A novel Cellvibrio mixtus family 10 xylanase that is both intracellular and expressed under non-inducing conditions

C. M. G. A. Fontes1, H. J. Gilbert2, G. P. Hazlewood3, J. H. Clarke3, J. A. M. Prates1, V. A. McKie2, T. Nagy2, T. H. Fernandes1 and L. M. A. Ferreira1

CIISA-Faculdade de Medicina Veterinária, Pólo Universitário do Alto da Ajuda, Rua Professor Cid dos Santos, 1300-477 Lisboa, Portugal1
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK2
Laboratory of Molecular Enzymology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK3

Author for correspondence: L. M. A. Ferreira. Tel: +351 213652800. Fax: +351 213533088. e-mail: luisferreira{at}fmv.utl.pt


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylo-oligosaccharides absorbed by the micro-organism. Cellvibrio mixtus, a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in {lambda}ZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptide with a deduced Mr of 43413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC identified a xynC homologue (designated xynG) encoding XylG; XylG and xynG were 67% and 63% identical to the corresponding C. mixtus sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on xynC supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an Mr of 41000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. C. mixtus was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that C. mixtus can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.

Keywords: family 10 xylanases, Cellvibrio mixtus, xylanase expression

Abbreviations: CBMs, carbohydrate-binding modules

The GenBank accession numbers for the sequences described in this paper are AF049493 and AF168359 for xynC and xynG, respectively.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Biological degradation of the main plant cell wall components, the polysaccharides cellulose and xylan, is a complex process that requires the synergistic interaction of a large consortium of microbial cellulases and xylanases (Tomme et al., 1995 ). Structural polysaccharides are insoluble polymers and, consequently, micro-organisms generally secrete plant cell wall hydrolases that are remarkably stable (Fontes et al., 1997 ). Extracellular cellulases and xylanases expressed by saprophytic aerobic soil bacteria, such as a Pseudomonas sp. (Hazlewood & Gilbert, 1998 ), Cellulomonas fimi (Meinke et al., 1994 ) and Cellvibrio mixtus (Fontes et al., 1998 ), bind individually to the plant cell wall via the action of specific non-catalytic domains designated carbohydrate-binding modules (CBMs). Therefore, most cellulases and xylanases derived from aerobic bacteria have a modular architecture, in which the catalytic domain is linked to one or more CBMs that are known to play a crucial role in enhancing the activity of the enzymes against crystalline cellulose and insoluble xylan, respectively (Hazlewood & Gilbert, 1998 ). In contrast, extracellular plant cell wall hydrolases from anaerobic organisms are modular enzymes that interact to form a large Mr enzyme complex, known as the cellulosome, that is bound to the microbial cell wall. Extracellular single-domain cellulases and xylanases have, however, been identified in both aerobes and anaerobes, and are thought to be involved in the hydrolysis of the soluble components of the cell wall. Recently, the view that all plant cell wall hydrolases are extracellular has been questioned by studies on the anaerobic bacterium Prevotella bryantii, in which the majority of the xylanase activity was shown to be located in the periplasm and is not exposed to the extracellular environment (Miyazaki et al., 1997 ). It was argued that the internal location of these enzymes might have an important role in allowing the bacterium to sequester the products of polysaccharide hydrolysis in energy-limiting densely populated gut ecosystems. It remains to be established whether aerobic bacteria, which can generate considerably more energy from pentose metabolism than anaerobic prokaryotes, also synthesize polysaccharidases that are not exported.

Plant cell wall degrading micro-organisms use a wide variety of carbohydrates as carbon and energy sources, and have therefore developed mechanisms to modulate the synthesis of polysaccharidases. Cellulases and xylanases are expressed when the organisms are grown in the presence of the structural polysaccharides and are subject to catabolite repression by readily metabolizable sugars such as glucose. However, some extracellular cellulases and xylanases have been shown to be constitutively expressed at very low levels by micro-organisms such as Trichoderma reesei (Zeilinger et al., 1996 ; Torigoi et al., 1996 ). In fungi, it is well established that these enzymes are crucial for triggering the expression of cellulases and xylanases; an initial attack on the cell wall by these plant cell wall hydrolases results in the absorption of the hydrolysis products by the organism and the consequent general induction of polysaccharidase expression by a mechanism which remains to be elucidated (Carle-Urioste et al., 1997 ). Pseudomonas fluorescens subsp. cellulosa was also shown to constitutively express polysaccharidases (Rixon et al., 1992 ), although their role in the regulation of gene expression in the pseudomonad is currently unknown.

