Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene

Abstract

SummaryA system where archaeal gene expression could be controlled by simple manipulation of growth conditions would enable the construction of conditional lethal mutants in essential genes, and permit the controlled overproduction of proteins in their native host. As tools for the genetic manipulation of Haloferax volcanii are well developed, we set out to identify promoters with a wide dynamic range of expression in this organism. Tryptophan is the most costly amino acid for the cell to make, so we reasoned that tryptophan-regulated promoters might be good candidates. Microarray analysis of H. volcanii gene expression in the presence and absence of tryptophan identified a tryptophanase gene (tna) that showed strong induction in the presence of tryptophan. qRT-PCR revealed a very fast response and an up to 100-fold induction after tryptophan addition. This result has been confirmed using three independent reporter genes (cct1, pyrE2 and bgaH). Vectors containing this promoter will be very useful for investigating gene function in H. volcanii and potentially in other halophilic archaea. To demonstrate this, we used the promoter to follow the consequences of depletion of the essential chaperonin protein CCT1, and to determine the ability of heterologous CCT proteins to function in H. volcanii.

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor

“…An incubation period of 4 h followed the second induction step. Induction of protein expression using L-tryptophan also induces the expression of the native tryptophanase gene tnaA; hence, H. volcanii is able to metabolise the added L-tryptophan (Large et al 2007 ) and correspondingly approach 6 gave the highest expression yields (Fig. 2 ).…”

“…The corresponding plasmid to this strain is pTA963 with the inducible tryptophanase promoter p.tna, allowing the induction of intracellular protein overexpression by L-tryptophan (Allers 2010 ;Allers et al 2004Allers et al , 2010Bitan-Banin et al 2003 ;Large et al 2007 ). The expression vector is based on the pHV2 origin which maintains the plasmid at a copy number of approximately 6 per genome equivalent (Allers 2010 ).…”

Bioinformatic prediction and experimental verification of sRNAs in the haloarchaeonHaloferax volcanii

“…Initially, the microarray was used to analyze the metabolic changes following a switch from casamino acids to glucose, 34 since then it has been successfully used e.g., to identify the genes involved in xylose metabolism, to discover a tryptophan-inducible promoter, to study translational regulation, and to compare the transcriptomes of deletion mutants with that of the wild-type. 35,39,46,47 The inclusion of a probe in the microarray and the detection of a signal in microarray analyses are listed. * 2 It is shown if the sRNA was detected by hTS and/or northern blot analysis.…”

“…Notably, deletion mutants can be constructed very easily, 32,33 it can be grown in microtiter plates enabling phenotyping of mutants, 31 transcriptome analysis using DNA microarrays has been established, 34,35 proteome analysis is routinely performed, 36,37 and many molecular genetic tools are available. [38][39][40] Here we report the application of two bioinformatic approaches to predict sRNA genes in the genome in H. volcanii by comparative genomics of intergenic regions. In addition, (differential) expression of the predicted putative sRNA genes was characterized by DNA microarray analysis and by comparison with the results of high throughput sequencing performed in a parallel study.…”

Overexpression and purification of halophilic proteins inHaloferax volcanii

“…volcanii, genes under control of the p.tnaA promoter show rapid and strong induction of expression upon addition of ≥1 mM tryptophan. 16 For expression of proteins with an N-terminal hexahistidine (6xHis) tag, a (CAC) 6 tract was incorporated in pTA963 (Fig. 1), and a polylinker ensures that cloning is facile.…”