Journal: Journal of Biological Chemistry

Article Title: Enoate Reductases of Clostridia

doi: 10.1074/jbc.m008656200

Figure Lengend Snippet: FIG. 2. Cloning of the enr gene from C. tyrobutyricum. Oligonu- cleotides used are given in Table II. E, EcoRI restriction site. FIG. 3. Cloning of the enr gene from C. thermoaceticum. Oligo- nucleotides used are given in Tables III and IV.

Article Snippet: TABLE I Microorganisms and plasmids used in this study Microorganism or plasmid Genotype or relevant characteristic Reference or source E. coli XL1-Blue RecA1, endA1, gyrA96, tht-1, hsdR17, supE44, relA1, lac, [F1, proAB, lacl1ZDM15, TnlC (t«t)] 21 M15(pREP4) Lac, ara, goI, mtl, recA1, uvr1, [pREP4, lacl, kam9] 23 Clostridium sp. C. kluyveri Ethanol-acetate fermentation DSM 555a C. tyrobutyricum Crotonate fermentation DSM 1460 C. thermoaceticum Homoacetate fermentation, thermophile DSM 1974 Plasmids pBluescript SKII High copy cloning vector Stratagene pBlue-EV-t Vector for direct cloning of PCR amplificates This study pBlueEno3/4 Vector containing a fragment of the enr gene of C. tyrobutyricum This study pUC18 High copy cloning vector Amersham Pharmacia Biotech pNCO113 High copy expression vector ATCC,b PTA-852 pNCO-ERCTYR Expression vector construct for the enr gene of C. tyrobutyricum This study pNCO-ERCTHERM Expression vector construct for the enr gene of C. thermoaceticum This study a DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany. b American type culture collection, patent deposit number.

Techniques: Cloning

Journal: Journal of Biological Chemistry

Article Title: Enoate Reductases of Clostridia

doi: 10.1074/jbc.m008656200

Figure Lengend Snippet: FIG. 4. Nucleotide sequence and predicted amino acid sequence of the enr gene from C. tyrobutyricum. EcoRI restriction sites are underlined. The putative ribosome-binding site, start and stop codons are shown in bold letters and underlined. Nucleotide residues forming the stem-loop of a putative terminator sequence are shown in bold letters. Peptide sequences obtained by Edman degradation are boxed.

Article Snippet: TABLE I Microorganisms and plasmids used in this study Microorganism or plasmid Genotype or relevant characteristic Reference or source E. coli XL1-Blue RecA1, endA1, gyrA96, tht-1, hsdR17, supE44, relA1, lac, [F1, proAB, lacl1ZDM15, TnlC (t«t)] 21 M15(pREP4) Lac, ara, goI, mtl, recA1, uvr1, [pREP4, lacl, kam9] 23 Clostridium sp. C. kluyveri Ethanol-acetate fermentation DSM 555a C. tyrobutyricum Crotonate fermentation DSM 1460 C. thermoaceticum Homoacetate fermentation, thermophile DSM 1974 Plasmids pBluescript SKII High copy cloning vector Stratagene pBlue-EV-t Vector for direct cloning of PCR amplificates This study pBlueEno3/4 Vector containing a fragment of the enr gene of C. tyrobutyricum This study pUC18 High copy cloning vector Amersham Pharmacia Biotech pNCO113 High copy expression vector ATCC,b PTA-852 pNCO-ERCTYR Expression vector construct for the enr gene of C. tyrobutyricum This study pNCO-ERCTHERM Expression vector construct for the enr gene of C. thermoaceticum This study a DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany. b American type culture collection, patent deposit number.

Techniques: Sequencing, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Enoate Reductases of Clostridia

doi: 10.1074/jbc.m008656200

Figure Lengend Snippet: FIG. 5. Predicted amino acid sequences of proteins similar to enoate reductase. Residues shared with enoate reductases of C. tyrobutyricum by at least three proteins are marked by contrast inversion. Conserved cysteine residues and putative binding domains for FAD and NADH/NADPH are indicated with asterisks. The positions and directions of the consensus primers for the cloning of C. thermoaceticum and C. kluyveri enr genes are indicated by arrows. A, 2,4-dienoyl-CoA reductase of E. coli; B, NADH:flavin oxidoreductase of Eubacterium. sp. strain VPI 12708; C, NADH:acceptor oxidoreductase of Thermoanaerobium brockii; D, putative NADH oxidase of Archaeoglobus fulgidus; E, enoate reductase of C. tyrobutyricum; F, trimethylamine dehydrogenase of Methylotrophus methylophilus (bacterium W3A1); G, dimethylamine dehydrogenase of Hyphomicrobium sp.