Studies in our laboratories have focused on the plant cell wall degrading systems of C. mixtus and Ps. fluorescens subsp. cellulosa. Although both organisms have been shown to express a large number of extracellular xylanases and cellulases that are subject to catabolite repression (Hazlewood et al., 1992 ), it remains to be established whether the two aerobic prokaryotes also synthesize non-extracellular xylanases. The objective of this study was to establish whether there is evidence, in C. mixtus, for xylanases that are both intracellular and constitutively expressed. Data presented in this paper show that a non-modular family 10 xylanase (XylC) from C. mixtus is primarily secreted into the periplasm and is produced when the organism grows in the presence of various carbon sources, including glucose. A XylC homologue was also detected in Ps. fluorescens subsp. cellulosa and the roles of these enzymes in plant cell wall hydrolysis are discussed.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, vectors and culture conditions.
Cellvibrio mixtus (NCIMB 8633) was cultured aerobically at 20 °C in Dubos mineral salts medium or on Dubos agar plates overlaid with filter paper (Millward-Sadler et al., 1995 ). Media were supplemented with sterile glucose (0·25%), after autoclaving, or CM-cellulose (medium viscosity), Avicel or oat spelt xylan, before autoclaving (all polysaccharides were used at 0·5% final concentration). Pseudomonas fluorescens subsp. cellulosa (NCIMB 10462) was cultured as described by Millward-Sadler et al. (1995) . Escherichia coli JM83 and XL-1 Blue were cultured at 37 °C in Luria broth (LB) or on LB-agar plates. Media were supplemented with 100 mg ampicillin l-1 or 2 mg 5-bromo-4-chloro-3-indolyl ß-D-galactoside l-1 to select for E. coli transformants and recombinants, respectively. Remazol Brilliant Blue R-xylan (Sigma) was added to solid media at a final concentration of 0·05% (w/v) to select for recombinant E. coli strains expressing xylanase activity. The phages and plasmids employed in this work were {lambda}ZAPII (Stratagene), pBluescript (Stratagene), pUC18 and pUC19 (Norrander et al., 1983 ).

General recombinant DNA procedures.
Plasmid DNA was prepared by the method of Birnboim & Doly (1979) , or by using Qiagen resin columns (Hybaid). Transformation of E. coli, agarose gel electrophoresis, Southern hybridization, slot blot hybridizations and the general use of nucleic acid modifying enzymes were as described by Sambrook et al. (1989) . C. mixtus and Ps. fluorescens subsp. cellulosa genomic DNA was isolated as described by Berns & Thomas (1965) . The genomic libraries were constructed in {lambda}ZAPII using the approach described by Clarke et al. (1991) . The libraries were screened for recombinants expressing xylanase activity by plating out the recombinant phage and host bacterium (E. coli XL-1 Blue) in soft agar poured onto NZY plates (NZY medium: 0·5% NaCl; 0·2% MgSO4 . 7H2O; 0·5% yeast extract; 1% NZ amine; pH adjusted to 7·5). The plaques generated were overlaid with agar containing 0·2% (w/v) xylan. After incubation at 37 °C for 16 h, xylanase-producing clones were identified by the appearance of clear haloes against a red background, after staining with Congo red (Teather & Wood, 1982 ). Plasmids [pBluescript SK(-)] containing genomic DNA inserts from C. mixtus and Ps. fluorescens subsp. cellulosa were excised from xylanase-positive recombinant phage and rescued into E. coli XL-1 Blue, as described in the Stratagene protocol. DNA hybridizations were performed using the fluorescein system from Amersham according to the manufacturer’s protocol.

Nucleotide sequencing.
To sequence xynC and xynG, nested deletions of the C. mixtus and Ps. fluorescens subsp. cellulosa chromosomal DNA [in pBluescript SK(-)], respectively, were created using the Exonuclease III/S1 nuclease method (Promega). Double-stranded plasmid DNA was sequenced manually by the dideoxy-chain-termination method of Sanger et al. (1977) , with the protocol recommended for the Sequenase DNA Sequencing kit (United States Biochemical/Amersham). Sequences were compiled and ordered using the computer software DNASIS from Hitachi. The complete sequences of xynC and xynG were determined in both strands.

Protein purification, production of antisera and Western blotting.
E. coli JM83 harbouring full-length xynC in plasmid pLMA2 was cultured for 16 h in LB broth containing ampicillin (100 mg l-1). A cell-free extract was prepared by sonicating the harvested cells and recovering the soluble fraction. Proteins were loaded onto a DEAE Trisacryl anion-exchange column, which was eluted in 10 mM Tris/HCl, pH 8·0, with a 0–400 mM NaCl gradient. Fractions expressing xylanase activity were further purified on a MonoQ column by anion-exchange chromatography. The purity of the xylanase fractions was confirmed by analysis through SDS-PAGE.

To produce polyclonal antisera against XylC, purified protein (approx. 500 µg) diluted to 1 ml with sterile distilled water was emulsified with Freund’s complete adjuvant (1 ml) and injected into New Zealand White male rabbits by intramuscular and subcutaneous routes. Second and third injections, with half as much protein mixed with Freund’s incomplete adjuvant, were made at 4-week intervals. Serum was collected 12 d after the last injection. Western blot analysis of C. mixtus proteins was performed essentially as described by Fontes et al. (1995) using the enhanced chemiluminescence system (Amersham).

