R. & Wittinghofer, A.(1990). The p-loop – a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci 15, 430-434.[CrossRef] [Google Scholar]
  36. Schägger, H. & von Jagow, G.(1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368-379.[CrossRef] [Google Scholar]
  37. Skovgaard, O., Oleson, K. & Wright, A.(1998). The central lysine in the p-loop motif of Escherichia coli DnaA protein is essential for initiating DNA replication from the chromosomal origin, oriC, and the F-factor origin, oriS, but is dispensable for initiation from the P1-plasmid origin, oriR. Plasmid 40, 91-99.[CrossRef] [Google Scholar]
  38. Smith, C. A. & Rayment, I.(1996). Active site comparisons highlight structural similarities between myosin and other p-loop proteins. Biophys J 70, 1590-1602.[CrossRef] [Google Scholar]
  39. Soppa, J., Link, T. A., Ruepp, A., Vatter, P. & zur Mühlen, A.(1998). Regulation of gene expression in Halobacterium salinarum: the arcRACB gene cluster and the TATA box-binding protein. In Microbiology and Biogeochemistry of Hypersaline Enviroments , pp. 249-263. Edited by A. Oren. Boca Raton, FL:CRC Press.
  40. Thomm, M.(1996). Archaeal transcription factors and their role in transcription initiation. FEMS Microbiol Rev 18, 159-171.[CrossRef] [Google Scholar]
  41. 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 Microbiol 33, 1081-1092.[CrossRef] [Google Scholar]
  42. Walsby, A. E.(1994). Gas vesicles. Microbiol Rev 58, 94-144. [Google Scholar]

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