1 Laboratory of Animal Science, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan
2 Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
Correspondence
Kohji Miyazaki
miyazaki{at}kpu.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Previous work demonstrated that the xynA and xynB genes are located in the same operon, together with xynD, which encodes a putative sodium-symporter that may be involved in xylo-oligosaccharide uptake (Miyazaki et al., 2003). Xylanase activity is induced in P. bryantii during growth on xylans, but is not induced by xylose (Gardner et al., 1995
; Miyazaki et al., 1997
). This induction is partly due to increased transcription of the xynA and xynB genes during growth on xylan, mediated by the product of the linked xynR regulatory gene (Miyazaki et al., 2003
). The cloned XynR product has been shown to bind just upstream of the xynABD operon and to stimulate transcription (Miyazaki et al., 2003
). XynR shows homology with two-component regulators (Miyazaki et al., 2003
) and is one of the first such regulators shown to govern expression of polysaccharide-degrading enzymes in bacteria. Little is known, however, about the nature of the inducer that stimulates xylanase gene expression in P. bryantii, or about the sensing mechanism that responds to it. This study reports investigations into the regulation of xylanase activity of P. bryantii B14 that demonstrate repression by glucose, and better define the nature of the xylan-derived induction signal. We report that in addition to xynA and xynB, the unlinked xynC gene is also subject to regulation by xylan.
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METHODS |
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Preparation of xylan fraction.
WS-X, and the acid-EtOH-insoluble (EtOH-insol) and -soluble (EtOH-sol) fractions from WS-X were prepared as described previously (Miyazaki et al., 1997). Ten grams of oat spelt xylan (Sigma) was added to 90 ml distilled water, and the mixture was shaken continuously at 39 °C for 2 h. After centrifugation (5000 g, 30 min, 4 °C), its supernatant was taken as WS-X. Three volumes of a cold ethanol/acetic acid solution (95 % ethanol, 5 % glacial acetic acid) was added to the WS-X, and the mixture was kept on ice for 30 min. This mixture was then centrifuged (16 000 g, 4 °C, 10 min), and the insoluble xylan pellet was dissolved in 50 ml sodium phosphate buffer (50 mM, pH 6·5) containing 2 mM DTT (EtOH-insol). The supernatant fluid was lyophilized, and dissolved in the same buffer (EtOH-sol). Each fraction was adjusted to 2 % (w/v) total pentose content.
Predigestion of EtOH-insol with cloned XynC.
Cloned XynC from P. bryantii B14 was prepared as previously described (Zhang & Flint, 1992; Garcia-Campayo et al., 1993
); 0·3 U of this enzyme preparation was added to 10 ml 2 % (w/v) EtOH-insol, and incubated at 37 °C for 4 h. A further 0·3 U of enzyme was then added to the digest and incubated for a further 4 h. After incubation, this digest was transferred to a 50 ml serum bottle, and the atmosphere of the bottle was replaced by 100 % CO2 before sealing. Such samples were designated XynC-digest.
Average degree of polymerization (DP) of xylan fraction.
The xylose equivalent of each xylan fraction was determined by using the orcinol reagent with xylose as the standard (Schneider, 1957). The reducing sugar content was measured by using the method of Nelson and Somogyi with xylose as the standard (Nelson, 1944
). Average DP was determined from the ratio of xylose equivalent to reducing sugar content.
Measurement of xylanase activity.
After incubation, the cell culture was kept on ice for 5 min. The culture was centrifuged at 10 000 g at 4 °C for 5 min, and the cell pellet was washed twice with sodium phosphate buffer (pH 6·8) containing 2 mM DTT. After dissolving this pellet in 1 ml of the same buffer, the cells were disrupted with an ultrasonicator (UD-201, Tomy). This mixture was kept at 20 °C until measurement. Xylanase activity was determined as described previously (Flint et al., 1991).
Total RNA extraction.
At 30 min after adding 0·05 % WS-X to mid-exponential-phase cells grown in RG medium, total RNA was extracted from the culture by Sepazol-RNA 1 super (Nacalai Tesque) according to the supplier's instructions. Total RNA was aliquoted and stored at 80 °C until required for analysis.
Dot blot analysis.
