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J Bacteriol. Mar 2012; 194(5): 1055–1064.
PMCID: PMC3294803

Ribulokinase and Transcriptional Regulation of Arabinose Metabolism in Clostridium acetobutylicum

Abstract

The transcription factor AraR controls utilization of l-arabinose in Bacillus subtilis. In this study, we combined a comparative genomic reconstruction of AraR regulons in nine Clostridium species with detailed experimental characterization of AraR-mediated regulation in Clostridium acetobutylicum. Based on the reconstructed AraR regulons, a novel ribulokinase, AraK, present in all analyzed Clostridium species was identified, which was a nonorthologous replacement of previously characterized ribulokinases. The predicted function of the araK gene was confirmed by inactivation of the araK gene in C. acetobutylicum and biochemical assays using purified recombinant AraK. In addition to the genes involved in arabinose utilization and arabinoside degradation, extension of the AraR regulon to the pentose phosphate pathway genes in several Clostridium species was revealed. The predicted AraR-binding sites in the C. acetobutylicum genome and the negative effect of l-arabinose on DNA-regulator complex formation were verified by in vitro binding assays. The predicted AraR-controlled genes in C. acetobutylicum were experimentally validated by testing gene expression patterns in both wild-type and araR-inactivated mutant strains during growth in the absence or presence of l-arabinose.

INTRODUCTION

The genus Clostridium is a diverse group of low-GC Gram-positive anaerobes that includes a large number of species important for cellulose degradation, development of renewable energy sources, and biotechnology. Many of these species are saprophytic organisms found in the soil (15). Among them, Clostridium acetobutylicum is one of the best-studied clostridia and was used to develop an industrial fermentation process for producing acetone and butanol (5, 22). This strain is known to utilize a broad range of monosaccharides, disaccharides, starches, and other polysaccharides (2, 20).

l-Arabinose is a major component of polysaccharides in plant cell walls, and its utilization pathway in bacteria has been investigated extensively (19). l-Arabinose can be transported into the cell through the ABC transport system AraFGH or arabinose-proton symporter AraE (Fig. 1A). l-Arabinose can also be obtained through hydrolysis of arabinosides. In some bacteria, such as Bacillus subtilis and Geobacillus stearothermophilus, arabinosides are transported into the cell and further degraded into l-arabinose by intracellular enzymes, including α-arabinofuranosidase AbfA and arabinosidase Arb43 (glycoside hydrolase family 43) (8, 36). l-Arabinose is consecutively converted to l-ribulose, l-ribulose-5-phosphate, and xylulose-5-phosphate by the action of l-arabinose isomerase AraA, l-ribulokinase AraB, and l-ribulose-5-phosphate 4-epimerase AraD, respectively, in many bacteria (e.g., B. subtilis). d-Xylulose-5-phosphate is further catabolized to form the central metabolic intermediates glyceraldehyde-3-phosphate and acetyl phosphate by the pentose phosphate pathway enzymes, including transketolase (Tkt), transaldolase (Tal), and phosphoketolase (Ptk).

Fig 1
Reconstruction of AraR regulons in Clostridium species. (A) Metabolic context of the reconstructed clostridial AraR regulons. Matching colors are used to mark components of three distinct pathways related to arabinose metabolism. The AraR regulon members ...

Our current knowledge of transcriptional regulation of l-arabinose utilization pathway in low-GC, Gram-positive bacteria is mostly based on the studies in B. subtilis. The arabinose utilization in B. subtilis is controlled by the transcription factor AraR, which consists of a GntR-type DNA-binding domain in the N-terminal region and a C-terminal effector-binding domain homologous to the GalR/LacI family of repressors (21, 32). In the absence of the effector arabinose, AraR represses the expression of 13 genes, including the araR gene, the genes involved in arabinose catabolism (araABD and araE) and arabinoside degradation (araNPQ, abfA, abnA, and abfA2), and two genes with unknown functions (araLM) (27). The DNA-binding sites of AraR in the promoter regions of these genes have been characterized in B. subtilis (9).

