Introduction

Plant polysaccharide degradation by fungi has been a topic of study for many decades due to the relevance of these enzymes for various industrial applications (food and feed, paper and pulp, detergents, beverages, bio-fuel, etc.). A detailed understanding of the regulatory systems that control the production of these enzymes in relevant fungi is essential to improve their production. For saprobic fungi, these systems are mainly aimed at liberating digestible carbon sources to support growth, and therefore, the hydrolysis of the polysaccharides is often linked to the metabolic conversion of the monomeric component. This is controlled at the transcriptional level by specific transcriptional activators and repressors, where the monomeric components of the polysaccharides are often the inducers of the transcriptional activators (de Vries and Visser 2001). Understanding the utilisation of the monomeric sugars will therefore also provide insight in the mechanism behind polysaccharide hydrolysis.

l-Arabinose and d-xylose are important components of the plant cell wall polysaccharides arabinan, arabinogalactan (substructures of pectin), xyloglucan and xylan. Aspergilli are able to release these pentoses from polysaccharides using a broad enzymatic system (reviewed by de Vries and Visser 2001). In Aspergilli, l-arabinose and d-xylose are catabolised through the pentose catabolic pathway (PCP; Witteveen et al. 1989). All corresponding genes and their regulation have been studied in Aspergillus niger (de Groot et al. 2007; Hasper et al. 2000; Mojzita et al. 2010a; Mojzita et al. 2010b; vanKuyk et al. 2001). Two regulatory systems that act in antagonistic fashion are involved in the PCP of A. niger, responding to d-xylose (xylanolytic regulator, XlnR; van Peij et al. 1998) and l-arabinose/l-arabitol (arabinolytic regulator, AraR; Battaglia et al. 2011).

XlnR regulates the first step (d-xylose reductase, xyrA) of the d-xylose pathway (Hasper et al. 2000). AraR predominantly regulates the l-arabinose pathway specific reactions; the l-arabitol dehydrogenase encoding gene (ladA) is under the control of AraR and indications that the l-arabinose reductase and l-xylulose reductase encoding genes (larA and lxrA) are under regulation of the arabinolytic system have been reported (Battaglia et al. 2011; de Groot 2005). AraR and XlnR both regulate the common steps of l-arabinose and d-xylose catabolism (xdhA, xkiA; Battaglia et al. 2011; de Groot et al. 2007).

The presence of the genes of the PCP as well as xlnR and araR is conserved in Aspergillus species (Battaglia et al. 2011; Flipphi et al. 2009; de Vries et al. 1994), but it is not clear if their expression and mechanism are identical in these fungi. In this study, we characterized AraR from A. nidulans and analysed its regulatory role, together with XlnR, in the PCP. In addition, we compared these results to those previously reported for A. niger to demonstrate the differences between these two species.

Materials and methods

Strains, media and growth conditions

The A. nidulans and A. niger strains used in this study are listed in Table 1. The A. nidulans AN031 strain was obtained from Prof. C. Scazzocchio (Institut de Genetique et Microbiologie, Orsay, France). A. niger and A. nidulans were grown in minimal medium (MM) or complete medium (CM; de Vries et al. 2004). For the growth of auxotrophic strains, the medium was supplemented with 0.2 g/l arginine, 0.2 g/l leucine, 0.2 g/l uridine and/or 1 mg/l nicotinamide. Agar was added at 1.5% (w/v) for solid medium. Liquid cultures were inoculated with 5 × 106 spores/ml and incubated on a rotary shaker at 220 rpm. Solid medium plates were inoculated with 1 × 103 spores. A. nidulans was cultivated at 37 °C and A. niger, at 30 °C.

