The transcription activator GvpE of halophilic Archaea and its interaction partners GvpD, TBP and TFB
Zusammenfassung der Projektergebnisse
Halobacterium salinarum strains NRC-1, PHH1 and PHH4 contain multiple TBP and TFB proteins required for the recruitment of RNA polymerase to the promoter. These three strains show differences in the number and expression of these genes, that are presumably the reason for variations in the gas vesicle phenotype when grown under non-standard conditions. Gas vesicle formation depends on the expression of 14 gvp genes arranged in two oppositely oriented gene clusters, gvpACNO and gvpDEFGHUKLM. The oppositely oriented promoters PA and RD drive the expression and differ in promoter strength and activity during growth. Both promoters are activated by the endogenous transcription activator GvpE, and the activation requires a 20-nt E-UAS adjacent to the TFB-binding site BRE. Both E-UAS elements partially overlap in the centre of the intergenic region, and mutations here affect the GvpE-induction of both promoters. Substitution of portions of these E-UAS sequences showed that E-UASA of PA is the stronger element, whereas E-UASD causes the weaker activity of PD. TATA box and/or BRE were exchanged between PA and PD and all elements of PA functioned well in the environment of "PD" and transfered the stronger PA activity to "PD". In contrast, BRED was not functional in the environment of "PA". An additional regulatory element was discovered in the region between TATAD and the transcription start site of gvpD. These results suggested that PD recruits another TFB than PA, which might explain the differences in promoter activities during growth. The regulation of gas vesicle formation also involves the repressing protein GvpD that counteracts GvpE. In the presence of GvpD the amount of GvpE is reduced. This effect was studied in vivo using the fluorescence of a haloadapted GFP protein as reporter. An N-terminal fusion of GFP to GvpE was reduced in amount in the presence of GvpD, and an even stronger reduction was observed in the presence of the super-repressor protein GvpD3-AAA. Several GvpE mutants were were tested using this system to determine regions required for this activity, and also different GvpD mutants were applied. A functional p-loop is required in GvpD, since p-loop mutants were unable to reduce the amount of GFP-GvpE. The basis for the super-repressor phenotype of mutant GvpD3-AAAwas defined; the alteration R494A Is responsible for this phenotype.
Projektbezogene Publikationen (Auswahl)
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(2008) Overlapping activator sequences determined for two oppositely oriented promoters in halophilic Archaea. Nucleic Acids Res 36(2): 598-606
Bauer M, Marschaus L, Reuff M, Besche V, Sartorius-Neef S, Pfeifer F
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(2008) Regulation of gvp genes encoding gas vesicle proteins in halophilic Archaea. Arch Microbiol 190(3): 333-340
Scheuch S, Marschaus L, Sartorius-Neef S, Pfeifer F
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(2008) Variations in the multiple tbp genes in different Halobacterium salinarum strains and their expression during growth. Arch Microbiol 190(3): 309-318
Teufel K, Bleiholder A, Griesbach T, Pfeifer F
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(2010) Interaction of transcription activator GvpE with TATA-box-binding proteins of Halobacterium salinarum. Arch Microbiol 192: 143-149
Teufel K, Pfeifer F
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(2011) Effect of anoxic conditions and temperature on gas vesicle formation in Halobacterium salinarum. In: Halophiles and Hypersaline Environments (Ed. Ventosa A, Oren A, Ma Y), Springer Verlag, Chapter 13, pp 237-248
Pfeifer F, Frommherz R, Faist K, Hechler T, Teufel K, Marschaus L
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(2011) Gasvesikel in Haloarchaea. Biospektrum 01-2011: 17-19
Pfeifer F
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(2012) Expression of multiple tfb genes in different Halobacterium salinarum strains and interaction of TFB with transcriptional activator GvpE. Arch Microbiol 194: 269-279
Bleiholder A, Frommherz R, Teufel K, Pfeifer F