Several Clostridium species have been shown to metabolize l-arabinose in early studies and our preliminary analysis (7, 26). The initial genomic survey of Clostridium acetobutylicum ATCC 824 identified all of the genes involved in arabinose utilization, except for the gene encoding ribulokinase AraB (22). Our previous study has tentatively predicted a candidate gene coding for an alternative ribulokinase (termed AraK), which is not orthologous to the known ribulokinases (30). However, this prediction has yet to be verified experimentally. Moreover, the regulatory mechanism of arabinose catabolism in Clostridium species remains unclear. Although the AraR-binding sites in the C. acetobutylicum genome have been previously predicted by bioinformatics analysis (30), the rapidly growing number of complete genomes in the Clostridium genus allows significant improvement of the accuracy of prediction of AraR-binding DNA motifs and expansion of AraR regulons. The bioinformatics predictions generated by such comparative genomic analyses can be confirmed by further experimental studies. Previously, we have applied this integrated approach to predict and validate novel metabolic enzymes and transcriptional regulons involved in xylose utilization in Clostridium (12).

In this study, we used a comparative genomic approach to identify AraR-binding DNA motifs and reconstruct AraR regulons in nine biotechnologically important Clostridium species. This approach combined metabolic reconstruction with analysis of conserved AraR-binding sites in multiple genomes (29). The novel ribulokinase AraK was identified in all analyzed Clostridium genomes, and this functional assignment was experimentally confirmed through a combination of genetic and biochemical techniques. The extension of the AraR regulon to the pentose phosphate pathway genes was identified in several Clostridium species and experimentally validated in C. acetobutylicum.

MATERIALS AND METHODS

Genome resources and bioinformatics tools.

Genome sequences of Clostridium spp. analyzed in this study were obtained from GenBank (http://www.ncbi.nlm.nih.gov/GenBank/). Identification of orthologs and gene neighborhood analysis were performed in MicrobesOnline (http://www.microbesonline.org/) (4). Functional annotations of genes involved in arabinose metabolism and related pathways were derived from the SEED comparative genomic database (http://theseed.uchicago.edu/FIG/index.cgi) (25). Phylogenetic trees were built using the maximum likelihood method in the PHYLIP package and visualized with Dendroscope (13). Sequence alignments were made with MUSCLE (6). Comparative genomic reconstruction of regulons was performed using the RegPredict web server (http://regpredict.lbl.gov) (24). Sequence logos for regulatory motifs were constructed using the WebLogo package (3).

Reconstruction of regulons.

For identification of regulatory DNA motifs of AraR, we started from training sets of known AraR-regulated genes in B. subtilis and their orthologs in multiple Clostridium genomes. An iterative motif detection algorithm implemented in RegPredict was used to identify common regulatory DNA motifs in upstream regions of these genes (reviewed in reference 29). For each clade of AraR proteins on the phylogenetic tree, a separate training gene set was used. A positional weight matrix was constructed for each identified motif and used to scan the genomes in this clade and identify candidate AraR-binding sites. Scores of candidate sites were calculated as the sum of positional nucleotide weights. The score threshold was defined as the lowest score observed in the training set. A gene having a site score over the threshold was included in the AraR regulon if its regulation was conserved in a set of related genomes. The details of the reconstructed AraR regulons are captured and displayed in the RegPrecise database (http://regprecise.lbl.gov) (23).

Strains and growth conditions.

The C. acetobutylicum strains and plasmids used in this study are given in Table 1. Escherichia coli strains DH5α (Invitrogen, Carlsbad, CA) and ER2275 (18) were used for gene cloning, and BL21(DE3)pLysS (Invitrogen, Carlsbad, CA) was used for protein overexpression. The E. coli K-12 strain that carries the plasmid harboring the araB (b0063) gene with the T5 promoter and the His6 tag was a kind gift from H. Mori at the Nara Institute of Science and Technology, Japan (16). E. coli strains were grown on LB medium. Kanamycin (30 μg ml−1), chloramphenicol (30 μg ml−1), spectinomycin (100 μg ml−1), and isopropyl-β-d-thiogalactopyranoside (IPTG; 0.2 mM) were added as appropriate. C. acetobutylicum strains were routinely cultured at 37°C in CGM medium (38). Erythromycin (30 μg ml−1) was added when needed. Solid medium was made with CGM medium containing 2% (wt/vol) agar A (Sangong Corp., Shanghai, China). For reverse transcription (RT)-PCR analysis of gene expression, C. acetobutylicum strains were grown in a medium without regulatory carbon sources, which contains (per liter) 1.5 g of K2HPO4, 1.5 g of KH2PO4, 2.2 g of CH3COONH4, 30 g of glycerol, 30 g of sodium pyruvate, 0.1 g of l-cysteine, 0.38 g of MgSO4 · H2O, 20 mg of MnSO4 · H2O, 20 mg of FeSO4 · 7H2O, 1 mg of p-aminobenzoic acid, 1 mg of vitamin B1, 1 mg of biotin, and 6 g of yeast extract. l-Arabinose was supplied at a concentration of 30 g liter−1 as indicated. For growth phenotype assays, C. acetobutylicum strains were cultivated at 37°C in triplicates in 60 ml of P2 minimal medium (1) supplemented with 5 g liter−1 l-arabinose or d-xylose as the sole carbon source. Cell growth was monitored spectrophotometrically at 600 nm. Arabinose and xylose were detected by high-pressure liquid chromatography (HPLC) using an Agilent model 1200 instrument equipped with a Waters Sugar Pak I column (6.5 by 300 mm) and a refractive index detector (Agilent). Double-distilled water was used as the mobile phase at a flow rate of 0.6 ml min−1, and the column was operated at 60°C.

