1 Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123182 Moscow, Russia
2 Research Group Microbial Biotechnology, Technische Universität München, Am Hochanger 4, D-85350 Freising-Weihenstephan, Germany
Correspondence
Wolfgang H. Schwarz
schwarz{at}mikro.biologie.tu-muenchen.de
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AJ275974.
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INTRODUCTION |
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To cope with these problems cellulolytic anaerobic bacteria have developed a defined arrangement of enzymes along a non-catalytic scaffolding protein, the cellulosome (Schwarz, 2001). This large multienzyme complex contains various cellulase components which differ in their modes of action (Lamed et al., 1983
; Bayer et al., 1998
). The cellulosome components are aligned on the scaffoldin (e.g. the CipA protein of Clostridium thermocellum) of the cellulosome with the aid of their dockerin modules which specifically recognize the CipA-cohesin modules (Béguin et al., 1998
). Due to the specificity of the cohesin-dockerin interaction, the presence of a dockerin module sequence in a protein is indicative of its localization in the cellulosome (Mechaly et al., 2000
; Salamitou et al., 1994
).
The bacterial cellulase systems investigated so far include a variety of non-processive endo--1,4-glucanases, producing new ends at random within a polysaccharide chain, and processive cellulases (exo-
-1,4-glucanases) which remain attached to one end of the substrate and split off cellobiose (cellobiohydrolases) or multimers of cellobiose (processive endocellulases) (Reverbel-Leroy et al., 1997
; Teeri, 1997
).
-1,4-Glucanases with a different mode of action work synergistically to effectively degrade the crystalline substrate (Barr et al., 1996
). The catalytic domains of cellulases have been assigned on the basis of sequence comparisons and hydrophobic cluster analysis to glycosyl hydrolase families (GHFs) (Henrissat et al., 1998
; CAZy server, http://afmb.cnrs-mrs.fr/
pedro/CAZY). In many cases the catalytic modules of processive and non-processive enzymes are closely related and only differ in the depth of their active site pocket or in additional helper modules' (Bayer et al., 2000
). The direction of processivity, i.e. activity on the reducing or the non-reducing end of the substrate, seems to be defined by the method of substrate binding and release (Parsiegla et al., 2000
).
Besides the presence of diverse catalytic modules, activity on crystalline cellulose also depends on the presence of non-catalytic modules which contribute tight substrate binding (Tomme et al., 1998). These carbohydrate-binding modules (CBMs) have a stimulating effect by increasing enzymesubstrate proximity, helping the enzyme to overcome the liquidsolid interface and enhancing the accessibility of the substrate surface by interacting with the crystal (Bolam et al., 1998
; Nutt et al., 1998
; Pagès et al., 1997
). They are indispensable in catalytic systems where the size relationship between substrate and enzyme(s) seriously limits the molecular dynamics of enzyme action.
Substrate-binding modules from bacteria have been divided into 26 families by sequence comparison [Carbohydrate-Active enZYmes and associated MODular Organization server (CAZyModO website), http://afmb.cnrs-mrs.fr/pedro/DB/db.html; Tomme et al., 1998
]. Different binding specificities promote the cellulolysis of different sites on the crystalline substrate (Carrard et al., 2000
). They have a size of 40180 aa. Family 3 plays an eminent role in bacterial cellulases. Three subforms are recognized: CBM3a, CBM3b and CBM3c, with CBM3a and CBM3b binding to crystalline or amorphous cellulose; CBM3c is connected exclusively to the catalytic module of GHF9 cellulases and as well as stabilizing it, may modulate the activity mode of an endoglucanase into a processive endoglucanase by binding a single cellulose molecule (Tormo et al., 1996
; Irwin et al., 1998
; Bayer et al., 2000
).
In this paper we report the detection and characterization of two new cellulosome components which form a gene cluster together with cellulase Cel9I on the genome of C. thermocellum F7. We show that Cel9N is an endoglucanase and is, in contrast to its close relative Cel9I, not processive. CseP is not hydrolytically active, but seems to be a structural component of the cellulosome.
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METHODS |
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Molecular biological methods.
The DNA sequence of clone pCU108 was determined from supercoiled double-stranded plasmid DNA on both strands (Thermosequenase Cycle Sequencing Kit; Amersham) with biotinylated oligonucleotide primers. DNA fragments were detected with a GATC 1500 Direct-Blotting Electrophoresis apparatus (GATC) using streptavidin-conjugated alkaline phosphatase and the chromogenic substrate NBT-BCIP (Promega). PCR was carried out using oligonucleotide primer pairs with chromosomal DNA as template and the Expand High Fidelity PCR System (Boehringer). The oligonucleotide primers were designed to truncate the gene at the N terminus for the leader peptide and to introduce a hexa-histidyl tag sequence for affinity chromatography. All primers introduced a restriction enzyme recognition site at a suitable position to ligate the PCR fragment into the vector in a predetermined way. The oligonucleotide primer sequences used are listed in Table 1. Restriction digests of DNA were done with restriction endonucleases (MBI Fermentas) as recommended by the manufacturer.
