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The Hypocrea jecorina (syn. Trichoderma reesei) lxr1 gene encodes a d -mannitol dehydrogenase and is not involved in l -arabinose catabolism
FEBS Letters 583 (2009) 1309–1313
journal homepage: www.FEBSLetters.org
The Hypocrea jecorina (syn. Trichoderma reesei) lxr1 gene encodes a D-mannitol dehydrogenase and is not involved in L-arabinose catabolism Benjamin Metz a,1, Ronald P. de Vries b,1, Stefan Polak a, Verena Seidl a, Bernhard Seiboth a,* a b
Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Vienna, Getreidemarkt 9, A-1060 Wien, Austria Microbiology, Utrecht University, Utrecht, The Netherlands
a r t i c l e
i n f o
Article history: Received 27 January 2009 Revised 5 March 2009 Accepted 13 March 2009 Available online 21 March 2009 Edited by Judit Ovádi Keywords: L-Arabinose L-Xylulose reductase D-Mannitol metabolism Short chain dehydrogenase/reductase Aspergillus niger
a b s t r a c t The Hypocrea jecorina LXR1 was described as the first fungal L-xylulose reductase responsible for NADPH dependent reduction of L-xylulose to xylitol in L-arabinose catabolism. Phylogenetic analysis now reveals that LXR1 forms a clade with fungal D-mannitol 2-dehydrogenases. Lxr1 and the orthologous Aspergillus niger mtdA are not induced by L-arabinose but expressed at low levels during growth on different carbon sources. Deletion of lxr1 does not affect growth on L-arabinose and Lxylulose reductase activity remains unaltered whereas D-mannitol 2-dehydrogenase activities are reduced. We conclude that LXR1 is a D-mannitol 2-dehydrogenase and that a true LXR1 is still awaiting discovery. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Fungi posses different metabolic pathways for the conversion of carbohydrates that are absent in other microorganisms. Examples of such fungal specific pathways are L-arabinose catabolism and D-mannitol metabolism (Fig. 1). Although both pathways are widespread over the fungal kingdom and of importance for the fungal lifestyle, they still await full elucidation. One reason for this is the fact that these pathways are not found in the model fungus Saccharomyces cerevisiae and received therefore less attention [1]. L-Arabinose is an abundant pentose found in different carbohydrate polymers of the plant cell wall [2]. Its degradation is therefore important for the cycling of carbon in our biosphere and has an impact on biotechnological processes which use plant materials as renewable carbon source for the production of e.g. biofuels. To ensure complete degradation of this pentose, the genes of the fungal L-arabinose catabolizing pathway were introduced in S. cerevisiae. This included the cloning of the first fungal gene encoding an L-xylulose reductase (LXR1) from the ascomycete Hypocrea jecorina (syn.: Trichoderma reesei) which together with other L-arabinose pathway genes enabled this S. cerevisiae strain to grow on L-arabinose [3]. A comparison of the amino acid sequence of the H. jecorina LXR1 to other proteins showed that highest similarity was * Corresponding author. Fax: +43 1 58801 17299. E-mail address: [email protected] (B. Seiboth). 1 B.M. and R.V. contributed equally to this article.
found to the Agaricus bisporus D-mannitol 2-dehydrogenase. The amino acid similarity was also reflected by its enzymatic properties: The enzyme catalyzed the NADPH/NADP+ specific reactions for L-xylulose/xylitol but also for D-fructose/D-mannitol, which is the typical substrate pair for D-mannitol 2-dehydrogenases (MDH). The polyol D-mannitol accumulates intracellularly to high levels in many fungal species and originally, a fungal D-mannitol cycle for NADPH regeneration was proposed [4]. However, recent genetic evidence suggests that a D-mannitol cycle does not exist and challenged also the proposed roles of D-mannitol in fungi [1,5]. The aim of the present study was therefore to assess the physiological role of the H. jecorina LXR1 in fungal L-arabinose catabolism and D-mannitol metabolism.
