INTRODUCTION

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Thermophilic anaerobic bacteria have attracted much interest for use in the bioconversion of industrial and agricultural lignocellulosic waste materials into value-added chemicals. The past two decades have witnessed a surge in exploiting microbial enzymes to unlock the potential energy in lignocellulosic biomass, such as agricultural byproducts and crop residues (31, 37). Besides the focus on polysaccharide depolymerases to hydrolyze plant cell wall polymers such as cellulose and hemicellulose and their subsequent fermentation to alcohol, there is also interest in the application of microbial enzymes to the paper industry (7, 41). Preliminary results show that the addition of xylanases and -mannanases can reduce the loading of chlorine-containing chemicals during the bleaching of pulp (7), hence reducing the cost of chemical waste disposal.

Endo-1,4--D-mannanases (EC 3.2.1.78) catalyze the random hydrolysis of the -D-1,4-mannopyranosyl linkages within the main chain of mannans and various polysaccharides consisting mainly of mannose, which are components of the plant cell wall. Thermostable mannanases have been purified in Bacillus stearothermophilus (36), as well as cloned and sequenced in Caldocellulosirruptor saccharolyticus (14, 24, 28). In order to utilize plant material as a source of carbon and energy, bacteria produce an array of hydrolytic enzymes with various specificities, which act cooperatively to convert these substrates to their constituent sugars (38). These enzymes commonly show a modular organization and consist usually of a single catalytic domain linked to one or more noncatalytic domains. However, bifunctional polysaccharidases comprising two dissimilar catalytic domains have been identified via gene cloning in Ruminococcus flavefaciens (13, 44), Clostridium thermocellum (1), C. saccharolyticus (14, 28), and Anaerocellum thermophilum (45).

We have recently isolated a number of thermophilic bacteria from the waste pile of a canning factory in Illinois. Among these isolates are two organisms closely related at the 16S ribosomal DNA level (98% similarity; GenBank accession numbers U40229 and U75993). These organisms, which have an optimum temperature for growth of 68°C, are highly saccharolytic and produce a number of polysaccharide-hydrolyzing enzymes. In this work, we describe the cloning, sequencing, and expression of a gene coding for a unique modular protein exhibiting both mannanase and endoglucanase activities from Thermoanaerobacterium polysaccharolyticum, one of these novel bacteria. Furthermore, we show that this large enzyme (119.6 kDa), which contains a unique catalytic region, duplicated cellulose-binding domains (CBD), and a triplicated surface-layer-like protein (SLH) domain within the same polypeptide, is encoded by a single gene.

	MATERIALS AND METHODS

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Bacterial strains and culture conditions. T. polysaccharolyticum was isolated from subsurface samples from the leachate of the waste pile from the canning factory in Hoopeston, Ill. The bacterium was routinely grown in a minimal medium with glucose as the energy source at 68°C. The isolation and characterization of T. polysaccharolyticum has been described elsewhere (8). Escherichia coli strains were grown at 37°C in Luria-Bertani (LB) medium supplemented with either ampicillin (100 µg/ml) or kanamycin sulfate (50 µg/ml). The reagents isopropyl--D-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) were added to the media where appropriate at concentrations of 125 and 80 µg/ml, respectively.

DNA isolation and manipulation. Genomic DNA from T. polysaccharolyticum was isolated by the method of Marmur (25). Recombinant plasmids were extracted as described by Birnboim and Doly (6). Isolation of recombinant plasmid for DNA sequence analysis was performed by using the Qiagen Midi-Prep System (Qiagen, Inc., Chatsworth, Calif.). Restriction enzymes were purchased from Stratagene (Stratagene Cloning Systems, La Jolla, Calif.) and were used according to the manufacturer's instructions.