Table 1 Aspergillus strains used in this study
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For transfer experiments, A. nidulans was grown for 16 h at 37 °C in CM with 1% d-fructose, after which mycelia aliquots were transferred to MM containing either 25 mM d-fructose, l-arabitol, d-xylose, xylitol or 25 mM l-arabinose. After 2 and/or 4 h, the mycelium was harvested, frozen in liquid nitrogen and stored at −80 °C. For chromosomal DNA isolation, the strains were grown overnight in CM containing 2% d-glucose and the necessary supplements. The mycelium was harvested and frozen in liquid nitrogen.

Molecular biology methods

General methods were performed according to standard procedures (Sambrook and Russell 2001), unless stated otherwise. For chromosomal DNA isolation, a standard phenol–chloroform extraction method was performed. For PCR and Southern blot analysis, 50–100 ng and 5–10 μg of chromosomal DNA were used, respectively. DNA fragments were amplified using an Expand High Fidelity PCR Amplification System (Roche). The 50 μl reaction mixture contained 5 μl of 10× Expand buffer, 3.5 units of the Expand DNA polymerase mixture, 200 μM dNTP mixture, 50 ng of a DNA template and 40 pmol of each primer. The ligations were performed using a Rapid Ligation Kit (Roche). The ligation mixtures were used to transform Escherichia coli One Shot® Top 10 competent cells. The transformants were selected on LB plates with 50 μg/ml of kanamycin. The plasmids and the primers used for DNA cloning are listed in Supplemental Table 1.

For the construction of plasmid pSFH1, araR was amplified with primers R1.7 T40 and R1.7 B3795. The PCR product was cloned into the pCR®-Blunt II-TOPO® (Invitrogen). To generate the deletion of ΔaraR, the araR 5′ flanking region was amplified with primers R1.7 SpeI T603 and R1.7 NdeI B4008. The PCR product was digested with SpeI/NdeI and used to replace the 546 bp SpeI/NdeI fragment of pSFH1. This construct was subsequently digested with NdeI/BstZ17I, and both ends were made blunt by Klenow DNA polymerase I fragment (Roche). The Aspergillus fumigatus pyrG gene (1.9 kb), digested by EcoRV from the pCDA21 vector (Chaveroche et al. 2000), was ligated in the NdeI/BstZ17I blunted vector, generating vector pSFH3. In the deletion construct, the pyrG gene replaces 214 bp of the 5′ flanking sequence upstream of the ATG codon and 301 bp of the coding part including the Zn2Cys6 binuclear cluster.

The deletion of xlnR coding region was performed by insertion of the argB gene between the flanking regions of xlnR. The 5′ and 3′ flanking regions (1.8 and 2 kb, respectively) were amplified by PCR with the primers xlnR T245 XhoI, xlnR B2067 KpnI, xlnR T3501 BamHI and xlnR B5510 NotI. The PCR product of the 5′ flanking region was digested with XhoI/KpnI, and subsequently ligated into pUC21 (Vieira and Messing 1991). The construct was digested with BamHI/NotI and used for the insertion of the 3′ xlnR PCR fragment. The resulting vector was digested with KpnI/BamHI and used for insertion of the A. nidulans argB gene (2.4 kb). This gene was amplified by PCR using primers argB T1 KpnI and argB B2690 BamHI and plasmid pIC20-arg as template DNA. The resulting plasmid pSFH4 was used to transform A. nidulans.

The primers used to generate probes for Northern analysis are listed in Supplemental Table 1. The probes were DIG-labelled using the PCR DIG Probe Syntheses Kit (Roche Applied Science) according to the supplier’s instructions.

Total RNA was isolated from powdered mycelium using a standard RNA isolation method with the TRIzol Reagent (Invitrogen). For Northern analysis, 3 μg total RNA was transferred to a Hybond-N+ membrane (Amersham Biosciences). Equal loading was determined by staining the blot for 5 min with 0.04% methylene blue, 0.5 M acetate pH 5.2 solution. The hybridization of the DIG-labelled probes was performed according to the DIG user’s manual (www.roche-applied-science.com). All blots were incubated overnight at 50 °C. The blots were exposed for 25 min up to 3 h to a Lumi-Film Chemiluminescent Detection Film (Roche Applied Science).

