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The transcriptional activators AraR and XlnR from Aspergillus niger regulate expression of pentose catabolic and pentose phosphate pathway genes
Research in Microbiology 165 (2014) 531e540 www.elsevier.com/locate/resmic
The transcriptional activators AraR and XlnR from Aspergillus niger regulate expression of pentose catabolic and pentose phosphate pathway genes Evy Battaglia a,b, Miaomiao Zhou b, Ronald P. de Vries a,b,* a
Microbiology & Kluyver Centre for Genomics of Industrial Fermentation, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands b CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CY, Utrecht, The Netherlands Received 27 January 2014; accepted 21 July 2014 Available online 31 July 2014
Abstract The pentose catabolic pathway (PCP) and the pentose phosphate pathway (PPP) are required for the conversion of pentose sugars in fungi and are linked via D-xylulose-5-phosphate. Previously, it was shown that the PCP is regulated by the transcriptional activators XlnR and AraR in Aspergillus niger. Here we assessed whether XlnR and AraR also regulate the PPP. Expression of two genes, rpiA and talB, was reduced in the DaraR/DxlnR strain and increased in the xylulokinase negative strain (xkiA1) on D-xylose and/or L-arabinose. Bioinformatic analysis of the 1 kb promoter regions of rpiA and talB showed the presence of putative XlnR binding sites. Combining all results in this study, it strongly suggests that these two PPP genes are under regulation of XlnR in A. niger. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Pentose phosphate pathway; Aspergillus niger; Regulation of gene expression; XlnR; AraR
1. Introduction In fungi, L-arabinose and D-xylose are metabolized through an oxidoreductive pathway named the pentose catabolic pathway (PCP) [1]. Both these pentose sugars go through oxidation, reduction and phosphorylation reactions to form Dxylulose-5-phosphate. This intermediate ketose sugar connects the PCP to the pentose phosphate pathway (PPP), one of the central metabolic pathways in primary metabolism. The PPP, first described by Horecker and Mehler, is important for the production of reducing power in the form of NADPH and precursors for nucleotide and amino acid biosynthesis [2]. Genetic engineering of the PCP and PPP in yeast and
* Corresponding author. CBS Fungal Biodiversity Centre Uppsalalaan 8 3584 CY Utrecht Netherlands. E-mail addresses: [email protected] (E. Battaglia), m.zhou@cbs. knaw.nl (M. Zhou), [email protected] (R.P. de Vries).
filamentous fungi has proven that the metabolic reactions of these pathways are highly interconnected during pentose utilization [3,4]. The production of D-xylulose-5-phosphate by the PCP not only connects the metabolic pathways by allowing an increased flux into the non-oxidative PPP, but also the (re) generation and demand of the redox cofactors NADH and NADPH in enzymatic reactions of both the PCP and oxidative PPP. Witteveen et al. demonstrated an increase in activity of the enzymes involved in the last two steps of the oxidative PPP during growth on L-arabinose and D-xylose, namely the activity of glucose-6-phosphate dehydrogenase (G6PD) and 6phosphogluconate dehydrogenase (6PGD) [1]. As metabolic pathways are interconnected, the molecular mechanisms regulating the expression of these metabolic genes are complex and only beginning to be unraveled in filamentous fungi. Regulation of the PCP has been studied in detail in Aspergilli and Trichoderma reesei [5e9]. In Aspergillus niger, the Zn2Cys6 transcriptional activator XlnR [10] regulates xyrA (encoding D-xylose reductase) the first step of
http://dx.doi.org/10.1016/j.resmic.2014.07.013 0923-2508/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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E. Battaglia et al. / Research in Microbiology 165 (2014) 531e540
D-xylose catabolism [7]. Analysis of two A. niger UV mutants provided evidence for the presence of a L-arabinose responsive regulator that together with XlnR controls PCP genes [6], which was later identified and named AraR [5]. Disruption of both xlnR and araR resulted in no expression of the PCP genes and an inability of this A. niger mutant to utilize D-xylose or Larabinose [5], indicating a complex regulatory interplay between these transcription factors in this pathway. In Magnaporthe oryzae, regulation of PPP was proven to be modulated by the levels of NADP, via trehalose-6-phosphate synthase (Tps1), an enzyme of trehalose biosynthesis [11]. No studies on the regulation of this central metabolic pathway during utilization of D-xylose or L-arabinose in filamentous fungi have been published. Interestingly, it has been reported that PPP genes are upregulated in A. niger during growth on D-xylose as a sole carbon source [12] and when other carbon sources such as starch and lactate [13] and complex sugar substrates are provided [14]. In Neurospora crassa, several PPP genes were upregulated on xylan compared to sucrose [15], and their expression was dependent on Xlr-1 (the N. crassa XlnR/Xyr1 homolog) on this substrate. In this study, the influence of the transcriptional activators XlnR and AraR on the expression of PCP and PPP genes was assessed in A. niger.
2. Materials and methods 2.1. Strains and growth conditions The A. niger strains, listed in Table 1, were grown in minimal medium (MM) or complete medium (CM) [16] at 30 C and 250 rpm in a rotary shaker. When necessary, 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. In transfer experiments, all strains were pre-grown in 200 ml CM containing 2% D-fructose in a 1 L Erlenmeyer flask. After 16 h of incubation, the mycelium was harvested without suction over a cheese cloth, washed twice with MM without carbon source and transferred to a 250 ml Erlenmeyer flask containing 50 ml MM containing 25 mM D-xylose or L-arabinose. After 2 h and 4 h of incubation the mycelium was harvested with suction, the mycelium was subsequently dried between tissue paper and directly frozen in liquid nitrogen.
Table 1 Aspergillus niger strains used in this study. Strain
Genotype
Reference
N402 N572 NW249 UU-A049.1
cspA1 cspA1, fwnA1, pyrA6, xkiA1, nicA1 cspA1, DargB, pyrA6, nicA1, leuA1 cspA1, pyrA6, nicA1, leuA1, DargB::pIM2101 (argBþ) cspA1, pyrA6, nicA1, leuA1, DaraR cspA1, DargB,nicA1, leuA1, DxlnR cspA1, nicA1, leuA1, DaraR, DxlnR
[33] [29] [34,35] [5]
UU-A033.21 UU-A062.10 UU-A063.22
[5] [5] [5]