Cloning of manA gene of T. polysaccharolyticum. Total genomic DNA was extracted from T. polysaccharolyticum cells grown to mid-exponential phase and was then partially digested with EcoRI. DNA fragments ranging from 3 to 12 kb were ligated into EcoRI-digested and dephosphorylated Lambda Zap Express arms (Stratagene). The resulting recombinant phage particles were packaged, and the products were incubated with E. coli XL-1 Blue cells {(mcrA)183 (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F' proAB lacI^qZM15 Tn10(Tet^r)]} at 37°C with gentle shaking. The mixture was plated on NZY medium in a soft agar containing the chromogenic substrate Ostazin brilliant red-hydroxyethyl cellulose (OBR-HEC) and incubated at 37°C overnight. The recombinant phages exhibiting endoglucanase activity produced halos within a red background. Endoglucanase-positive phages were cored and purified, and the circularized phagemid with the DNA insert coding for the ManA of T. polysaccharolyticum was recovered through an f1 helper phage-mediated in vivo excision and recircularization in E. coli XLOLR cells {(mcrA)183, (mcrCB-hsdSMR-mrr)173 endA1 thi-1 recA1 gyrA96 relA1 lac [F' proAB lacI^q ZM15 Tn10(Tet^r)] Su (nonsuppressing) ^r (lambda resistant)}.

DNA sequencing. The nucleotide sequences of both strands of the DNA insert (3.4 kb) were determined by the University of Illinois Biotechnology Center by using an Applied Biosystems 373A automated DNA sequencer (Applied Biosystems, Foster City, Calif.) with both dye primers and dye dideoxynucleotide chemistries. Computer analysis of DNA sequences was performed by using the DNA sequence analysis software packages Geneworks 2.45 (Intelligenetics, Inc., Mountain View, Calif.) and CLUSTAL V (16). Homology searches in the GenBank were carried out by using the BLAST program (2).

Genome-walking PCR. Genome-walking PCR was used to obtain sequence downstream from of the 3.4-kb insert since sequence analysis of the 3.4-kb insert did not reveal an in-frame termination codon. The procedure for genome-walking PCR was as previously described (28). A primer targeted from bases 2896 to 2916 (5'-GGATGGCCTGATTGGGGTTAT-3') of the T. polysaccharolyticum genomic DNA insert was designed to perform a genome-walking PCR to obtain a sequence from downstream of the 3.4-kb insert. An aliquot (500 ng in a 50-µl reaction) of genomic DNA from T. polysaccharolyticum was completely digested with BamHI. Two complementary oligonucleotides, the upper linker with a BamHI sticky end (5'-GATCGCGCAGGAAACAGCTATGACCGGT-3') and the lower linker (5'-ACCGGTCATAGCTGTTTCCTGCGC-3') were synthesized. Portions (75 pmol) of each oligonucleotide in a 50-µl reaction mixture were assembled into a BamHI linker by denaturation at 94°C and annealing at 50°C for 30 min. The linker library was constructed by ligating 1 µl of the linker to 5 µl of BamHI-digested genomic DNA in a volume of 10 µl overnight at 4°C. The volume of the library was increased to 50 µl, and 5 µl was used as the template to amplify, via PCR, the region downstream of the 3.4-kb insert. The PCR mixture contained 250 mM concentrations of each deoxynucleoside triphosphate, 2 mM MgCl₂, and other ingredients as described by the manufacturer (LA PCR kit; TaKaRa Shuzo Co., Ltd., Kyoto, Japan). The oligonucleotides were synthesized by standard methods with an automated DNA/RNA synthesizer (Applied Biosystems). PCR was performed in a programmable thermal controller (PTC-100; MJ Research, Inc., Watertown, Mass.). The thermal profiles involved 29 cycles of denaturation at 94°C for 30 s, annealing at a temperature of 58°C for 50 s, and extension at 72°C for 1.5 min. The PCR fragment obtained was ligated into a pGEM-T vector DNA (Promega, Madison, Wis.), and the product was used in transforming E. coli JM109 cells through electroporation. The insert was subsequently sequenced as described above. The amplification of the complete manA gene was achieved by using the oligonucleotides 5'-TGTTGAGCCGGCCTAGCGACAACG-3' (bases 86 to 103) and 5'-CCGCCTGAAACCTTTATGCC-3' (bases 4120 to 4101) as the forward and reverse primers, respectively. T. polysaccharolyticum genomic DNA served as the template.