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Preparation of cellulosomes.
Cellulosomes were purified from 0·5 l cultures of C. thermocellum F7 in GS-2 medium (Johnson et al., 1981) containing Avicel with the affinity digestion method (Morag et al., 1992
) as described by Zverlov et al. (1999)
.
Enzyme assays.
Enzyme aliquots in standard assays were incubated in MES buffer (50 mM) containing 5 mM CaCl2 at the optimum pH and temperature. The concentration of substrates was 1 % for soluble and 2 % (w/v) for insoluble polysaccharides. Reducing sugars released from polymeric substrates were detected by the 3,5-dinitrosalicylic acid method (Wood & Bhat, 1988), assuming that 1 U enzyme liberates 1 µmol glucose equivalent min-1 and (mg protein)-1. Specific activities were determined in the linear range of the reaction. Protein concentration was determined with Coomassie brilliant blue (Sedmak & Grossberg, 1977
). p-Nitrophenol liberated from pNP-glycosides was measured by its absorption in alkaline solution (0·6 M Na2CO3) at 395 nm. 1 U of activity is defined as the amount of enzyme producing 1 µmol p-nitrophenol min-1 (0·013
OD395=1 nmol). All determinations were performed in triplicate.
The optimum pH was determined by measuring the specific activity of the enzyme at a given pH (MES buffer). The optimum temperature was the temperature at which the highest activity of the enzyme occurred during incubation for a given time.
The release of reducing sugars in the soluble and the insoluble fraction of cellulose was performed with a preparation of phosphoric acid swollen cellulose (PASC) (see below) that pellets easily on centrifugation. Enzyme reactions were set up as described above and incubated. Probes were taken at different time points and centrifuged. The pellet was washed two times with MES buffer and resuspended in 1 vol. buffer. Reducing sugars were determined in the supernatant of the enzyme reaction and the washed pellet separately as described. The pellet probes were centrifuged again after boiling and the optical density of the clear supernatants was determined.
Denaturing gel electrophoresis (SDS-PAGE) and Western blot.
SDS-PAGE was performed in 10 % polyacrylamide slab gels in the presence of 0·1 % SDS after boiling the proteins in the presence of 1·25 % (v/v) mercaptoethanol (Laemmli, 1970).
To elicit polyvalent antibodies against CseP, 0·25 mg purified hCsePd1 protein in Freund's adjuvant was used to immunize white rabbits. The antibodies were purified using a serum IgG purification column. Western blotting from SDS-PAGE gel slabs on nitrocellulose membranes was done by standard techniques using donkey anti-rabbit serum conjugated to horseradish peroxidase (Amersham) and 4-chloro-1-naphtol as substrate.
Sequence analysis.
Sequence data were analysed with the DNASIS/PROSIS software package (Hitachi Software Engineering). Nucleotide and protein sequence databases were screened using BLAST software at the EBI server at EMBL (http://www.ebi.ac.uk). The CAZY server (http://afmb.cnrs-mrs.fr/pedro/CAZY) was used for determining the GHF or the binding domain family [Carbohydrate-Active enZYmes and associated MODular Organization server (CAZyModO website), http://afmb.cnrs-mrs.fr/
pedro/DB/db.html].
TLC.
Polymeric and oligomeric substrates were hydrolysed to completion under the conditions described above. Hydrolysis products were separated on 0·2 mm aluminium sheet silica gel 60 plates (Merck) with acetronitrile/water as eluent (80 : 20, v/v). Sugars were detected by spraying the plates with a freshly prepared mixture of 10 ml stock solution with 1 ml o-phosphoric acid, followed by heating the plates at 120 °C until colour developed. The stock solution consisted of 1 g diphenylamine and 1 ml aniline dissolved in 100 ml acetone.
Substrates.
Birchwood xylan, Avicel CF1, carboxymethyl-cellulose (CM-cellulose, low viscosity) and pNP-glycosides were obtained from Sigma-Aldrich, cellodextrins from Merck, barley -glucan from Megazym and pustulan from Roth. PASC was prepared from Avicel CF1 according to Wood (1988)
.
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RESULTS |
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Hydrolytic mode
To gather more information on the hydrolytic mode of action, the degradation products were analysed by TLC (Fig. 3). hCelI degraded G4 slowly to G3 and glucose (G1), less to cellobiose (G2), whereas hCelN yielded mainly G2 and much less G3 and G1. G5 was completely degraded by hCelI and hCelN to either G4+G1 or G3+G2, and hCelN split G4 almost completely to G2. The amorphous polymer PASC was hydrolysed by hCelI to G4 which subsequently was degraded to G3+G1 and less to G2 (Fig. 4
); no larger cellodextrins could be detected even early in the reaction. This suggests a processive action mode of CelI. In contrast, hCelN produced larger cellodextrins first from PASC, which were later on degraded to G4 (and subsequently to G2) and less to G3 and G1. This indicates a non-processive endohydrolytic action mode for CelN.