Table 1
C. acetobutylicum strains and plasmids used in this study

Gene disruption in C. acetobutylicum.

Gene disruption in C. acetobutylicum ATCC 824 was performed by using group II intron-based targetron technology as described previously (35). Briefly, the 350-bp fragments for retargeting introns to insert within the araK (CAC1344) or araR (CAC1340) genes, respectively, were generated by one-step assembly PCR using the primers shown in Table S1 in the supplemental material according to the protocol of the TargeTron gene knockout system (Sigma). The PCR products were then digested and ligated to a targetron plasmid, pWJ1 (39), yielding the plasmids pWJ1-araK and pWJ1-araR. Each plasmid was methylated in vivo in E. coli ER2275(pAN1) (18) and electroporated into C. acetobutylicum ATCC 824. The transformants were selected on CGM plates supplemented with erythromycin. The resulting mutants with intron insertion in araK or araR genes were confirmed by PCR (see Fig. S1 in the supplemental material). Southern blot analysis with probes against the intron was used to confirm that the intron was incorporated only once into the genome (see Fig. S1 in the supplemental material).

For genetic complementation experiments, the araK gene from C. acetobutylicum was cloned into the pSY9 vector (28) under the control of the constitutive Pptb promoter (37). PCR was carried out using the C. acetobutylicum ATCC 824 genomic DNA and the primers shown in Table S1 in the supplemental material. The obtained plasmid, pSY9-araK, was electroporated into the araK-inactivated mutant, generating the araK-complemented strain. As a negative control, the pSY9 vector was also expressed in the araK-inactivated mutant.

RNA isolation and real-time RT-PCR.

Total RNA was isolated from C. acetobutylicum ATCC 824 grown in medium without regulatory carbon sources or with addition of arabinose, as described above. Cells were harvested at mid-exponential growth phase (~24 h), frozen immediately in liquid nitrogen, and ground into powder. RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) by following the manufacturer's instructions. Contaminant DNA was removed by DNase I (Takara) digestion, which was verified by performing the PCR under identical conditions without adding reverse transcriptase. cDNA was generated by reverse transcription reactions using random hexamers as primers, 1 μg purified RNA, and Moloney murine leukemia virus (MMLV) (RNase H) reverse transcriptase (Takara). The cDNA was amplified using the Applied Biosystems 7300 real-time PCR system. The reaction mixture (20 μl) contained 50 to 100 ng cDNA, 0.2 μM gene-specific primers (as shown in Table S1 in the supplemental material), and Power SYBR green PCR master mix (Applied Biosystems). The PCR parameters were 1 cycle of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 31 s. Melt curves were analyzed to ensure specificity of primer annealing and lack of primer secondary structures. Data analysis was performed with the 7300 system software (Applied Biosystems). The CAC2679 gene encoding a pullulanase was used an internal control for normalization, because its transcript level did not change when C. acetobutylicum was grown on various carbohydrates, including arabinose (34). The expression level of each gene was presented as the average of six measurements from two biological replicates, with the corresponding standard deviation.

Northern blot analysis.

Total RNA was isolated from C. acetobutylicum ATCC 824 grown in P2 minimal medium supplemented with 30 g liter−1 l-arabinose. The RNA (20 μg) was subjected to electrophoresis on 1.5% agarose gel containing 0.55 M formaldehyde and transferred to positively charged nylon membranes (Roche Applied Science) according to standard methods (31). Digoxigenin-labeled DNA probes for araK or tal were prepared by PCR using the primers shown in Table S1 in the supplemental material and the Roche digoxigenin (DIG) gel shift kit (Roche Applied Science). After hybridization and washes, an anti-DIG antibody was incubated with the blots and then detected by chemiluminescence with the CDP-STAR reagent kit (Roche Applied Science).

