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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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
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 manufacturers 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 0400 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 Freunds 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 Freunds 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)
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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 37, 50 mM phosphate citrate; pH 89, 50 mM sodium barbitone), as described above.
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RESULTS |
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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 stemloop with a
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).
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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.
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DISCUSSION |
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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 enzymes 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 enzymes 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 organisms 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.
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ACKNOWLEDGEMENTS |
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Received 3 March 2000;
revised 17 May 2000;
accepted 22 May 2000.