2.2. PCP and PPP genes collection All PCP genes were selected and corresponding enzymes were biochemically characterized previously (see Table 2 for references). PPP genes were selected based on two previously published papers in which the authors both computationally and manually annotated these metabolic genes from A. niger CBS 513.88 and ATCC 1015 [17,18]. 2.3. Micro-array and quantitative real-time PCR analysis Total RNA was isolated from mycelium that was ground in a microdismembrator (B Braun). Approximately ~100 mg of ground mycelium was homogenized in 1 ml Trizol reagent (Invitrogen). After addition of 200 ml chloroform and phase separation by centrifugation, 70% ethanol was added to the aqueous phase, which was subsequently transferred on a silica spin column from NucleoSpin RNA kit (Macherey-Nagel). RNA was further purified according to the manufacturer's instructions. The quality of RNA was checked using gel electrophoresis and RNA was quantified using a UV spectrophotometer. cDNA was synthesized from 2.5 mg of total RNA using the Thermoscript transcriptase according to the manufacturer's instructions (Invitrogen). The RNase H treatment was followed by an ethanol precipitation. cDNA was dissolved in 10 ml H2O and checked using gel electrophoresis. All qPCR primers pairs were designed with Primer3 Plus [19] and experimentally validated with two quality control (specificity and efficiency) assays. The optimal primer concentrations were tested by dissociation curve analysis to check for gene-specific signals and nonspecific signals caused by primer dimers. Combinations of 50 nM, 300 nM and 900 nM (final concentration) per primer pair were used. The efficiency of each primer pair (>90%) was assessed by plotting the cycle threshold value (Ct) against the logarithm of dilution of A. niger genomic DNA samples, ranging from 10 ng to 1 pg. The sequences of all primer pairs, optimal concentrations and amplicon sizes are listed in Supplemental Table 1. qPCR was performed using the ABI 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA, USA). RT-PCR mixtures (20 ml) contained: 10 ml ABI Fast SYBR Master Mix (Applied Biosystems, Foster City, CA, USA), the forward and reverse primer pair (2 ml of each primer, at optimal concentration), 2 ml of 100 fold diluted cDNA and 4 ml H2O. The cycle conditions were as follows: 95 C for 2 s, 95 C for 3 s and 60 C for 30 s (40 cycles). The A. niger histone H2B gene (An11g11310) was used as a reference gene for normalization. The results were analyzed using the comparative CT (2-DDCT) method [20]. Graphpad Prism version 6 was used to calculate the mean, standard error of the mean and significance between the reference and mutant samples using two sample t tests. The quality of the RNA samples for Affimetrix microarray analysis was assessed using the Agilent 2100 Bioanalyzer. Microarray data was analyzed using the Bioconductor Affy tool package (http://www.bioconductor.org/) under the
Table 2 Transcriptional levels of PCP and PPP genes in the DxlnR strain and the wild type strain N402 on D-xylose, and the DaraR strain and reference strain N402 L-arabinose. araR/arae
FDR
733 ± 53.9
8287.6 ± 274.9
¡11.31
1.36E-10
5047.8 ± 234.6
3535.1 ± 56.7
PCP
352 ± 7.5
4678.9 ± 379.6
¡13.29
6.65E-10
2946.7 ± 151.5
An01g03740 An00g00176_at [7]
PCP
8789.1 ± 423.8
8913.5 ± 530.8
1.01
0.91
An08g01930 An00g03632_at [36]
PCP
150.6 ± 28.4
7275.6 ± 162.7
¡48.31
5.73E-11
An11g01120 An00g06977_at [37]
PCP
1634.7 ± 296.5
12063 ± 18.7
An12g00030 An00g09559_at [6] An11g02040 An00g10295_at [4]
PCP PPP
5922.4 ± 418.7 2972.4 ± 52.2
An02g12140 An00g00194_at [38]
PPP
1394.6 ± 178.7
An14g00160 An01g05150 An15g07500 An09g03450
[18] [18] [17] [18]
PPP PPP PPP PPP
589.7 933.9 351.9 69.5
An11g00470 An00g10887_at [18]
PPP
535.3 ± 15.3
988.2 ± 10.7
An02g02930 An00g00058_at [18]
PPP
4988.7 ± 138.3
4524.9 ± 388.4
An07g03850 An07g03160 An08g06570 An02g06430 An14g03500
[18] [18] [4] [18] [17]
PPP PPP PPP PPP PPP
4338.5 943.8 4661.3 19 46.1
An11g06120 An00g10294_at [17]
PPP
Gene ID
ladA
An01g10920 An00g11747_at [6]
PCP
An07g03140 An00g08027_at [29]
L-arabitol
dehydrogenase xkiA D-xylulose kinase xyrA D-xylose reductase lxrA L-xylo-3-hexulose reductase larA NADPH-dependent aldehyde reductase xdhA xylitol dehydrogenase gndA 6-phosphogluconate dehydrogenase gsdA glucose-6-phosphate 1-dehydrogenase rbtA D-ribulokinase pglA 6-phosphogluconolactonase rbkA ribokinase rpeA D-ribulose-phosphate-3 epimerase rpiA ribose 5-phosphate isomerase rpiB ribose 5-phosphate isomerase talA transaldolase talB transaldolase tktA transketolase tktB transketolase tktC dihydroxyacetone synthase gndB phosphogluconate dehydrogenase (decarboxylating)
An00g08020_at An00g11069_at An00g10879_at An00g10897_at
An00g11820_at An00g11821_at An00g11869_at An00g06454_at An00g11348_at
Ref
± ± ± ±
± ± ± ± ±
9.7 23.9 0.4 0.6
31.9 39.6 506.2 1.5 4
19.3 ± 0.3
xlnRc
xyld
xlnR/xyle
FDR
1.43
4.37E-06
4421.9 ± 131.2
1.50
1.12E-05
5751.8 ± 251.6
11107.9 ± 164.2
¡1.93
6.16E-09
4750.6 ± 13.7
2908.4 ± 94.3
1.63
1.57E-07
¡7.38
2.17E-09 13258.7 ± 90.7
9233.2 ± 67.7
1.44
1.62E-07
12305 ± 358.3 7121.4 ± 544
¡2.08 ¡2.40
4.11E-06 12886.4 ± 427.1 5.38E-06 10158.3 ± 130.6
9321.5 ± 145.3 12785.5 ± 267.5
1.38 1.26
1.68E-06 2.26E-05
4220.1 ± 327
¡3.03
7.28E-06
6332.9 ± 50.7
9899.7 ± 9.5
¡1.56
4.38E-08
1.45 1.25 ¡1.61 ¡2.25
0.02 0.15 0.01 0.00
464.5 1047.3 511 302.4
13.7 58.9 0.2 6.2
1.03 ¡1.77 2.18 ¡2.69
0.67 1.89E-06 4.19E-07 2.78E-08
¡1.85
0.00
1006 ± 63
928.6 ± 26.6
1.08
0.34
1.10
0.49
4489 ± 138.4
1.39
8.07E-06
1.03 ¡8.70 1.05 ¡8.79 1.05
0.41 5.72E-11 0.14 4.04E-10 0.56
407.4 1171.6 565 156.7
8630.8 840.1 9331.3 23.1 39
± ± ± ±
± ± ± ± ±
26.8 132.2 68.7 22.8
385.4 65.8 193.8 0.8 0.9
19.1 ± 0.7
¡1.99 1.12 ¡2.00 1.21 1.18 1.01
± ± ± ±
10.3 2.9 21.4 9.2
6.42E-06 12430.9 ± 223.3 0.43 245.6 ± 9.1 8.61E-06 12075.3 ± 233.4 0.01 18.7 ± 0.2 0.14 31 ± 1.8 0.90
20 ± 0.1
450.1 1856.7 234.2 813.5
± ± ± ±
3222.3 ± 7.5 12789.9 2138.1 12718.2 164.7 29.6
± ± ± ± ±
57.5 88.5 99.3 25.3 1.5
19.1 ± 0.6
1.05
E. Battaglia et al. / Research in Microbiology 165 (2014) 531e540
arab
Probe number
Metabolic araRa pathway
Gene Activity
0.30
PPP: pentose phosphate pathway and PCP: pentose catabolic pathway. The mean expression levels of duplicate samples for DaraR 2 h L-ara, reference 2 h L-ara, DxlnR 2 h D-xyl and reference 2 h D-xyl, respectively. e The fold change ratio calculated by the expression levels of the disruption strain versus reference strain for DaraR/ref and DxlnR/ref, respectively. Genes with significant differential expression (fold change >1.5 and fdr value <0.05) are marked bold.
