Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene.
A 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.
Large
Andrew
A
School of Biosciences, The University of Birmingham, Edgbaston B15 2TT, UK.
Stamme
Claudia
C
Lange
Christian
C
Duan
Zhenhong
Z
Allers
Thorsten
T
Soppa
Jörg
J
Lund
Peter A
PA
D019843:Archaeal Proteins / Q000235:genetics*; D020869:Gene Expression Profiling; D019848:Gene Expression Regulation, Archaeal; D020043:Genes, Essential; D017930:Genes, Reporter; D005822:Genetic Vectors; D019622:Haloferax volcanii / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D018832:Molecular Chaperones / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D019642:RNA, Archaeal / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014364:Tryptophan / Q000378:metabolism; D014368:Tryptophanase / Q000235:genetics
D019843:Archaeal Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D019642:RNA, Archaeal; D012333:RNA, Messenger; D014364:Tryptophan; D014368:Tryptophanase