Protein overexpression and purification.

The araR (CAC1340) and araK (CAC1344) genes from C. acetobutylicum were PCR amplified (primers shown in Table S1 in the supplemental material) and cloned into the expression vector pET28a. The recombinant proteins were overexpressed as N-terminal fusions with a His6 tag in E. coli BL21(DE3)pLysS. The cells were grown on LB medium to an optical density at 600 nm (OD600) of 0.8 at 37°C, induced by 0.2 mM IPTG, and harvested after 12 h of shaking at 16°C. Protein purification was performed using a rapid Ni-nitrilotriacetic acid (NTA) agarose minicolumn protocol as described previously (40). Briefly, harvested cells were resuspended in 20 mM HEPES buffer (pH 7) containing 100 mM NaCl, 0.03% Brij-35, 2 mM β-mercaptoethanol, and 2 mM phenylmethylsulfonyl fluoride. Lysozyme was added to a concentration of 1 mg ml−1, and the cells were lysed by freeze-thawing followed by sonication. After centrifugation, the supernatant was loaded onto a Ni-NTA agarose column (0.2 ml). After bound proteins were washed with 50 mM Tris-HCl buffer (pH 8) containing 1 M NaCl, 0.3% Brij-35, and 2 mM β-mercaptoethanol, they were eluted with 0.3 ml of the same buffer supplemented with 250 mM imidazole. The buffer was then changed to 10 mM Tris-HCl (pH 7.4) containing 0.3 mM dithiothreitol (DTT), 1 mM EDTA, and 10% glycerol by using Bio-Spin columns (Bio-Rad). The purified proteins were run on a 12% sodium dodecyl sulfate-polyacrylamide gel to monitor their size and purity (see Fig. S2 in the supplemental material).

EMSA.

For the electrophoretic mobility shift assay (EMSA), the 180-bp DNA fragments from the upstream region of individual genes were PCR-amplified from C. acetobutylicum ATCC 824 genomic DNA using the primers shown in Table S1 in the supplemental material. Both forward and reverse primers were Cy5 fluorescence labeled at the 5′ end (Sangong Corp., Shanghai, China). The PCR products were purified with the PCR purification kit, and their concentration was determined spectrophotometrically. The fluorescence-labeled DNA (1 nM) was incubated with the indicated amount of purified AraR protein in 20 μl of binding buffer containing 20 mM Tris (pH 7.5), 0.25 mM DTT, 10 mM MgCl2, 5% glycerol, 0.8 μg bovine serum albumin (BSA), and 1 μg salmon sperm DNA (nonspecific competitor). As potential effectors of AraR-DNA binding, l-arabinose or d-xylose was added as indicated. After incubation at 30°C for 20 min, the reaction mixture was electrophoresed at 4°C on a 6% native polyacrylamide gel in 0.5× Tris-borate-EDTA for 1.5 h at 100 V. Fluorescence-labeled DNA on the gel was then detected with the Starion FLA-9000 (FujiFilm, Japan). Specificity of the AraR-DNA interactions was tested by including a 400-fold molar excess of nonlabeled target DNA (specific competitor) in binding reaction mixtures.

Enzyme assays.

Ribulokinase activity was assayed by coupling the formation of ADP to the oxidation of NADH to NAD+ via pyruvate kinase and lactate dehydrogenase. Briefly, 0.5 μg of purified enzyme was added to 200 μl of 50 mM Tris buffer (pH 7.5) containing 20 mM MgCl2, 1 mM ATP, 2 mM phosphoenolpyruvate, 0.3 mM NADH, 1.2 U of pyruvate kinase, 1.2 U of lactate dehydrogenase, and 1 mM l-ribulose (Sigma-Aldrich). The change in NADH absorbance was monitored at 340 nm at 30°C by using a Beckman DU-800 spectrophotometer. Another protein (CAC1342) overexpressed and purified in the same way was used as a negative control. To test substrate specificity, l-ribulose was replaced by 5 mM d-glucose, d-fructose, d-ribose, and d-xylulose in the assay mixture. For determination of the apparent kcat and the Km values for l-ribulose, its concentration was varied in the range of 0.5 to 10 mM in the presence of 2 mM (saturating) ATP. Kinetic data were analyzed using Graphpad Prism 5.0 software. Protein concentrations were measured by using the Bradford reagent (Sangong Corp., Shanghai, China) with BSA as a standard.