a, b, c, d
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statistical environment R [21]. The probe intensities were normalized for background by using the Robust Multiarray Average (RMA) algorithm [22] for the Affimetrix CEL files with only the perfect match (PM) probes. The quantiles algorithm [23] was used to perform normalization and gene expression values were calculated by the medianpolish summary method [22]. The normalized gene expression values were then processed with the statistical tool Cyber-T [24] to detect differential significance, using the BayesANOVA algorithm. Gene expression variation with P-value <0.05 and fold change >1.5 were considered significant. 2.4. Northern analysis The Minifold II slot blot apparatus (Schleicher & Schuell) was used for the transfer of 3 mg total RNA to a Hybond-Nþ membrane (Amersham Biosciences). Equal loading of total RNA was controlled by soaking the blot for 5 min in 0.04% methylene blue, 0.5 M acetate pH 5.2 solution. The methylene blue stained membrane is included in Figs. 1 and 2 as the loading control. The primers used to generate the probes for Northern analysis are listed in Supplemental Table 1. The probes were DIG-labeled using the PCR DIG Probe Syntheses Kit (Roche Applied Science) according to the supplier's instructions. A cDNA library [6] or genomic DNA (obtained from A. niger N402) was used as a template in the PCR reactions for synthesis of the probes. Hybridization of the DIGlabeled probes to the blot and their detection was performed according to the DIG user's manual (www.roche-appliedscience.com). 2.5. Transcription factor binding motifs in regulated genes The DNA sequences of 1000 nucleotide upstream of each PCP and PPP gene were extracted from their genomes according to their gene calling. The upstream DNA stretches were fed to MEME for detection of conserved motifs using parameters of 1E-5, minimal length 2, maximum length 15, and allowing any numbers of repetition [25]. The figures of motifs were generated by Weblogo [26]. The predicted motifs with more than 2 sites were validated with known XlnR/AraRregulated enzymes by MAST using the default setting [27]. The known XlnR motif was searched on DNA sequences using PatScan. Search of known transcriptional factor binding site was done by JASPAR fungi core motifs with relative profile score threshold of 90% [28]. 3. Results 3.1. Deletion of the Zn2Cys6 transcriptional activators XlnR and AraR in A. niger results in down-regulation of both the pentose catabolic and phosphate pathway during growth on pentose sugars Expression of PCP and PPP genes was compared in the wild type and the DxlnR and DaraR strains (Table 2). The
expression of five out of six genes encoding enzymes of the PCP (ladA, xdhA, xkiA, lxrA, larA) showed significant reduction in the DaraR strain on L-arabinose. The D-xylose reductase gene xyrA had, as reported before [5], an unchanged expression level in the DaraR on L-arabinose and a reduced expression level in the DxlnR strain on D-xylose. Expression levels of four of the other genes (larA, ladA, xdhA, xkiA) were unaffected in the DxlnR strain during growth on D-xylose, whereas lxrA expression was moderately increased. Seven out of fourteen PPP genes showed significant reduced expression levels in the DaraR strain on L-arabinose (Table 2). The expression level of only one PPP gene, Dribulose-5-phosphate 3-epimerase (rpeA), was significantly altered in both strains. The expression of one transaldolase (talB) and one transketolase (tktB) gene was significantly decreased in the DxlnR strain on D-xylose but not significantly affected in the DaraR strain on L-arabinose. The expression of four PPP genes (rbtA, rpiB, tktC and gndB) was less than 1.5fold altered, so unaffected in both the regulator mutants on Larabinose or D-xylose (Table 2). Based on the micro-array results, a subset of the genes ( gndA, rpeA, rpiA, talA, talB and tktA) was selected for confirmation by Northern analysis. The reference strain, the DaraR and DxlnR strains, as well as a strain in which both araR and xlnR were deleted (UU-A063.22), were incubated for 2 h in MM with L-arabinose or D-xylose. The reduced expression levels of rpiA, talA, tktA and gndA in DaraR on L-arabinose and talB in DxlnR on D-xylose confirmed the microarray data (Fig. 1A). In DaraR on D-xylose an increased expression level of talB was observed, while reduced expression was observed for gndA and rpiA in DxlnR on L-arabinose compared to the reference strain. In contrast, expression of rpiA was increased in the DaraR and DxlnR strains on D-xylose. In the DaraR/DxlnR strain, expression of five PPP genes ( gndA, rpiA, talA, talB and tktA) was reduced on L-arabinose and of four PPP genes ( gndA, talA, talB and tktA) on D-xylose (Fig. 1A). 3.2. Expression pattern of PPP genes in the A. niger xkiA1 mutant strain indicates regulation by AraR and/or XlnR Expression of the PPP genes was analyzed in an xkiA1 mutant strain to determine whether expression of these PPP genes is changed by regulation by AraR and/or XlnR or indirectly by altered levels of D-xylulose-5-phosphate. In this strain, D-xylulose kinase activity is absent, resulting in an arrest of pentose catabolism at D-xylulose and no formation of Dxylulose-5-phosphate [29]. Genes regulated by AraR and/or XlnR should show increased expression in this strain due to accumulation of the inducers of these regulators, while genes affected by reduced D-xylulose 5-phosphate levels should show decreased expression. The reference strain and the xkiA1 mutant (N572) were grown for 2 and 4 h on L-arabinose and D-xylose. Two genes of the PPP, talB and rpiA, showed increased expression levels in the xkiA mutant after 4 h of growth on D-xylose and L-arabinose, respectively (Fig. 2A), which is a similar pattern as that
E. Battaglia et al. / Research in Microbiology 165 (2014) 531e540
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Fig. 1. A) Northern blot analysis of pentose phosphate pathway genes. The reference strain and the DaraR, DxlnR and DaraR/DxlnR strains were grown for 2 on 25 mM L-arabinose or 25 mM D-xylose. Methylene blue staining of total RNA is included as a loading control. B) Quantitative gene expression analysis of the transaldolase gene (talB) and the ribose-5-isomerase gene (rpiA). For both genes, the expressions are normalized against the expression of histone h2b. Error bars indicate the standard deviation of two biological and two technical repeats. R ¼ reference, DX ¼ DxlnR, DA ¼ DaraR, and DADX ¼ DaraR/DxlnR strain. Significance tests were performed between reference and regulator mutant strains. p < 0.01 (**), p < 0.05 (*).
observed for two PCP genes (ladA and xkiA), suggesting regulation by AraR or XlnR. These two PPP genes (rpiA and talB) and the two PCP genes (ladA and xkiA) were not induced in the reference strain on D-fructose. Reduced or similar expression levels were observed for the other genes ( gndA, rpeA, talA, and tktA) in the xkiA1 mutant on both substrates, compared to the reference strain (Fig. 2A).
To conclude, our Northern blot results suggest the rpiA and talB genes are regulated by AraR and/or XlnR. RT-qPCR analysis was carried out to have an accurate quantification of the differences in gene expression levels, and thus confirm observed changes for the rpiA and talB genes. The RT-qPCR results are presented in both Figs. 1 and 2 as the b-sections of the figures. The data obtained with RT-qPCR did not fully
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E. Battaglia et al. / Research in Microbiology 165 (2014) 531e540
Fig. 2. A) Northern blot analysis of pentose catabolic pathway genes and pentose phosphate pathway genes in the reference strain and the xkiA1 mutant. Both strains were grown for 2 and 4 h on 25 mM D-fructose (F), L-arabinose (A) or D-xylose (X). Methylene blue staining of total RNA is included as a loading control. B) Quantitative gene expression analysis of the D-xylulose kinase genes (xkiA), the transaldolase gene (talB) and the ribose-5-isomerase gene (rpiA) in the reference strain and the xkiA1 mutant. For both genes, the expressions are normalized against the expression of histone h2b. Error bars indicate the standard deviation of two biological and two technical repeats. R ¼ reference, DX ¼ DxlnR, DA ¼ DaraR, and DADX ¼ DaraR/DxlnR strain. Significance tests were performed between reference and regulator mutant strains. p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), p < 0.05 (*).
E. Battaglia et al. / Research in Microbiology 165 (2014) 531e540
correlate with our Northern blot results, even for the control gene xkiA that we included. Nevertheless, the quantitative results did indeed confirm changes in expression levels between the reference and mutants strains that suggest regulation by the transcription factors, and thus not alter our overall conclusion. Expression levels of rpiA were significantly reduced in the DaraR/DxlnR on D-xylose, while expression increased on D-xylose (4h) in the xkiA1 mutant (Figs. 1B and 2B). Expression levels of the talB gene were reduced in the DxlnR and DaraR/DxlnR strain on both L-arabinose and Dxylose, while expression was increased on both sugars (4 h) in the xkiA1 mutant. 3.3. Transcription factor binding motifs in PCP and PPP genes The 1 kb upstream regions of the PCP and PPP genes listed in Table 2 were analyzed for conserved elements using the MEME software (see Materials and Methods for our search parameters) [25]. The six motifs with the highest significance scores are depicted in Fig. 3. The genes together with the conserved motif sequence, significance value and location are listed in Supplemental File 2a. Most PCP and PPP genes contained motif 1 and 2 (Fig. 3 and Supplemental File 2a). Motif 3 and 6 were mainly present in PPP genes and only one or two PCP gene(s). Motif 4 was only found in PCP genes that were down-regulated in the araR deletion mutant. The promoter regions of 5 out of 6 PCP genes contained this motif. Motif 5 was present in only two PCP (xdhA and xkiA) and two PPP genes (rbtA and tktA). All six motifs that we found were not identical to known fungal transcriptional factor binding sites according to the JASPAR database [28] (Supplementary File 3). This strongly suggests the motifs are not similar to known/yeast motifs and these sequences are novel. All these conserved motifs are candidate binding sites for AraR, because the results showed that the 1 kb promoter regions of both known AraR-regulated PCP, endo-arabinanase and a-arabinofuranosidase genes (abnA, abfA and abfB) genes contained these motifs (Supplemental File 2a and 2b). The characterized XlnR binding site G-G-C-T-A-[AG] [ATCG] was searched against all PCP and PPP genes together with known XlnR-regulated genes (b-xylosidase gene: xlnD; feruloyl esterase gene: faeA; endo-glucanase gene: eglA) (Supplemental File 2c). Besides the presence in eglA, faeA, xlnD, the XlnR binding site was also found in three known XlnR-regulated PCP genes (xdhA, xyrA and xkiA) and six PPP genes ( pglA, rpeA, rpiA, talA, talB and tktA). If we combine these results with the gene expression experiments, this strongly suggests XlnR plays a role in regulation of talB and rpiA. 4. Discussion The PCP and PPP enable complete conversion of D-xylose and L-arabinose to central carbon catabolism. In this research, expression profiles of six PCP genes in A. niger confirmed that
537
the regulation of the complete PCP is under control of AraR and XlnR in this fungus, with heavier influences from AraR than XlnR as reported previously [5]. This study also aimed to elucidate whether these transcription factors regulate PPP genes. After an initial screen of transcriptome data, a subset of genes was selected for more detailed analysis to confirm whether the observed changes in gene expression levels in the regulatory mutants were caused by control of the regulators or indirect changes in D-xylulose-5-phosphate levels. We used a xylulose kinase deficient (xkiA1) strain to determine regulation by AraR and/or XlnR. As the xkiA1 mutant accumulates the inducers for AraR and XlnR, genes under control of these regulators have increased expression in the xkiA1 mutant compared to the wild type, as was previously shown for PCP genes [5,29]. Expression of the ribose-5-phosphate isomerase encoding gene (rpiA) was reduced in the DaraR/DxlnR strain and increased in the xkiA1 mutant on D-xylose, as assessed by Northern blot and qPCR analysis. In the 1 kb promoter region of the rpiA gene, a putative binding site for XlnR was found. Combining the expression and promoter results, it strongly suggests this PPP gene is under control of XlnR. However, the data of this study also show that rpiA is not only dependent on XlnR and AraR on D-xylose, because expression was still observed in the DaraR/DxlnR strain on this substrate. Expression levels of rpiA were detected on D-fructose by qPCR analysis suggesting this gene is not specifically induced on pentoses. A second putative ribose-5-phosphate isomerase encoding gene (rpiB) is present in the A. niger genome and seven other Aspergilli [18]. Expression of this gene was unaffected by deletion of xlnR or araR and this gene was therefore not studied in detail. Two transaldolase genes are present in the A. niger genome. The expression data indicate that A. niger talA is constitutively expressed and not under regulation of XlnR or AraR. In Pichia stipitis and Trichoderma reesei, the ortholog of talA is also constitutively expressed and the corresponding enzyme of these fungi is involved in the metabolism of many carbon sources [30,31]. The expression profiles suggest that only talB is under control of XlnR during growth on L-arabinose and D-xylose. This was shown using the same experimental set up as rpiA. The A. niger talB gene is predominantly induced on D-xylose and L-arabinose and not on D-fructose (this study) or maltose [12]. Based on the expression levels in the DxlnR, DaraR/ DxlnR and xkiA1 deficient strain on D-xylose, talB expression appears to be predominantly under regulation of XlnR. XlnR regulation of talB is supported by the presence of a XlnR binding site in its promoter [32]. It should be noted that for the rpiA gene the Northern blot and micro-array changes between the A. niger reference and regulator deletion strains on L-arabinose do not completely correlate with the mRNA changes measured by qPCR analysis. If the expression of rpiA or talB is influenced by AraR as well, remains unclear. This may suggest that the regulatory effect is less pronounced than XlnR and differences in
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Fig. 3. Conserved motifs in the promoter regions of PCP and PPP genes. The promoter regions (1 kb) of the PCP and PPP genes listed in Table 2 were used in our search for conserved sequences. The six best candidate motifs for the AraR binding site are shown. Right panel: standard orientation. Left panel: reverse complement.
