Section Molecular Genetics of Industrial Micro-organisms, Wageningen University, Dreijenlaan 2, NL-6703HA Wageningen, The Netherlands
Correspondence
Marco J. L. de Groot
Marco.de.Groot{at}BPRC.nl
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ABSTRACT |
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Present address: Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, NL-6709 PD Wageningen, The Netherlands.
Present address: Microbiology, Utrecht University, Padualaan 8, NL-3584CH Utrecht, The Netherlands.
||Present address: Leiden University, Institute Biology Leiden, Clusius Laboratory, Dept of Fungal Genetics and Metabolomics, NL-2333 AL Leiden.
¶Present address: Department of Clinical Genetics, Leiden University Medical Centre, PO Box 9600, NL-2300RC Leiden, The Netherlands.
#Present address: Fungal Genetics and Technology Consultancy, PO Box 396, NL-6700AJ Wageningen, The Netherlands.
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INTRODUCTION |
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Arabinan is another polysaccharide found in plant cell wall heteropolysaccharides as a side chain of pectin (de Vries & Visser, 2001). A. niger is able to degrade arabinan to L-arabinose using its arabinanolytic system. To date three arabinanases have been characterized: two
-L-arabinofuranosidases (ABFA and ABFB) and an arabinan 1,5-
-L-arabinanase (ABNA) (van der Veen et al., 1991
). L-arabinose can be used by the fungus as a carbon source via the L-arabinose catabolic pathway. This pathway converts L-arabinose to D-xylulose 5-phosphate, which is further metabolized via the non-oxidative pentose phosphate pathway. Two A. niger genes involved in pentose metabolism have previously been cloned and characterized: xyrA, encoding D-xylose reductase (Hasper et al., 2000
), and xkiA, encoding D-xylulose kinase (vanKuyk et al., 2001
).
Little is known about the regulation of the arabinanase system. Arabinanolytic genes are specifically induced when A. niger is grown on arabinan-containing substrates or the monomeric compounds L-arabinose and L-arabitol (Flipphi et al., 1994; Gielkens et al., 1997
, 1999
; Ramon et al., 1993
; van der Veen et al., 1991
, 1993
). One of the intermediates of the L-arabinose catabolic pathway, L-arabitol, is believed to be the low-molecular-mass inducer of the system (van der Veen et al., 1993
). In Aspergillus nidulans it has been shown that increased intracellular arabitol accumulation correlates with higher production of the enzymes involved in arabinan breakdown (de Vries et al., 1994
), suggesting that L-arabitol is the true inducer of this system. The genes encoding three arabinan-degrading enzymes, abfA, abfB and abnA, have been cloned and characterized (Flipphi et al., 1993a
, b
, c
). Expression analysis of these genes showed that they are co-ordinately expressed. Moreover, insertion of additional copies of one of the arabinanase genes in A. niger resulted in lower expression of the other two genes, suggesting a titration effect at the level of a single specific transcriptional activator (Flipphi et al., 1994
).
The aim of this study was to find genetic evidence for a positive-acting specific regulatory system involved in the induction of arabinanases in A. niger. Furthermore we wanted to assess whether the same system regulates induction of the intracellular enzymes of the L-arabinose catabolic pathway, and investigate the interaction between the regulation of the arabinanase and xylanase systems.
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METHODS |
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Genetic localization of the araA4 and araB2 mutations was determined by somatic recombination using test strain NW148 as described by Bos et al. (1988).
Complementation tests of the araA4 mutation with other mutations were performed using heterokaryons. Strain NW320 was isolated from somatic recombination experiments using strains NW148//NW316. Heterokaryons were selected from NW320 with some of the other ara mutants isolated from NW315. Balanced heterokaryons, recognizable by a good mixture of fawn-coloured and black conidiospores, were tested for growth on MM with nicotinamide and L-arabinose, and MM with nicotinamide and D-glucose. NW320 and NW322 were used as negative controls and N423 as a positive control.
Preparation of cell extracts and protein determination.
Mycelia were harvested and disrupted as described previously (Witteveen et al., 1989). Cell extracts were obtained by suspending disrupted frozen mycelium in extraction buffer (10 mM Bistris, 5 mM MgCl2, 1 mM 2-mercaptoethanol, 0·5 mM EDTA pH 6·3) followed by centrifugation at 15 00020 000 g in order to remove cell debris. The entire procedure was performed at 0 to +4 °C. After denaturation with sodium deoxycholate and precipitation of protein with trichloroacetic acid (Bensadoun & Weinstein, 1976
) protein concentrations were determined using the bicinchoninic acid method as described by the manufacturer (Sigma).
Fractionation of cell extracts.