The reaction product of ribulokinase was identified by HPLC using an Agilent model 1200 system. The reaction mixture (200 μl) containing 200 mM HEPES (pH 7.5), 20 mM MgCl2, 1 mM ATP, and 5 mM l-ribulose, and the purified ribulokinase (5 μg) was incubated for 2 h at 37°C. Samples were applied at 70°C into a Waters Sugar Pak I column linked to a refractive index detector and eluted with double-distilled water at a flow rate of 0.6 ml min−1.

RESULTS

Comparative genomic reconstruction of AraR regulons in Clostridium spp.

AraR has been characterized in B. subtilis as a repressor of the l-arabinose metabolic operons (32). Orthologs to the B. subtilis araR were identified by BLAST searches against a nonredundant set of sequenced bacterial genomes (Fig. 1). A strong tendency of araR genes to cluster on the chromosome with arabinose utilization genes was observed. The phylogenetic tree was constructed for representative AraR proteins from Bacillales, Lactobacillales, and Clostridiales (Fig. 1B). Two separate groups were observed for clostridial AraR. AraR proteins in C. acetobutylicum and Clostridium ljungdahlii were located on a distinct branch from AraR in other clostridia such as Clostridium beijerinckii and Clostridium cellulolyticum. Two diverged AraR paralogs in Clostridium carboxidivorans belonged to these two clostridial branches, respectively.

To reconstruct the AraR regulons in Clostridium species, we applied the integrative comparative genomics approach (as implemented in RegPredict web server) that combines identification of candidate transcription factor-binding sites with cross-genomic comparison of regulons and with the functional context analysis of candidate target genes. The upstream regions of arabinose utilization genes in nine Clostridium genomes were analyzed using a motif recognition program to identify conserved AraR-binding DNA motifs. After construction of a positional weight matrix for each identified motif, we searched for additional AraR-binding sites in the analyzed Clostridium genomes and finally performed a consistency check or cross-species comparison of the predicted AraR regulons.

As shown in Fig. 1B, the identified palindromic AraR-binding DNA motif in C. acetobutylicum and C. ljungdahlii was partially similar to the AraR-binding sequences experimentally determined in B. subtilis (5′-ATTTGTACGTACAAAT-3′) (9). A relatively divergent binding motif was identified for AraR from other Clostridium species, including C. beijerinckii, C. cellulolyticum, and Clostridium saccharolyticum (5′-ATGTTGTACGTACAAGTT-3′), although the central core region of the motif was conserved. The obtained AraR-binding motifs were used to detect candidate members of the AraR regulons in the nine Clostridium genomes (Fig. 1; see Table S2 in the supplemental material). The most conserved part of the AraR regulons included the arabinose utilization genes araA, araB, araD, and transporter genes araE or araFGH. The presence of AraR-binding sites in the upstream region of araR gene suggested autoregulation of its expression in Clostridium.

Interestingly, a conserved member of AraR regulons in the nine Clostridium species was a candidate gene encoding an alternative ribulokinase, termed AraK. This novel ribulokinase is a nonorthologous replacement of the ribulokinase AraB from B. subtilis. Both AraK and AraB proteins belong to the FGGY sugar kinase family (Pfam accession number PF00294), which displays remarkable diversity in substrate specificity (Fig. 2). Phylogenetic analysis of the FGGY kinase family showed that the branches containing orthologs of AraB and AraK, respectively, were highly diverged from each other (Fig. 2). Because of the homology with some xylulokinases, the AraK protein in C. acetobutylicum (CAC1344) was incorrectly annotated as xylulokinase in the public databases (e.g., GenBank). Here the functional assignment of ribulokinase in the nine Clostridium genomes was supported by chromosomal clustering with arabinose utilization genes and by identification as AraR regulon members (Fig. 1). Additionally, an alternative arabinose isomerase (AraA-II) from the fucose isomerase FucI family was predicted for C. cellulolyticum, C. carboxidivorans, and Clostridium papyrosolvens (Fig. 1).

Fig 2
Maximum likelihood phylogenetic tree of selected FGGY family sugar kinases. The colors reflect chromosomal clustering of kinase genes with the genes from the respective sugar utilization pathways. Experimentally characterized sugar kinases are indicated. ...

