Use of GFP-GvpE fusions to quantify the GvpD-mediated reduction of the transcriptional activator GvpE in haloarchaea
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ORIGINAL PAPER
Use of GFP-GvpE fusions to quantify the GvpD-mediated reduction of the transcriptional activator GvpE in haloarchaea Ina Schmidt · Felicitas Pfeifer
Received: 23 January 2013 / Revised: 5 March 2013 / Accepted: 7 March 2013 / Published online: 16 April 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract Gas vesicle formation of Halobacterium salinarum is regulated by the transcriptional activator GvpE, and in the presence of the repressing protein GvpD, the amount of GvpE is strongly reduced. The green fluorescence protein was used to report this GvpD-mediated reduction of GvpE in vivo in Haloferax volcanii transformants. Both N- or C-terminal fusions of GFP to GvpE were tested, but only the N-terminal fusion reported the reduction. The fluorescence of GFP-GvpE was 62 % reduced with GvpD wild type (DWT), 78 % with the super-repressor D3-AAA, and only 10 % with the repression defect DMut6. Further analysis of D3-AAA indicated that the super-repression was due to the alteration R496A. GFP-GvpE variants defect in promoter activation was tested in the presence of DWT, D3-AAA and DMut6, and two of them were more stable. Overall, the GFPGvpE fusion was suitable to study and quantify the amount of GvpE in vivo. Keywords Gas vesicle regulator · Archaea · Green fluorescent protein · Transcriptional activation
Introduction Gas vesicles are intracellular structures produced by some bacteria and archaea allowing the cells to thrive in their aqueous environment (Walsby 1994; Pfeifer 2012). Fully gas vacuolated cells are able to float towards the surface of the water body. Gas vesicles are hollow protein structures
I. Schmidt · F. Pfeifer (*) Fachbereich Biologie, Mikrobiologie und Archaea, Technische Universität Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt, Germany e-mail: [email protected]
filled by diffusion with gases dissolved in the cytoplasm, and many of them constitute the gas vacuole. The gas vesicle wall is solely constituted of protein with the major structural protein GvpA forming the ribbed wall that is strengthened by a second protein, GvpC, attached to the outside. Fourteen gvp genes arranged in two oppositely oriented gene clusters, gvpACNO and gvpDEFGHIJKLM, are involved in the gas vesicle formation in Halobacterium salinarum or Haloferax mediterranei (Englert et al. 1992a), and the endogenous GvpD and GvpE proteins influence the activity of the two promoters PA and PD. The 21-kDa GvpE acts as transcriptional activator, whereas the 60-kDa GvpD represses gas vesicle formation. The presence of GvpD leads to a significantly reduced amount of GvpE (Englert et al. 1992b; Zimmermann and Pfeifer 2003). The gvp gene expression also depends on environmental factors such as oxygen availability, light, high cell densities or chemical factors, but the signal transductions influencing gvp gene expression are not yet known (Pfeifer 2012). The GvpE-mediated activation of PA and PD has been investigated in Haloferax volcanii transformants harbouring construct pEex (gvpE expressed under
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