Enzyme assays.
Periplasmic and cell-free extracts were prepared as described by Ferreira et al. (1990) with a 200 ml culture. Enzyme assays were performed in 50 mM potassium phosphate/12 mM citric acid buffer, pH 6·5 (PC buffer) at 37 °C, using 0·2% (w/v) of the appropriate plant structural polysaccharide, unless otherwise stated. Reducing sugar was measured with the dinitrosalicylic acid reagent (Miller, 1959 ). Insoluble xylan was prepared from oat spelt xylan as described by Fernandes et al. (1999) . One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol reducing sugar min-1. HPLC analysis of hydrolysis products was performed as described by Black et al. (1994) . Total protein was measured by the Lowry method with BSA as standard. SDS-PAGE analysis was carried out as described by Laemmli (1970) .

Proteolysis, thermostability and pH optimum experiments.
E. coli fractions containing XylC, at a concentration of approximately 10 g total protein l-1, were incubated with porcine pancreatin at a final concentration of 10 g l-1 at 37 °C. At regular time intervals, an aliquot of the reaction mixture was removed and assayed for enzyme activity as described above. For the thermostability experiments, the xylanase-containing extracts were incubated for 15 min at temperatures ranging from 37 to 85 °C, cooled on ice for 5 min and, after centrifugation at 13000 g for 10 min, assayed for residual xylanase activity. Extracts containing XylC were assayed for xylanase activity in buffers with different pH values (pH 3–7, 50 mM phosphate citrate; pH 8–9, 50 mM sodium barbitone), as described above.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Production and cellular location of xylanases in C. mixtus
To establish the growth pattern of C. mixtus in the presence of various carbon sources, the bacterium was cultured at 20 °C in minimal media containing glucose (0·25%) or oat spelt xylan (0·5%; Sigma) as the sole carbon source, and the OD600 of the cultures was monitored over a 7 d period. The results (not shown) demonstrated that for the substrate concentrations used, C. mixtus reached stationary phase faster when grown on glucose (36 h) than on xylan (84 h). The general pattern of xylanase expression by C. mixtus, grown in the presence of xylan or glucose, was determined using late-exponential-phase cells. The data, presented in Table 1, demonstrate that most of the xylanase activity in xylan-grown cells was extracellular, while no apparent activity was detected in the culture supernatant of glucose-grown C. mixtus. However, C. mixtus cells grown on xylan or glucose contained considerable internal xylanase activity, which was located mainly in the periplasm. Under these experimental conditions, the majority of malate dehydrogenase activity, alkaline phosphatase and arabinase (cell membrane enzyme; unpublished data) activities were located in the cytoplasm, periplasm and cell envelope, respectively, suggesting that the cell fractions had been adequately separated. Collectively, these data suggest that C. mixtus expresses internal xylanase(s) which, in both glucose- and xylan-grown cells, are predominantly located in the periplasmic space.


View this table:
[in this window]
[in a new window]
 
Table 1. Localization of xylanase, malate dehydrogenase, alkaline phosphatase and arabinase activities in C. mixtus grown on different carbon sources, and in E. coli harbouring the plasmid pLMA4

 
Isolation and characterization of xylanase genes that encode potential intracellular enzymes
To isolate genes encoding non-extracellular xylan-degrading enzymes, a C. mixtus genomic library constructed in {lambda}ZAPII was screened for xylanase activity. Xylanase-positive phages were isolated at a frequency of 1 in 200 clones. The C. mixtus genomic inserts from 12 recombinant clones were excised into pBluescript SK(-) and their similarity with the previously described C. mixtus xylanase genes, xynA and xynB, was determined by Southern hybridization (Millward-Sadler et al., 1995 ). The results (not shown) revealed that out of the 12 clones, six C. mixtus sequences cross-hybridized and did not hybridize with xynA or xynB, indicating that the six plasmids contained a novel xylanase gene. The xylanase activity expressed by E. coli cells harbouring one of the six recombinant plasmids (pLMA4) was found predominantly in the cytoplasm, suggesting that the encoded recombinant xylanase was not efficiently exported by E. coli (Table 1). A restriction map of the C. mixtus genomic fragment containing the new xylanase gene, designated xynC, is presented in Fig. 1. Deletion and subcloning experiments located the position of xynC within the C. mixtus DNA fragment.



View larger version (7K):
[in this window]
[in a new window]
 
Fig. 1. Restriction map of recombinant plasmids containing xynC from C. mixtus. Restriction enzyme sites are as follows: H, HindIII; C, ClaI; Sp, SphI; S, SalI; E, EcoRI; P, PstI. The large arrow indicates the position and orientation of xynC within the C. mixtus sequence. The fragment cloned in plasmid pLMA3 was cloned from C. mixtus genomic DNA by inverse PCR. The xylanase phenotype of E. coli harbouring the respective plasmids is indicated by + and -.