2. Materials and methods 2.1. Strains and culture conditions H. jecorina QM9414 (ATCC 26921) was maintained on potato dextrose agar. Strains were grown in 1 l Erlenmeyer flasks on a rotary shaker (250 rpm) at 28 °C in 250 ml medium [6] with the respective carbon source (1% w/v). For transfer cultures mycelia were transferred after 22 h of pregrowth on glycerol to flasks containing the indicated carbon source. Aspergillus niger strains N402 (cspA1) [7] and NW321 (cspA1, fwnA6, leuA1, araA4) [8] are both derived from A. niger N400 (CBS120.49). Transfer experiments were
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.03.027
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B. Metz et al. / FEBS Letters 583 (2009) 1309–1313
Fig. 1. Fungal L-arabinose catabolism and D-mannitol metabolism.
performed as described previously [8]. Escherichia coli strain JM109 (Promega) was used for plasmid propagation. For growth tests plates were inoculated with a small piece of agar in the centre. In submerged cultures, the increase in dry biomass was recorded by washing mycelial samples and drying them to constant weight at 80 °C. 2.2. Plasmid constructions and H. jecorina transformation For the lxr1 deletion cassette the upstream region of lxr1 was amplified using the primers lxrXba 50 -CAATGTTCTAGAGCATATTGACC-30 and lxrHind 50 -GATGGGCGAAGCTTAGGATG-30 . The XbaI/ HindIII restricted amplicon was ligated with the HindIII/XhoI restricted hph expression cassette [9] into a XbaI/XhoI restricted pBluescript SK(+) (Stratagene). The downstream region was amplified with the primers lxrXho 50 -CACTACTCGAGAAAGAAATAATG-30 and lxrAcc65 50 -CATTAGAGCGGTACCTCTTCC-30 and introduced via XhoI/Acc65I. From the resulting vector the 5.9 kb XbaI/Acc65I fragment was used in fungal transformations [9]. 2.3. Nucleic acid isolation and hybridization DNA and RNA extraction, electrophoresis, blotting and hybridization of nucleic acids with random primed probes was described previously [10,11]. Probes were a 1.6 kb lxr1 upstream region fragment, a 900 bp lxr1 amplicon containing the coding region (LXRgexfw 50 -CATCGGGATCCATGCCTCAGCCTGTC-30 and LXRgexrv 50 -CATTGAATTCTTATCGTGTAGTGTAACCT-30 ) and a 300 bp 18S rRNA fragment [12]. Transcript analysis and probes for A. niger were described [8,13,14].
MEGA 2.1 [17] using Neighbour Joining as a distance algorithmic method. Stability of clades was evaluated by 1000 bootstrap rearrangements. Bootstrap values lower than 50% are not displayed. 2.5. Enzyme assays and mannitol determination Cell-free extracts were prepared as described [12]. For enzyme activities the rate of change in absorbance at 340 nm for NAD(P)H oxidation or NAD(P)+ reduction at 30 °C was determined. D-Mannitol assays consisted of 100 mM Tris–HCl (pH 9.0), 0.5 mM MgCl2, 2 mM NAD(P)+ and 100 lg cell free extract and were started by adding D-mannitol (final conc. 100 mM). D-Fructose (L-xylulose) reduction was measured in 100 mM HEPES–NaOH (pH 7.0), 2 mM MgCl2, 0.2 mM NAD(P)H and 100 lg cell free extract and started by adding the sugar (final conc. 250 mM [100 mM]). Activities are expressed as nkat and are given as specific activities (nkat/mg protein). D-Mannitol was determined by HPLC using a Biorad Aminex HPX-87H Ion Exclusion column. The sugars were separated under isocratic conditions with 10 mM sulfuric acid at a flow rate of 0.5 ml/min and 40 °C. A range of single sugar standards were run before the analysis. 2.6. Microscopical analysis Samples were examined by using differential interference contrast optics on an inverted Nikon TE2000 microscope and imaged with a Nikon DXM1200F digital camera. 3. Results
2.4. Phylogenetic analysis
3.1. Phylogeny of putative D-mannitol dehydrogenases and L-xylulose reductases
Protein sequences were aligned with CLUSTALX 1.8 [15], visually adjusted using GENEDOC 2.6 [16] and phylogenetically analyzed in
A BLASTP search with the H. jecorina LXR1 as query against the NCBI database revealed that it has about equally similar sequence
B. Metz et al. / FEBS Letters 583 (2009) 1309–1313
identities (2e127–3e108) to different other fungal proteins which are either annotated as LXRs or MDHs. For most of these proteins a functional characterization is not available and we therefore assume that they were simply annotated according to their similarity as either LXRs or MDHs. The phylogenetic relationship of this group showed that the different LXRs and MDHs group closely together (Fig. 2). This analysis also demonstrates that all of these proteins are orthologous with the exception of Penicillium chrysogenum where two proteins are found. Gene duplication might have been one possible explanation since the resulting paralogous often evolve new functions after their duplication. 3.2. Regulation of lxr1/mtdA transcription by carbon sources Transcript profiling on a variety of carbon sources showed that low levels of transcripts of H. jecorina lxr1 were found on all tested carbon sources only after long exposure times (Fig 3A), but – in contrast to what would be typical for a gene involved in the L-arabinose pathway – no enhanced transcript levels were observed during growth on L-arabinose or L-arabitol. Notably, we also were not able to find higher transcript levels on D-mannitol as carbon source.
