The Hypocrea jecorina (Trichoderma reesei) hypercellulolytic mutant RUT C30 lacks a 85 kb (29 gene-encoding) region of the wild-type genome
- Verena Seidl†1,
- Christian Gamauf†1,
- Irina S Druzhinina1,
- Bernhard Seiboth1,
- Lukas Hartl1 and
- Christian P Kubicek1Email author
© Seidl et al; licensee BioMed Central Ltd. 2008
Received: 28 October 2007
Accepted: 11 July 2008
Published: 11 July 2008
The hypercellulolytic mutant Hypocrea jecorina (anamorph Trichoderma reesei) RUT C30 is the H. jecorina strain most frequently used for cellulase fermentations and has also often been employed for basic research on cellulase regulation. This strain has been reported to contain a truncated carbon catabolite repressor gene cre1 and is consequently carbon catabolite derepressed. To date this and an additional frame-shift mutation in the glycoprotein-processing β-glucosidase II encoding gene are the only known genetic differences in strain RUT C30.
In the present paper we show that H. jecorina RUT C30 lacks an 85 kb genomic fragment, and consequently misses additional 29 genes comprising transcription factors, enzymes of the primary metabolism and transport proteins. This loss is already present in the ancestor of RUT C30 – NG 14 – and seems to have occurred in a palindromic AT-rich repeat (PATRR) typically inducing chromosomal translocations, and is not linked to the cre1 locus. The mutation of the cre1 locus has specifically occurred in RUT C30. Some of the genes that are lacking in RUT C30 could be correlated with pronounced alterations in its phenotype, such as poor growth on α-linked oligo- and polyglucosides (loss of maltose permease), or disturbance of osmotic homeostasis.
Our data place a general caveat on the use of H. jecorina RUT C30 for further basic research.
Due to its carbon catabolite derepressed phenotype, H. jecorina RUT C30 has frequently been used as a reference strain in studies on the regulation of gene expression [16–18] or cell biology . In a similar type of study, we have recently observed that the transcript of a gene was completely absent from RUT C30, and subsequently we found that also the gene was absent from this strain (Christian Gamauf, Christian P. Kubicek and Bernhard Seiboth, unpublished data). In the attempt to identify the reason for the absence of this gene, we discovered that H. jecorina RUT C30 lacks a large (85 kb) segment of genes present on scaffold 15 of the genomic sequence of the wild-type strain H. jecorina QM6a http://genome.jgi-psf.org/Trire2/Trire2.home.html, . The identification of these genes, and their correlation with changes in the phenotype of H. jecorina RUT C30 compared to strains QM6a and QM9414, are reported in this paper.
Fungal Strains and culture conditions
The Hypocrea jecorina strains QM6a (wild-type; ATCC 13631), QM9414 (early cellulase overproducing mutant; ATCC 26921), NG 14 (ATCC 56767) and RUT C30 (ATCC 56765) were used throughout this study. They were maintained on PDA slants (potato dextrose agar; Difco, Franklin Lakes, NJ, USA), and stock cultures kept at -80°C.
For shake flask cultures, 200 ml of Mandels Andreotti (MA) medium  with carbon sources added as given at the respective results sections, was suspended into 1 L Erlenmeyer flasks, inoculated with 5 × 107 spores, and incubated on a rotary shaker at 28°C and 200 rpm. To induce polyol dehydrogenases, glycerol 1% (w/v) was used as a carbon source. Cultures were harvested after 24 hrs by gentle filtration, and replaced onto fresh MA medium with either L-arabinose, erythritol or xylitol as an inducer (10 mM), and incubated for further 12 hrs. At this time they were harvested and used to prepare cell-free extracts (see below).
Detection production of antimicrobial agents
Secretion of potential antimicrobial polyketides was tested by an agar diffusion method and plate confrontation tests. For the former, culture filtrates from various time points during growth- and stationary phase on D-glucose and lactose as a carbon source were sampled, proteins denatured by heating (100°C, 5 min), and the samples then concentrated to a tenth of their volume in a Speed Vac. They were then filtered through 20 μ filters (Millipore, Billerica, MA, USA) and pipetted into 8 mm holes punched into agar plates containing inocula of Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae. In the plate confrontation tests, 8 mm diameter agar plugs of mycelia of the two strains of H. jecorina were placed 5 cm apart from a respective colony of the same microbes. The presence of an antimicrobial component was indicated in both methods by a clearing zone.