 
To determine whether xynC was reiterated within the Cellvibrio genome, genomic DNA was subjected to Southern hybridization using the DNA insert from the recombinant plasmid containing xynC (pLMA2) as the probe. The data, not shown, indicate that xynC is present as a single copy in the C. mixtus genome. It was previously demonstrated that xynA and xynB from C. mixtus share considerable sequence identity with the xylanase genes xynE and xynF from Ps. fluorescens subsp. cellulosa (Millward-Sadler et al., 1995 ). To assess whether a homologue to xynC was present in the pseudomonad genome, xynC derived from pLMA2 was used to probe Ps. fluorescens subsp. cellulosa DNA. The results (not shown) revealed the presence of a single locus in the Ps. fluorescens genome exhibiting extensive homology with xynC. The Ps. fluorescens subsp. cellulosa xynC homologue, designated xynG, was isolated from a {lambda}ZAPII genomic library and shown to encode a functional xylanase defined as XylG (data not shown).

Nucleotide sequence of xynC and xynG and the primary structures of XylC and XylG
The nucleotide sequences of the genomic fragments containing xynC and xynG were determined in both strands. The data revealed ORFs of 1137 and 1134 bp, for xynC and xynG, respectively, encoding polypeptides with predicted Mr of 43413 and 43167. The codon usage of the ORFs was very similar to other C. mixtus and Ps. fluorescens plant cell wall hydrolases (Fontes et al., 1997 , 1998 ; Millward-Sadler et al., 1994 , 1995 ). The proposed ATG translational start codons were preceded (7 bp) by the sequence GAGGA, which exhibits strong similarity to the ribosome-binding motif most frequently found in genes from Gram-negative bacteria. The presence of translational stop codons in all three reading frames of the 5' flanking sequence of xynC and xynG provides further support for the validity of the putative translational start. The N-terminal sequences of XylC and XylG contain a basic N-terminus followed by 12 small hydrophobic residues. These sequence motifs exhibit similarities to bacterial signal peptides, although the hydrophobic region of the corresponding secretion signals from other Ps. fluorescens and C. mixtus polysaccharidases tends to be longer (Fontes et al., 1998 ). The two genes terminated at a TAA codon followed by translational stop codons in all three reading frames. A DNA palindromic sequence capable of forming a stem–loop with a {Delta}G of -22·9 kcal (-96·18 kJ) was noted downstream of xynC and xynG. This structure was followed by an A+T-rich region, which is characteristic of a rho-independent transcription termination sequence.

Deletion of the 45 N-terminal or 35 C-terminal amino acids, respectively, from XylC resulted in the complete loss of xylanase activity, suggesting that the protein is a single-domain enzyme. This was supported by homology studies, which revealed extensive sequence identity between both XylC and XylG and the catalytic domains of family 10 glycosyl hydrolases, as defined by Henrissat & Bairoch (1993) . The sequence with the highest level of homology to XylC and XylG was XylA (43 and 45% identity, respectively) from Bacteroides ovatus (Whitehead, 1995 ), followed by XylA from Prev. bryantii (39 and 40% identity, respectively; Gasparic et al., 1995 ), xylanase A from Bacillus strain N137 (37 and 36% identity, respectively; Tabernero et al., 1995 ) and XylX from Aeromonas caviae (36 and 36% identity, respectively; accession no. 3299808). Interestingly, all these enzymes are non-modular xylanases, suggesting that this subset of family 10 enzymes (Henrissat & Bairoch, 1993 ) may share a common ancestral origin. Together, these results suggest that XylC and XylG are family 10 single-domain xylanases.

Biochemical properties of XylC
XylC was purified from the cytoplasmic fraction of E. coli cells harbouring pLMA2 and its biochemical properties were evaluated. The enzyme had an Mr of 41000 (Fig. 2) and displayed activity over a limited pH range, with a maximum at pH 7·5 (not shown). XylC was thermolabile, displaying considerable loss in activity at temperatures in excess of 40 °C and with a half-life of less than 10 min at 50 °C. XylC was also rapidly inactivated when incubated with pancreatic proteinases, demonstrating very high susceptibility to proteolysis (only less than 10% of XylC residual activity was recovered after a 3 min incubation with proteinases). The addition or removal of 5 mM CaCl2, which has been shown to stabilize at least one family 10 xylanase (Spurway et al., 1997 ), did not affect the thermal stability or the proteolytic sensitivity of XylC. Collectively, these results suggest that XylC is particularly sensitive to proteinase inactivation, which is in sharp contrast to previously characterized extracellular xylanases, from both mesophilic and thermophilic organisms, which are completely resistant to proteolytic inactivation over a 3 h incubation period with pancreatic proteinases (Fontes et al., 1995 ; Spurway et al., 1997 ). Analysis of the substrate specificities of XylC revealed that the enzyme hydrolysed both the soluble and the insoluble fractions of oat spelt xylan [443 and 189 U (mg protein)-1] and exhibited slight activity against ß-glucan [1·4 U (mg protein)-1] and CM-cellulose [0·03 U (mg protein)-1]. The xylanase was unable to hydrolyse crystalline forms of cellulose such as filter paper and Avicel, even after prolonged incubation for 24 h, or 1,3-ß-glucans such as laminarin. Very low activity against soluble cellulosic substrates has been reported for other family 10 xylanases, exemplified by Cex from Cellulomonas fimi (Gilkes et al., 1984 ). To evaluate whether full-length XylC was able to bind insoluble polysaccharides, the recombinant enzyme was mixed with Avicel and insoluble oat spelt xylan and the retention of xylanase activity in the pellets was assessed. The data indicated that XylC is unable to bind significantly to either cellulose or xylan (not shown).