The AraR regulons in C. acetobutylicum, C. carboxidivorans, C. beijerinckii, and C. cellulovorans extended to the pentose phosphate pathway in the central carbon metabolism, in addition to the known AraR targets in B. subtilis (Fig. 1). Candidate AraR-binding sites were identified in the regulatory region of tkt, tal, and ptk genes that were involved in conversion of xylulose-5-phosphate to central metabolic intermediates glyceraldehyde-3-phosphate and/or acetyl phosphate (Fig. 1A). The AraR regulon members also include the genes involved in uptake and degradation of arabinosides (araT, abfA, and arb43). A member of the aldose 1-epimerase family in the AraR regulon was tentatively assigned the functional role of arabinose mutarotase (Epi), which interconverts alpha and beta anomers of l-arabinose.

In summary, the comparative genomics analysis allowed us to reconstruct the AraR regulons in C. acetobutylicum and eight other Clostridium genomes and to tentatively assign the candidate genes for an alternative ribulokinase. We then performed experimental validation of the predicted function of araK and characterization of the AraR regulon in C. acetobutylicum as described below.

Experimental validation of the predicted ribulokinase in C. acetobutylicum.

To validate the inferred function of the araK gene in C. acetobutylicum, we disrupted this gene by inserting an intron (confirmed by PCR and Southern blot analysis, as shown in Fig. S1 in the supplemental material) and tested the ability of the mutant to grow on arabinose as the sole carbon source. Inactivation of the araK gene severely impaired the growth on arabinose (Fig. 3A). The growth rate of the araK-inactivated mutant (0.0014 OD h−1) was about 90-fold lower than that of the wild type (0.13 OD h−1). Complementation of the araK-inactivated mutant by using a plasmid construct constitutively expressing araK restored the growth on arabinose. On the other hand, the growth rate of the araK-inactivated mutant on xylose was not diminished (Fig. 3B). Therefore, the phenotype of the C. acetobutylicum araK-inactivated mutant confirmed the predicted physiological role of the araK gene in arabinose utilization. There may be a second low-activity kinase that could also be used for arabinose utilization.

Fig 3
Effect of araK gene disruption on cell growth of C. acetobutylicum on l-arabinose (A) and d-xylose (B). Cells were grown in P2 minimal medium containing 5 g liter−1 of l-arabinose or d-xylose as the sole carbon source. The optical density at 600 ...

To extend the genetic findings and provide biochemical evidence to the proposed gene assignment, we used the recombinant AraK from C. acetobutylicum, which was overexpressed in E. coli with the N-terminal His6 tag and purified using Ni-NTA affinity chromatography, to test for ribulokinase activity (Fig. 4A). As expected, the C. acetobutylicum AraK displayed a ribulokinase activity with the kcat of 41 s−1. The apparent Km value for l-ribulose (0.96 mM) was higher than the value reported for E. coli ribulokinase AraB (0.14 mM) (17). Furthermore, the substrate specificity of C. acetobutylicum AraK was tested. It was highly specific to l-ribulose and incapable of phosphorylating d-xylulose (Fig. 4A), d-glucose, d-fructose, and d-ribose (data not shown). Finally, the conversion of l-ribulose to l-ribulose 5-phosphate by AraK was directly detected using HPLC (Fig. 4B). The reaction mixture contained l-ribulose, ATP, and purified recombinant AraK from C. acetobutylicum, and the formation of l-ribulose 5-phosphate was identified by HPLC. The ribulokinase from E. coli was assayed in parallel as a control. These biochemical assays provided an independent verification of the predicted enzymatic activity of AraK from C. acetobutylicum.

Fig 4
Biochemical characterization of a recombinant AraK protein. (A) Ribulokinase activity was assayed by coupling the formation of ADP to the oxidation of NADH to NAD+ and monitoring the absorbance at 340 nm. For testing substrate specificity, l-ribulose ...

Experimental characterization of the predicted AraR regulon in C. acetobutylicum.

To validate the regulatory role of the inferred araR gene in l-arabinose metabolism and the predicted AraR regulon, the C. acetobutylicum araR-inactivated mutant was constructed. Total RNA was isolated from both the wild-type and araR-inactivated mutant strains grown in the medium without regulatory carbon sources (as described in Materials and Methods) or with addition of arabinose. By using quantitative RT-PCR, we examined the relative transcript levels of predicted AraR-controlled genes in both strains during growth in the absence or presence of arabinose (Fig. 5A). In the absence of arabinose, the expression levels of the araK (CAC1344), araA (CAC1342 and CAC1346), araD (CAC1341), and araE (CAC1339 and CAC1345) genes in the araR-inactivated mutant were elevated more than 17-fold compared to that of the wild-type strain. Several of these genes were expressed at high levels in the araR-inactivated mutant regardless of the presence or absence of arabinose, whereas addition of arabinose to the culture caused a significant induction of the genes' expression in the wild-type strain.