Nucleic acid isolation and hybridisation
PCR Primers used throughout this work
Sequence (5' → 3')
Determination of the are of deletion
Determination of the downstream end of the deletion
To identify the 5' end of the deletion, the GenomeWalker™ Universal Kit (Clontech, Mountain View, CA, USA; ) was used. Briefly, this method first constructs pools of uncloned, adaptor-ligated genomic DNA fragments. Then, two PCR amplifications are preformed per library: the first uses the outer adaptor primer (AP1, provided by the manufacturer) provided in the kit and the outer, gene-specific primer (GWRUT C30gsp1; Table 1). The resulting PCR mixture is then used as a template for a secondary or "nested" PCR with the nested adaptor primer (AP2, provided by the manufacturer) and the nested gene-specific primer (GWRUT C30gsp2; Table 1). The DNA fragments were then cloned and sequenced. PCR amplifications were performed using the Long PCR Enzyme Mix (Fermentas, St.Leon-Rot, Germany). Distinct PCR products were amplified from libraries constructed with Dra I and Stu I endonucleases and sequenced (MWG Biotech, Ebersberg, Germany).
Amplification and sequencing of the cre1 locus in H. jecorina RUT C30
The wild-type H. jecorina cre1 locus is located on scaffold 2, and its open reading frame (ORF) spans from 786955–789433 (ID 120117). Oligonucleotides used for the amplification of the cre1.1 mutation in strain RUT C30 and are given in Table 1.
Enzyme extraction and assays
Preparation of cell free extracts and assay of xylitol and L-arabinitol dehydrogenases was performed essentially as described previously [26, 27]. Erythritol dehydrogenase was measured in the same way as L-arabinitol dehydrogenase, but using 100 mM erythritol as a substrate.
Conidida from 7 – 10 day old cultures were collected and suspended in liquid Mandels Andreotti medium  containing either 1% or 10% (w/v) glucose and cultivated at 28°C. 50 μl drops of conidial suspension were placed on large cover slips and examined at room temperature by using differential interference contrast optics with a 60× (1.2 numerical aperture [NA]) water immersion plan apo objective on an inverted Nikon TE2000 microscope (Nikon, Kingston-Upon-Thames, UK). Images were captured with a Nikon DXM1200F digital camera and transferred into Adobe Photoshop software (version 10.0; Adobe Systems Inc., San Jose, CA, USA) for further processing.
Biolog Phenotype Microarray analysis
Global carbon assimilation patterns were investigated using Biolog FF MicroPlate™ (Biolog Inc., Hayward, CA, USA), using the protocol published recently . Briefly, H. jecorina strains were pregrown on 20 g·l-1 malt extract agar, and 90 μl of a conidial suspension from them (75 ± 2% transmission at 590 nm) dispensed into each of the wells of a Biolog FF MicroPlate™ (Biolog Inc., Hayward, CA, USA). Inoculated microplates were incubated in the dark at 30°C, and percent absorbance determined after 12, 18, 24, 36, 42, 48, 66 and 72 h at 750 nm. Analyses were repeated at least three times for each strain.
Basic statistical methods such as multiple regression analysis and analysis of variance (ANOVA) as well as multivariate exploratory techniques (cluster and factor analyses) were performed using STATISTICA 6.1 (StatSoft, Inc., Tulsa, OK, USA) data analysis software system.
Sequence analysis and phylogeny
The genome sequence of H. jecorina is available . To screen the genome for genes missing in strain RUT C30, the "browse" function was used. Genes are identified by their protein ID number (search → gene models → protein id). Sequence analysis of the genes identified to be missing in H. jecorina RUT C30 was performed with InterProScan ) and SMART (/; ). Proteins with most similar sequences were identified by BLASTX. For phylogenetic analysis, protein sequences were aligned using CLUSTALX 1.83 , the alignment edited with GENEDOC 2.6  and the phylogenetic analysis performed in MEGA 3.1 .
Identification of a genome fragment missing in H. jecorina RUT C30
The 3' end of the gap identified by genome walking corresponded to the region identified by PCR amplification and specified it at +86603 bp of scaffold 15 in the 5' nontranscribed area of the nitrilase-encoding gene (see above). Thus, this analysis provides evidence that H. jecorina RUT C30 contains an approximately 85 kb large gap on scaffold 15, which in H. jecorina QM6a  contains 29 ORFs (Fig. 3b) and that most of these genes are not present in the genome anymore.
