Robert L. Mach

Xyr1 (Xylanase Regulator 1) Regulates both the Hydrolytic Enzyme System and d-Xylose Metabolism in Hypocrea jecorina

ABSTRACT Xyr1 (xylanase regulator 1) of the ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) was recently demonstrated to play an essential role in the transcriptional regulation of the xyn1 (xylanase 1-encoding) gene expression. Consequently, this study reports on the deletion of the xyr1 gene from the H. jecorina genome. Comparative studies of the growth behavior of the different mutant strains (deleted and retransformed xyr1) grown on various carbon sources pointed to the strongly reduced ability of the xyr1 deletion strain to utilize d-xylose and xylan. Transcriptional analysis of the xyl1 (d-xylose reductase 1-encoding) gene as well as measurements of corresponding enzymatic activities gave evidence that Xyr1 takes part in the control of the fungal d-xylose pathway, in particular in the regulation of d-xylose reductase. It could be demonstrated that the uptake of d-xylose into the fungal cell is uninfluenced in the Δxyr1 strain. Furthermore, transcriptional regulation of the major hydrolytic enzyme-encoding genes xyn1 and xyn2 (xylanases 1 and 2), cbh1 and cbh2 (cellobiohydrolases 1 and 2), and egl1 (endoglucanase 1) is strictly dependent on Xyr1. Regulation of the respective genes via Xyr1 is not affected by the substances mediating induction (xylose, xylobiose, and sophorose) and is indispensable for all modes of gene expression (basal, derepressed, and induced). Moreover, Xyr1, it was revealed, activated transcriptional regulation of inducer-providing enzymes such as β-xylosidase BXLI and β-glucosidase BGLI but was not shown to be involved in the regulation of BGLII.

Crel, the carbon catabolite repressor protein from Trichoderma reesei

In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR‐primers were designed to amplify a 0.7‐bp fragment of the crel gene, which was used to clone the entire gene. It encodes a 402‐amino acid protein with a calculated M r, of 43.6 kDa. Its aa‐sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A. niger, respectively. Similarity was restricted to the aa‐region containing the C2H2 zinc finger and several aa‐regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins. Another aa‐region rich in the SPXX‐motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors. The presence of the crel translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (±2) kDa in cell‐free extracts from T. reesei. A Cre1 protein fragment from the two zinc fingers to the region similar to the aa‐sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S‐transferase. EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5′‐GCGGAG‐3′, which matches well with the A. nidulans consensus sequence for CreA binding (5′‐SYGGRG‐3′). Cell‐free extracts of T. reesei formed different complexes with DNA‐fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated. We conclude that T. reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.

Regulation of transcription of cellulases- and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei)

The filamentous fungi Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) have been the subject of many studies investigating the mechanism of transcriptional regulation of hemicellulase- and cellulase-encoding genes. The transcriptional regulator XlnR that was initially identified in A. niger as the transcriptional regulator of xylanase-encoding genes controls the transcription of about 20–30 genes encoding hemicellulases and cellulases. The orthologous xyr1 (xylanase regulator 1-encoding) gene product of H. jecorina has a similar function as XlnR, although at points, the mechanisms seems to be different. Specifically in H. jecorina, the interaction of Xyr1 and the co-regulators Ace1 and Ace2 in the regulation of transcription of xylanases and cellulases has been studied. This paper describes the similarities and differences in the transcriptional regulation of expression of hemicellulases and cellulases in A. niger and H. jecorina.

Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals

Trichoderma reesei was transformed to hygromycin B resistance using a novel vector, which contains the E. coli hygromycin B phosphotransferase gene (hph) fused between promoter and terminator elements of the homologous Trichoderma pkil (coding for pyruvate kinase) and cbh2 (coding for cellobiohydrolase II) genes, respectively. Transformation frequencies of over 1800–2500 transformants/μg DNA were obtained, which is a 15–20-fold increase over that with pAN7-1, which contains hph between A. nidulans expression signals. Mitotically-stable transformants contained the hph gene and the regulatory sequences of the pkil promoter and the cbh2 terminator integrated into the genome. Evidence for preferentially ectopic integration is given.

Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei

The filamentous fungus Trichoderma reesei forms two specific, xylan‐inducible xylanases encoded by xyn1 and xyn2 to degrade the β‐1,4‐d‐xylan backbone of hemicelluloses. This enzyme system is formed in the presence of xylan, but not glucose. The molecular basis of the absence of xylanase I formation on glucose was the purpose of this study. Northern blotting of the xyn1 transcript as well as the use of the Escherichia coli hygromycin B phosphotransferase‐encoding gene (hph) as a reporter consistently showed that the basal expression of xyn1 was affected by glucose, whereas its induction by xylan remained uninfluenced. The repression of basal xyn1 transcription is mediated by the carbon catabolite repressor protein Cre1, which in vivo binds to two of four consensus sites (5‐SYG‐GRG‐3) in the xyn1 promoter, which occurred in the form of an inverted repeat. T. reesei strains, bearing a xynlv. hph reporter construct, in which four nucleotides from the middle of the inverted repeat had been removed, expressed hph on glucose at a level comparable to that observed during growth on a carbon catabolite derepressing carbon source. Northern analysis of xynl expression in a T. reesei mutant strain (RUT C‐30), which contains a truncated, non‐functional crel gene, also confirmed basal transcription of xyn1. In this strain, xyn1 transcription was still inducible by xylose or xylan to an even higher degree than in the wild‐type strain, suggesting that induction overcomes glucose repression at the level of xynl expression. Based on these data, we postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1‐mediated carbon catabolite repression.

Transcriptional Regulation of xyr1, Encoding the Main Regulator of the Xylanolytic and Cellulolytic Enzyme System in Hypocrea jecorina

ABSTRACT In Hypocrea jecorina, Xyr1 (xylanase regulator 1) is the main transcription activator of hydrolase-encoding genes, such as xyn1, xyn2, bxl1, cbh1, cbh2, egl1, and bgl1. Even though Xyr1 mediates the induction signal for all these genes derived from various inducing carbon sources and compounds, xyr1 transcription itself is not inducible by any of these substances. However, cultivation on glucose as the carbon source provokes carbon catabolite repression of xyr1 transcription mediated by Cre1. In addition, xyr1 transcription is repressed by the specific transcription factor Ace1. Moreover, Xyr1 is permanently available in the cell, and no de novo synthesis of this factor is needed for a first induction of xyn1 transcription. The constitutive expression of xyr1 leads to a significant elevation/deregulation of the xyn1, xyn2, and bxl1 transcription compared to what is seen for the parental strain. Overall, the corresponding xylanolytic enzyme activities are clearly elevated in a constitutively xyr1-expressing strain, emphasizing this factor as an auspicious target for genetically engineered strain improvement.

Improvement of the Fungal Biocontrol Agent Trichoderma atroviride To Enhance both Antagonism and Induction of Plant Systemic Disease Resistance

ABSTRACT Biocontrol agents generally do not perform well enough under field conditions to compete with chemical fungicides. We determined whether transgenic strain SJ3-4 of Trichoderma atroviride, which expresses the Aspergillus niger glucose oxidase-encoding gene, goxA, under a homologous chitinase (nag1) promoter had increased capabilities as a fungal biocontrol agent. The transgenic strain differed only slightly from the wild-type in sporulation or the growth rate. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted. SJ3-4 had significantly less N-acetylglucosaminidase and endochitinase activities than its nontransformed parent. Glucose oxidase-containing culture filtrates exhibited threefold-greater inhibition of germination of spores of Botrytis cinerea. The transgenic strain also more quickly overgrew and lysed the plant pathogens Rhizoctonia solani and Pythium ultimum. In planta, SJ3-4 had no detectable improved effect against low inoculum levels of these pathogens. Beans planted in heavily infested soil and treated with conidia of the transgenic Trichoderma strain germinated, but beans treated with wild-type spores did not germinate. SJ3-4 also was more effective in inducing systemic resistance in plants. Beans with SJ3-4 root protection were highly resistant to leaf lesions caused by the foliar pathogen B. cinerea. This work demonstrates that heterologous genes driven by pathogen-inducible promoters can increase the biocontrol and systemic resistance-inducing properties of fungal biocontrol agents, such as Trichoderma spp., and that these microbes can be used as vectors to provide plants with useful molecules (e.g., glucose oxidase) that can increase their resistance to pathogens.