expression levels are smaller to detect and reproduce by different techniques. By using bioinformatics analysis, we found six novel conserved transcription factor binding sites in the promoter regions of both PCP and PPP genes, that are conserved as well
in known AraR-regulated CWDE genes. One of these sequences (motif 1) is found in three (mainly) AraR-regulated PCP genes (ladA, xdhA, xkiA) and all three AraR-regulated CWDE genes (abnA, abfA and abfB). The AraR binding site is still unknown, but this sequence may be a promising
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539
Fig. 4. Regulatory model for PCP and PPP in A. niger. Gene expression values are presented under the genes and indicated by color gradient. Fold change of gene expression (mutant over reference strain) is visualized by bars next to the corresponding genes. Decreases of expression are marked by green bars pointing downwards and increased expressions are colored red pointing upwards. The length of the bars is in scale of the fold change. Abbreviations for the PPP genes are listed in Table 2. AraR regulated genes are in blue. XlnR regulated genes are in orange. Genes regulated by AraR and XlnR are in green. Genes for which regulation by AraR and XlnR was not confirmed are indicated in black and genes that remain to be analyzed are underlined.
candidate binding site for this transcription factor. Interestingly, the promoter regions of both rpiA and talB contained this motif. In future, it is important to first experimentally identify the AraR binding site in A. niger. Second, potential putative AraR binding sites must be functionally tested by Chip-PCR to have evidence for direct regulation of these PPP genes by AraR. Our experimental evidence suggests regulation of the rpiA and talB genes by XlnR in A. niger. This demonstrates that the role of XlnR involves not only pentose release and initial conversion, but extends into more central parts of carbon catabolism in A. niger. Based on the data presented in this study, the previously published regulatory model of the PCP in A. niger was confirmed and expanded to also include the PPP (Fig. 4). Most likely, XlnR controls specific PPP genes to ensure a sufficient flux through this pathway during growth on pentoses. As the PPP is also required during growth on other carbon sources, it is likely that other regulators also affect the expression of the PPP genes, which could be the reason why
the expression profiles of the PPP genes are more complex than those of the PCP genes. More detailed studies into the individual PPP genes will be needed to fully reveal the regulatory control by AraR. Conflict of interest The authors declare no conflicts of interest. Acknowledgments E. Battaglia was supported by a grant of the Dutch Technology Foundation STW, Applied Science division of NWO and the Technology Program of the Ministry of Economic Affairs UGC 07063 to R.P. de Vries. M. Zhou was supported by a grant from the Netherlands Organisation for Scientific Research (NWO) and the Netherlands Genomics Initiative 93511035 to R. P. de Vries. We thank Mikael R. Andersen for inspiring discussion and suggestions on microarray data
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analysis. Special thanks to Prof. Jack Leunissen for PatScan software support.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.resmic.2014.07.013. References [1] Witteveen CFB, Busink R, Van de Vondervoort PJI, Dijkema C, Swart K, Visser J. L-arabinose and D-xylose catabolism in Aspergillus niger. J Gen Microbiol 1989;135:2163e71. [2] Horecker BL, Mehler AH. Carbohydrate metabolism. Annu Rev Biochem 1955;24:207e74. [3] Hahn-Hagerdal B, Karhumaa K, Jeppsson M, Gorwa-Grauslund MF. Metabolic engineering for pentose utilization in Saccharomyces cerevisiae. Adv Biochem Eng/Biotechnol 2007;108:147e77. [4] Poulsen BR, Nohr J, Douthwaite S, Hansen LV, Iversen JJ, Visser J, et al. Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger. FEBS J 2005;272:1313e25. [5] Battaglia E, Visser L, Nijssen A, van Veluw J, W€osten HAB, de Vries RP. Analysis of regulation of pentose utilisation in Aspergillus niger reveals evolutionary adaptations in the Eurotiales. Stud Mycol 2011;69:31e8. [6] de Groot MJL, van den Dool C, Wosten HAB, Levisson M, vanKuyk PA, Ruijter GJG, et al. Regulation of pentose catabolic pathway genes of Aspergillus niger. Food Technol Biotechnol 2007;45:134e8. [7] Hasper AA, Visser J, de Graaff LH. The Aspergillus niger transcriptional activator XlnR, which is involved in the degradation of the polysaccharides xylan and cellulose, also regulates D-xylose reductase gene expression. Mol Microbiol 2000;36:193e200. [8] Seiboth B, Metz B. Fungal arabinan and L-arabinose metabolism. Appl Microbiol Biotechnol 2011;89:1665e73. [9] Stricker AR, Grosstessner-Hain K, Wurleitner E, Mach RL. Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina. Eukaryot Cell 2006;5:2128e37. [10] van Peij NN, Visser J, de Graaff LH. Isolation and analysis of xlnR, encoding a transcriptional activator co-ordinating xylanolytic expression in Aspergillus niger. Mol Microbiol 1998;27:131e42. [11] Wilson RA, Gibson RP, Quispe CF, Littlechild JA, Talbot NJ. An NADPH-dependent genetic switch regulates plant infection by the rice blast fungus. Proc Nat Acad Sci U S A 2010;107:21902e7. [12] Jorgensen TR, Goosen T, Hondel CA, Ram AF, Iversen JJ. Transcriptomic comparison of Aspergillus niger growing on two different sugars reveals coordinated regulation of the secretory pathway. BMC Genom 2009;10:44. [13] Sorensen LM, Lametsch R, Andersen MR, Nielsen PV, Frisvad JC. Proteome analysis of Aspergillus niger: lactate added in starchcontaining medium can increase production of the mycotoxin fumonisin B2 by modifying acetyl-CoA metabolism. BMC Microbiol 2009;9:255. [14] de Souza WR, de Gouvea PF, Savoldi M, Malavazi I, de Souza Bernardes LA, Goldman MH, et al. Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse. Biotechnol Biofuels 2011;4:40. [15] Sun J, Tian C, Diamond S, Glass NL. Deciphering transcriptional regulatory mechanisms associated with hemicellulose degradation in Neurospora crassa. Eukaryot Cell 2012;11:482e93. [16] de Vries RP, Burgers K, van de Vondervoort PJI, Frisvad JC, Samson RA, Visser J. A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production. Appl Environ Microbiol 2004;70:3954e9.