Fig 5
Transcriptional analysis of AraR regulon genes. (A) Quantitative RT-PCR analysis of gene expression. Total RNA was isolated from C. acetobutylicum ATCC 824 wild-type and araR-inactivated mutant strains grown in the medium without regulatory carbon sources ...

In addition to the genes of arabinose utilization pathway, the transcription of the pentose phosphate pathway genes tkt (CAC1348), tal (CAC1347), and ptk (CAC1343) and the arabinoside degradation genes epi (CAC1349) and arb43 (CAC1529) was also controlled by AraR (Fig. 5A). In fact, the most prominent effect of araR mutation was observed for the ptk gene, which was upregulated over 1,000-fold in the araR-inactivated mutant compared to the wild type when grown without arabinose. The expression levels of the tkt and tal genes in the araR-inactivated mutant in the absence of arabinose were over 23-fold higher than those in the wild-type strain grown in the same medium and about 3-to 4-fold higher than the levels measured in the arabinose-grown wild-type cells. All of the genes studied, especially the tkt and tal genes, in the araR-inactivated mutant exhibited reduced expression levels when arabinose was present in the medium. Although the underlying mechanisms are yet to be studied, these genes may also be controlled by another transcriptional factor. The impaired growth of the araR-inactivated mutant on arabinose was observed (data not shown), which may be caused by accumulation of toxic metabolic intermediates due to constitutive expression of arabinose utilization genes. Therefore, the quantitative RT-PCR results confirm that AraR is a negative regulator of 11 genes involved in the arabinose utilization, arabinoside degradation, and pentose phosphate pathway in C. acetobutylicum.

To determine whether the araK-araE-araA-tal-tkt-epi cluster constituted a transcriptional unit, Northern blot analysis was performed. Total RNA was isolated from wild-type C. acetobutylicum grown with arabinose as the sole carbon source and was hybridized to DNA probes for araK or tal (Fig. 5B). A transcript of about 8 kb was obtained, which matched the expected size of the full-length araK-araE-araA-tal-tkt-epi transcript. In addition, the probes also hybridized to smaller transcripts, suggesting that there might be internal promoters within the gene cluster. The cotranscription of araK with araE, araA, tal, tkt, and epi genes is in line with the quantitative RT-PCR results.

To confirm the ability of AraR protein to specifically bind to the predicted DNA sites, EMSA was performed using the purified recombinant AraR protein from C. acetobutylicum. Five predicted target DNA fragments (180 bp) from the upstream region of the C. acetobutylicum araE, araR, araD, ptk, and araK genes, respectively, were tested by EMSA (Fig. 6A to E). A shifted band was observed upon incubation of AraR protein with the target DNA fragments, and its intensity was enhanced in the presence of increasing amounts of AraR protein. The band shift for all five target sites was essentially complete at an AraR concentration above 0.5 μM. Different binding affinities of AraR protein to individual DNA fragments were observed. The AraR protein exhibited higher affinity for the tested upstream fragments of ptk and araK genes than for that of the araR gene. However, AraR may bind to multiple sites in the regulatory region between divergently transcribed araR and araD genes (Fig. 1C), and the cooperative binding could result in a high level of repression (21). To test the effector of AraR-DNA binding, different concentrations of arabinose were used in EMSA. As shown in Fig. 6F, the AraR protein was released from the AraR-DNA complex when the arabinose concentration was higher than 1 mM, whereas xylose (20 mM) had no effect on the binding of AraR to target DNA (see Fig. S3 in the supplemental material). The formation of the AraR-DNA complex was suppressed in the presence of 400-fold excess unlabeled DNA fragments but not in the presence of nonspecific competitor, salmon sperm DNA (see Fig. S3 in the supplemental material). These observations confirm that AraR is an arabinose-responsive regulator that binds specifically to the predicted DNA-binding sites in the C. acetobutylicum genome.

Fig 6
Binding of AraR to the promoter region of the ptk (A), araK (B), araE (C), araR (D), and araD (E) genes. EMSA was performed by incubating the indicated amounts of purified C. acetobutylicum AraR protein with a Cy5 fluorescence-labeled 180-bp DNA fragment ...