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In A. niger, regulatory mutants for the L-arabinose pathway have been described and it was demonstrated that genes encoding Larabitol dehydrogenase and xylulokinase have reduced or absent transcript levels in these mutants during growth on L-arabinose or L-arabitol [8,14]. We therefore included a transcriptional analysis of the orthologous A. niger mtdA. Similarly, it exhibits low expression levels on L-arabinose and L-arabitol which were not affected in the araA mutant (Fig. 3B). 3.2. Loss of lxr1 does not influence L-arabinose catabolism but affects 2-dehydrogenase activities
D-mannitol
The role of lxr1 was investigated by targeted gene deletion. Thereby the coding region of lxr1 was replaced by a hygromycin B expression cassette. About 10% of the H. jecorina transformants were deleted in lxr1 (Fig. 4A). All of these Dlxr1 strains grew normally on all carbon sources tested including L-arabinose and Larabitol but also D-mannitol either on solid (Fig. 4B) or in liquid medium (data not shown). The absence of growth retardation in these strains could be due to the possibility that another enzyme replaces at least partially the function of LXR1 or that the activity of LXR1 is not a limiting factor in the catabolism of these carbohydrates. Therefore we compared the NADPH dependent LXR
Fig. 2. Phylogeny of putative L-xylulose reductases and D-mannitol dehydrogenases. Phylogenetic analyses were performed using Neighbour Joining. Numbers below nodes indicate the bootstrap value. The bar marker indicates the genetic distance, which is proportional to the number of amino acid substitutions. GenBank accession numbers are given in brackets.
Fig. 3. Northern analysis of H. jecorina lxr1 after 6 h (A) and A. niger mtdA after 2 h (B) following the transfer of pregrown mycelia to new media with different carbon sources. The mtdA transcript profile is compared to the L-arabinose pathway gene ladA as reported previously [14].
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activities of cell free extracts grown on L-arabinose. Similar activities were found in Dlxr1 strains and the parental QM9414 (100 ± 10 nkat/mg). However, there was a significant reduction in NADP+ dependent D-mannitol 2-dehydrogenase activity in the Dlxr1 strains during growth on different carbon sources (Fig. 4C). We conclude that lxr1 is not involved in the L-arabinose catabolism but that it encodes a D-mannitol 2-dehydrogenase which is not necessary for D-mannitol catabolism.
3.3. Mannitol metabolism MDH activity is present in conidiospores of different Aspergilli and was implicated in mannitol metabolism during germination [1,5]. We therefore investigated the effect of the lxr1 deletion on germination (Fig. 5). In QM9414, lxr1 transcript levels were shortly upregulated during the initial stages of germination (data not shown) which is reflected by an increase in NADP+ dependent
Fig. 4. (A) Southern analysis of Dlxr1 strains. EcoRV digested chromosomal DNA was probed with a 1.6 kb lxr1 fragment. Deletion of lxr1 led to an increase of the original 4.6 kb lxr1 fragment to 6.5 kb. (B) Growth comparison of a Dlxr1 strain to the parental QM9414 after 3.5 days. (C) Comparison of NADP+ dependent D-mannitol 2dehydrogenase activities during growth on different carbon sources after 24 h.
Fig. 5. Comparison of morphology (bars = 10 lm), mannitol levels and NADP+ dependent MDH activity formation during germination in QM9414 and Dlxr1 during shake flask cultivation on D-glucose.
