Genome-wide binding analysis of the transcriptional regulator TrmBL1 in Pyrococcus furiosus

Abstract

BackgroundSeveral in vitro studies document the function of the transcriptional regulator TrmBL1 of Pyrococcus furiosus. These data indicate that the protein can act as repressor or activator and is mainly involved in transcriptional control of sugar uptake and in the switch between glycolysis and gluconeogenesis. The aim of this study was to complement the in vitro data with an in vivo analysis using ChIP-seq to explore the genome-wide binding profile of TrmBL1 under glycolytic and gluconeogenic growth conditions.ResultsThe ChIP-seq analysis revealed under gluconeogenic growth conditions 28 TrmBL1 binding sites where the TGM is located upstream of coding regions and no binding sites under glycolytic conditions. The experimental confirmation of the binding sites using qPCR, EMSA, DNase I footprinting and in vitro transcription experiments validated the in vivo identified TrmBL1 binding sites. Furthermore, this study provides evidence that TrmBL1 is also involved in transcriptional regulation of additional cellular processes e.g. amino acid metabolism, transcriptional control or metabolic pathways. In the initial setup we were interested to include the binding analysis of TrmB, an additional member of the TrmB family, but western blot experiments and the ChIP-seq data indicated that the corresponding gene is deleted in our Pyrococcus strain. A detailed analysis of a new type strain demonstrated that a 16 kb fragment containing the trmb gene is almost completely deleted after the first re-cultivation.ConclusionsThe identified binding sites in the P. furiosus genome classified TrmBL1 as a more global regulator as hitherto known. Furthermore, the high resolution of the mapped binding positions enabled reliable predictions, if TrmBL1 activates (binding site upstream of the promoter) or represses transcription (binding site downstream) of the corresponding genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2360-0) contains supplementary material, which is available to authorized users.

CopR, a global regulator of transcription to maintain copper homeostasis inPyrococcus furiosus

“…Immunoprecipitation: We used an adaption of a ChIP-seq protocol that was established previously for P. furiosus (48). P. furiosus cells were grown under anaerobic conditions in serum bottles containing 40 ml ½ SME medium at 95°C as described earlier.…”

Conserved principles of transcriptional networks controlling metabolic flexibility in archaea

“…(B) An example TMnet from archaea. When glucose is added to the medium, the TF TrmB is released from DNA binding, de-activating genes encoding enzymes in gluconeogenesis and de-repressing glycolytic genes [10][11][12][13]. Given that archaeal subnetworks are less understood, here we explore the conservation of the topological and dynamical properties of such TMnets across bacteria and archaea.…”

“…In archaea, the TrmB TF is a widely conserved regulator of central metabolic pathways [41]. In many species of euryarchaea, TrmB activates gluconeogenic pathways and represses glycolytic pathways [11,12,42]. Recent metabolic modeling suggests that the TrmB TMnet functions as a metabolic switch between gluconeogenesis and glycolysis in Halobacterium salinarum [13] ( Figure 1B).…”

“…The global regulons governed by the TrmB family of TFs have been characterized across several species [11,12,68]. These TFs typically regulate gene-encoding enzymes in glycolysis and gluconeogenesis [41].…”

A Global Characterisation of the Archaeal Transcription Machinery

“…at work, including gene-specific regulators. The molecular mechanisms of several archaeal metabolic and stress response regulators have been elucidated in vitro, and their regulons characterised by ChIP-chip and ChIP-seq methods(Liu et al 2016;Nguyen-Duc et al 2013;Reichelt et al 2016;Rudrappa et al 2015;Tonner et al 2015;Wilbanks et al 2012). Archaeal regulators operate by a range of different mechanisms including repression by promoter occlusion and activation by enhancing the recruitment of the PIC; the mode of action of the same factor can depend on the location of the binding site relative to the promoter(Aravind and Koonin 1999;Charoensawan et al 2010;Dahlke and Thomm 2002;Geiduschek and Ouhammouch 2005;Kanai et al 2007;Lee et al 2008;Lipscomb et al 2009;Ochs et al 2012;Peeters et al 2013;Peeters et al 2015; Perez-Rueda and Janga 2010).…”