Transformation procedure and sexual crossing

The A. nidulans pyrG89 argB2 recipient (AN031) strain was used for deletion of araR and xlnR. A standard method of sorbitol/PEG transformation was applied (Tilburn et al. 1983). Transformants were screened for impaired growth on l-arabinose or a loss of β-d-xylosidase activity in a plate test with 4-methylumbelliferyl-β-d-xyloside for ΔaraR and ΔxlnR deletions, respectively. The sexual cross of the strains ΔaraR (argB2) and ΔxlnR (pyrG89) was performed as described previously (Todd et al. 2007). Prototrophs were isolated as ΔaraRΔxlnR double deletions for further analysis.

Genomic cluster comparison

The conservation of the genomic region flanking araR across ten Aspergillus genomes was visualized using Sybil, a web-based software for comparative genomics (Crabtree et al. 2007). The genomic sequence data and the Sybil comparative visualization tool were accessed through the Aspergillus genome database (AspGD; http://www.aspgd.org). The Aspergillus genomes used in the comparison comprised A. nidulans FGSC A4, A. niger CBS 513.88, A. niger ATCC 1015, Aspergillus terreus NIH2624, Aspergillus oryzae RIB40/ATCC 42149, Aspergillus flavus NRRL 3357, Aspergillus clavatus NRRL 1, Neosartorya fischeri NRRL 181, A. fumigatus A1163 and A. fumigatus Af293.

Enzymatic assays

The cell-free extract was prepared by adding 1 ml extraction buffer (50 mM K2HPO4, 5 mM MgCl2, 5 mM 2-mercaptoethanol, 0.5 mM EDTA) to the powdered mycelium. The mixtures were centrifuged for 10 min at 12,000 rpm at 4 °C. Intracellular enzyme measurements and activity calculations were performed as described previously (Battaglia et al. 2011). l-Xylulose reductase (Lxr) activity was determined using 50 mM Tris buffer (pH 7.8), 0.2 mM NADPH and 4.5 mM l-xylulose. The absorbance changes were measured at 340 nm in a Spectronic Unicam UV1 spectrophotometer.

Absorbance measurements were performed in triplicate. One unit of activity equals the conversion of 1 mmol of substrate per minute. Total protein concentrations were determined using a BCA protein assay kit (Pierce).

Results

Identification of A. nidulans AraR

BlastP analysis of A. niger AraR (An04g08600) revealed a close ortholog (AN0388.4) in A. nidulans. AN0388.4 is found on contig 7, which is located on chromosome VIII. BLAST alignment of A. niger and A. nidulans AraR revealed 70% DNA and 77% amino acid identity.

The genomic region surrounding this gene is conserved in the eight Aspergilli that were analysed (Fig. 1). The alignment of AraR proteins from eight Aspergilli showed highly conserved regions, particularly in the DNA-binding domain and C-terminal region (Fig. 2). The region between the second and third cysteine is nearly identical between AraR from A. niger (C2HARRVRC3) and A. nidulans (C2HSRRVRC3; Fig. 2) and differs significantly from this region in XlnR in both species (C2NQLRTKC3). This region was demonstrated to determine DNA-binding specificity in several zinc cluster proteins (Marmorstein et al. 1992; Marmorstein and Harrison 1994; Swaminathan et al. 1997; Walters et al. 1997).

