Transcript analysis of the c-vac region and differential synthesis of the two regulatory gas vesicle proteins GvpD and GvpE in Halobacterium salinarium PHH4

Abstract

Halobacterium salinarium PHH4 synthesizes gas vesicles in the stationary growth phase by the expression of 14 gvp genes arranged in two clusters. The chromosomal gvpACNO (c-gvpACNO) gene cluster (encoding the major structural gas vesicle protein GvpA and the minor structural protein GvpC) was transcribed as three mRNA species starting at one promoter during the stationary phase of growth. The second gene cluster, c-gvpDEFGHIJKLM, was transcribed during all stages of growth as a relatively unstable, single mRNA with a maximal length of 6 kb. In addition, a 1.7-kb c-gvpD transcript was synthesized during stationary growth starting at the same promotor as that of the c-gvpDEFGHIJKLM mRNA. The expression of the first two genes located in this unit (c-gvpD and c-gvpE) was also monitored by Western blot (immunoblot) analyses using antisera raised against these proteins synthesized in Escherichia coli. While the cGvpD protein was present only during early exponential growth and disappeared during gas vesicle formation, the cGvpE protein was present during cGvpA and gas vesicle synthesis in the early stationary phase of growth. Previous data indicated that cGvpD is involved in repression of gas vesicle formation, whereas cGvpE is a transcriptional activator for the c-gvpA promoter. The appearance of both proteins during the growth cycle is in line with the functions of these proteins in gas vesicle synthesis. The mechanism of the differential translation of cGvpD and cGvpE from the c-gvpDEFGHIJKLM mRNA still has to be elucidated, but antisense RNAs complementary to the 5 terminus as well as the 3 portion of the c-gvpD mRNA might be involved in this regulation. Such RNAs occurred during early stationary growth when the cGvpD protein level decreased and may possibly inhibit the translation of the c-gvpD mRNA.

Expression of multiple tfb genes in different Halobacterium salinarum strains and interaction of TFB with transcriptional activator GvpE

“…7 Binding studies using TFB His or GvpH His and cGvpE. Western analyses were performed with proteins derived from the Ni-NTA aYnity chromatography using an antiserum raised against cGvpE (Krüger and Pfeifer 1996). Ni-NTA matrices loaded with 400 g of the TFB His protein were incubated with 2 mg of soluble proteins of Hfx.…”

“…The faint band at 66 kDa is due to PitA, an endogenous, multiple histidine residues containing protein that always co-puriWes on Ni-NTA matrices (Bab-Dinitz et al 2006). Western analyses using an antiserum raised against cGvpE (Krüger and Pfeifer 1996) detected GvpE in all samples derived from TFB His matrices (Fig. 7, lane 2 each).…”

Gas Vesicles of Archaea and Bacteria

“…the c-gvpDEFGHIJKLM operon, is also transcribed in this strain. However, only cGvpD involved in the repression of gas vesicle formation is detectable, whereas the transcriptional activator cGvpE required for the activation of the P cA promoter is absent (Krüger and Pfeifer 1996). The presence of the other cGvp proteins encoded by this operon has not yet been determined.…”

“…GvpE is a transcriptional activator, whereas GvpD has a role in the repression of gas vesicle formation (Offner and Pfeifer 1995). Most of the gvp promoters are induced by GvpE (Krüger and Pfeifer 1996;Zimmermann and Pfeifer 2003;Hofacker et al 2004). Archaeal promoters consist of a TATA-box centred around position-28 upstream of the transcriptional start site, and the transcription initiation factor TFB, the TATA-box binding protein TBP and a multicomponent RNA polymerase (RNAP) related to the eukaryotic RNAP II are required for transcription initiation (Bell and Jackson 1998).…”

Eight of Fourteen gvp Genes Are Sufficient for Formation of Gas Vesicles in Halophilic Archaea

“…Such transformation experiments showed that (i) the entire p-vac region (construct M-O) leads to gas vesicle formation in H. volcanii (10), and (ii) the p-gvpACNO (A-O) and p-gvpDEF GHIJKLM (D-M) gene clusters present on different vector constructs (or A-O/F-M ϭ ⌬DE) allow the formation of spindle-shaped gas vesicles in H. volcanii transformants (28). The GvpD and GvpE proteins are involved in the regulation of gas vesicle formation: GvpE is a transcriptional activator required for gvpA promoter activity, whereas GvpD is involved in the repression of gas vesicle formation (11,23,24,36).…”