View larger version (122K):
[in this window]
[in a new window]
 
Fig. 2. Purification of XylC from C. mixtus. SDS-PAGE analysis using a 10% (w/v) polyacrylamide gel of cell-free extract from E. coli JM83 (lane 2) and recombinant E. coli harbouring pLMA2 (lane 3). Lane 4 contains XylC purified from E. coli harbouring pLMA2. Lanes 1 and 5 contain Sigma low-Mr markers. The Mr of purified XylC is shown.

 
To evaluate the mode of action of XylC, the products generated by the action of the enzyme against oligosaccharides were analysed by HPLC. The data revealed that XylC displayed a typical endo-mode of activity against xylo-oligosaccharides. For example, xylohexose was cleaved to mainly xylotriose and small amounts of xylobiose and xylotetraose; xylopentaose to xylobiose and xylotriose; xylotetraose was hydrolysed exclusively to xylobiose; the enzyme did not cleave xylobiose. The relative activities of XylC against xylotriose, xylotetraose, xylopentaose and xylohexaose were 1:603:3038:3050, respectively. The activity of XylC and that reported for C. fimi Cex by Charnock et al. (1998) against these xylo-oligosaccharides were very similar. These data suggest that XylC is not an exo-acting enzyme and that the enzyme has a substrate-binding site that accommodates five xylose units. The products of xylopentaose hydrolysis showed that two and three xylose-binding sites must be located on either site of the nucleophile and the acid–base residues. From the above discussion, it is apparent that the biochemical properties of XylC are very similar to other family 10 xylanases, which have been shown to have substrate-binding clefts ranging from five (C. fimi Cex) to seven (Pseudomonas Xyn10A; Charnock et al., 1998 ) sugar-binding subsites.

Cellular location of XylC from C. mixtus
To determine the cellular location of XylC in its original host, C. mixtus was grown to stationary phase in minimal medium containing either glucose (0·25%), oat spelt xylan (0·5%), CM-cellulose (0·5%), Avicel (0·5%), CMC/xylan (0·25% each) or Avicel/xylan (0·25% each) as sole carbon source, and the proteins present in the culture supernatant (not shown) and bacterial cell pellet were probed with anti-XylC polyclonal antibodies by Western blot analysis. The results, presented in Fig. 3, showed that a protein of Mr 41000, which was immunoreactive with anti-XylC antiserum, was present exclusively in the cell pellet (negative Western blot results with the culture media are not shown). To test the possibility of XylC being inactivated by proteinases potentially present in the culture media, the enzyme was incubated with media collected from stationary phase cells over a 3 h period. The data (not shown) demonstrated that XylC was completely resistant to inactivation when incubated under the conditions described. The similar size of XylC produced by C. mixtus and the recombinant form of the enzyme suggested that the xylanase was not subject to post-translational modifications in its endogenous host (Fig. 3). In addition, the results showed that XylC is not subject to catabolite repression, but is expressed when C. mixtus grows in the presence of glucose. To evaluate the localization of XylC in C. mixtus cells, the bacterium was grown to late-exponential phase in minimal media supplemented with glucose (0·25%) or oat spelt xylan (0·5%) and periplasmic, cytoplasmic and membrane envelope fractions were prepared as described by Ferreira et al. (1990) . To verify that fractions were correctly prepared, samples were assayed for periplasmic, cytoplasmic and cell envelope enzymes (data not shown). The data, in Fig. 4, show that XylC was present predominantly in the periplasm of C. mixtus and is expressed when the organism is cultured on glucose or xylan. Although purely qualitative, the Western blot data of Fig. 4 confirm that significant amounts of XylC are expressed by glucose-grown cells.



View larger version (78K):
[in this window]
[in a new window]
 
Fig. 3. Production of XylC by C. mixtus. The bacterium was grown in LB (lane 1) or in minimal media containing Avicel (lane 2), glucose (lane 3), oat spelt xylan (lane 4), CMC (lane 5), CMC/xylan (lane 6) and Avicel/xylan (lane 7), as described in Methods, until stationary phase. Total cellular proteins were fractionated by SDS-PAGE. Lane 8 contains total proteins from E. coli harbouring pLMA4. Western blotting was carried out using antisera raised against purified recombinant XylC as described in Methods. The Mr of the immunoreactive polypeptides is indicated.

