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

Abstract

Background: Several 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.

Results: The 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.

Conclusions: The 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.

Fig. 1

Western blot analysis using anti-TrmBL1 IgG and anti-TrmB IgG. 100 ng each recombinant TrmB (lane 1), TrmBL1 (lane 2) and TrmBL2 (lane 3) were used and 20 μg crude extract obtained from P. furiosus cells, which were grown under gluconeogenic (pyruvate, lane 4) or glycolytic (starch, lane 5) conditions. a using purified antibodies raised against recombinant TrmBL1 (anti-TrmBL1 IgG, 1:2000) for detection. b using purified antibodies raised against recombinant TrmB (anti-TrmB IgG, 1:2000) for detection

Fig. 2

TrmBL1 binds to the genome under gluconeogenic growth conditions. TrmBL1 ChIP-seq experiments with P. furiosus cells grown under gluconeogenic (pyruvate 1) and glycolytic (starch 1) conditions were done and mapped TrmBL1 immunoprecipitation (IP 1) and input reads were visualized for the whole genome of P. furiosus using the IGV genome browser [67]. Prominent peaks found under gluconeogenic conditions are announced

Fig. 3

ChIP-qPCR validation of selected TrmBL1 binding sites identified by ChIP-Seq. ChIP enrichment is presented as % input. The mean with SD of at least three replicates of IP is shown for all analysed genomic loci. a TrmBL1 ChIP of cells grown under gluconeogenic condition (pyruvate 1). The genes PF1882 (aaa + atpase) and PF1602 (gdh) represent the negative controls for the anti-TrmBL1 IgG. b TrmBL1 ChIP of cells grown under glycolytic growth condition (starch 1). The genes PF1882 (aaa + atpase) and PF1602 (gdh) represent the negative controls for the anti-TrmBL1 IgG. c ChIP with a Phr specific antibody using cells grown on pyruvate (gluconeogenic conditions). The gene PF1882 (aaa + atpase; [29]) is the positive control for the anti-Phr IgG, whereas the genes PF1784 (pfk) and PF1602 (gdh) are the negative controls for the anti-Phr IgG. d ChIP with a Phr specific antibody using cells grown on starch (glycolytic conditions). The gene PF1882 (aaa + atpase) is the positive control for the anti-Phr IgG, whereas the genes PF1784 (pfk) and PF1602 (gdh) are the negative controls for the anti-Phr IgG

Fig. 4

TrmBL1 preferentially binds to TGM containing regions in the P. furiosus genome. a Sequence logo (T [TG] [TC] A [TC] CAC [CT] [ATC] [AG] [CA] [AG] [GA] TGA [TA] [AT]); E-value = 1.1E-024) of the TGM based on 34 TrmBL1 binding sites identified by ChIP-seq and De-novo motif discovery using MEME. b Central motif enrichment of the TGM in the detected TrmBL1 binding sites (E –value = 1.9E-15; region width = 54) analysed by Centrimo

Fig. 5

TrmBL1 DNase I footprint regions enclose the TGM. DNaseI footprints were done in the presence (+) or absence (−) of TrmBL1 using various identified binding sites. DNA ladders and orientations of the used template strands (transcribed or non-transcribed) are marked by 5’ and 3’. TrmBL1 footprint regions are written in bold letters and the TGMs are highlighted as grey box. Translational start sites, if present, are shown as black arrow and positions of the BREs and TATA-boxes are announced

Fig. 6

TrmBL1 functions as transcriptional repressor and activator. In vitro transcription assays were done using DNA templates containing the TGM downstream or upstream of the promoter (A -I). The concentrations of TrmBL1 were indicated on top of each lane. Template PF1602 contains the gdh promoter without TGM as a negative control (J).

Fig. 7

Deletion of a 16 kb fragment encoding the TM system and TrmB. a southern blot analysis using BamHI and SmaI digested P. furiosus genomic DNA. DNA was isolated from cells after recultivation and adaption to glycolytic (starch) or gluconeogenic (pyruvate) growth conditions. The result of two independent recultivations is shown. The probe, specific for PF1753, detects a 3 kb fragment, while the PF1743 specific probe binds to a 7 kb fragment. b copy number analysis of the genes PF1743 and PF1753 in P. furiosus cells grown under the following conditions: glycolytic (starch and maltose) or gluconeogenic (pyruvate). The gene PF1784 was used as calibrator and the recultivation culture as reference. The mean of three biological replicates including SD is shown

Fig. 8

TrmBL1 controls expression of genes involved in functionally-linked metabolic pathways by its own or in concert with secondary regulators. TrmBL1 mediated repression or activation is depicted in red or blue. Complete colouring indicates validation of repression or activation by cell-free transcription. Banded colouring indicates suggested transcriptional effects. Genes differentially expressed in a microarray analysis of P. furiosus grown on maltose or peptides are underlined [54]. Gene products: PF0132, proposed α-glucosidase; PF0196, phosphoglucose isomerase; PF0272, proposed 4-α-glucanotransferase; PF0287, pyrolysin; PF0464, glycerinaldehyde 3-phosphate:ferredoxin oxidoreductase; PF0477, proposed extracellular α-amylase. PF0478, proposed extracellular cyclomaltodextrin glucano-transferase; PF0505, putative DNA binding protein; PF0588, phosphoglucose mutase; PF0853, 5'-methylthioadenosine phosphorylase; PF0874, membrane dipeptidase; PF0967-PF0965, pyruvate ferredoxin oxidoreductase; PF1109/1110, extracellular starch-binding protein; PF1350, proposed single-component transport protein; PF1476, putative PadR-like regulator; PF1535, maltodextrin phosphorylase; PF1539, dihydroorotate dehydrogenase 1b; PF1784, phosphofructokinase; PF1874 glycerinaldehyde 3-phosphate dehydroghenase; PF1920, triosephosphate isomerase; PF1935, amylo-pullulanase; PF1938-PF1933 maltodextrin-specific ABC transporter (MD-system); PF1956 phosphoglycerate mutase; PF1959, fructose-1,6-bisphosphate aldolase; PF2016, preprotein translocase subunit SecG; PF2047, L-asparaginase. AA; amino acid