The 85-kb deletion is unlinked to the cre1 mutation
In silico identification of the genes lacking in H. jecorina RUT C30
Identification of genes located on the 5' end of scaffold 15*
Location on scaffold
247 – 3990
4265 – 4550
Peptidase S26, signal peptidase
Glycerol dehydrogenase GLD2
Multidrug resistance protein
carbohydrate esterase (family 4), imidase
Heterokaryon incompatibility protein het-6
Glucan endo-1,6-β-glucosidase (GH5)
Ankyrin repeat protein
Protein of the cytochrome P450 CYP2 family (phenylacetate-2 hydroxylase)
Aromatic and unpolar amino acid permease
N2, N2-dimethylguanosine tRNA methyl transferase
Hypothetical protein, poorly conserved
Cytochrome P450-dependent alkane hydroxylase
Unknown protein, poorly conserved
Acid trehalase GH65
Polyketide synthase class 1, reducing
Hypothetical protein, well conserved
Cys6 transcription factor
Sexual development inhibiting protein LsdA
Cys6 transcription factor
H. jecorina RUT C30 is impaired in the assimilation of α-glucans and -glucosides
H. jecorina RUT C30 displays several alterations in carbon source assimilation
Polyol dehydrogenase activities in H. jecorina QM 9414 and RUT C30
0.03 [± 0,025]
0.09 [± 0.01]
0.52 [± 0.03]
2.51 [± 0.04]
1.1 [± 0.05]
2.4 [± 0.3]
0,14 [± 0.02]
0.018 [± 0.01]
0,021 [± 0.006]
0.3 [± 0.02]
1.15 [± 0.03]
0.45 [± 0.03]
0.75 [± 0.05]
0.05 [± 0.01]
0.23 [± 0.04]
0.25 [± 0.04]
H. jecorina RUT C30 favors high osmotic pressure
Spore volume increase during germination in H. jecorina RUT C30
H. jecorina RUT C30 lacks pigment formation
An intriguing observation during the cultivation experiments was that strain RUT C30 does not form the yellow pigment, which is characteristic for H. jecorina and other Trichoderma spp. from section Longibrachiatum . This difference was observed both in late submerged cultures as well as during plate growth. We suspected that this could be due to the absence of the class I polyketide synthase ID 65172. In order to test this presumption, we subjected its amino acid sequence to phylogenetic analysis (NJ) with other polyketide synthases investigated by Kroken et al. . In this analysis (data not shown), the H. jecorina polyketide synthase was determined to be member of clade I from the reducing polyketide synthases, thereby clustering most closely to Bipolaris mayidis PKS5, whose function is not known. Since none of the members of this cluster is known to be responsible for pigment formation, but some of them (e.g. the lovastatin synthase) synthesize antimicrobial polyketides, we also tested whether RUT C30 would be deficient in formation of an antimicrobial compound. However, using the agar diffusion assay and the confrontation assay, we could not detect any such compound in strain QM9414 and consequently also not in RUT C30 (data not shown). While the use of more sensitive methods such as MS may detect differences in secondary metabolite production between H. jecorina QM6a and RUT C30, our data show that the loss of this class I polyketide synthase does not influence the antimicrobial activity of H. jecorina.
H. jecorina NG 14 has a full-size cre1 but lacks the 85 kb fragment
Both, H. jecorina RUT C30 and its ancestor NG 14, are mutants that underwent mutagenesis by nitrosoguanidin and were selected for growth on cellulose in the presence of glycerol (NG 14) and 2-desoxiglucose (RUT C30). We therefore wondered whether the loss of the 85 kb fragment and the truncation of cre1 were the result of one or both of these mutation steps.
In order to test for the presence or absence of the 85-kb gene fragment, which is missing in RUT C30, the gene specific primers for ORFs 3, 4, 5, 10, 20 and 26 (table 1) were used. By means of these primers, we were unable to amplify a PCR product from strains NG 14 and RUT C30, whereas amplicons were obtained in the control with QM9414 (data not given), indicating that the large chromosome lesion is already present in the ancestor of RUT C30.
In the present work we have shown that the hypercellulolytic mutant H. jecorina RUT C30, in which only two mutations (in the carbon catabolite repressor protein CRE1 and the processing β-glucosidase II [12, 13] had been described so far, carries a major deletion in its genome which comprises 85.048 bp including 29 open reading frames. Although this finding had not been detected so far, it is in accordance with earlier karyotyping results, which showed that the size and number of chromosomes in H. jecorina strain RUT C30 differed significantly from QM6a and QM9414 [14, 15]. Unfortunately, none of the marker genes that were used in these studies was located on scaffold 15, and we were thus unable to identify the chromosome on which the 85 kb fragment described in this paper is lacking. However, despite of the fact that these 0.085 Mbp are a significant lesion, they are small compared to the changes in chromosome size determined by these authors. While the size determination in contour-clamped homogeneous electric field (CHEF) gel is not sensitive enough to distinguish between 0.1 and 0.2 Mbp, it is nonetheless possible that more deletions may be present in the genome of RUT C30.