Fig. 1
figure 1

Orthologous gene cluster of AraR. Graphic presentation of the genomic region of A. nidulans araR and comparison to other Aspergilli (Crabtree et al. 2007). AN0384 conserved serine rich protein, AN0385 putative branched-chain amino acid transaminase, AN0386 conserved hypothetical protein, AN0387 DNA photolyase, AN0388 AraR, AN0389 and AN0390 conserved hypothetical proteins, AN0391 NACHT domain-containing protein. The araR genes are connected by pink-shaded boxes. Nearby genes (in the 10-kb region on each side of araR) are shown, and those that are conserved are connected by grey-shaded boxes

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Fig. 2
figure 2figure 2

Multiple sequence alignment of AraR. The following amino acid sequences are aligned by ClustalW (Chenna et al. 2003): Neosartorya fischeri (NFIA023070), Aspergillus fumigatus (Afu1g01590), Aspergillus clavatus (ACLA032900), Aspergillus niger (An04g08600), Aspergillus nidulans (AN0388.3), Aspergillus oryzae (AO090003001292), Aspergillus flavus (AFL2G01779.2) and Aspergillus terreus (ATEG04909.1). Conserved amino acid residues are shown in grey. Single asterisk indicates identical residues. Semi-colon indicates conservative substitutions. Period indicate less conservative substitutions. Cysteine residues are indicated in green. Fungal-specific transcription-factor domain of unknown function (Suarez et al. 1995) is shown in yellow

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Phenotypic comparison of A. niger and A. nidulans ΔaraR and ΔaraR/ΔxlnR

We constructed araR, xlnR and double (araR/xlnR) disruptant strains and verified them by Southern analysis (Suppl. Fig. 1). The growth was analysed in these strains on d-glucose, l-arabinose, l-arabitol, d-xylose and xylitol. The deletion of xlnR in both A. nidulans and A. niger did not affect growth on the tested carbon sources. The growth of the other deletion strains revealed significant differences between A. niger and A. nidulans (Fig. 3). The growth of the A. nidulans ΔaraR strain was nearly impaired on l-arabinose, but only a small growth reduction was observed on l-arabitol. In contrast, the A. niger ΔaraR strain showed only a small growth decrease on l-arabinose, while the growth on l-arabitol was strongly reduced. Both A. nidulans and A. niger ΔaraR/ΔxlnR strains showed strongly impaired growth on d-xylose. Surprisingly, the growth of the A. nidulans ΔaraR/ΔxlnR strain was only slightly affected on l-arabinose, whereas the growth was absent on this carbon source in the A. niger ΔaraR/ΔxlnR strain. Furthermore, the absence of AraR and/or XlnR in A. nidulans did not affect the growth on xylitol, whereas the growth was reduced on this carbon source in the A. niger ΔaraR/ΔxlnR strain.

Fig. 3
figure 3

Growth of the A. niger and A. nidulans reference, ΔaraR, ΔxlnR and ΔaraR/ΔxlnR strains on 25 mM d-glucose, l-arabinose, l-arabitol, d-xylose and xylitol. The A. niger growth data was represented from our previous study (Battaglia et al. 2011) with permission from the publisher

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Transcriptional analysis of the pentose catabolic pathway genes

To study the regulation of the pentose catabolic pathway in A. nidulans, a transcriptional profile of the PCP genes was compared in the A. nidulans reference, ΔaraR, ΔxlnR and ΔaraR/ΔxlnR strains. To this end, a transfer experiment was performed (see the “Materials and methods” section) using the strains and the carbon sources indicated in Fig. 4. In the presence of xylitol, no or low induction was detected for any of the genes in any of the strains tested. In the reference strain, the expression of the l-arabinose specific PCP genes (ladA, larA, lxrA) was induced on l-arabinose and l-arabitol, but not on d-xylose (Fig. 4). The expression of xyrA (d-xylose specific) was only observed on d-xylose, whereas the genes encoding the last two steps common in both pathways (xdhA, xkiA) were induced by both l-arabinose and d-xylose.

Fig. 4
figure 4

Expression analysis of the A. nidulans pentose catabolic pathway genes. All A. nidulans strains were transferred to 25 mM of the indicated carbon source for 2 h of growth. ladA l-arabitol dehydrogenase, larA l-arabinose reductase, xdhA xylitol dehydrogenase, xkiA xylulose kinase, xyrA d-xylose reductase, LC loading control. 1 d-fructose, 2 l-arabinose, 3 l-arabitol, 4 d-xylose, 5 xylitol

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No xyrA expression was observed on d-xylose in the ΔxlnR strain, while genes of the l-arabinose pathway (ladA, larA and lxrA) were induced. A reduced level of xdhA was observed on both l-arabinose and d-xylose in the ΔxlnR strain, while a higher expression level of both xdhA and xkiA was detected on l-arabitol.

