Strains and growth conditions
The ascomycete H. jecorina (T. reesei) QM9414 (ATCC 26921; a cellulase hyper-producing mutant derived from the wild-type strain QM6a ) was used throughout this study and maintained on malt agar. For replacement experiments, mycelia were pre-cultured in 1-L-Erlenmeyer flasks on a rotary shaker (250 rpm) at 30°C for 18 h in 250 mL of Mandels-Andreotti (MA) medium  supplemented with 1% (w/v) glycerol as a sole carbon source. 108 conidia per litre (final concentration) were used as the inoculum. Pre-grown mycelia were washed and then equal amounts were resuspended in MA media containing 1% (w/v) glucose, glycerol, xylose or xylan as the sole carbon source or supplemented with 1.5 mM sophorose or xylobiose or without any carbon source.
E. coli JM109 (Promega, Wisconsin, US) was used for the propagation of plasmid vectors, and the strain BL21-Gold (Stratagene, La Jolla, CA) was used as the host for the production of GST (Glutathion S-transferase) fusion proteins.
Electrophoretic mobility shift assay (EMSA)
Radioactive EMSA synthetic oligonucleotides (VBC, Vienna, Austria) (Table 1) were used. After annealing, double stranded oligonucleotides were end-labelled with (*-32P)-dCTP using Klenow Polymerase (Promega) and purified with non-denaturing PAGE (polyacrylamide gel electrophoresis). The binding assays and PAGE experiments were performed essentially as described in . Binding was achieved by incubating 100 μg of cell-free extract protein with 5 ng of labelled fragment for 15 min on ice. Cell-free extracts were prepared as described previously .
For fluorescent EMSA, the synthetic FAM-labelled oligonucleotides (MWG Biotech, Ebersberg, Germany) were annealed with their complementary oligonucleotides (Table 1) by cooking them in 200 mM Tris/HCl (pH 7.5) for 5 min and then letting them cool slowly to room temperature. The binding assay and PAGE experiments were performed as described previously . Binding was achieved by incubating 100 ng of the GST fusion proteins or 60 ng of in vitro translated, unlabelled 2488prp (putative repressor protein) with 15 ng of labelled fragment for 15 min on ice. In vitro translation of the complete 2488prp protein was performed using the TNT Quick Coupled Transcription/Translation System (Promega) according to the manufacturer's instructions starting from the plasmid pMPF2488, which inserts the 2488prp structural gene into the vector pTNT (Promega). To verify the completion of the translation process by a SDS-PAGE, both 2488prp and luciferase (a control template DNA provided by Promega) were labelled using FluoroTect™Green (Promega). Gels (EMSA and SDS-PAGE) were analysed using a Typhoon 8600 variable mode imager (Amersham Bioscience, part of GE Healthcare, CT, US).
RNA-extraction, reverse transcription, transcript analyses, quantitative PCR (qPCR)
Harvested mycelia were homogenized in 1 mL peqGOLD TriFast DNA/RNA/protein purification system (PEQLAB Biotechnologie, Erlangen, Germany) using a FastPrep FP120 BIO101 ThermoSavant cell disrupter (Qbiogene, Carlsbad, US). RNA was isolated according to the manufacturer's instructions.
The synthesis of cDNA from mRNA was achieved with the RevertAid™H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany).
All PCRs for checking transcription were performed in an iCycler iQ, Real-Time Detection System (Bio-Rad, Hercules, US). The reactions were performed in a 25 μl volume containing 1 × buffer (Promega), 2.5 mM MgCl2, 0.1 μM forward primer, 0.1 μM reverse primer, 0.25 U Taq-polymerase (Go-taq, Promega), and a mixture of glucose and xylose-derived cDNAs (10-fold diluted) as template. The primer sequences (transkr2488f/r, transkr3151f/r, transkr3151f/r, transkr7236f/r) are given in Table 1. The PCR run included a blank (sterile, bi-distilled water instead of sample). The following PCR protocol was used: 3 min initial denaturation at 95°C, followed by 30 cycles of 15 s at 95°C, 15 s at 59°C, and 20 s at 72°C.
All qPCRs were performed in a Mastercycler® ep realplex2 (Eppendorf, Hamburg, Germany). The software realplex 2.2 was used to compile PCR protocols and define plate set-ups. All reactions were performed in triplicate. To analyze 2488prp transcription, a SYBR Green assay with an actin reference was performed using 1 × iQ SYBR Green Supermix (Bio-Rad), 0.1 μM forward primer, 0.1 μM reverse primer, and cDNA as template. Primer pairs are given in Table 1. The following PCR program was used: 3 min initial denaturation at 95°C, followed by 45 cycles of 15 s at 95°C, 15 s at 59°C and 15 s at 72°C. The data are expressed relative to the transcription of the actin gene. The data in the figures are means of three independent experiments. Error bars indicate standard deviations. These amounts always refer to one reference sample within an experiment, which is marked in the figure with an asterisk.