[17] Andersen MR, Nielsen ML, Nielsen J. Metabolic model integration of the bibliome, genome, metabolome and reactome of Aspergillus niger. Mol Syst Biol 2008;4:178. [18] Flipphi M, Sun J, Robellet X, Karaffa L, Fekete E, Zeng AP, et al. Biodiversity and evolution of primary carbon metabolism in Aspergillus nidulans and other Aspergillus spp. Fung Genet Biol 2009;46(Suppl. 1):S19e44. [19] Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA. Primer3Plus, an enhanced web interface to Primer3. Nucl Acids Res 2007;35:W71e4. [20] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402e8. [21] R Development Core Team. R: a language and environment for statistical computing. R Foundation for Statistical Computing; 2009. [22] Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostat 2003;4:249e64. [23] Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinform 2003;19:185e93. [24] Kayala MA, Baldi P. Cyber-T web server: differential analysis of highthroughput data. Nucl Acids Res 2012;40:W553e9. [25] Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, et al. MEME SUITE: tools for motif discovery and searching. Nucl Acids Res 2009;37:W202e8. [26] Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res 2004;14:1188e90. [27] Bailey TL, Gribskov M. Combining evidence using p-values: application to sequence homology searches. Bioinform 1998;14:48e54. [28] Mathelier A, Zhao X, Zhang AW, Parcy F, Worsley-Hunt R, Arenillas DJ, et al. JASPAR 2014: an extensively expanded and updated open-access database of transcription factor binding profiles. Nucl Acids Res 2014;42:D142e7. [29] vanKuyk PA, de Groot MJL, Ruijter GJG, de Vries RP, Visser J. The Aspergillus niger D-xylulose kinase gene is co-expressed with genes encoding arabinan degrading enzymes and is essential for growth on arabinose and xylose. Eur J Biochem 2001;268:5414e23. [30] Fan JX, Yang Q, Liu ZH, Huang XM, Song JZ, Chen ZX, et al. The characterization of transaldolase gene tal from Pichia stipitis and its heterologous expression in Fusarium oxysporum. Mol Biol Rep 2011;38:1831e40. [31] Wang T, Merja P, Gao P. Expression of xylose-metabolic key genes of Trichoderma reesei on various carbon sources measured by a series of Northern hybridizations. Acta Microbiol Sin 1999;39:503e9. [32] van der Veen D, Oliveira JM, van den Berg WA, de Graaff LH. Analysis of variance components reveals the contribution of sample processing to transcript variation. Appl Environ Microbiol 2009;75:2414e22. [33] Bos CJ, Debets AJ, Swart K, Huybers A, Kobus G, Slakhorst SM. Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger. Curr Genet 1988;14:437e43. [34] Jalving R, van de Vondervoort PJI, Visser J, Schaap PJ. Characterization of the kexin-like maturase of Aspergillus niger. Appl Environ Microbiol 2000;66:363e8. [35] vanKuyk PA, Diderich JA, MacCabe AP, Hererro O, Ruijter GJG, Visser J. Aspergillus niger mstA encodes a high-affinity sugar/Hþ symporter which is regulated in response to extracellular pH. Biochem J 2004;379:375e83. [36] Mojzita D, Vuoristo K, Koivistoinen OM, Penttila M, Richard P. The 'true' L-xylulose reductase of filamentous fungi identified in Aspergillus niger. FEBS Lett 2010;584:3540e4. [37] Mojzita D, Penttila M, Richard P. Identification of an L-arabinose reductase gene in Aspergillus niger and its role in L-arabinose catabolism. J Biol Chem 2010;285:23622e8. [38] van den Broek P, Goosen T, Wennekes B, van den Broek H. Isolation and characterization of the glucose-6-phosphate dehydrogenase encoding gene ( gsdA) from Aspergillus niger. Mol Gen Genet 1995;247:229e39.
The transcriptional activators AraR and XlnR from Aspergillus niger regulate expression of pentose catabolic and pentose phosphate pathway genes Evy Battaglia a,b, Miaomiao Zhou b, Ronald P. de Vries a,b,* a
Microbiology & Kluyver Centre for Genomics of Industrial Fermentation, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands b CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CY, Utrecht, The Netherlands Received 27 January 2014; accepted 21 July 2014 Available online 31 July 2014
Abstract The pentose catabolic pathway (PCP) and the pentose phosphate pathway (PPP) are required for the conversion of pentose sugars in fungi and are linked via D-xylulose-5-phosphate. Previously, it was shown that the PCP is regulated by the transcriptional activators XlnR and AraR in Aspergillus niger. Here we assessed whether XlnR and AraR also regulate the PPP. Expression of two genes, rpiA and talB, was reduced in the DaraR/DxlnR strain and increased in the xylulokinase negative strain (xkiA1) on D-xylose and/or L-arabinose. Bioinformatic analysis of the 1 kb promoter regions of rpiA and talB showed the presence of putative XlnR binding sites. Combining all results in this study, it strongly suggests that these two PPP genes are under regulation of XlnR in A. niger. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Pentose phosphate pathway; Aspergillus niger; Regulation of gene expression; XlnR; AraR
1. Introduction In fungi, L-arabinose and D-xylose are metabolized through an oxidoreductive pathway named the pentose catabolic pathway (PCP) [1]. Both these pentose sugars go through oxidation, reduction and phosphorylation reactions to form Dxylulose-5-phosphate. This intermediate ketose sugar connects the PCP to the pentose phosphate pathway (PPP), one of the central metabolic pathways in primary metabolism. The PPP, first described by Horecker and Mehler, is important for the production of reducing power in the form of NADPH and precursors for nucleotide and amino acid biosynthesis [2]. Genetic engineering of the PCP and PPP in yeast and
* Corresponding author. CBS Fungal Biodiversity Centre Uppsalalaan 8 3584 CY Utrecht Netherlands. E-mail addresses: [email protected] (E. Battaglia), m.zhou@cbs. knaw.nl (M. Zhou), [email protected] (R.P. de Vries).
filamentous fungi has proven that the metabolic reactions of these pathways are highly interconnected during pentose utilization [3,4]. The production of D-xylulose-5-phosphate by the PCP not only connects the metabolic pathways by allowing an increased flux into the non-oxidative PPP, but also the (re) generation and demand of the redox cofactors NADH and NADPH in enzymatic reactions of both the PCP and oxidative PPP. Witteveen et al. demonstrated an increase in activity of the enzymes involved in the last two steps of the oxidative PPP during growth on L-arabinose and D-xylose, namely the activity of glucose-6-phosphate dehydrogenase (G6PD) and 6phosphogluconate dehydrogenase (6PGD) [1]. As metabolic pathways are interconnected, the molecular mechanisms regulating the expression of these metabolic genes are complex and only beginning to be unraveled in filamentous fungi. Regulation of the PCP has been studied in detail in Aspergilli and Trichoderma reesei [5e9]. In Aspergillus niger, the Zn2Cys6 transcriptional activator XlnR [10] regulates xyrA (encoding D-xylose reductase) the first step of
http://dx.doi.org/10.1016/j.resmic.2014.07.013 0923-2508/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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D-xylose catabolism [7]. Analysis of two A. niger UV mutants provided evidence for the presence of a L-arabinose responsive regulator that together with XlnR controls PCP genes [6], which was later identified and named AraR [5]. Disruption of both xlnR and araR resulted in no expression of the PCP genes and an inability of this A. niger mutant to utilize D-xylose or Larabinose [5], indicating a complex regulatory interplay between these transcription factors in this pathway. In Magnaporthe oryzae, regulation of PPP was proven to be modulated by the levels of NADP, via trehalose-6-phosphate synthase (Tps1), an enzyme of trehalose biosynthesis [11]. No studies on the regulation of this central metabolic pathway during utilization of D-xylose or L-arabinose in filamentous fungi have been published. Interestingly, it has been reported that PPP genes are upregulated in A. niger during growth on D-xylose as a sole carbon source [12] and when other carbon sources such as starch and lactate [13] and complex sugar substrates are provided [14]. In Neurospora crassa, several PPP genes were upregulated on xylan compared to sucrose [15], and their expression was dependent on Xlr-1 (the N. crassa XlnR/Xyr1 homolog) on this substrate. In this study, the influence of the transcriptional activators XlnR and AraR on the expression of PCP and PPP genes was assessed in A. niger.