B. Metz et al. / FEBS Letters 583 (2009) 1309–1313
MDH activity followed by a sharp decrease of activity after around 12 h of growth. The high conidial mannitol levels decreased rapidly in initial stages of germination before they were replenished. Deletion of lxr1 did neither affect the germination process nor the hyphal morphology. Although MDH activities were almost not detectable in Dlxr1 strains, the mannitol levels were comparable to QM9414 in conidiospores and during the first hours of germination including the initial mannitol catabolism which was followed by mannitol biosynthesis. Only during later stages mannitol levels were slightly lower in Dlxr1 strains. Deletion of lxr1 did also not affect the amount of conidiospores produced using either complete or minimal media (data not shown). 4. Discussion We provide conclusive evidence that LXR1 is not the L-xylulose reductase involved in fungal L-arabinose catabolism as previously claimed [3] but that it is a D-mannitol 2-dehydrogenase. Consequentially, also protein entries in the NCBI database based on sequence similarity to H. jecorina LXR1 (conf. Fig. 2) or follow-up publications [18] should be revised. MDHs belong to the short chain dehydrogenase/reductase superfamily which have a broad substrate spectrum [19]. These characteristics make it obviously impossible to predict a correct functional assignment based on enzymatic properties only. But which protein is now the true LXR? Unfortunately, only one further fungal LXR was described which is NAD+ dependent [22] and NADP+ dependent LXRs are only known from mammals [20,21]. Therefore further efforts will be necessary to assess if the true fungal NADP+ dependent LXR is related to one of these proteins. From our results and other studies [1,23] the exact physiological role of MDHs remains unclear: D-mannitol metabolism occurs at least via two different routes involving MDH or mannitol 1phosphate dehydrogenase (MPD; Fig. 1). In addition to a residual NADP+ dependent MDH activity in Dlxr1 strains on some carbon sources, we also found evidence for a NAD+ dependent MDH activity (unpublished results) which would represent a third option for D-mannitol metabolism. Genetic evidence from studies in plant pathogenic fungi suggests that the two different routes can at least partially compensate for the lack of the other one. Stagonospora nodorum Dmdh1 strains were significantly hindered in their growth on D-mannitol whereas loss of MPD1 prevented growth at all. Mpd1 deletion affected conidial mannitol levels and Dmdh1Dmpd1 strains produced only traces of mannitol. In Alternaria alternata growth on D-mannitol was affected in Dmpd1 and Dmdh1Dmpd1 strains but D-mannitol biosynthesis depended on both genes. For the saprobe A. niger MPD deficient strains do not accumulate any mycelial D-mannitol but still produce a third of the conidial wild-type levels [24] whereas growth on D-mannitol was not affected. Therefore only a comparison of the respective single and double deletion strains will give us further clues about the relevance of LXR1 in D-mannitol metabolism in H. jecorina. Acknowledgements This study was supported by the Austrian Science Foundation (P19421) and RV by the Dutch Technology Foundation STW, Applied Science Division of NWO and the Technology Program of the Ministry of Economic Affairs, Project No. 07063.
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References [1] Solomon, P.S., Waters, O.D. and Oliver, R.P. (2007) Decoding the mannitol enigma in filamentous fungi. Trends Microbiol. 15, 257–262. [2] O’Neil, M.A. and York, W.S. (2003) in: The Plant Cell Wall (Rose, J.K.C., Ed.), pp. 1–54, Blackwell, Oxford. [3] Richard, P., Putkonen, M., Vaananen, R., Londesborough, J. and Penttilä, M. (2002) The missing link in the fungal L-arabinose catabolic pathway, identification of the L-xylulose reductase gene. Biochemistry 41, 6432–6437. [4] Hult, K. and Gatenbeck, S. (1978) Production of NADPH in the mannitol cycle and its relation to polyketide formation in Alternaria alternata. Eur. J. Biochem. 88, 607–612. [5] Dijksterhuis, J. and de Vries, R.P. (2006) Compatible solutes and fungal development. Biochem. J. 399, e3–5. [6] Mandels, M.M. and Andreotti, R.E. (1978) The cellulose to cellulase fermentation. Proc. Biochem. 13, 6–13. [7] Bos, C.J., Debets, A.J., Swart, K., Huybers, A., Kobus, G. and Slakhorst, S.M. (1988) Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger. Curr. Genet. 14, 437–443. [8] de Groot, M.J., van de Vondervoort, P.J., de Vries, R.P., vanKuyk, P.A., Ruijter, G.J. and Visser, J. (2003) Isolation and characterization of two specific regulatory Aspergillus niger mutants shows antagonistic regulation of arabinan and xylan metabolism. Microbiology 149, 1183–1191. [9] Mach, R.L., Schindler, M. and Kubicek, C.P. (1994) Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals. Curr. Genet. 25, 567–570. [10] Seiboth, B., Hartl, L., Pail, M., Fekete, E., Karaffa, L. and Kubicek, C.P. (2004) The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on D-galactose. Mol. Microbiol. 