 


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 4. Cellular localization of XylC from C. mixtus. Cells grown in the presence of glucose (a) and xylan (b) were grown to late-exponential phase (for 30 and 60 h, respectively) and treated as described in Methods for isolation of cell-bound (lane 1), cytoplasmic (lane 2) and periplasmic (lane 3) proteins. Lane 4 contains extracellular polypeptides and lane 5 the sucrose released protein. SDS-PAGE, performed using 10 µg protein, and Western analysis were performed according to the legend of Fig. 3. The Mr of the immunoreactive polypeptide is indicated.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
It is well-established that plant cell wall degrading organisms secrete extensive consortia of modular cellulases and hemicellulases containing non-catalytic CBMs, suggesting the existence of a strong selective pressure for the retention of these modules. Cellulose, and more recently, xylan binding domains have been shown to play a pivotal role in the hydrolysis of cell wall polysaccharides, both by promoting the interactions between the enzymes and the substrates and, in some instances, by contributing to the physical disruption of the substrates (Din et al., 1991 , 1994 ; Millward-Sadler et al., 1994 ; Bolam et al., 1998 ; Sun et al., 1998 ; Fernandes et al., 1999 ). Despite the strong selection pressure for modular plant cell wall hydrolases, single-domain cellulases and xylanases are expressed by micro-organisms, suggesting that these enzymes also play an important role in plant cell wall hydrolysis. It could be argued that the anchoring of enzymes to the polysaccharides would limit the hydrolysis of soluble oligo- and polysaccharides released from the cell wall and thus non-modular enzymes would primarily be involved in the hydrolysis of soluble substrates (Fontes et al., 1998 ). Evidence presented in this study shows that C. mixtus expresses a protease-sensitive xylanase, XylC, when grown in the presence of various carbon sources and directs the secretion of the enzyme into the periplasm. XylC and its homologue in Ps. fluorescens subsp. cellulosa, XylG, exhibited highest identities with non-modular family 10 xylanases, particularly those from Prev. bryantii and B. ovatus. Furthermore, studies on the biophysical properties of XylC showed that the enzyme was unusually sensitive to proteolytic inactivation, while, to our knowledge, extracellular xylanases are resistant to proteinase attack (Fontes et al., 1995 ).

In this report, we show that C. mixtus expresses low levels of periplasmic xylanase activity when grown on glucose, but on xylan produces high levels of extracellular xylanase activity, and also increased internal xylanase expression. One of the components responsible for the periplasmic xylanase activity seems to be XylC, although it remains to be elucidated if it is unique. The presence of an N-terminal sequence that resembles a signal peptide in both XylC and XylG suggests that the enzymes are subject to a protein sorting mechanism. However, the size of the hydrophobic domain is smaller than that of signal peptides from other previously characterized extracellular polysaccharidases of both C. mixtus and Ps. fluorescens subsp. cellulosa. The periplasmic location of XylC suggests that the enzyme’s primary targets are xylo-oligosaccharides that have been transported into the bacterium. XylC preferentially hydrolyses oligosaccharides with DP >5 and therefore C. mixtus can either import relatively large xylo-oligosaccharides (xylopentaose or larger) or the enzyme is hydrolysing xylotriose and xylotetraose relatively slowly. The concept that microbial xylanases can be intracellular is supported by a recent study which showed that approximately 80% of xylanase activity expressed by Prev. bryantii is located in the periplasm (Miyazaki et al., 1997 ).

We propose that the protected periplasmic environment of XylC has removed the selective pressures for the enzyme to become highly stable – a general feature of extracellular xylanases is resistance to proteinases and thermal denaturation, for example. As the majority of Prev. bryantii xylanase activity was found in the periplasm, it is likely that XynA from this bacterium is also periplasmic, which would be consistent with the enzyme’s designation as highly thermolabile (Gasparic et al., 1995 ). This view is supported by the observation that XynA, when expressed in Bacteroides vulgartus, is intracellular (H. J. Flint, personal communication). Based on the data presented in this paper, and results reported by Gasparic et al. (1995) , it is likely that within glycosyl hydrolase family 10, there is a subset of non-extracellular xylanases that includes XylC (C. mixtus), XylG (Ps. fluorescens), XynA (Prev. bryantii) and XylI (B. ovatus), which are particularly labile. Whether these enzymes have all evolved from an ancestral family 10 enzyme that was particularly thermolabile and sensitive to proteolytic attack, or exhibit these properties because of their intracellular location remains to be elucidated.

In contrast with the general pattern of microbial cellulase and hemicellulase synthesis, XylC was shown to be expressed when C. mixtus was grown on glucose. In fungi, constitutively expressed extracellular xylanases play an important role in regulating polysaccharidase gene expression (Zeilinger et al., 1996 ). Furthermore, the production of the general cellulase inducer, sophorose, was shown to be mediated by a constitutive intracellular ß-glucosidase which generated the inducer by catalysing the transglycosylation of cellobiose. Constitutive expression of a bacterial xylanase has also been reported in Streptomyces cyaneus (Wang et al., 1992 ): one of that organism’s three xylanases (xylanase III) was expressed in the presence of glucose, and it was suggested that this enzyme acts as a ‘xylan’ sensor probably involved in the regulation of other xylanase genes. In view of the expression of XylC when the organism grows on different carbon sources, it is tempting to speculate that this enzyme plays a key role in generating signals, from absorbed xylo-oligosaccharides, that induce xylanase expression in C. mixtus. Clearly this hypothesis can only be viewed as tentative until the role of XylC in xylanase gene expression is analysed in more detail.