The reason for this gene loss in RUT C30 is unclear. The genealogy leading to mutant RUT C30 involved three mutagenesis steps from the wild type strain (Fig. 1), of which the first one was simple UV mutagenesis whereas the subsequent two (from M7 to NG 14, and from NG 14 to RUT C30) involved mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine . However, both agents usually do not lead to chromosome alterations or translocations. The results from genome walking have shown that the 5' site of the deletion is located in a region containing an about 1600 nt long palindromic AT-rich region (PATRR). PATRRs have been found to mediate genomic instability, thereby contributing to translocations, deletions and amplifications [46, 47]. Carter et al.  have speculated that the lack of a sexual cycle and the need for mitotic pairing of chromosomes arising from there may increase the tolerance of mitosporic fungi to chromosome rearrangements. In the light of the above reasoning, PATRRs may be preferred regions for this. In N. crassa, this has been shown to be due to escape from het-c incompatibility . The possibility that such rearrangements may regularly occur in H. jecorina would be consistent with similar data from other fungi , and also be consistent with results from the analysis of the genome of H. jecorina QM6a  which revealed a number of gene conversion events. In addition, such events could also occur during the regeneration of protoplasts after transformation with DNA (as shown for Nectria haematococca ), which would explain the high phenotypic diversity in H. jecorina transformants . Our results with H. jecorina NG 14 show that the loss of the 85 kb gene fragment already occurred before the origin of RUT C30, and such an event must therefore have taken place in this or even an earlier mutant strain.
The structure of the gene encoding the rhodanese-like protein also supports such a scenario: this gene does not have any orthologues in fungi, but shows similarity to flavibacterial rhodanese-like proteins. It is conceivable that this gene arrived by horizontal gene transfer in an instable region, which led to the insertion to this unsual high number (14) of introns. The gene seems to be active, though, as the database lists 15 ESTs for it.
We were not able to predict the putative function of all genes which have been lost in RUT C30. Although we could therefore not to relate all of these genes to distinct phenotypic properties, for some of them clear correlations were obtained. One of them was the inability to grow on α-linked oligo- and polysaccharides, which we interpret to be due to the loss of the maltose permease gene (ID 65191). This finding implies that H. jecorina RUT C30 may not be a good source of enzyme production on carbon sources containing starch and other α-linked glycans, unless this deficiency is complemented by the corresponding gene from QM 9414.
Another intriguing finding during this study was that the loss of glycerol dehydrogenase GLD2 does not lead to an impaired osmotolerance. Consistent findings have been reported with a gldB-knock out strain of A. nidulans , where it was shown that this strain failed to accumulate glycerol during osmotic stress, but instead accumulated other polyols including D-mannitol, L-arabinitol and L-erythritol. It is therefore possible that other polyol dehydrogenase genes of H. jecorina can compensate for the loss of GLD2. However, the microscopic findings from this study, i.e. that RUT C30 displays a significant swelling of its conidia before it starts to germinate, indicate that osmotic homeostasis is impaired in this strain. A possible explanation for this finding would be that the compensating polyols (L-arabinitol, L-erythritol) are less fast metabolized, and thus lead to an increased osmotic pressure in the spores and delayed germination. The carbon source assimilation experiments also revealed that strain RUT C30 shows an enhanced growth rate on a number of simple carbon sources such as glycerol, N-acetylglucosamine, D-mannitol, D-fructose, D-trehalose, D-mannose, and D-ribose. Interestingly, there is evidence that some of them act as catabolite repressing carbon sources in H. jecorina (e.g. glycerol, ; fructose, ; mannose, unpublished data by L. Hartl, C.P. Kubicek and B. Seiboth). The phenotype in RUT C30 may be related to the loss of function of CRE1, and be due to the relief of catabolite repression by these carbon sources within their own catabolic pathways, most likely at the level of uptake. Sugar permeases of H. jecorina and other mitosporic fungi are known to be repressed by elevated levels of their substrates [52, 53]. This property enables strain RUT C30 to grow faster at high sugar concentrations such as 6% lactose, a condition employed to make use of its superior cellulase forming capacity .
In conclusion, we have identified a major genomic alteration in the hypercellulolytic mutant strain H. jecorina RUT C30, and have been able to correlate several of them with not yet apparent phenotypes of this strain. Likely, insights provided in this paper may only just be the beginning, and further such changes may be found when its genome would be subjected to a more thorough investigation. While the differences between the parent strain and RUT C30 do not interfere with the use of RUT C30 in biotechnology for the production of cellulases, it is clear that the use of this strain for basic research in physiology or molecular genetics is flawed. This is especially true for its use as a "carbon catabolite derepressed" mutant, because the truncation in its CRE1 protein clearly is only one of several more changes compared to its wild-type parent. Such a comparison may only be valid, if the results are compared to the mutant strain NG 14 in which the cre1 truncation has not yet occurred.
This work was supported by grants from the Austrian Science Foundation (FWF P-19143 and FWF P-19421) to CPK and BS, respectively. The H. jecorina/T. reesei genome sequencing project was funded by the United States Department of Energy.
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