51, 1015– 1025. [11] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (2008)Current Protocols in Molecular Biology, Wiley Interscience, New York. [12] Hartl, L., Kubicek, C.P. and Seiboth, B. (2007) Induction of the gal pathway and cellulase genes involves no transcriptional inducer function of the galactokinase in Hypocrea jecorina. J. Biol. Chem. 282, 18654–18659. [13] vanKuyk, P.A., de Groot, M.J., Ruijter, G.J., de Vries, R.P. and Visser, J. (2001) The Aspergillus niger D-xylulose kinase gene is co-expressed with genes encoding arabinan degrading enzymes, and is essential for growth on D-xylose and Larabinose. Eur. J. Biochem. 268, 5414–5423. [14] de Groot, M.J.L., van den Dool, C., Wösten, H.A.B., Levisson, M., vanKuyk, P.A., Ruijter, G.J.G. and de Vries, R.P. (2007) Regulation of pentose catabolic pathway genes of Aspergillus niger. Food Technol. Biotechnol. 45, 134–138. [15] Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. [16] Nicholas Jr., H.B. and McClain, W.H. (1987) An algorithm for discriminating sequences and its application to yeast transfer RNA. Comput. Appl. Biosci. 3, 177–181. [17] Kumar, S., Tamura, K. and Nei, M. (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 5, 150–163. [18] Nair, N. and Zhao, H. (2007) Biochemical characterization of an L-xylulose reductase from Neurospora crassa. Appl. Environ. Microbiol. 73, 2001–2004. [19] Kallberg, Y., Oppermann, U., Jornvall, H. and Persson, B. (2002) Short-chain dehydrogenase/reductase (SDR) relationships: a large family with eight clusters common to human, animal, and plant genomes. Protein Sci. 11, 636–641. [20] Ishikura, S., Isaji, T., Usami, N., Kitahara, K., Nakagawa, J. and Hara, A. (2001) Molecular cloning, expression and tissue distribution of hamster diacetyl reductase. Identity with L-xylulose reductase. Chem. Biol. Interact. 130–132, 879–889. [21] Nakagawa, J. et al. (2002) Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney. J. Biol. Chem. 277, 17883–17891. [22] Verho, R., Putkonen, M., Londesborough, J., Penttilä, M. and Richard, P. (2004) A novel NADH-linked L-xylulose reductase in the L-arabinose catabolic pathway of yeast. J. Biol. Chem. 279, 14746–14751. [23] Velez, H., Glassbrook, N.J. and Daub, M.E. (2007) Mannitol metabolism in the phytopathogenic fungus Alternaria alternata. Fungal Genet. Biol. 44, 258–268. [24] Ruijter, G.J., Bax, M., Patel, H., Flitter, S.J., van de Vondervoort, P.J., de Vries, R.P., vanKuyk, P.A. and Visser, J. (2003) Mannitol is required for stress tolerance in Aspergillus niger conidiospores. Eukaryot. Cell 2, 690–698.
journal homepage: www.FEBSLetters.org
The Hypocrea jecorina (syn. Trichoderma reesei) lxr1 gene encodes a D-mannitol dehydrogenase and is not involved in L-arabinose catabolism Benjamin Metz a,1, Ronald P. de Vries b,1, Stefan Polak a, Verena Seidl a, Bernhard Seiboth a,* a b
Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Vienna, Getreidemarkt 9, A-1060 Wien, Austria Microbiology, Utrecht University, Utrecht, The Netherlands
a r t i c l e
i n f o
Article history: Received 27 January 2009 Revised 5 March 2009 Accepted 13 March 2009 Available online 21 March 2009 Edited by Judit Ovádi Keywords: L-Arabinose L-Xylulose reductase D-Mannitol metabolism Short chain dehydrogenase/reductase Aspergillus niger
a b s t r a c t The Hypocrea jecorina LXR1 was described as the first fungal L-xylulose reductase responsible for NADPH dependent reduction of L-xylulose to xylitol in L-arabinose catabolism. Phylogenetic analysis now reveals that LXR1 forms a clade with fungal D-mannitol 2-dehydrogenases. Lxr1 and the orthologous Aspergillus niger mtdA are not induced by L-arabinose but expressed at low levels during growth on different carbon sources. Deletion of lxr1 does not affect growth on L-arabinose and Lxylulose reductase activity remains unaltered whereas D-mannitol 2-dehydrogenase activities are reduced. We conclude that LXR1 is a D-mannitol 2-dehydrogenase and that a true LXR1 is still awaiting discovery. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Fungi posses different metabolic pathways for the conversion of carbohydrates that are absent in other microorganisms. Examples of such fungal specific pathways are L-arabinose catabolism and D-mannitol metabolism (Fig. 1). Although both pathways are widespread over the fungal kingdom and of importance for the fungal lifestyle, they still await full elucidation. One reason for this is the fact that these pathways are not found in the model fungus Saccharomyces cerevisiae and received therefore less attention [1]. L-Arabinose is an abundant pentose found in different carbohydrate polymers of the plant cell wall [2]. Its degradation is therefore important for the cycling of carbon in our biosphere and has an impact on biotechnological processes which use plant materials as renewable carbon source for the production of e.g. biofuels. To ensure complete degradation of this pentose, the genes of the fungal L-arabinose catabolizing pathway were introduced in S. cerevisiae. This included the cloning of the first fungal gene encoding an L-xylulose reductase (LXR1) from the ascomycete Hypocrea jecorina (syn.: Trichoderma reesei) which together with other L-arabinose pathway genes enabled this S. cerevisiae strain to grow on L-arabinose [3]. A comparison of the amino acid sequence of the H. jecorina LXR1 to other proteins showed that highest similarity was * Corresponding author. Fax: +43 1 58801 17299. E-mail address: [email protected] (B. Seiboth). 1 B.M. and R.V. contributed equally to this article.