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CseP: a non-catalytic cellulosome component
The fifth reading frame in the sequenced genomic fragment was called cseP (cellulosomal element P) due to the presence of a dockerin module type I in its C terminus. cseP is separated from the preceding gene, celN, by a potential bidirectional transcription terminator and is transcribed from the opposite DNA strand. The complete gene was expressed in E. coli (hCseP) and the gene product was purified by His-tag affinity chromatography. All cellulosome components containing a dockerin type I module investigated so far had hydrolytic activity. The sequence of CseP was compared to the databases, but not even a weak sequence similarity to any known hydrolytic enzyme could be detected. Nevertheless, hCseP was incubated with 10 polysaccharides, 14 aryl-glycosides and pNP-acetate (Table 4). No hydrolytic activity could be detected with these substrates, neither by release of the chromophore aglycon nor with reducing sugars. CseP presumably is not hydrolytically active.
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To verify the expression of cseP in C. thermocellum and to prove the presence of CseP in the cellulosome, polyclonal antibodies were raised against hCsePd1. This mutant protein is stripped of the dockerin domain which is present in all cellulosomal components and might cause cross-reactions. The antibodies were used to selectively label the CseP protein in a denaturing SDS-PAGE of cellulosomal proteins and as a control, the purified hCsePd1 protein (Fig. 6). Among the separated cellulosomal proteins only one band of 61 kDa was recognized by the antibody which was in accordance with the deduced molecular mass for native CseP (61 532 Da). This indicated that the antibody was specific and that CseP was present in the cellulosome.
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DISCUSSION |
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CtF7-Cel9I is identical to CtNCIB-CelI from C. thermocellum NCIB 10682 (Hazlewood et al., 1993) except for an accidental deletion of a 53 bp fragment between two 6 bp direct repeats near the 3' end of the gene (TTTCAA; bp 28372890 in L04735), possibly due to an error in sequence alignment in the CtNCIB-CelI sequence. This introduced a frameshift in the C-terminal part of the protein. The high degree of sequence identity between large stretches of DNA derived from different strains of C. thermocellum (3625 bp) emphasizes the close relationship between different bacterial isolates on different continents at least in the Northern hemisphere. Such high similarity was also found with other genes when sequenced from two strains, like celA-chiA, celC, licA, licB and others (Zverlov et al., 2002
; K. P. Fuchs, V. V. Zverlov, G. A. Velikodvorskaya, F. Lottspeich & W. H. Schwarz, unpublished results). Because of the identity with the already described CtATTC-Cel9I sequence, CtF7-Cel9I was investigated only so far as to compare it with the newly identified CtF7-Cel9N.
Next to themselves, the sequences of Cel9I and Cel9N are most similar to the 1,4--glucanases CelA from Anaerocellum thermophilum (GenBank accession no. Z86105, 47 % identity), CelA and CelE from Caldicellulosiruptor sp. Tok7B.1 (L32742 and AF078042, 46 %), CelF from C. thermocellum (X60545, 45 %), a cellulase from Bacillus sp. BP-23 (AJ133614, 46 %) and Avicelase I (CelZ) from Clostridium stercorarium (S12021, 45 %). All these enzymes have a GH9-CBM3c arrangement of modules and show a comparable substrate specificity, being slightly active on amorphic and crystalline cellulose, but with the highest activity on barley
-glucan. Within the GH9-CBM3c group of cellulases the hydrolytic mode of the E4-glucanase of Thermobifida fusca has been investigated in the greatest detail (Irwin et al., 1998
). These authors suggested an accessory role for the CBM3c module in holding a single cellulose chain and feeding it into the active site pocket of E4, after the endoglucanolytic activity of the catalytic centre has produced a new nick in the cellulose molecule. This makes the enzyme a processive endoglucanase, a trait not yet tested for the other members of that family.
The processive endoglucanase type of hydrolytic activity could be verified for CtF7-Cel9I by assaying the release of reducing ends in the soluble versus the insoluble fraction of digested PASC. However, no processivity could be detected with Cel9N. This was surprising in the light of the interpretation of the three-dimensional data from endoglucanase E4 which would suggest a similar function of the CBM3c module for the other enzymes with an equivalent module arrangement. However, the accessory function of the CBM3c module depends on correct spacing and orientation towards the active site pocket of the GH9 module which could be easily modified by a very limited number of mutations. With respect to substrate specificity, the CBM3c module might have a different or no function in Cel9N.
