Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA.
BACKGROUND
Archaea are prokaryotic organisms with simplified versions of eukaryotic transcription systems. Genes coding for the general transcription factors TBP and TFB are present in multiple copies in several Archaea, including Halobacterium sp. NRC-1. Multiple TBP and TFBs have been proposed to participate in transcription of genes via recognition and recruitment of RNA polymerase to different classes of promoters.


RESULTS
We attempted to knock out all six TBP and seven TFB genes in Halobacterium sp. NRC-1 using the ura3-based gene deletion system. Knockouts were obtained for six out of thirteen genes, tbpCDF and tfbACG, indicating that they are not essential for cell viability under standard conditions. Screening of a population of 1,000 candidate mutants showed that genes which did not yield mutants contained less that 0.1% knockouts, strongly suggesting that they are essential. The transcriptomes of two mutants, Delta tbpD and DeltatfbA, were compared to the parental strain and showed coordinate down regulation of many genes. Over 500 out of 2,677 total genes were regulated in the Delta tbpD and DeltatfbA mutants with 363 regulated in both, indicating that over 10% of genes in both strains require the action of both TbpD and TfbA for normal transcription. Culturing studies on the Delta tbpD and DeltatfbA mutant strains showed them to grow more slowly than the wild-type at an elevated temperature, 49 degrees C, and they showed reduced viability at 56 degrees C, suggesting TbpD and TfbA are involved in the heat shock response. Alignment of TBP and TFB protein sequences suggested the expansion of the TBP gene family, especially in Halobacterium sp. NRC-1, and TFB gene family in representatives of five different genera of haloarchaea in which genome sequences are available.


CONCLUSIONS
Six of thirteen TBP and TFB genes of Halobacterium sp. NRC-1 are non-essential under standard growth conditions. TbpD and TfbA coordinate the expression of over 10% of the genes in the NRC-1 genome. The Delta tbpD and DeltatfbA mutant strains are temperature sensitive, possibly as a result of down regulation of heat shock genes. Sequence alignments suggest the existence of several families of TBP and TFB transcription factors in Halobacterium which may function in transcription of different classes of genes.
Coker
James A
JA
University of Maryland Biotechnology Institute, Center of Marine Biotechnology, 701 East Pratt Street, Baltimore, MD 21202, USA. coker@umbi.umd.edu
DasSarma
Shiladitya
S
D019843:Archaeal Proteins / Q000235:genetics*; D019848:Gene Expression Regulation, Archaeal; D019766:Genes, Archaeal; D006217:Halobacterium / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010802:Phylogeny; D035181:TATA-Box Binding Protein / Q000235:genetics*; D035581:Transcription Factor TFIIB / Q000235:genetics*; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; C106466:transcription factor B, Archaea