found to the Agaricus bisporus D-mannitol 2-dehydrogenase. The amino acid similarity was also reflected by its enzymatic properties: The enzyme catalyzed the NADPH/NADP+ specific reactions for L-xylulose/xylitol but also for D-fructose/D-mannitol, which is the typical substrate pair for D-mannitol 2-dehydrogenases (MDH). The polyol D-mannitol accumulates intracellularly to high levels in many fungal species and originally, a fungal D-mannitol cycle for NADPH regeneration was proposed [4]. However, recent genetic evidence suggests that a D-mannitol cycle does not exist and challenged also the proposed roles of D-mannitol in fungi [1,5]. The aim of the present study was therefore to assess the physiological role of the H. jecorina LXR1 in fungal L-arabinose catabolism and D-mannitol metabolism.
2. Materials and methods 2.1. Strains and culture conditions H. jecorina QM9414 (ATCC 26921) was maintained on potato dextrose agar. Strains were grown in 1 l Erlenmeyer flasks on a rotary shaker (250 rpm) at 28 °C in 250 ml medium [6] with the respective carbon source (1% w/v). For transfer cultures mycelia were transferred after 22 h of pregrowth on glycerol to flasks containing the indicated carbon source. Aspergillus niger strains N402 (cspA1) [7] and NW321 (cspA1, fwnA6, leuA1, araA4) [8] are both derived from A. niger N400 (CBS120.49). Transfer experiments were
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.03.027
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B. Metz et al. / FEBS Letters 583 (2009) 1309–1313
Fig. 1. Fungal L-arabinose catabolism and D-mannitol metabolism.
performed as described previously [8]. Escherichia coli strain JM109 (Promega) was used for plasmid propagation. For growth tests plates were inoculated with a small piece of agar in the centre. In submerged cultures, the increase in dry biomass was recorded by washing mycelial samples and drying them to constant weight at 80 °C. 2.2. Plasmid constructions and H. jecorina transformation For the lxr1 deletion cassette the upstream region of lxr1 was amplified using the primers lxrXba 50 -CAATGTTCTAGAGCATATTGACC-30 and lxrHind 50 -GATGGGCGAAGCTTAGGATG-30 . The XbaI/ HindIII restricted amplicon was ligated with the HindIII/XhoI restricted hph expression cassette [9] into a XbaI/XhoI restricted pBluescript SK(+) (Stratagene). The downstream region was amplified with the primers lxrXho 50 -CACTACTCGAGAAAGAAATAATG-30 and lxrAcc65 50 -CATTAGAGCGGTACCTCTTCC-30 and introduced via XhoI/Acc65I. From the resulting vector the 5.9 kb XbaI/Acc65I fragment was used in fungal transformations [9]. 2.3. Nucleic acid isolation and hybridization DNA and RNA extraction, electrophoresis, blotting and hybridization of nucleic acids with random primed probes was described previously [10,11]. Probes were a 1.6 kb lxr1 upstream region fragment, a 900 bp lxr1 amplicon containing the coding region (LXRgexfw 50 -CATCGGGATCCATGCCTCAGCCTGTC-30 and LXRgexrv 50 -CATTGAATTCTTATCGTGTAGTGTAACCT-30 ) and a 300 bp 18S rRNA fragment [12]. Transcript analysis and probes for A. niger were described [8,13,14].