Cell extracts were fractionated by anion-exchange chromatography. Two millilitres of cell extract was loaded on a ResourceQ column with a bed volume of 1 ml (Pharmacia Biotech). The column was washed with 8 ml extraction buffer. Elution was started with a gradient of 00·5 M NaCl over 18 column volumes. The flow rate used was 3 ml min-1. Fractions of 0·5 ml were collected during this gradient and kept on ice until they were used in the enzyme activity measurements.
Enzyme assays.
All enzyme assays were performed at 30 °C. L-Arabitol dehydrogenase and xylitol dehydrogenase activities were determined using 100 mM glycine pH 9·6, 0·2 mM NAD+ and 40 mM L-arabitol or xylitol, respectively. L-arabinose reductase and D-xylose reductase activities were determined using 50 mM sodium phosphate pH 7·8, 0·2 mM NADPH and 40 mM L-arabinose or D-xylose, respectively. Absorbance changes at 340 nm ( =6·22 mM-1 cm-1) were measured using a Cobas Bio autoanalyser (Roche) or a UV-2501PC spectrophotometer (Shimadzu Scientific Instruments).
Extracellular hydrolytic activities were assayed as follows using p-nitrophenyl -L-arabinofuranoside (pnp-a) or p-nitrophenyl
-D-xylopyranoside (pNP-X) as substrates: 0·01 % pNP-substrate, 2040 µl sample and 25 mM sodium acetate pH 5·0 were used in a total volume of 100 µl, incubated for 1 h at 30 °C (pNP-A) or 50 °C (pNP-X) and the reaction was subsequently stopped by adding 100 µl 0·25 M Na2CO3. Absorbance was measured at 405 nm in a microtitre platereader (Molecular Devices). The activity was calculated using a standard curve ranging from 0 to 80 nmol p-nitrophenol per assay volume.
Polyol extraction and determination.
Intracellular polyols were extracted as described previously (Witteveen et al., 1994). Polyol concentrations were measured by HPAEC (Dionex) with a Carbopac MA1 column using isocratic elution with 0·48 M NaOH.
Statistical analysis.
Statistical analysis of the polyol and enzyme levels was carried out using the Student's standard t-test with a reliability interval of 95 %.
Expression analysis.
Total RNA was isolated from powdered mycelium using TRIzol Reagent (Life Technologies), according to the supplier's instructions. For Northern blot analysis 3 µg total RNA was incubated with 3·3 µl 6 M glyoxal, 10 µl DMSO and 2 µl 0·1 M sodium phosphate pH 7 in a total volume of 20 µl for 1 h at 50 °C to denature the RNA. RNA electrophoresis was performed on a 1·5 % agarose gel using 0·01 M sodium phosphate buffer pH 7 and transferred to Hybond-N filters (Amersham) by capillary blotting. Filters were hybridized at 42 °C in a solution of 50 % (v/v) formamide, 10 % (w/v) dextran sulphate, 0·9 M NaCl, 90 mM trisodium citrate, 0·2 % (w/v) Ficoll, 0·2 % (w/v) polyvinylpyrrolidone, 0·2 % (w/v) bovine serum albumin, 0·1 % (w/v) SDS and 100 µg single-stranded herring sperm DNA ml-1. Washing was performed under stringent conditions using 30 mM NaCl, 3 mM trisodium citrate and 0·5 % (w/v) SDS at 68 °C. Probes used were: a 2·0 kb PstI fragment of abfA (Flipphi et al., 1994); a 2·8 kb PstI fragment of abfB (Flipphi et al., 1994
); a 3·1 kb HindIII fragment of abnA (Flipphi et al., 1994
); a 0·5 kb EcoRV/XhoI fragment of aguA (de Vries et al., 2001
); a 2·8 kb PstI/NsiI fragment of xlnD (van Peij et al., 1997
); a 0·3 kb SalI/XhoI fragment of xyrA (Hasper et al., 2000
); a 4·0 kb SstI fragment of xkiA (vanKuyk et al., 2001
) and a 0·7 kb EcoRI fragment from the gene encoding the 18S rRNA subunit (Melchers et al., 1994
). The 18S probe was used as an RNA loading control.
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RESULTS |
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Out of 105 spores of the xkiA1 strain NW315 which were irradiated, 20 colonies were purified. First, we tested these mutants for growth on D-xylose to exclude reversion of the xkiA1 mutation. Subsequently, derepression of growth on D-gluconate or L-alanine in the presence of L-arabinose, D-xylose or xylitol was tested. Three mutants (NW316, NW318 and NW319) showed increased growth on L-alanine or D-gluconate with L-arabinose and not with D-xylose or xylitol.