DISCUSSION

In this study, we performed comparative genomic reconstruction of AraR regulons in nine biotechnologically important Clostridium species by combining the identification of candidate AraR-binding sites with functional analysis of target genes. Although two different types of AraR-binding DNA motifs were identified for Clostridium species, the consensus sequences shared the conserved bases (G9, A11, and T16) that have been shown to be critical for AraR binding in B. subtilis (9). The combined bioinformatics with in vitro and in vivo characterization of AraR regulon in C. acetobutylicum confirmed that AraR repressed the expression of genes involved in arabinose utilization, arabinoside degradation, and the pentose phosphate pathway in the absence of arabinose. When arabinose was added to the culture, binding of arabinose to AraR inhibited the binding of AraR to target DNAs, leading to derepression of transcription of the above genes.

Based on the reconstructed AraR regulons, we have identified a novel ribulokinase AraK present in all analyzed Clostridium species, which is a nonorthologous replacement of previously characterized ribulokinase AraB. Disruption of the araK gene in C. acetobutylicum and detailed biochemical characterization of purified recombinant AraK allowed us to confirm the predicted function. Interestingly, unlike in other Clostridium species, where AraK is the only ribulokinase, both AraK and AraB are present in C. carboxidivorans. This organism also possesses two AraR paralogs, and their DNA-binding sites were detected in the upstream region of the araK and araB genes, respectively. AraK and AraB, together with E. coli glycerol kinase, fucolokinase, gluconokinase, xylulokinase, and rhamnulokinase, belong to the FGGY sugar kinase family, which represents a striking example of variations in substrate specificity. Comparative sequence analysis and structural modeling could help us identify specificity-determining regions and to assess evolution of substrate specificity in this important yet poorly explored family (Y. Zhang, personal communication).

The CAC1339 to -1349 gene cluster in the C. acetobutylicum genome includes genes for two AraA paralogs, CAC1342 and CAC1346. Previous DNA microarray analysis has showed that the CAC1346 gene was induced by both arabinose and xylose, while the CAC1342 gene was strongly induced by arabinose and weakly induced by xylose (34). Here our results indicated that both arabinose isomerase genes are controlled by AraR. Moreover, we overexpressed both genes in E. coli, purified the recombinant proteins, and performed the biochemical assays. Both AraA proteins were highly specific to l-arabinose and cannot convert d-xylose to d-xylulose (data not shown). In addition, the CAC1339 to -1349 region also contains genes for two putative arabinose proton-symporters (CAC1339 and CAC1345), which are considered to be responsible for arabinose uptake. Our previous genetic mutagenesis and complementation experiments have showed that the CAC1345 gene product may also contribute to xylose transport (12). The exact interpretation of the observed functional redundancy in C. acetobutylicum requires further investigation.

Due to the absence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, the pentose phosphate pathway in C. acetobutylicum mainly serves to synthesize biomass precursors, ribose-5-phosphate and erythrose-4-phosphate (33). Regulation of pentose phosphate pathway genes by AraR allows the cells to activate this pathway and catabolize arabinose efficiently when arabinose is available in the culture. Moreover, expression of arabinose transporters and metabolic enzymes in C. acetobutylicum is also under the control of AraR. Thus, the coordinated control of different interrelated pathways by AraR could result in efficient arabinose utilization. On the other hand, the transcriptional factor XylR controls the expression of only metabolic enzymes XylAB for xylose utilization, while the xylose transporter gene and pentose phosphate pathway genes are not members of the XylR regulon in C. acetobutylicum (12). This difference between AraR and XylR regulons may partly explain the much shorter lag phase of C. acetobutylicum grown on arabinose compared to growth on xylose. In addition to AraR regulation, expression of arabinose utilization genes in B. subtilis is also subject to catabolite repression mediated by the transcriptional factor CcpA, which binds to the catabolite responsive element (cre) (10, 14). The inhibition of arabinose metabolism in the presence of glucose (26) and induction of arabinose utilization genes upon glucose depletion (11) were also observed for C. acetobutylicum. Studies on the involvement of CcpA and cre in regulation of clostridial arabinose metabolism are now under way.

Supplementary Material

Supplemental material:

ACKNOWLEDGMENTS

This work was supported in part by the National Natural Science Foundation of China (31070033 and 31121001), the National Basic Research Program of China (973: 2012CB721101), and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-G-5 and KSCX1-YW-11C3). The work of D.A.R and S.A.L was supported by the U.S. Department of Energy (DE-SC0004999) and Russian Academy of Sciences (program “Molecular and Cellular Biology”).

Footnotes

Published ahead of print 22 December 2011

Supplemental material for this article may be found at http://jb.asm.org/.

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