2. Materials and methods 2.1. Strains and growth conditions The A. niger strains, listed in Table 1, were grown in minimal medium (MM) or complete medium (CM) [16] at 30 C and 250 rpm in a rotary shaker. When necessary, 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. In transfer experiments, all strains were pre-grown in 200 ml CM containing 2% D-fructose in a 1 L Erlenmeyer flask. After 16 h of incubation, the mycelium was harvested without suction over a cheese cloth, washed twice with MM without carbon source and transferred to a 250 ml Erlenmeyer flask containing 50 ml MM containing 25 mM D-xylose or L-arabinose. After 2 h and 4 h of incubation the mycelium was harvested with suction, the mycelium was subsequently dried between tissue paper and directly frozen in liquid nitrogen.
Table 1 Aspergillus niger strains used in this study. Strain
Genotype
Reference
N402 N572 NW249 UU-A049.1
cspA1 cspA1, fwnA1, pyrA6, xkiA1, nicA1 cspA1, DargB, pyrA6, nicA1, leuA1 cspA1, pyrA6, nicA1, leuA1, DargB::pIM2101 (argBþ) cspA1, pyrA6, nicA1, leuA1, DaraR cspA1, DargB,nicA1, leuA1, DxlnR cspA1, nicA1, leuA1, DaraR, DxlnR
[33] [29] [34,35] [5]
UU-A033.21 UU-A062.10 UU-A063.22
[5] [5] [5]
2.2. PCP and PPP genes collection All PCP genes were selected and corresponding enzymes were biochemically characterized previously (see Table 2 for references). PPP genes were selected based on two previously published papers in which the authors both computationally and manually annotated these metabolic genes from A. niger CBS 513.88 and ATCC 1015 [17,18]. 2.3. Micro-array and quantitative real-time PCR analysis Total RNA was isolated from mycelium that was ground in a microdismembrator (B Braun). Approximately ~100 mg of ground mycelium was homogenized in 1 ml Trizol reagent (Invitrogen). After addition of 200 ml chloroform and phase separation by centrifugation, 70% ethanol was added to the aqueous phase, which was subsequently transferred on a silica spin column from NucleoSpin RNA kit (Macherey-Nagel). RNA was further purified according to the manufacturer's instructions. The quality of RNA was checked using gel electrophoresis and RNA was quantified using a UV spectrophotometer. cDNA was synthesized from 2.5 mg of total RNA using the Thermoscript transcriptase according to the manufacturer's instructions (Invitrogen). The RNase H treatment was followed by an ethanol precipitation. cDNA was dissolved in 10 ml H2O and checked using gel electrophoresis. All qPCR primers pairs were designed with Primer3 Plus [19] and experimentally validated with two quality control (specificity and efficiency) assays. The optimal primer concentrations were tested by dissociation curve analysis to check for gene-specific signals and nonspecific signals caused by primer dimers. Combinations of 50 nM, 300 nM and 900 nM (final concentration) per primer pair were used. The efficiency of each primer pair (>90%) was assessed by plotting the cycle threshold value (Ct) against the logarithm of dilution of A. niger genomic DNA samples, ranging from 10 ng to 1 pg. The sequences of all primer pairs, optimal concentrations and amplicon sizes are listed in Supplemental Table 1. qPCR was performed using the ABI 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA, USA). RT-PCR mixtures (20 ml) contained: 10 ml ABI Fast SYBR Master Mix (Applied Biosystems, Foster City, CA, USA), the forward and reverse primer pair (2 ml of each primer, at optimal concentration), 2 ml of 100 fold diluted cDNA and 4 ml H2O. The cycle conditions were as follows: 95 C for 2 s, 95 C for 3 s and 60 C for 30 s (40 cycles). The A. niger histone H2B gene (An11g11310) was used as a reference gene for normalization. The results were analyzed using the comparative CT (2-DDCT) method [20]. Graphpad Prism version 6 was used to calculate the mean, standard error of the mean and significance between the reference and mutant samples using two sample t tests. The quality of the RNA samples for Affimetrix microarray analysis was assessed using the Agilent 2100 Bioanalyzer. Microarray data was analyzed using the Bioconductor Affy tool package (http://www.bioconductor.org/) under the
Table 2 Transcriptional levels of PCP and PPP genes in the DxlnR strain and the wild type strain N402 on D-xylose, and the DaraR strain and reference strain N402 L-arabinose. araR/arae
FDR
733 ± 53.9
8287.6 ± 274.9
¡11.31
1.36E-10
5047.8 ± 234.6
3535.1 ± 56.7
PCP
352 ± 7.5
4678.9 ± 379.6
¡13.29
6.65E-10
2946.7 ± 151.5
An01g03740 An00g00176_at [7]
PCP
8789.1 ± 423.8
8913.5 ± 530.8
1.01
0.91
An08g01930 An00g03632_at [36]
PCP
150.6 ± 28.4
7275.6 ± 162.7
¡48.31
5.73E-11
An11g01120 An00g06977_at [37]
PCP
1634.7 ± 296.5
12063 ± 18.7
An12g00030 An00g09559_at [6] An11g02040 An00g10295_at [4]
PCP PPP
5922.4 ± 418.7 2972.4 ± 52.2
An02g12140 An00g00194_at [38]
PPP
1394.6 ± 178.7
An14g00160 An01g05150 An15g07500 An09g03450
[18] [18] [17] [18]
PPP PPP PPP PPP
589.7 933.9 351.9 69.5
An11g00470 An00g10887_at [18]
PPP
535.3 ± 15.3
988.2 ± 10.7
An02g02930 An00g00058_at [18]
PPP
4988.7 ± 138.3
4524.9 ± 388.4
An07g03850 An07g03160 An08g06570 An02g06430 An14g03500
[18] [18] [4] [18] [17]
PPP PPP PPP PPP PPP
4338.5 943.8 4661.3 19 46.1
An11g06120 An00g10294_at [17]
PPP
Gene ID
ladA
An01g10920 An00g11747_at [6]
PCP
An07g03140 An00g08027_at [29]
L-arabitol
dehydrogenase xkiA D-xylulose kinase xyrA D-xylose reductase lxrA L-xylo-3-hexulose reductase larA NADPH-dependent aldehyde reductase xdhA xylitol dehydrogenase gndA 6-phosphogluconate dehydrogenase gsdA glucose-6-phosphate 1-dehydrogenase rbtA D-ribulokinase pglA 6-phosphogluconolactonase rbkA ribokinase rpeA D-ribulose-phosphate-3 epimerase rpiA ribose 5-phosphate isomerase rpiB ribose 5-phosphate isomerase talA transaldolase talB transaldolase tktA transketolase tktB transketolase tktC dihydroxyacetone synthase gndB phosphogluconate dehydrogenase (decarboxylating)
An00g08020_at An00g11069_at An00g10879_at An00g10897_at
An00g11820_at An00g11821_at An00g11869_at An00g06454_at An00g11348_at
Ref
± ± ± ±
± ± ± ± ±
9.7 23.9 0.4 0.6
31.9 39.6 506.2 1.5 4
19.3 ± 0.3
xlnRc
xyld
xlnR/xyle
FDR
1.43
4.37E-06
4421.9 ± 131.2
1.50
1.12E-05
5751.8 ± 251.6
11107.9 ± 164.2
¡1.93
6.16E-09
4750.6 ± 13.7
2908.4 ± 94.3
1.63
1.57E-07
¡7.38
2.17E-09 13258.7 ± 90.7
9233.2 ± 67.7
1.44
1.62E-07
12305 ± 358.3 7121.4 ± 544
¡2.08 ¡2.40
4.11E-06 12886.4 ± 427.1 5.38E-06 10158.3 ± 130.6
9321.5 ± 145.3 12785.5 ± 267.5
1.38 1.26
1.68E-06 2.26E-05
4220.1 ± 327
¡3.03
7.28E-06
6332.9 ± 50.7
9899.7 ± 9.5
¡1.56
4.38E-08
1.45 1.25 ¡1.61 ¡2.25
0.02 0.15 0.01 0.00
464.5 1047.3 511 302.4
13.7 58.9 0.2 6.2
1.03 ¡1.77 2.18 ¡2.69
0.67 1.89E-06 4.19E-07 2.78E-08
¡1.85
0.00
1006 ± 63
928.6 ± 26.6
1.08
0.34
1.10
0.49
4489 ± 138.4
1.39
8.07E-06
1.03 ¡8.70 1.05 ¡8.79 1.05
0.41 5.72E-11 0.14 4.04E-10 0.56
407.4 1171.6 565 156.7
8630.8 840.1 9331.3 23.1 39
± ± ± ±
± ± ± ± ±
26.8 132.2 68.7 22.8
385.4 65.8 193.8 0.8 0.9
19.1 ± 0.7
¡1.99 1.12 ¡2.00 1.21 1.18 1.01
± ± ± ±
10.3 2.9 21.4 9.2
6.42E-06 12430.9 ± 223.3 0.43 245.6 ± 9.1 8.61E-06 12075.3 ± 233.4 0.01 18.7 ± 0.2 0.14 31 ± 1.8 0.90
20 ± 0.1
450.1 1856.7 234.2 813.5
± ± ± ±
3222.3 ± 7.5 12789.9 2138.1 12718.2 164.7 29.6
± ± ± ± ±
57.5 88.5 99.3 25.3 1.5
19.1 ± 0.6
1.05
E. Battaglia et al. / Research in Microbiology 165 (2014) 531e540
arab
Probe number
Metabolic araRa pathway
Gene Activity
0.30
PPP: pentose phosphate pathway and PCP: pentose catabolic pathway. The mean expression levels of duplicate samples for DaraR 2 h L-ara, reference 2 h L-ara, DxlnR 2 h D-xyl and reference 2 h D-xyl, respectively. e The fold change ratio calculated by the expression levels of the disruption strain versus reference strain for DaraR/ref and DxlnR/ref, respectively. Genes with significant differential expression (fold change >1.5 and fdr value <0.05) are marked bold.