Genetic analysis of the ara mutations
Two ara mutants (NW316 and NW318) were subjected to a somatic recombination experiment with tester strain NW148 for several purposes. Firstly, to gather information on the genetic localization of the ara mutations and secondly to remove the xkiA1 mutation. From the somatic recombination of NW316 (araA4) and NW148, 85 recombinants were analysed. Recombination frequencies of the genetic markers on chromosome V with araA4 were 28 % for argB13, 31 % for nicA1 and 31 % for metB10. Recombination frequencies between araA4 and markers on the other seven chromosomes ranged from 37 % (trpB2) to 62 % (fwnA1). Recombination frequencies higher than 25 % are not considered to be absolute evidence for linkage, but since none of the markers on the other chromosomes showed a better linkage, we concluded that the araA4 mutation is located on chromosome V. From the somatic recombination of NW318 (araB3) and NW148, 109 recombinants were analysed. No convincing linkage was found with any of the markers used. Best linkage of araB3 was found with pyrA6 and bioA1, both located on chromosome III, with 38 % recombination. Since recombination percentages with the markers on the other chromosomes ranged from 43 % to 57 %, araB3 is probably located on chromosome III.
Complementation of the araA4 mutation with two other ara mutations was tested. Heterokaryons of strains NW318 (araB3) and NW319 (araA2) with NW320 (araA4) were isolated and tested for growth on L-arabinose and D-glucose. NW318 was found to complement the araA4 defect. NW319 did not complement araA4, indicating that the mutations designated araA2 and araA4 are allelic. NW315 and the mutants derived from it are pentose non-utilizing due to the xkiA1 mutation. Growth tests of xkiA+ recombinants of the original mutants showed that the araA and araB mutations resulted in strongly reduced growth on L-arabinose and L-arabitol and a slight decrease in growth on xylitol, whereas growth on D-xylose was not affected (Table 2).
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In order to visualize the relative level of L-arabinose reductase and D-xylose reductase a ratio of the activities was calculated (Fig. 1c). Both these enzymes are able to reduce L-arabinose and D-xylose, and they elute at almost the same salt concentration during the fractionation of cell extracts. As D-xylose reductase activity is not induced in the
xlnr strain on D-xylose (Hasper et al., 2000
) we concluded that a ratio of L-arabinose reductase/D-xylose reductase activity of 0·5 to 0·6 represents a situation in which D-xylose reductase is mostly present, and as one would expect to find only L-arabinose reductase when inducing the wild-type on L-arabinose a ratio of 1·9 to 2·2 represents a situation in which L-arabinose reductase is predominantly present. In the wild-type strain L-arabinose reductase is induced on L-arabinose and D-xylose reductase on D-xylose. In the
xlnR strain the L-arabinose reductase/D-xylose reductase ratio was 2 on both L-arabinose and D-xylose, which is consistent with the absence of expression of xyrA encoding D-xylose reductase (Hasper et al., 2000
). The L-arabinose reductase/D-xylose reductase ratio in the araa and arab strains after incubation on L-arabinose and D-xylose resembled that of the wild-type grown on D-xylose. In other words only D-xylose reductase can be detected and L-arabinose reductase was not present in the ara mutant strains.
-L-Arabinofuranosidase is absent in the araA and araB strains
Extracellular -L-arabinofuranosidase activity levels (Fig. 2
a) were strongly decreased in the araA and araB strains compared to the wild-type and
xlnR strains when grown on L-arabinose. There also appeared to be some production of
-L-arabinofuranosidase by the wild-type when grown on D-xylose, although not to the same level as when grown on L-arabinose. In NW321 (araA), NW322 (araB) and NW199 (
xlnR) there was no production of
-L-arabinofuranosidase on D-xylose apart from the basal level observed on D-glucose.
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The araA and araB strains accumulate arabitol
Intracellular arabitol levels (Fig. 3a) were at least fivefold higher in the araA and araB strains grown on L-arabinose than in the wild-type and
xlnR strains. Arabitol was present at a low level in all four strains on D-xylose and D-glucose. The arabitol produced during growth on D-glucose is most likely D-arabitol produced from an intermediate of the pentose phosphate pathway. Xylitol levels (Fig. 3b
) were lower in the araA and araB mutants compared to the wild-type strain on L-arabinose, but comparable to the level in the wild-type on D-xylose. Xylitol was not detected in mycelium grown on D-glucose in any of the four strains.
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Expression of genes of the arabinanase system is decreased in the ara mutants
Expression of the known L-arabinose-induced genes, abfA and abfB, was severely decreased or completely absent in the araA and araB strains on L-arabinose or L-arabitol compared to the expression in the wild-type (Fig. 4). The data show that xkiA is regulated by both L-arabinose and D-xylose induction. Expression of xkiA is normal in the araA and araB strains on D-xylose. In the
xlnR strain a higher expression level of the arabinanase-encoding genes and xkiA was detected. Conversely, the expression of the XLNR-regulated genes, xlnD, aguA and xyrA, was decreased or absent in the
xlnR strain and elevated in the two ara mutants. Expression of xyrA in the
xlnR strain is less than in the wild-type strain but not zero.