MEGA 2.1 [17] using Neighbour Joining as a distance algorithmic method. Stability of clades was evaluated by 1000 bootstrap rearrangements. Bootstrap values lower than 50% are not displayed. 2.5. Enzyme assays and mannitol determination Cell-free extracts were prepared as described [12]. For enzyme activities the rate of change in absorbance at 340 nm for NAD(P)H oxidation or NAD(P)+ reduction at 30 °C was determined. D-Mannitol assays consisted of 100 mM Tris–HCl (pH 9.0), 0.5 mM MgCl2, 2 mM NAD(P)+ and 100 lg cell free extract and were started by adding D-mannitol (final conc. 100 mM). D-Fructose (L-xylulose) reduction was measured in 100 mM HEPES–NaOH (pH 7.0), 2 mM MgCl2, 0.2 mM NAD(P)H and 100 lg cell free extract and started by adding the sugar (final conc. 250 mM [100 mM]). Activities are expressed as nkat and are given as specific activities (nkat/mg protein). D-Mannitol was determined by HPLC using a Biorad Aminex HPX-87H Ion Exclusion column. The sugars were separated under isocratic conditions with 10 mM sulfuric acid at a flow rate of 0.5 ml/min and 40 °C. A range of single sugar standards were run before the analysis. 2.6. Microscopical analysis Samples were examined by using differential interference contrast optics on an inverted Nikon TE2000 microscope and imaged with a Nikon DXM1200F digital camera. 3. Results
2.4. Phylogenetic analysis
3.1. Phylogeny of putative D-mannitol dehydrogenases and L-xylulose reductases
Protein sequences were aligned with CLUSTALX 1.8 [15], visually adjusted using GENEDOC 2.6 [16] and phylogenetically analyzed in
A BLASTP search with the H. jecorina LXR1 as query against the NCBI database revealed that it has about equally similar sequence
B. Metz et al. / FEBS Letters 583 (2009) 1309–1313
identities (2e127–3e108) to different other fungal proteins which are either annotated as LXRs or MDHs. For most of these proteins a functional characterization is not available and we therefore assume that they were simply annotated according to their similarity as either LXRs or MDHs. The phylogenetic relationship of this group showed that the different LXRs and MDHs group closely together (Fig. 2). This analysis also demonstrates that all of these proteins are orthologous with the exception of Penicillium chrysogenum where two proteins are found. Gene duplication might have been one possible explanation since the resulting paralogous often evolve new functions after their duplication. 3.2. Regulation of lxr1/mtdA transcription by carbon sources Transcript profiling on a variety of carbon sources showed that low levels of transcripts of H. jecorina lxr1 were found on all tested carbon sources only after long exposure times (Fig 3A), but – in contrast to what would be typical for a gene involved in the L-arabinose pathway – no enhanced transcript levels were observed during growth on L-arabinose or L-arabitol. Notably, we also were not able to find higher transcript levels on D-mannitol as carbon source.
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In A. niger, regulatory mutants for the L-arabinose pathway have been described and it was demonstrated that genes encoding Larabitol dehydrogenase and xylulokinase have reduced or absent transcript levels in these mutants during growth on L-arabinose or L-arabitol [8,14]. We therefore included a transcriptional analysis of the orthologous A. niger mtdA. Similarly, it exhibits low expression levels on L-arabinose and L-arabitol which were not affected in the araA mutant (Fig. 3B). 3.2. Loss of lxr1 does not influence L-arabinose catabolism but affects 2-dehydrogenase activities
D-mannitol
The role of lxr1 was investigated by targeted gene deletion. Thereby the coding region of lxr1 was replaced by a hygromycin B expression cassette. About 10% of the H. jecorina transformants were deleted in lxr1 (Fig. 4A). All of these Dlxr1 strains grew normally on all carbon sources tested including L-arabinose and Larabitol but also D-mannitol either on solid (Fig. 4B) or in liquid medium (data not shown). The absence of growth retardation in these strains could be due to the possibility that another enzyme replaces at least partially the function of LXR1 or that the activity of LXR1 is not a limiting factor in the catabolism of these carbohydrates. Therefore we compared the NADPH dependent LXR
Fig. 2. Phylogeny of putative L-xylulose reductases and D-mannitol dehydrogenases. Phylogenetic analyses were performed using Neighbour Joining. Numbers below nodes indicate the bootstrap value. The bar marker indicates the genetic distance, which is proportional to the number of amino acid substitutions. GenBank accession numbers are given in brackets.
Fig. 3. Northern analysis of H. jecorina lxr1 after 6 h (A) and A. niger mtdA after 2 h (B) following the transfer of pregrown mycelia to new media with different carbon sources. The mtdA transcript profile is compared to the L-arabinose pathway gene ladA as reported previously [14].