Article Snippet: TABLE I Microorganisms and plasmids used in this study Microorganism or plasmid Genotype or relevant characteristic Reference or source E. coli XL1-Blue RecA1, endA1, gyrA96, tht-1, hsdR17, supE44, relA1, lac, [F1, proAB, lacl1ZDM15, TnlC (t«t)] 21 M15(pREP4) Lac, ara, goI, mtl, recA1, uvr1, [pREP4, lacl, kam9] 23 Clostridium sp. C. kluyveri Ethanol-acetate fermentation DSM 555a C. tyrobutyricum Crotonate fermentation DSM 1460 C. thermoaceticum Homoacetate fermentation, thermophile DSM 1974 Plasmids pBluescript SKII High copy cloning vector Stratagene pBlue-EV-t Vector for direct cloning of PCR amplificates This study pBlueEno3/4 Vector containing a fragment of the enr gene of C. tyrobutyricum This study pUC18 High copy cloning vector Amersham Pharmacia Biotech pNCO113 High copy expression vector ATCC,b PTA-852 pNCO-ERCTYR Expression vector construct for the enr gene of C. tyrobutyricum This study pNCO-ERCTHERM Expression vector construct for the enr gene of C. thermoaceticum This study a DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany. b American type culture collection, patent deposit number.

Techniques: Binding Assay, Cloning

Journal: Cell reports

Article Title: Identification of the main barriers to Ku accumulation in chromatin

doi: 10.1016/j.celrep.2024.114538

Figure Lengend Snippet:

Article Snippet: The pUC18-GFP plasmid (deposited on Addgene) was produced by cloning using a EcoRI+BamHI digestion a minimal transcription unit for GFP expression, consisting of the small SV40 promoter, the GFP ORF and the BGH polyadenylation signal, generated by PCR assembly.

Techniques: Generated, Recombinant, Plasmid Preparation, Modification, Software

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: Expression levels of major cellulase genes when Avicel is used as the carbon source. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the transcriptional levels of four major cellulase genes, cbh1 ( A ), cbh2 ( B ), egl1 ( C ), and egl2 ( D ), and two major xylanase genes xyn1 ( E ) and xyn2 ( F ). Conidia of T. reesei were inoculated into MA liquid medium with 1% (w/v) Avicel as the sole carbon source. Sampling was done at 36, 48, and 60 h. The data are normalized to expression of QM6a at 36 h for each tested gene, with the sar1 gene used as an endogenous control in all samples. Values are represented as mean ± SD of the results from three independent experiments. Asterisks (*) indicate significant differences compared to the parental strain (Student’s t -test, * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001)

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Sampling, Control

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: rsr1 negatively regulates cellulase production. A – E Enzyme activities, including p NPCase ( A ), CMCase ( B ), FPase ( C ), and xylanase activity ( D ), and extracellular protein concentration ( E ) in Trichoderma reesei strains. ( F ) Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the secretomes in supernatants of T. reesei strains. The measurements were made during culture with 1% (w/v) Avicel as the sole carbon source. Values are represented as mean ± standard deviation (SD) of the results from three independent experiments. Asterisks (*) indicate significant differences compared to the parental strain (Student’s t -test, * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001)

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques: Activity Assay, Protein Concentration, Polyacrylamide Gel Electrophoresis, SDS Page, Standard Deviation

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: Effects of rsr1 deletion on cellulase production in the T. reesei industrial strain, RUT-C30. The activities of p NPCase ( A ) and CMCase ( B ) of parental strain RUT-C30 and rsr1 deletion strain C30- rsr1 after culture in liquid 2 × Mandels’ medium containing 2% lactose. Values are the means ± SD of the results from three independent experiments. Asterisks (*) indicate a significant difference (Student’s t -test, ** P < 0.01; **** P < 0.0001)

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques:

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: Effect of the loss of rsr1 on the ACY1-cAMP-PKA pathway. A , B Intracellular cyclic adenosine monophosphate (cAMP) content of the original strain (QM6a), deletion strain (Δ rsr1 ), and complementation strain (RC- rsr1 ) were measured when 1% (w/v) Avicel ( A ) and 2% glucose ( B ) were the sole carbon source, respectively. Fresh conidia were generated from hyphae in MA liquid medium and intracellular cAMP content was measured at 2 days, as described in the Methods. ( C – F ) Transcriptional levels of acy1 ( C ), pkac1 ( D ), pkac2 ( E ), and pkar1 ( F ) detected by RT-qPCR in QM6a, Δ rsr1 , and RC- rsr1 strains with 1% (w/v) Avicel as the sole carbon source. Values are represented as mean ± SD of the results from three independent experiments. Asterisks (*) indicate significant differences compared to the parental strain (Student’s t -test, *** P < 0.001; **** P < 0.0001; ns represents no significant difference)