a, b, c, d
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statistical environment R [21]. The probe intensities were normalized for background by using the Robust Multiarray Average (RMA) algorithm [22] for the Affimetrix CEL files with only the perfect match (PM) probes. The quantiles algorithm [23] was used to perform normalization and gene expression values were calculated by the medianpolish summary method [22]. The normalized gene expression values were then processed with the statistical tool Cyber-T [24] to detect differential significance, using the BayesANOVA algorithm. Gene expression variation with P-value <0.05 and fold change >1.5 were considered significant. 2.4. Northern analysis The Minifold II slot blot apparatus (Schleicher & Schuell) was used for the transfer of 3 mg total RNA to a Hybond-Nþ membrane (Amersham Biosciences). Equal loading of total RNA was controlled by soaking the blot for 5 min in 0.04% methylene blue, 0.5 M acetate pH 5.2 solution. The methylene blue stained membrane is included in Figs. 1 and 2 as the loading control. The primers used to generate the probes for Northern analysis are listed in Supplemental Table 1. The probes were DIG-labeled using the PCR DIG Probe Syntheses Kit (Roche Applied Science) according to the supplier's instructions. A cDNA library [6] or genomic DNA (obtained from A. niger N402) was used as a template in the PCR reactions for synthesis of the probes. Hybridization of the DIGlabeled probes to the blot and their detection was performed according to the DIG user's manual (www.roche-appliedscience.com). 2.5. Transcription factor binding motifs in regulated genes The DNA sequences of 1000 nucleotide upstream of each PCP and PPP gene were extracted from their genomes according to their gene calling. The upstream DNA stretches were fed to MEME for detection of conserved motifs using parameters of 1E-5, minimal length 2, maximum length 15, and allowing any numbers of repetition [25]. The figures of motifs were generated by Weblogo [26]. The predicted motifs with more than 2 sites were validated with known XlnR/AraRregulated enzymes by MAST using the default setting [27]. The known XlnR motif was searched on DNA sequences using PatScan. Search of known transcriptional factor binding site was done by JASPAR fungi core motifs with relative profile score threshold of 90% [28]. 3. Results 3.1. Deletion of the Zn2Cys6 transcriptional activators XlnR and AraR in A. niger results in down-regulation of both the pentose catabolic and phosphate pathway during growth on pentose sugars Expression of PCP and PPP genes was compared in the wild type and the DxlnR and DaraR strains (Table 2). The
expression of five out of six genes encoding enzymes of the PCP (ladA, xdhA, xkiA, lxrA, larA) showed significant reduction in the DaraR strain on L-arabinose. The D-xylose reductase gene xyrA had, as reported before [5], an unchanged expression level in the DaraR on L-arabinose and a reduced expression level in the DxlnR strain on D-xylose. Expression levels of four of the other genes (larA, ladA, xdhA, xkiA) were unaffected in the DxlnR strain during growth on D-xylose, whereas lxrA expression was moderately increased. Seven out of fourteen PPP genes showed significant reduced expression levels in the DaraR strain on L-arabinose (Table 2). The expression level of only one PPP gene, Dribulose-5-phosphate 3-epimerase (rpeA), was significantly altered in both strains. The expression of one transaldolase (talB) and one transketolase (tktB) gene was significantly decreased in the DxlnR strain on D-xylose but not significantly affected in the DaraR strain on L-arabinose. The expression of four PPP genes (rbtA, rpiB, tktC and gndB) was less than 1.5fold altered, so unaffected in both the regulator mutants on Larabinose or D-xylose (Table 2). Based on the micro-array results, a subset of the genes ( gndA, rpeA, rpiA, talA, talB and tktA) was selected for confirmation by Northern analysis. The reference strain, the DaraR and DxlnR strains, as well as a strain in which both araR and xlnR were deleted (UU-A063.22), were incubated for 2 h in MM with L-arabinose or D-xylose. The reduced expression levels of rpiA, talA, tktA and gndA in DaraR on L-arabinose and talB in DxlnR on D-xylose confirmed the microarray data (Fig. 1A). In DaraR on D-xylose an increased expression level of talB was observed, while reduced expression was observed for gndA and rpiA in DxlnR on L-arabinose compared to the reference strain. In contrast, expression of rpiA was increased in the DaraR and DxlnR strains on D-xylose. In the DaraR/DxlnR strain, expression of five PPP genes ( gndA, rpiA, talA, talB and tktA) was reduced on L-arabinose and of four PPP genes ( gndA, talA, talB and tktA) on D-xylose (Fig. 1A). 3.2. Expression pattern of PPP genes in the A. niger xkiA1 mutant strain indicates regulation by AraR and/or XlnR Expression of the PPP genes was analyzed in an xkiA1 mutant strain to determine whether expression of these PPP genes is changed by regulation by AraR and/or XlnR or indirectly by altered levels of D-xylulose-5-phosphate. In this strain, D-xylulose kinase activity is absent, resulting in an arrest of pentose catabolism at D-xylulose and no formation of Dxylulose-5-phosphate [29]. Genes regulated by AraR and/or XlnR should show increased expression in this strain due to accumulation of the inducers of these regulators, while genes affected by reduced D-xylulose 5-phosphate levels should show decreased expression. The reference strain and the xkiA1 mutant (N572) were grown for 2 and 4 h on L-arabinose and D-xylose. Two genes of the PPP, talB and rpiA, showed increased expression levels in the xkiA mutant after 4 h of growth on D-xylose and L-arabinose, respectively (Fig. 2A), which is a similar pattern as that
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Fig. 1. A) Northern blot analysis of pentose phosphate pathway genes. The reference strain and the DaraR, DxlnR and DaraR/DxlnR strains were grown for 2 on 25 mM L-arabinose or 25 mM D-xylose. Methylene blue staining of total RNA is included as a loading control. B) Quantitative gene expression analysis of the transaldolase gene (talB) and the ribose-5-isomerase gene (rpiA). For both genes, the expressions are normalized against the expression of histone h2b. Error bars indicate the standard deviation of two biological and two technical repeats. R ¼ reference, DX ¼ DxlnR, DA ¼ DaraR, and DADX ¼ DaraR/DxlnR strain. Significance tests were performed between reference and regulator mutant strains. p < 0.01 (**), p < 0.05 (*).
observed for two PCP genes (ladA and xkiA), suggesting regulation by AraR or XlnR. These two PPP genes (rpiA and talB) and the two PCP genes (ladA and xkiA) were not induced in the reference strain on D-fructose. Reduced or similar expression levels were observed for the other genes ( gndA, rpeA, talA, and tktA) in the xkiA1 mutant on both substrates, compared to the reference strain (Fig. 2A).
To conclude, our Northern blot results suggest the rpiA and talB genes are regulated by AraR and/or XlnR. RT-qPCR analysis was carried out to have an accurate quantification of the differences in gene expression levels, and thus confirm observed changes for the rpiA and talB genes. The RT-qPCR results are presented in both Figs. 1 and 2 as the b-sections of the figures. The data obtained with RT-qPCR did not fully
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Fig. 2. A) Northern blot analysis of pentose catabolic pathway genes and pentose phosphate pathway genes in the reference strain and the xkiA1 mutant. Both strains were grown for 2 and 4 h on 25 mM D-fructose (F), L-arabinose (A) or D-xylose (X). Methylene blue staining of total RNA is included as a loading control. B) Quantitative gene expression analysis of the D-xylulose kinase genes (xkiA), the transaldolase gene (talB) and the ribose-5-isomerase gene (rpiA) in the reference strain and the xkiA1 mutant. For both genes, the expressions are normalized against the expression of histone h2b. Error bars indicate the standard deviation of two biological and two technical repeats. R ¼ reference, DX ¼ DxlnR, DA ¼ DaraR, and DADX ¼ DaraR/DxlnR strain. Significance tests were performed between reference and regulator mutant strains. p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), p < 0.05 (*).