Quantitative PCR Method for Sensitive Detection of Ruminant Fecal Pollution in Freshwater and Evaluation of This Method in Alpine Karstic Regions

ABSTRACT A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 × 109 marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 105 marker equivalents per liter).

Different Inducibility of Expression of the Two Xylanase Genes xyn1 and xyn2 in Trichoderma reesei

Regulation of formation of the extracellular xylanase system of Trichoderma reesei QM 9414 during growth on xylan, cellulose, and replacement onto a number of soluble inducers was investigated by Northern analysis of xyn1 and xyn2 transcripts and by the use of the Escherichia coli hph (hygromycin B-phosphotransferase-encoding) gene as a reporter. Whereas the xyn1 promoter is active in the presence of xylan and xylose, and virtually silenced in the presence of glucose, the xyn2 promoter enables basal transcription at a low level, but is enhanced in the presence of xylan and xylobiose and also of sophorose or cellobiose. The respective regulatory nucleotide regions were localized on a 221-base pair fragment and a 55-base pair fragment of the xyn1 and xyn2 5′-upstream noncoding sequences, respectively. Electrophoretic mobility shift assays, using cell-free extracts, identified induction-specific protein-DNA complexes: one complex of high mobility was observed under basal, noninduced conditions (glucose) with xyn2, which was in part replaced by a slow-migrating complex upon induction by xylan or sophorose. Both complexes bound to a CCAAT box. With xyn1, the induced complex also binds to a CCAAT box, but this binding is not observed in the presence of the carbon catabolite repressor Cre1, which binds to a nearby located consensus motif.

Transcriptional Regulation of xyn1, Encoding Xylanase I, in Hypocrea jecorina

ABSTRACT Two major xylanases (XYN I and XYN II) of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) are simultaneously expressed during growth on xylan but respond differently to low-molecular-weight inducers. In vivo footprinting analysis of the xylanase1 (xyn1) promoter revealed three different nucleotide sequences (5′-GGCTAAATGCGACATCTTAGCC-3′ [an inverted repeat of GGCTAA spaced by 10 bp], 5′-CCAAT-3′, and 5′-GGGGTCTAGACCCC-3′ [equivalent to a double Cre1 site]) used to bind proteins. Binding to the Cre1 site is only observed under repressed conditions, whereas binding to the two other motifs is constitutive. Applying heterologously expressed components of the H. jecorina cellulase regulators Ace1 and Ace2 and the xylanase regulator Xyr1 suggests that Ace1 and Xyr1 but not Ace2 contact both GGCTAA motifs. H. jecorina transformants containing mutated versions of the xyn1 promoter, leading to elimination of protein binding to the left or the right GGCTAA box revealed either strongly reduced or completely eliminated induction of transcription. Elimination of Cre1 binding to its target released the basal transcriptional level from glucose repression but did not influence the inducibility of xyn1 expression. Mutation of the CCAAT box prevents binding of the Hap2/3/5 complex in vitro and is partially compensating for the loss of transcription caused by the mutation of the right GGCTAA box. Finally, evidence for a competition of Ace1 and Xyr1 for the right GGCTAA box is given. These data prompted us to hypothesize that xyn1 regulation is based on the interplay of Cre1 and Ace1 as a general and specific repressor with Xyr1 as transactivator.