A reduced level of ladA expression was observed on l-arabinose and l-arabitol in the ΔaraR strain, and no expression of larA and lxrA was detected. For xdhA and xkiA, expression on l-arabinose was decreased in the absence of AraR. An increase of xdhA expression was observed on l-arabitol, while both xdhA and xkiA expression levels remained unaffected on d-xylose.

Reduced, but detectable expression levels of ladA, larA and xdhA were observed in the ΔaraRΔxlnR strain on l-arabinose and l-arabitol, while xdhA was also expressed on d-xylose in the ΔaraRΔxlnR strain. No xkiA specific transcript was detected in a double-knockout strain under all the conditions tested.

Analysis of the activity of pentose catabolic pathway enzymes

The enzymatic activities for l-arabitol dehydrogenase (Lad), xylitol dehydrogenase (Xdh), l-arabinose reductase (Lar), d-xylose reductase (Xyr) and l-xylulose reductase (Lxr) were measured in the mycelium samples of a transfer experiment to l-arabinose and d-fructose (see the “Materials and methods” section) using the strains indicated in Fig. 5.

Fig. 5
figure 5

The comparison of intracellular enzyme activities in A. nidulans and A. niger. l-2-keto-3-deoxyarabonatearabitol dehydrogenase (Lad), xylitol dehydrogenase (Xdh), l-2-keto-3-deoxyarabonatearabinose reductase (Lar), d-xylose reductase (Xyr) and l-2-keto-3-deoxyarabonatexylulose reductase (Lxr) activity were measured in the A. nidulans and A. niger reference, ΔaraR and ΔaraR/ΔxlnR strains. The A. niger enzyme activity data was represented from our previous study (Battaglia et al. 2011) with permission of the publisher. The enzyme activities are given in units per milligramme of the total protein

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After 2 h of growth on l-arabinose, the reduced activities of Lad, Xdh, Lar, Xyr and Lxr were detected in the A. nidulans ΔaraR strain. The results showed no Lad and Xdh activity in A. nidulans ΔaraRΔxlnR (Fig. 5). After 4 h of growth on l-arabinose, increased activities of Xdh, Lar and Xyr were detected in the A. nidulans ΔaraR strain, while Lxr activity was still lower than the reference strain. Only Lxr activity was decreased in the A. nidulans ΔaraRΔxlnR strain after 4 h on l-arabinose, while the other activities were similar to the reference strain (Fig. 5). Very low activities of Lad, Xdh, Lar, Xyr and Lxr were observed on d-fructose in any of the strains (data not shown).

Discussion

In this study, we analysed the function of the arabinolytic regulator (AraR) in A. nidulans and compared it to the recently reported A. niger AraR (Battaglia et al. 2011). Plate growth tests revealed reduced growth of the A. nidulans ΔaraR strain on l-arabinose and l-arabitol, confirming a role for AraR in l-arabinose catabolism in this species. In addition, the expression analysis confirmed that AraR predominantly controls the A. nidulans PCP genes, including the recently identified genes encoding l-arabinose reductase (larA; Mojzita et al. 2010a) and l-xylulose reductase (lxrA; Mojzita et al. 2010b). However, the data presented in this paper reveals that the functional role of AraR in the PCP differs significantly in A. nidulans and A. niger.

Comparative analysis clearly shows differences between growth on l-arabinose and the PCP in the A. nidulans and A. niger ΔaraR and ΔaraR/ΔxlnR strains. The results observed in these A. nidulans strains can be explained by either an additional regulatory system or an alternative pathway for l-arabinose catabolism.