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DISCUSSION |
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L-Arabitol, the most potent inducer of arabinanase biosynthesis and L-arabinose catabolism, accumulates to much higher levels in the araA and araB strains than in the wild-type, indicating that the lack of expression and production of the arabinanase system is not due to lack of inducer accumulation. Therefore, we propose that araA and araB are mutations in components of the arabinanolytic regulatory system. These mutations are either at the level of the transcriptional activator itself, or at the level of a second element which modulates the transcriptional activator activity. The intracellular accumulation of arabitol in the araA and araB mutants in the presence of L-arabinose can be explained by the ability of D-xylose reductase to reduce L-arabinose to L-arabitol and the low L-arabitol dehydrogenase activity.
The accumulation of arabitol also indicates that L-arabinose is still taken up in the araA and araB mutants. This indicates that L-arabinose uptake is not strictly regulated by araA or araB. If araA and araB affect not only L-arabinose catabolism, but also an L-arabinose transport system, this would indicate the presence of a second transporter for L-arabinose that is not under control of araA or araB. Alternatively, L-arabinose uptake might be completely independent of araA and araB.
The expression of xkiA, encoding D-xylulose kinase, was previously shown not to be controlled by XLNR (vanKuyk et al., 2001), even though expression was observed on both L-arabinose and D-xylose. This suggests that expression of xkiA on L-arabinose is induced by the regulatory system that also activates the expression of the arabinanolytic genes.
Genes of the xylanolytic system, such as xlnD and aguA, were not expressed in the xlnR disruptant (Fig. 3 xlnD, aguA), which is in agreement with previous results (van Peij et al., 1998a
). The expression of xyrA, encoding D-xylose reductase, was previously reported to be controlled by XLNR (Hasper et al., 2000
) and absence of expression of xyrA in a xlnR disruption strain was reported in that study. In our investigation, however, expression of xyrA in the same xlnR disruption strain was reduced compared to wild-type but not absent. XLNR control of xyrA expression is therefore not absolute. The differences in expression level between the two studies in the xlnR disruption strain can be explained by the different D-xylose concentrations used. It was demonstrated previously (de Vries et al., 1999
) that apart from being an inducer of xylanolytic gene expression mediated by XLNR, D-xylose also represses the expression of these genes. This repression is stronger at higher xylose concentrations. In our study we used 15 mM D-xylose whereas Hasper et al. (2000)
used 50 mM, resulting in lower expression levels for xyrA.
Expression of xlnD and aguA was increased in the araA and araB mutants, especially on L-arabinose, compared to the expression in the wild-type. Conversely, expression of abfA, abfB, abnA and xkiA was higher in the xlnR disruptant compared to the wild-type. This suggests an interaction between the xlnR and the ara system and may be explained by a relief of repression when either regulatory system is not functional or by a change in intracellular inducer concentration. The latter was observed for L-arabitol; however, the L-arabitol concentration was much higher in the araA and araB mutants compared to the wild-type, which suggests a high level of induction if the regulatory system was present (de Vries et al., 1994; van der Veen et al., 1993
). The intracellular concentration of D-xylose, which is believed to be the inducer of the XLNR system, cannot be measured accurately. This supports a model (Fig. 5
) in which the repression caused by the presence of D-xylose or L-arabinose is relieved and that XLNR and the arabinose regulatory system are involved in the signalling of this repression. In the presence of L-arabinose, araA and araB play a role in the activation of carbon catabolite repression of other catabolic systems, e.g. xylanolytic functions. Mutations in araA or araB relieve this repression. This model explains how the mutants have been selected, i.e. selecting for a relief of repression of L-arabinose towards D-gluconate or L-alanine. This catabolite repression could be mediated either directly by the ara or XLNR system, or indirectly by the changes in metabolite levels.
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The function of the two specific inducers (L-arabitol and D-xylose), of the ara and XLNR systems respectively, both as an inducer for their own targets, and as carbon catabolite repressors mediated by CREA or by a more specific system, enable A. niger to respond specifically to the presence of different pentoses and hemicelluloses. Since the occurrence of these two pentoses in hemicellulose is never completely separate, such a system would function to fine-tune the regulation of the arabinanase- and xylanase-encoding genes. The metabolism of L-arabitol and D-xylose is linked, both by enzymic equilibrium and by the broad substrate specificity of the enzymes involved, and always leads to induction of both the arabinan- and xylan-degrading systems. The bias created by the inductionrepression system proposed reduces the production of unnecessary enzymes and is therefore beneficial to the organism.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 September 2002;
revised 31 December 2002;
accepted 24 January 2003.
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