Deletion of CBD1 and CBD2. The two oligonucleotides 5'-TGTTGAGCCGGCCTAGCGACAACG-3' (nucleotides 86 to 103) and 5'-GTCTTGCCAGCTGTCCAGACCGTC-3' (nucleotides 2479 to 2455) as forward and reverse primers, respectively, were used to amplify a fragment of manA with both CBDs and the surface-layer-protein-like sequences (SLH) deleted. The PCR fragment was cloned into pGEM-T vector as described above. Four positive clones harboring the insert were used to assay for both cellulose binding and enzymatic activities.

Purification of recombinant mannanase and enzyme assay. E. coli JM109 cells [endA1 recA1 gyrA96 thi hsdR17 relA1 supE44 (lac-proAB) (F' traD36 proAB lacI^q ZM15)] containing the manA gene were grown overnight to saturation in 1.5 liters of LB broth supplemented with ampicillin at 100 µg/ml. The cells were harvested by centrifugation at 10,000 × g for 20 min at 4°C followed by suspension in 10 ml of 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 mM phenylmethylsulfonyl fluoride. The disruption of cells to release recombinant protein was achieved by two passages through a French pressure cell at 16,000 lb/in². Cell debris was removed by centrifugation at 10,000 × g for 20 min at 4°C. E. coli proteins were partially removed by heat precipitation at 70°C for 10 min followed by centrifugation as described above. The supernatant from the heat-precipitated sample was applied to a column packed with Sigmacell Type 50 (Sigma Chemical Co., St. Louis, Mo.) which had been equilibrated with 50 mM sodium phosphate buffer (pH 5.8). To elute unbound proteins, the column was washed continuously with the equilibration buffer while monitoring the protein elution at 280 nm by spectroscopy (Beckman DU7500). The elution of bound protein was carried out with distilled water, and the proteins were concentrated (Centricon 10; Amicon, Beverly, Mass.) and resuspended in a volume of 50 mM sodium phosphate buffer (pH 7.0).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detection of enzyme activity. The detection of activity by plate assay was done by growing E. coli cells harboring the recombinant plasmids with the manA gene on LB plates supplemented with the appropriate antibiotic. The cells were grown overnight and lysed in a chloroform chamber for 4 to 5 min. The plates were then flooded with a top agar containing 0.8% agarose and 0.5% carboxymethyl cellulose (CMC; Sigma). After a setting period, the plates were incubated upright at 65°C overnight and stained with a solution of 0.1% congo red for 2 min. This was followed by destaining through repeated washings with 1 M NaCl.

N-terminal sequencing. N-terminal amino acid sequencing was done by the University of Illinois Biotechnology Center with an Applied Biosystems model 477A protein sequencer with a model on-line PTH analyzer by using Edman degradation chemistry (10).

Nucleotide sequence and accession number. The DNA sequence reported here has been submitted to the GenBank database and has been assigned the accession number U82255.

	RESULTS AND Discussion

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Cloning and sequencing of manA. Six thousand plaques were screened for endoglucanase activity by using a chromogenic substrate, OBR-HEC. Eight recombinant phages exhibiting endoglucanase activity were detected and purified. The phagemids isolated from these phages were further analyzed by using endoglucanase assays at both 37 and 65°C. Two restriction endonucleases, EcoRI and PstI, were used to digest the phagemids to examine the restriction patterns of the DNA inserts. The results indicated that seven recombinant phagemids harbored the same segment of the T. polysaccharolyticum genomic DNA in one orientation, while one contained an identical fragment in the opposite orientation. One of the seven similar recombinant phagemids was selected and used for further analysis. This phagemid harbored a DNA insert of approximately 3.4 kb (Fig. 1), which was nucleotide sequenced. The results revealed a large open reading frame (ORF) lacking a termination codon, which indicated an incomplete gene sequence. Using a genome-walking PCR method, an overlapping segment (1.5 kb) of T. polysaccharolyticum genomic DNA was amplified and cloned into a pGEM-T plasmid and nucleotide sequenced to obtain the entire gene. The integrity of the cloned PCR fragment was confirmed through Southern hybridization by using a ³²P-labeled fragment amplified from the cloned DNA insert (data not shown).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Restriction map and domain organization of ManA and its derivatives, ManA-SL and ManA-SL-CBD. The full length of ManA contains the signal peptide and three other domains. CBD1 and CBD2 are not separated by a Pro-Ser-Thr-rich linker. Sp, signal peptide. The putative mannanase domain is also the catalytic domain. Note that all proteins from T. polysaccharolyticum used in the biochemical analyses have the signal peptide processed.