As A. nidulans appears to have AraR/XlnR independent and l-arabinose-specific induction of several PCP genes (larA, ladA and xdhA), this suggests the involvement of an as-yet-unknown regulator. That this effect is not caused by a low constitutive expression level of these genes is demonstrated by the absence of expression on d-fructose. This unknown regulator may be activated by l-arabinose or its intermediate product, but appears to be dependent on the intracellular accumulation of this inducer. A similar effect was observed for XlnR (Battaglia et al, unpublished results). In the A. nidulans ΔaraR strain on l-arabinose, XlnR activates the expression of xylanase genes, which does not occur in a wild-type strain. This suggests that XlnR responds to the presence of accumulated concentrations of l-arabinose or a conversion product. Such a sophisticated mode of regulation, where a pathway-specific regulator can trigger the transcription of target genes in the presence of a threshold level of an inducing molecule not directly linked to this metabolic pathway may be used by A. nidulans as a rescue strategy and give an additional advantage to the fungus.

BlastP analysis of AraR and XlnR to the A. nidulans genome gave three significant hits, AraR and XlnR itself and a third unknown regulator (data not shown). The function of this regulator is currently under investigation.

No expression was detected of the l-xylulose reductase encoding gene (lxrA) and xylulose kinase encoding gene (xkiA), suggesting a partially active PCP in ΔaraR/ΔxlnR strain on l-arabinose. For the utilisation of this substrate, other enzymes that can compensate for the loss of LxrA and XkiA activity must be involved. We propose that these unknown genes are not induced in the presence of XlnR because of the impaired growth of the ΔaraR strain on l-arabinose. It is currently unknown if isoenzymes exist that compensate for the reactions of the PCP in the A. nidulans. Putative paralogs of several PCP genes are present in the A. nidulans genome (Flipphi et al. 2009), but their biochemical function is not determined. We observed similar activities for 4 of the tested enzymes (Lar, Xyr, Lad, Xdh) in the A. nidulans ΔaraR/ΔxlnR and the reference strain after 4 h on l-arabinose, while expression of these genes was significantly reduced in the ΔaraR/ΔxlnR strain. This suggests that other enzymes can in part compensate for the absence of the PCP-specific enzymes. However, this was not the case for Lxr, suggesting that growth on l-arabinose in the A. nidulans ΔaraR/ΔxlnR strain is not likely caused by isoenzymes generating a similar pathway as the normal PCP. It is therefore more likely that an alternative pathway can convert l-arabinose that may in part use similar activities as the PCP.

There is currently no evidence for the existence of an alternative fungal l-arabinose pathway and the data of our study suggests that should such a pathway exists, it is not dependent on AraR and is only activated in the absence of XlnR. Alternative pathways for carbon utilisation have been shown to exist in fungi and bacteria. A second pathway for d-galactose catabolism (the oxido-reductive pathway) was previously found in A. nidulans (Fekete et al. 2004). In bacteria, two l-arabinose pathways are known: the arabinose (araBAD) operon (Lee et al. 1986) and an alternative pathway through l-2-keto-3-deoxyarabonate (Watanabe et al. 2006a; Watanabe et al. 2006b).

The comparison of growth, expression and activity profiles demonstrates that the role of XlnR in the regulation of the PCP is similar in A. nidulans and A. niger. In both Aspergillus species, it has been demonstrated that XlnR also share the same functional regulatory role with respect to extracellular d-xylose releasing enzymes (Tamayo et al. 2008; van Peij et al. 1998).

In summary, this study demonstrates that the regulatory role of AraR in the PCP differs between two Aspergilli, while this is not the case for XlnR. This may indicate that the two species optimized their carbon source utilisation by subtle, but significant variations in the catabolic pathway itself and its regulatory mechanism. It demonstrates that the influence of homologous regulatory systems in different (even related) fungi can differ significantly. This also implies that manipulating regulatory systems to improve the production of polysaccharide degrading enzymes, may give different results in different industrial fungi.