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correlate with our Northern blot results, even for the control gene xkiA that we included. Nevertheless, the quantitative results did indeed confirm changes in expression levels between the reference and mutants strains that suggest regulation by the transcription factors, and thus not alter our overall conclusion. Expression levels of rpiA were significantly reduced in the DaraR/DxlnR on D-xylose, while expression increased on D-xylose (4h) in the xkiA1 mutant (Figs. 1B and 2B). Expression levels of the talB gene were reduced in the DxlnR and DaraR/DxlnR strain on both L-arabinose and Dxylose, while expression was increased on both sugars (4 h) in the xkiA1 mutant. 3.3. Transcription factor binding motifs in PCP and PPP genes The 1 kb upstream regions of the PCP and PPP genes listed in Table 2 were analyzed for conserved elements using the MEME software (see Materials and Methods for our search parameters) [25]. The six motifs with the highest significance scores are depicted in Fig. 3. The genes together with the conserved motif sequence, significance value and location are listed in Supplemental File 2a. Most PCP and PPP genes contained motif 1 and 2 (Fig. 3 and Supplemental File 2a). Motif 3 and 6 were mainly present in PPP genes and only one or two PCP gene(s). Motif 4 was only found in PCP genes that were down-regulated in the araR deletion mutant. The promoter regions of 5 out of 6 PCP genes contained this motif. Motif 5 was present in only two PCP (xdhA and xkiA) and two PPP genes (rbtA and tktA). All six motifs that we found were not identical to known fungal transcriptional factor binding sites according to the JASPAR database [28] (Supplementary File 3). This strongly suggests the motifs are not similar to known/yeast motifs and these sequences are novel. All these conserved motifs are candidate binding sites for AraR, because the results showed that the 1 kb promoter regions of both known AraR-regulated PCP, endo-arabinanase and a-arabinofuranosidase genes (abnA, abfA and abfB) genes contained these motifs (Supplemental File 2a and 2b). The characterized XlnR binding site G-G-C-T-A-[AG] [ATCG] was searched against all PCP and PPP genes together with known XlnR-regulated genes (b-xylosidase gene: xlnD; feruloyl esterase gene: faeA; endo-glucanase gene: eglA) (Supplemental File 2c). Besides the presence in eglA, faeA, xlnD, the XlnR binding site was also found in three known XlnR-regulated PCP genes (xdhA, xyrA and xkiA) and six PPP genes ( pglA, rpeA, rpiA, talA, talB and tktA). If we combine these results with the gene expression experiments, this strongly suggests XlnR plays a role in regulation of talB and rpiA. 4. Discussion The PCP and PPP enable complete conversion of D-xylose and L-arabinose to central carbon catabolism. In this research, expression profiles of six PCP genes in A. niger confirmed that
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the regulation of the complete PCP is under control of AraR and XlnR in this fungus, with heavier influences from AraR than XlnR as reported previously [5]. This study also aimed to elucidate whether these transcription factors regulate PPP genes. After an initial screen of transcriptome data, a subset of genes was selected for more detailed analysis to confirm whether the observed changes in gene expression levels in the regulatory mutants were caused by control of the regulators or indirect changes in D-xylulose-5-phosphate levels. We used a xylulose kinase deficient (xkiA1) strain to determine regulation by AraR and/or XlnR. As the xkiA1 mutant accumulates the inducers for AraR and XlnR, genes under control of these regulators have increased expression in the xkiA1 mutant compared to the wild type, as was previously shown for PCP genes [5,29]. Expression of the ribose-5-phosphate isomerase encoding gene (rpiA) was reduced in the DaraR/DxlnR strain and increased in the xkiA1 mutant on D-xylose, as assessed by Northern blot and qPCR analysis. In the 1 kb promoter region of the rpiA gene, a putative binding site for XlnR was found. Combining the expression and promoter results, it strongly suggests this PPP gene is under control of XlnR. However, the data of this study also show that rpiA is not only dependent on XlnR and AraR on D-xylose, because expression was still observed in the DaraR/DxlnR strain on this substrate. Expression levels of rpiA were detected on D-fructose by qPCR analysis suggesting this gene is not specifically induced on pentoses. A second putative ribose-5-phosphate isomerase encoding gene (rpiB) is present in the A. niger genome and seven other Aspergilli [18]. Expression of this gene was unaffected by deletion of xlnR or araR and this gene was therefore not studied in detail. Two transaldolase genes are present in the A. niger genome. The expression data indicate that A. niger talA is constitutively expressed and not under regulation of XlnR or AraR. In Pichia stipitis and Trichoderma reesei, the ortholog of talA is also constitutively expressed and the corresponding enzyme of these fungi is involved in the metabolism of many carbon sources [30,31]. The expression profiles suggest that only talB is under control of XlnR during growth on L-arabinose and D-xylose. This was shown using the same experimental set up as rpiA. The A. niger talB gene is predominantly induced on D-xylose and L-arabinose and not on D-fructose (this study) or maltose [12]. Based on the expression levels in the DxlnR, DaraR/ DxlnR and xkiA1 deficient strain on D-xylose, talB expression appears to be predominantly under regulation of XlnR. XlnR regulation of talB is supported by the presence of a XlnR binding site in its promoter [32]. It should be noted that for the rpiA gene the Northern blot and micro-array changes between the A. niger reference and regulator deletion strains on L-arabinose do not completely correlate with the mRNA changes measured by qPCR analysis. If the expression of rpiA or talB is influenced by AraR as well, remains unclear. This may suggest that the regulatory effect is less pronounced than XlnR and differences in
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Fig. 3. Conserved motifs in the promoter regions of PCP and PPP genes. The promoter regions (1 kb) of the PCP and PPP genes listed in Table 2 were used in our search for conserved sequences. The six best candidate motifs for the AraR binding site are shown. Right panel: standard orientation. Left panel: reverse complement.
expression levels are smaller to detect and reproduce by different techniques. By using bioinformatics analysis, we found six novel conserved transcription factor binding sites in the promoter regions of both PCP and PPP genes, that are conserved as well
in known AraR-regulated CWDE genes. One of these sequences (motif 1) is found in three (mainly) AraR-regulated PCP genes (ladA, xdhA, xkiA) and all three AraR-regulated CWDE genes (abnA, abfA and abfB). The AraR binding site is still unknown, but this sequence may be a promising
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Fig. 4. Regulatory model for PCP and PPP in A. niger. Gene expression values are presented under the genes and indicated by color gradient. Fold change of gene expression (mutant over reference strain) is visualized by bars next to the corresponding genes. Decreases of expression are marked by green bars pointing downwards and increased expressions are colored red pointing upwards. The length of the bars is in scale of the fold change. Abbreviations for the PPP genes are listed in Table 2. AraR regulated genes are in blue. XlnR regulated genes are in orange. Genes regulated by AraR and XlnR are in green. Genes for which regulation by AraR and XlnR was not confirmed are indicated in black and genes that remain to be analyzed are underlined.
candidate binding site for this transcription factor. Interestingly, the promoter regions of both rpiA and talB contained this motif. In future, it is important to first experimentally identify the AraR binding site in A. niger. Second, potential putative AraR binding sites must be functionally tested by Chip-PCR to have evidence for direct regulation of these PPP genes by AraR. Our experimental evidence suggests regulation of the rpiA and talB genes by XlnR in A. niger. This demonstrates that the role of XlnR involves not only pentose release and initial conversion, but extends into more central parts of carbon catabolism in A. niger. Based on the data presented in this study, the previously published regulatory model of the PCP in A. niger was confirmed and expanded to also include the PPP (Fig. 4). Most likely, XlnR controls specific PPP genes to ensure a sufficient flux through this pathway during growth on pentoses. As the PPP is also required during growth on other carbon sources, it is likely that other regulators also affect the expression of the PPP genes, which could be the reason why
the expression profiles of the PPP genes are more complex than those of the PCP genes. More detailed studies into the individual PPP genes will be needed to fully reveal the regulatory control by AraR. Conflict of interest The authors declare no conflicts of interest. Acknowledgments E. Battaglia was supported by a grant of the Dutch Technology Foundation STW, Applied Science division of NWO and the Technology Program of the Ministry of Economic Affairs UGC 07063 to R.P. de Vries. M. Zhou was supported by a grant from the Netherlands Organisation for Scientific Research (NWO) and the Netherlands Genomics Initiative 93511035 to R. P. de Vries. We thank Mikael R. Andersen for inspiring discussion and suggestions on microarray data
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analysis. Special thanks to Prof. Jack Leunissen for PatScan software support.
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