The different hydrolysis mode of Cel9I and Cel9N was corroborated by the analysis of the occurrence of hydrolysis products. By its processive action, Cel9I produces mainly cellotetraose which in a second, non-processive step is slowly hydrolysed further. The products of Cel9N initially are long oligosaccharides which are continuously hydrolysed to smaller ones. On small oligosaccharides, such as cellotetraose, Cel9N obviously has a higher activity compared with Cel9I.
Another hint at the differences in processivity comes from the effect calcium ions have on the hydrolytic activity. Whereas the activity of hCelI on PASC was inhibited by removing Ca2+ ions, no or little effect was observed on barley -glucan. In contrast, Ca2+ depletion had no effect with hCelN (data not shown). CBM family 3 is known to depend in its binding function on the presence of Ca2+ ions, and binding a cellulose strand supposedly is an integral part of Cel9I action on cellulose, but presumably not on mixed-linkage
-1,3-1,4-glucan. Abolishing the binding to cellulose would thereby negatively influence the activity on PASC. The endo-mode of Cel9N may be entirely independent of the activity of a binding module and thus was not influenced by Ca2+. Measurements with Avicel have shown that the CBM3c module in hCelN does not bind to crystalline cellulose (data not shown). The so-called helper-module, CBM3c, thus may not play a role of in cellulose hydrolysis by Cel9N.
However, according to the three-dimensional model of endoglucanase E4 from Thermobifida fusca, the CBM3c module is tightly attached to the catalytic module and seems to stabilize the integrity of the catalytic protein module. The protection from denaturation could be a consequence of this close association, as seen by the thermolability of the deletion proteins hCelId2 and hCelNd2. The function of a binding module as a thermostabilizing domain was also observed in CelZ (C. stercorarium; Riedel et al., 1998) in the same enzyme family and in other enzymes like a xylanase from Thermotoga neapolitana and Thermotoga maritima (Zverlov et al., 1996
; Meissner et al., 2000
).
It is interesting to note that all bacterial cellulolytic enzyme systems contain, in addition to the indispensable GHF48 enzyme, one or more GHF9 enzymes (Schwarz, 2001). A variety of GHF9-based endoglucanases seem to be necessary to fulfil the needs of effectively attacking a crystalline cellulosic substrate which displays a multitude of topologies. This variety in enzymes is verified by different module combinations, and the greatest diversity is seen in the GHF9 enzyme family. In the cellulosome of C. thermocellum so far seven GHF9 enzymes have been identified and investigated: two theme C enzymes (CelD, CelJ), two theme D enzymes (CbhA, CelK) and three theme B enzymes (CelF, CelQ, CelN), according to the classification of Bayer et al. (2000)
. An additional GHF9 enzyme, Cel9I, is a free cellulolytic component which might assist in the process of attacking different substrate topologies. Cel9I therefore is equipped with a substrate-binding module of its own (CBM3b) which makes it independent of the cellulosomal substrate-binding capacity, whereas Cel9N can rely on the strongly binding CBM3 attached to the CipA component of the cellulosome. The differences between the closely related enzymes Cel9I and Cel9N reflect a potential for an even greater variability in the ability of the GHF9 cellulases to hydrolyse different topologies in their substrate cellulose.
The gene cseP, which is also present in the sequenced 10 kb fragment, can be predicted to be transcribed independently of celI and celN. The protein CseP consists of an N-terminal module without any homology to hydrolytic enzymes in the databases and a dockerin type I module, which is regarded as an indicator for binding to the cohesin modules located on the cellulosomal core protein CipA. The gene product, CseP, was further investigated as a potentially interesting new cellulosome component. Our investigations with 25 substrates representing a wide range of glycans and glycosides did not reveal any hydrolytic activity. However, CseP showed significant homology to CotH from Bacillus subtilis, which is a structural component of the spore and is needed for proper formation of the spore coat (Zilhao et al., 1999). In addition, sequence similarity with other reading frames from genomic sequences of spore- or cellulosome-forming bacteria was observed. It was also similar to an uncharacterized protein of a single archaeon, Methanosarcina acetivorans, which is unique among the Archaea in forming complex multi-cellular structures (Galagan et al., 2002
). This implies the involvement of this family of proteins in the building of multi-protein structures.
One could argue that the occurrence of cseP was a mishap in the evolution of a cellulosomal protein, created by unsuccessful module shuffling. Such a gene would not be expected to be expressed. However, Western blots with anti-hCsePd1 antibodies verified that cseP is expressed and the protein successfully incorporated into the cellulosome. It was present in the cellulosome as a single band in significant amounts. These data indicate a potential role of CseP in the structural integrity of the assembly of the cellulosome and open new perspectives for the investigation of cellulosomal structure and function.
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ACKNOWLEDGEMENTS |
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Received 23 August 2002;
revised 18 October 2002;
accepted 28 October 2002.