Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection

Abstract

The sequence and spacing requirements of the archaeal "distal promoter element' (DPE) were examined by randomizing positions -19 to -32 upstream of the transcriptional start site of the ferredoxin (fdx) promoter of Halobacterium salinarium. This randomized promoter library containing 4(14) entries was cloned in front of the dihydrofolate reductase (DHFR) reporter gene and transformed into Haloferax volcanii. Two approaches were used to characterize these synthetic promoters. First, 1040 independent clones were randomly chosen and their degrees of trimethoprim resistance were determined. The sequences of 20 clones that were either sensitive, partially resistant or very resistant, respectively, were determined. Secondly, the transformed library was screened by direct selection for high-activity promoters by growing transformants in the presence of trimethoprim. Both approaches produced the following consensus sequence for a halobacterial promoter: (Formula: see text) (where R = A or G; Y = C or T; W = A or T; S = G or C; N = A, C, G or T). Further characterization of two sensitive, two partially resistant, and two very resistant clones verified that DHFR activity and cell phenotype are directly correlated. Sensitive clones did not contain detectable dhfr mRNA, whereas partially resistant clones contained a 700 nucleotide (nt)-long transcript, and very resistant clones contained both the 700nt-long transcript and a second, more abundant, 500nt-long truncated transcript. Quantification of the dhfr mRNA and DHFR enzyme activity suggests that the 3'-untranslated region of the dhfr transcript, missing from the shorter transcript, functions as a negative regulator of translation.

Post-translational Secretion of Fusion Proteins in the Halophilic Archaea Haloferax volcanii

“…Thus, the pulsechase radiolabeling studies support the view that post-translational translocation of the SP-bearing CBD occurs in the transformed H. volcanii cells. The cellular fraction also contained a substantial amount of SP-CBD-derived breakdown products, in agreement with earlier studies addressing the expression of fusion proteins by H. volcanii (29,32).…”

“…This scenario is, however, unlikely due to several reasons. The plasmid employed for transformation of H. volcanii cells includes the non-inducible constitutive PrR16 promotor (29,32). Accordingly, no differences were noted in the growth rates or cell yields of the background and recombinant strains, effects that would be expected upon massive protein overproduction.…”

SSoNΔ and SsoNΔlong: two thermostable esterases from the same ORF in the archaeon Sulfolobus solfataricus?

“…These automatic methods of gene detection are based on the statistical relevance of the critical constraints that define a gene, start codon and translational signals, and were originally developed for the analysis of bacterial genomes. A recent comparative analysis of the available archaeal genomes has confirmed the presence of specific translational signals (Torarinsson et al 2005); in particular the BRE motif constituted by two to four A/Ts (Bell et al, 1999) at position -32, which follows an AT-rich peak between positions -29 and -23, called the Box A motif (Hain et al 1992, Palmer and Daniels 1995, Danner and Soppa 1996. These signals are prevalent in leaderless transcripts, which mostly correspond to unique genes or to the first gene of an operon (Type 1 genes).…”

The transcriptional activator GvpE for the halobacterial gas vesicle genes resembles a basic region leucine-zipper regulatory protein

“…In the case of halophilic archaea, the molecular tools for the investigation of gene functions are well developed. Several vector plasmids, including expression vectors, and a transformation system are available and have been used for the study of promoters and gene expression in halobacteria (Gropp et al, 1995;Pfeifer et al, 1994;Palmer & Daniels, 1995;Danner & Soppa, 1996;Ro È der & Pfeifer, 1996).…”