DNA sequence analysis of the manA gene. The cloned gene is designated manA because of its unusually high specific activity on galactomannan (locust bean gum). Figure 1a shows a schematic map of the manA gene. The gene is preceded by the carboxy terminus of a truncated protein showing 54 and 53% homology to a 51-amino-acid stretch of -fructofuranosidases from Beta vulgaris (33) and Vicia faba (39), respectively. The start codon, ATG, of manA is located at position 586, which is 22 nucleotides after the termination codon of the truncated ORF, whereas the termination codon, a TAG, is at position 3,877 (Fig. 2). The entire ORF is therefore composed of 3,291 bases and codes for a preprotein of 1,097 amino acid residues with an estimated molecular mass of 119,672 Da. The start codon is preceded by a putative ribosome binding site (TAAGGCGGTG) nine bases upstream and a putative 10 (TAAAAT) and 35 (TTCGC) promoter region occurring within the truncated protein. With the exception of one base, the putative ribosome binding site is a perfect complement of a sequence found at the 3' end of E. coli 16S ribosomal RNA (3'-AUUCCUCCAC-5') which plays a crucial role in bringing the 30S ribosome to the initiator codon. Furthermore, an AT-rich spacer region is found between the putative Shine-Dalgarno sequence and the initiator (ATG), which is also followed immediately by the sequence AAAA. The requirements for optimal transcription in E. coli (9) are fulfilled by the putative promoter region of the manA gene; hence, the gene is constitutively expressed in E. coli. The production of enzyme from plasmids harboring the insert in both orientations indicated that the promoter sequence of manA was recognized by the E. coli transcriptional apparatus. The G+C content of the region upstream of manA was 51.3%, whereas the region coding for ManA was 46.2%, which reflected the overall G+C content of T. polysaccharolyticum genomic DNA (46%). On the other hand, the region which does not contain an ORF located immediately downstream of manA had a G+C content of 39.7%.

View larger version (138K): [in this window] [in a new window]   FIG. 2.   Nucleotide and amino acid sequences of manA, the mannanase gene of T. polysaccharolyticum. The locations of the 35 and 10 sites and ribosome binding sites are shown. The sequences of the signal peptide () the CBDs (), and SLH () domains are also demarcated. The asterisk shows the stop codon.

Domain analysis of ManA. The N-terminal amino acid sequence of ManA has the features of a typical signal peptide (Fig. 2), a characteristic of extracellular enzymes. When the N-terminal region of ManA is compared with the cleavage sites of other signal peptides, there appears to be a typical signal peptidase I Ala-X-Ala processing site at amino acid residue 32 (22). The N-terminal amino acid sequence of recombinant ManA purified from E. coli was AGTSGDGRFHV, which corresponds to the amino acid sequence from positions 33 to 43 and suggests that this protein is secreted by both E. coli and T. polysaccharolyticum. The residues of the signal peptide of ManA show high homology to those of several proteins in the GenBank, notably 77 and 64% homology to the major outer sheath signal peptide of Treponema denticola (11) and that of the -lactamase of Lactococcus lactis (35), respectively.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Alignment of family A5 domain of T. polysaccharolyticum ManA, B. subtilis DLG End2 (29), C. acetobutylicum Egl (43), B. subtilis CK2 Cel (21), B. subtilis endo--1,4-glucanase (32), B. fibrisolvens CelA (15), and Bacillus sp. strain CelB (34). The boldface letters show the conserved residues containing the catalytic center of cellulase family A5. The asterisks indicate amino acid conservation in all enzymes compared. Adjacent amino acids are separated by dashes, where necessary, for optimal alignment. All sequences are aligned from Met-1 of the peptide.