   ACKNOWLEDGEMENTS
 
We wish to thank Fundação para a Ciência e Tecnologia (FCT) for supporting this work (Praxis XXI/C/AGR/11042/98).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Berns, K. I. & Thomas, C. A. (1965). Isolation of high molecular weight DNA from Haemophilus influenza.J MoI Biol11, 476-490.

Birnboim, B. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA.Nucleic Acids Res7, 1513-1515.[Abstract]

Bolam, D. N., Ciruela, A., McQueen-Mason, S., Simpson, P., Williamson, M. P., Rixon, J. E., Boraston, A., Hazlewood, G. P. & Gilbert, H. J. (1998). Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity.Biochem J331, 775-781.[Medline]

Black, G. W., Hazlewood, G. P., Shou, G.-P., Orpin, C. G. & Gilbert, H. J. (1994). Xylanase B from Neocalimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide. Biochem J299, 381-387.[Medline]

Carle-Urioste, J. C., Escobar-Vera, J., El-Gogary, S., Renrique-Silva, F., Torigoi, E., Crivellaro, O., Berrera-Estreta, A. & EI-Dorry, R. (1997). Cellulase induction in Trichoderma reesei by cellulose requires its own basal expression. J Biol Chem272, 10169-10174.[Abstract/Free Full Text]

Charnock, S. J., Spurway, T. D., Xie, H., Beylot, M. H., Virden, R., Warren, R. A., Hazlewood, G. P. & Gilbert, H. J. (1998). The topology of the substrate binding clefts of glycosyl hydrolase family 10 xylanases are not conserved.J Biol Chem273, 32187-32199.[Abstract/Free Full Text]

Clarke, J. H., Laurie, J. I., Gilbert, H. J. & Hazlewood, G. P. (1991). Multiple xylanases of Cellulomonas fimi are encoded by distinct genes. FEMS Microbiol Lett83, 305-310.

Din, N., Gilkes, N. R., Tekant, B., Miller, R. C., Warren, R. A. J. & Kilburn, D. G. (1991). Non-hydrolytic disruption of cellulose fibers by the binding domain of a bacterial cellulase.Bio/Technology9, 1096-1099.

Din, N., Damude, H. J., Gilkes, N. R., Miller, R. C., Warren, R. A. J. & Kilburn, D. G. (1994). C1-Cx revisited: intramolecular synergism in a cellulase.Proc Natl Acad Sci U S A91, 11383-11387.[Abstract/Free Full Text]

Fernandes, A. C., Fontes, C. M. G. A., Gilbert, H. J., Hazlewood, G. P., Fernandes, T. H. & Ferreira, L. M. A. (1999). Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes. Biochem J342, 105-110.[Medline]

Ferreira, L. M. A., Durrant, A. J., Rixon, J., Hazlewood, G. P. & Gilbert, H. J. (1990). Spatial separation of protein domains is not essential for catalytic activity or substrate binding in a xylanase.Biochem J269, 261-264.[Medline]

Fontes, C. M. G. A., Hazlewood, G. P., Morag, E., Hall, J., Hirst, B. H. & Gilbert, H. J. (1995). Evidence for a general role for non-catalytic thermostabilizing domains in xylanases from thermophilic bacteria.Biochem J307, 151-158.[Medline]

Fontes, C. M. G. A., Clarke, J. H., Hazlewood, G. P., Fernandes, T. H., Gilbert, H. J. & Ferreira, L. M. A. (1997). Possible roles for a non-modular, thermostable and proteinase-resistant cellulase from the mesophilic aerobic soil bacterium Cellvibrio mixtus. Appl Microbiol Biotechnol48, 473-479.[Medline]

Fontes, C. M. G. A., Clarke, J. H., Hazlewood, G. P., Fernandes, T. H., Gilbert, H. J. & Ferreira, L. M. A. (1998). Identification of tandemly repeated type VI cellulose-binding domains in an endoglucanase from the aerobic soil bacterium Cellvibrio mixtus. Appl Microbiol Biotechnol49, 552-559.[Medline]

Gasparic, A., Marinsek, L. R., Martin, J., Wallace, R. J., Nekrep, F. V. & Flint, H. J. (1995). Isolation of genes encoding beta-D-xylanase, beta-D-xylosidase and alpha-L-arabinofuranosidase activities from the rumen bacterium Prevotella ruminicola B1(4).FEMS Microbiol Lett125, 135-141.[Medline]

Gilkes, N. R., Langsford, M. L., Kilburn, D. G., Miller, R. C. & Warren, R. A. J. (1984). Mode of action and substrate specificities of cellulases from cloned bacterial genes.J Biol Chem259, 10455-10459.[Abstract/Free Full Text]