Isolation of DNA-binding proteins with streptavidin affinity chromatography
Biotinylated oligonucleotides (VBC, Vienna, Austria) (Bxyn2p250f/r, see Table 1) were annealed (95°C, 5 min followed by a slow cool to 35°C and then ice) and incubated with 200 μL μMACS Streptavidin Micro Beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) on ice for 15 min. Afterwards, 800 μL of binding mix (200 mM KCl, 200 ng/μL poly(dIdC), 200 ng/μL CKT067/CKT068, 8 mM Spermidine) and then 880 μL of cell-free extract protein were added and incubated on ice for 15 min first and then for 10 min. CKT067/CKT068 is a double-stranded DNA fragment used to titrate proteins that nonspecifically interact with DNA. This fragment and poly(dIdC) is used in a ten-fold excess of the specific probe . Cell-free extracts were prepared as described previously . Magnetic separation was performed according to the manufacturer's instructions after equilibration and subsequent saturation with CKT067/068 was achieved. Elution was performed with 150 μL of cold (room temperature) SDS loading buffer followed by 150 μL of hot (60 to 70°C) SDS loading buffer. The eluate was placed on ice immediately.
Nano-HPLC-separation and identification of target proteins
Prior to MS analysis, reversed phase (RP) nano-HPLC separation of the eluted peptide mixtures from an in gel digest was performed. An UltiMate™HPLC System equipped with a FAMOS autosampler, Switchos and UV detector (all Dionex, Sunnyvale, CA, US) was used. After concentration and desalting on a trapping column (Acclaim PepMap100 C18, 5 μm, 100 Å, 300 μm i.d. × 5 mm, Dionex), peptides were separated on a PepMap 100 (C18) nanocolumn (Acclaim PepMap100 C18, 3 μm, 100 Å 75 μm i.d. × 15 cm, Dionex) at a flow-rate of 275 nL/min. Peptides were eluted with a linear gradient from 0% to 50% B in 30 min formed by mixing the two solvents A (5% ACN, 0.1% FA) and B (80% ACN, 0.08% FA), followed by a high organic wash (4 min at 90% B). The quality of separation was monitored by UV absorption at 214 nm.
The outlet of the nano-HPLC system was directly coupled to a Thermo™LTQ linear ion trap mass spectrometer (Thermo Electron Corp., Waltham, MA, US). Mass spectra were acquired in positive ionization mode. The applied method consisted of seven scans; the first was used to determine the precursor ions that were investigated in the following tandem mass scans by CAD (collisionally activated dissociation) fragmentation.
Tandem mass spectra were extracted by extract-msn (Thermo). Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.2.04). Mascot was set up to search the treeseiV2_FrozenGeneCatalog20081022.proteins.fasta.gz database (unknown version, 9143 entries), using predictions for a trypsin digest. Mascot was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 1.5 Da. The iodoacetamide derivative of cysteine was specified in Mascot as a fixed modification. S-carbamoylmethylcysteine cyclization of the N-terminus and methionine oxidation were specified in Mascot as variable modifications.
Criteria for protein identification
Scaffold (version Scaffold-01_07_00, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm . Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least three identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm . Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
Vectors for expressing the DNA-binding domains (DBD) as GST fusion proteins were constructed as follows. PCR was used to amplify the DBD coding regions using 25 μl reaction-mixtures containing 1 × buffer with MgCl2 (Fermentas), 0.1 μM forward primer, 0.1 μM reverse primer, 0.5 U High Fidelity DNA Polymerase (Fermentas), and genomic DNA as the template. Primer sequences (Exp2488F/R, Exp3151F/R, and Exp7236F/R) are given in Table 1. The following PCR protocol was followed: 3 min initial denaturation at 95°C, followed by 30 cycles of 15 s at 95°C, 15 s at 59°C, and 15 s at 72°C. Derived amplicons (165 bp, 219 bp, and 258 bp) were inserted into plasmid pGEX-4T-2 (Amersham) via the restriction enzymes BamHI and XhoI to obtain p2488DBD, p3151DBD, and p7236DBD, respectively.
The plasmid for expressing the 2488prp full length protein as an in vitro translation product was constructed as follows. PCR was used to amplify full length cDNA as described above, but in this case, the template was a mixture of cDNAs obtained under repressing conditions (glucose). The primer sequences (fltn2488f/r) are given in Table 1. The PCR protocol was as follows: 3 min initial denaturation at 95°C, followed by 30 cycles of 15 s at 95°C, 15 s at 59°C, and 2 min at 72°C. The derived amplicon (1,479 bp) was inserted into plasmid pTNT (Promega) via the restriction enzyme KpnI and NotI to obtain pMPF2488.
Purification of the DBDs of 2488prp, 3151prp, and 7236prp as GST fusion proteins
All heterologously expressed proteins used in this study were produced as GST fusions with the pGEX system (Amersham), following the manufacturer's guidelines and using the plasmids p2488DBD, p3151DBD, and p7236DBD as templates. Expression products purified via glutathione-sepharose-columns (Amersham) were verified by SDS-PAGE (using the Mini-PROTEAN System, Bio-Rad) followed by SYPRO Ruby (Bio-Rad) staining. Thrombin cleavage was performed for 1 h at 37°C with 1 U of thrombin (Amersham), 150 mM NaCl, 2.5 mM CaCl2, and 25 mM Tris/HCl (pH 8) plus 20% Glycerin to remove the GST moiety.