View larger version (40K): [in this window] [in a new window]   FIG. 4.   Multiple sequence alignment of the SLH domains of T. polysaccharolyticum ManA (TpolmanA), Thermoanaerobacterium endoxylanase (TmSpXyn [22]), T. thermosulfurigenes pullulanase (TmTamypul [26]), T. saccharolyticum endoxylanase (TmSxyn [18]), and C. thermocellum exoglucanase (CITexoglu; accession number P38535). SLH-1, SLH-2, and SLH-3 show the triplication of this domain. The asterisks indicate conservation. Numbering for each protein starts from the putative initiation codons.

Purification and characterization of manA. In order to clone the complete manA gene in E. coli, primers were designed to amplify the complete ORF of manA by PCR with T. polysaccharolyticum genomic DNA as the template. The recombinant protein produced was purified from E. coli cells harboring the gene by a two-step purification program, a heating step, and a cellulose affinity step (affinity chromatography). Heat treatment of cell extracts increased the activity of ManA 5.2-fold through the denaturation of host proteins. Application of this partially purified protein to a column of Sigmacell followed by elution with water yielded two protein bands migrating to approximately 116 and <97 kDa on SDS-PAGE (Fig. 5). The 116-kDa protein corresponded well with the molecular mass estimated from the nucleotide sequence of manA. Zymogram analysis indicated that both protein bands have mannanase activity (Fig. 6). The results of N-terminal amino acid sequencing showed that both proteins possess the same N-terminal sequence (AGTSGDGRFHV), suggesting that the smaller size protein (<97-kDa band) was a result of truncation at the carboxy terminus. A derivative of ManA with a processed signal peptide and the SLH domains deleted will be approximately 95 kDa in mass. It is our estimation that the truncated protein lacks the C-terminal S-layer repeats and probably also a segment of CBD2. The ability to bind to cellulose and the possession of enzymatic activity confirmed our assumption that the SLH repeats are not required for binding to substrate and enzymatic activity.

View larger version (62K): [in this window] [in a new window]   FIG. 5.   Purification steps of recombinant ManA from E. coli cells. Proteins were loaded on and SDS-10% polyacrylamide gel and stained with Coomassie brilliant blue. Lanes: 1, Bio-Rad molecular mass markers; 2, total cell extract; 3, supernatant after heat treatment at a 70°C for 15 min; 4, purified ManA and a truncated derivative (ManA-SL) eluted from a Sigmacell column with water. Molecular mass markers are indicated on the left in kilodaltons.

View larger version (30K): [in this window] [in a new window]   FIG. 6.   A zymogram showing hydrolysis of locust bean gum (galactomannan) by both proteins eluted from a Sigmacell column (Fig. 5, lane 4). Equal amounts of eluted proteins were loaded in each lane (1 to 4) to show the consistency of hydrolysis.

View larger version (12K): [in this window] [in a new window]   FIG. 7.   Effect of pH and temperature on the hydrolysis of substrates by ManA. (a) Enzyme assays were carried out at 65°C for 10 min in 50 mM sodium phosphate buffer at pH 4.5 to 8.0. Reaction mixtures (total volume, 500 µl) contained 450 µl of 0.5% locust bean gum and 50 µl of enzyme. The final concentration of enzyme was 200 ng/ml, and the pH was measured at 65°C. (b) Enzyme activity was assayed in 50 mM phosphate buffer at pH 5.7 at 40 to 80°C. The final concentration of enzyme in the assay for mannanase () and CMCase () activities were 200 ng/ml and 1.8 µg/ml, respectively. Relative activity was defined as a proportion of the highest absorbance at 410 nm.