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activities of cell free extracts grown on L-arabinose. Similar activities were found in Dlxr1 strains and the parental QM9414 (100 ± 10 nkat/mg). However, there was a significant reduction in NADP+ dependent D-mannitol 2-dehydrogenase activity in the Dlxr1 strains during growth on different carbon sources (Fig. 4C). We conclude that lxr1 is not involved in the L-arabinose catabolism but that it encodes a D-mannitol 2-dehydrogenase which is not necessary for D-mannitol catabolism.
3.3. Mannitol metabolism MDH activity is present in conidiospores of different Aspergilli and was implicated in mannitol metabolism during germination [1,5]. We therefore investigated the effect of the lxr1 deletion on germination (Fig. 5). In QM9414, lxr1 transcript levels were shortly upregulated during the initial stages of germination (data not shown) which is reflected by an increase in NADP+ dependent
Fig. 4. (A) Southern analysis of Dlxr1 strains. EcoRV digested chromosomal DNA was probed with a 1.6 kb lxr1 fragment. Deletion of lxr1 led to an increase of the original 4.6 kb lxr1 fragment to 6.5 kb. (B) Growth comparison of a Dlxr1 strain to the parental QM9414 after 3.5 days. (C) Comparison of NADP+ dependent D-mannitol 2dehydrogenase activities during growth on different carbon sources after 24 h.
Fig. 5. Comparison of morphology (bars = 10 lm), mannitol levels and NADP+ dependent MDH activity formation during germination in QM9414 and Dlxr1 during shake flask cultivation on D-glucose.
B. Metz et al. / FEBS Letters 583 (2009) 1309–1313
MDH activity followed by a sharp decrease of activity after around 12 h of growth. The high conidial mannitol levels decreased rapidly in initial stages of germination before they were replenished. Deletion of lxr1 did neither affect the germination process nor the hyphal morphology. Although MDH activities were almost not detectable in Dlxr1 strains, the mannitol levels were comparable to QM9414 in conidiospores and during the first hours of germination including the initial mannitol catabolism which was followed by mannitol biosynthesis. Only during later stages mannitol levels were slightly lower in Dlxr1 strains. Deletion of lxr1 did also not affect the amount of conidiospores produced using either complete or minimal media (data not shown). 4. Discussion We provide conclusive evidence that LXR1 is not the L-xylulose reductase involved in fungal L-arabinose catabolism as previously claimed [3] but that it is a D-mannitol 2-dehydrogenase. Consequentially, also protein entries in the NCBI database based on sequence similarity to H. jecorina LXR1 (conf. Fig. 2) or follow-up publications [18] should be revised. MDHs belong to the short chain dehydrogenase/reductase superfamily which have a broad substrate spectrum [19]. These characteristics make it obviously impossible to predict a correct functional assignment based on enzymatic properties only. But which protein is now the true LXR? Unfortunately, only one further fungal LXR was described which is NAD+ dependent [22] and NADP+ dependent LXRs are only known from mammals [20,21]. Therefore further efforts will be necessary to assess if the true fungal NADP+ dependent LXR is related to one of these proteins. From our results and other studies [1,23] the exact physiological role of MDHs remains unclear: D-mannitol metabolism occurs at least via two different routes involving MDH or mannitol 1phosphate dehydrogenase (MPD; Fig. 1). In addition to a residual NADP+ dependent MDH activity in Dlxr1 strains on some carbon sources, we also found evidence for a NAD+ dependent MDH activity (unpublished results) which would represent a third option for D-mannitol metabolism. Genetic evidence from studies in plant pathogenic fungi suggests that the two different routes can at least partially compensate for the lack of the other one. Stagonospora nodorum Dmdh1 strains were significantly hindered in their growth on D-mannitol whereas loss of MPD1 prevented growth at all. Mpd1 deletion affected conidial mannitol levels and Dmdh1Dmpd1 strains produced only traces of mannitol. In Alternaria alternata growth on D-mannitol was affected in Dmpd1 and Dmdh1Dmpd1 strains but D-mannitol biosynthesis depended on both genes. For the saprobe A. niger MPD deficient strains do not accumulate any mycelial D-mannitol but still produce a third of the conidial wild-type levels [24] whereas growth on D-mannitol was not affected. Therefore only a comparison of the respective single and double deletion strains will give us further clues about the relevance of LXR1 in D-mannitol metabolism in H. jecorina. Acknowledgements This study was supported by the Austrian Science Foundation (P19421) and RV by the Dutch Technology Foundation STW, Applied Science Division of NWO and the Technology Program of the Ministry of Economic Affairs, Project No. 07063.
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