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques: Generated, Quantitative RT-PCR

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: Effect of ACY1-cAMP-PKA pathway on rsr1 -mediated cellulase overexpression. p NPCase activity ( A ) and CMCase activity ( B ) of T. reesei QM6a, Δ acy1 , Δ rsr1 , Δ rsr1 Δ acy1 , QM6a-OE acy1 and Δ rsr1 -OE acy1 cultured in MA with 1% (w/v) Avicel at 2, 3, and 4 days. The transcript levels of cbh1 ( C ), cbh2 ( D ), egl1 ( E ), and egl2 ( F ) in T. reesei QM6a, Δ acy1 , Δ rsr1 , Δ rsr1 Δ acy1 , QM6a - OE acy1 and Δ rsr1- OE acy1 strains cultured in MA with 1% (w/v) Avicel at 36 and 48 h. Values are represented as mean ± SD of the results from three independent experiments. Asterisks (*) indicate significant differences compared to the parental strain (Student’s t -test, * P < 0.05)

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques: Over Expression, Activity Assay, Cell Culture

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: Transcriptome analysis results. A Volcano plot of the differential level of gene expression. The genes that were significantly upregulated and downregulated are denoted by red and green dots, respectively. Gray dots indicate genes whose expression was not significantly changed. B Gene Ontology (GO) annotations analysis of differentially expressed genes in the Δ rsr1 and QM6a strains. Significantly enriched categories are shown (adjusted P < 0.05). C KEGG enrichment analysis for the Δ rsr1 and QM6a strains. D Correlation between RT-qPCR and RNA sequencing results

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, RNA Sequencing

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: Log 2 fold change (Log 2 fc) of characterized transcriptional factors involved in the regulation of lignocellulase genes in Δ rsr1 and QM6a strains

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques:

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: Log 2 fold change (Log 2 fc) of characterized transporters in Δ rsr1 and QM6a strains

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques:

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: Log 2 fold change (Log 2 fc) of G-protein coupled receptors in Δ rsr1 and QM6a strains

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques:

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: The role of three G-protein coupled receptors (GPCRs) on rsr1 -mediated cellulase overproduction. p NPCase activity ( A ) and CMCase activity ( B ) of T. reesei QM6a, Δ tre62462 , Δ tre58767 , Δ tre53238 , Δ rsr1 , Δ rsr1 Δ tre62462 , Δ rsr1 Δ tre58767 , and Δ rsr1 Δ tre53238 cultured in MA with 1% (w/v) Avicel at 2, 3, and 4 d. The transcript levels of cbh1 ( C ), cbh2 ( D ), egl1 ( E ) and egl2 ( F ) in T. reesei QM6a, Δ tre62462 , Δ tre58767 , Δ tre53238 , Δ rsr1 , Δ rsr1 Δ tre62462 , Δ rsr1 Δ tre58767 , and Δ rsr1 Δ tre53238 strains cultured in MA with 1% (w/v) Avicel at 36 and 48 h. Values are represented as mean ± SD of the results from three independent experiments. Asterisks (*) indicate significant differences compared to the parental strain (Student’s t -test, * P < 0.05)

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques: Activity Assay, Cell Culture

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

doi: 10.1186/s13068-023-02341-z

Figure Lengend Snippet: Putative mechanism for RSR1 regulating cellulase production in T. reesei . GPCRs are shaded in green, ACY1-cAMP-PKA pathway in blue, cellulose molecule in purple, cellulase gene in red, and RSR1 in yellow. Positive regulation is represented by a solid line with arrowheads. Negative regulation is represented by a line ending in a vertical line. Dotted arrows in both directions represent unknown connections. The solid arrows indicate data supported by our results, and the dashed arrows indicate undefined regulation. After receiving signal stimulation from extracellular cellulose molecules, three GPCRs on the membrane may transmit signals to RSR1, promoting the expression of ACY1-cAMP-PKA pathway-related genes, increasing intracellular cAMP content, negatively regulating the expression of ACE3 and XYR1, promoting the expression of CRE1, and ultimately repressing cellulase production

Article Snippet: T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains [ ].

Techniques: Membrane, Expressing