Hazlewood, G. P. & Gilbert, H. J. (1998). Structure and function analysis of Pseudomonas plant cell wall hydrolases. Biochem Soc Trans26, 185-190.[Medline]

Hazlewood, G. P., Laurie, J. I., Ferreira, L. M. A. & Gilbert, H. J. (1992). Pseudomonas fluorescens subsp. cellulosa: an alternative model for bacterial cellulase.J Appl Bacteriol72, 244-251.[Medline]

Henrissat, B. & Bairoch, A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities.Biochem J280, 309-316.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature227, 680-685.[Medline]

Meinke, A., Gilkes, N. R., Kwan, E., Kilburn, D. G., Warren, R. A. J. & Miller, R. C. (1994). Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a ß-1,4-exocellobiohydrolase analogous to Trichoderma reesei CBHII. Mol Microbiol12, 423-432.[Medline]

Miller, G. L. (1959). The use of dinitrosalicylic acid reagent for determination of reducing sugar.Anal Chem31, 426-428.

Millward-Sadler, S. J., Poole, D. M., Henrissat, B., Hazlewood, G. P., Clarke, J. H. & Gilbert, H. J. (1994). Evidence for a general role for high-affinity non-catalytic cellulose binding domains in microbial plant cell wall hydrolases.Mol Microbiol11, 375-382.[Medline]

Millward-Sadler, S. J., Clarke, J. H., Davidson, K., Hazlewood, G. P., Black, G. W. & Gilbert, H. J. (1995). Novel cellulose-binding domains, NodB homologues and conserved modular architecture in xylanases from the aerobic soil bacteria Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus. Biochem J312, 39-48.[Medline]

Miyazaki, K., Martin, J. C., Marinsek-Logar, R. & Flint, H. J. (1997). Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii (formerly P-ruminicola subsp. brevis).Anaerobe3, 373-381.

Norrander, J., Kemp, T. & Messing, J. (1983). Construction of improved M13 vectors using oligonucleotide-directed mutagenesis.Gene26, 101-106.[Medline]

Rixon, J. E., Ferreira, L. M. A., Durrant, A. J., Laurie, J. L., Hazlewood, G. P. & Gilbert, H. J. (1992). Characterization of the gene celD and its encoded product the 1,4-D-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa.Biochem J285, 947-955.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors.Proc Natl Acad Sci U S A474, 5463-5467.

Spurway, T. D., Morland, C., Cooper, A., Sumner, L., Hazlewood, G. P., O’Donnell, A. G., Pickersgill, R. W. & Gilbert, H. J. (1997). Calcium protects a mesophilic xylanase from proteinase inactivation and thermal unfolding. J Biol Chem272, 17523-17530.[Abstract/Free Full Text]

Sun, J. L., Sakka, K., Karita, S., Kimura, T. & Ohmiya, K. (1998). Adsorption of Clostridium stercorarium xylanase A to insoluble xylan and the importance of the CBD to xylan hydrolysis.J Ferment Bioeng85, 63-68.

Tabernero, C., Sánebez, T. J., Pérez, P. & Santamaría, I. L. (1995). Cloning and DNA sequencing of xynA, a gene encoding an endo-beta-1,4-xylanase from an alkalophilic Bacillus strain (N137).Appl Environ Microbiol61, 2420-2424.[Abstract]

Teather, R. M. & Wood, P. J. (1982). Use of Congo Red-polysaccharide interactions to the enumeration and characterization of cellulolytic bacteria from the bovine rumen.Appl Environ Microbiol43, 777-782.[Medline]

Tomme, P., Warren, R. A. & Gilkes, N. R. (1995). Cellulose hydrolysis by bacteria and fungi.Adv Microb Physiol37, 1-81.[Medline]

Torigoi, E., Henrique-Silva, F., Escobar-Vera, J., Carle-Uríoste, J. C., Crivellaro, O., El-Dorry, H. & El-Gogary, S. (1996). Mutants of Trichoderma reesei are defective in cellulose induction, but not basal expression of cellulase-encoding genes.Gene173, 199-203.[Medline]

Wang, P., Ali, S., Mason, J. C., Sims, P. F. G. & Broda, P. (1992). Xylanases from Streptomyces cyaneus. In Xylans and Xylanases, pp. 225-234. Edited by J. Visser, G. Beldman, M. A. Kusters-van Someren & A. G. J. Voragen. Amsterdam: Elsevier.

Whitehead, T. R. (1995). Nucleotide sequences of xylan-inducible xylanase and xylosidase-arabinosidase genes from Bacteroides ovatus V975.Biochim Biophys Acta1244, 239-241.[Medline]

Zeilinger, S., Mach, K. L., Sebindler, M., Rerzog, P. & Kubicek, C. (1996). Different inducibility of expression of the two xylanase genes xyn1 and xyn2 in Trichoderma reesei. J Biol Chem271, 25624-25629.[Abstract/Free Full Text]

Received 3 March 2000; revised 17 May 2000; accepted 22 May 2000.