Total RNA samples of 0·252·0 µg were used for dot blot analysis. Total RNA was transferred to positively charged nylon membrane (Hybond-N+, Amersham Pharmacia Biotech) by using a dot blotter (Multimicro filter FLE348, Advantec MFS) according to the supplier's instructions. Nucleotides were fixed to the membrane by UV irradiation.
Probes.
For the detection of the target mRNA, digoxigenin (DIG)-labelled probes were prepared by PCR using the DIG Probe Synthesis Kit (Roche Diagnostics). The primers used were XA-F (5'-CAGCCTACGATGAAGGATG-3') and XA-R (5'-CCTCGTTAACGACATCCC-3') for the xynA probe, XB-F (5'-GAACAGGATGCCAAGGACTG-3') and XB-R (5'-GGAGCCTCAGCAAACTGC-3') for the xynB probe, and XC-F (5'-GATGAGCCTGCTGAGGTTATC-3') and XC-R (5'-CTCACGATCCCAGTTGTTGG-3') for the xynC probe, corresponding to a 403 bp, a 416 bp and a 420 bp fragment, respectively.
Detection of DIG-labelled probes.
The membrane was washed with 0·1 % SDS/2x SSC for 15 min at room temperature, followed by 0·1 % SDS/0·2x SSC for 15 min at 50 °C. Additionally, it was shaken for 30 min in skim milk solution [3 g skim milk per 100 ml TBS (pH 7·4); TBS contained 137 mM NaCl, 2·68 mM KCl, 25 mM Tris/HCl]. After washing three times with TBS, the membrane was incubated at 37 °C for 1 h in anti-DIG solution [1 µl anti-digoxigenin-AP (Boehringer Mannheim) in 3 ml TBS]. The membrane was shaken vigorously twice for 15 min in T-TBS solution (0·05 % Tween 20 in TBS). After soaking it for 5 min in AP 9·5 solution [100 mM Tris buffer (pH 9·5), 100 mM NaCl, 5 mM MgCl2], the membrane was incubated for 5 min with CDP-Star (Amersham Life Science). Chemiluminesent signals were detected by exposure to X-ray film (Medical X-ray Film MXJB-1, Kodak). Non-specific hybridization of each probe was checked by using yeast RNA (Ambion). Cross-hybridization experiments between the three xylanase genes were performed by applying plasmid DNA of each xylanase gene. Neither non-specificity nor cross-hybridization was observed.
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RESULTS |
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DISCUSSION |
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The inability to induce xylanase activity in P. bryantii by addition of WS-xylan during early growth stages in the presence of glucose is strongly suggestive of catabolite repression, or inducer exclusion. In Escherichia coli and Bacillus sp. catabolite repression acts through well-established mechanisms involving binding of CRP (Pedersen et al., 1995; Kristensen et al., 1997
) or CcpA (Heuck et al., 1995
; Henkin, 1996
; Monedero et al., 1997
) repressor proteins to operator sites in response to cAMP levels. Another well-known mechanism is inhibition of uptake of sugars other than glucose by the glucose-specific components of phosphotransferase systems, which can result in inducer exclusion (Postma et al., 1993
). Because no phosphotransferase system activity (Martin & Russell, 1986
) and a very low concentration of cAMP (Cotta et al., 1994
) were detected in P. bryantii B14, it seemed that previously defined catabolite regulatory mechanisms could not explain the regulation of xylanase, as Fields & Russell (2001)
suggested for
-glucanase induction.
The P. bryantii xynA and xynB xylanase genes, together with the xynD putative oligosaccharide transporter, were previously shown to be positively regulated at the transcriptional level by the multidomain regulatory protein XynR, which has homology to bacterial two-component regulators (Miyazaki et al., 2003). In addition, xynR mRNA expression was itself regulated in response to xylan (Miyazaki et al., 2003
). There must be a mechanism, presumably involving a sensor protein domain, mediating the response to xylo-oligosaccharides, most likely present in the periplasmic space, or outside the cell. These oligosaccharides will be generated by the initial hydrolysis of the xylan polymer, probably by the enzyme XynC, which seems to be located on the cell envelope and has a preference for substrates with a chain length greater than 8. The induction signal must then be communicated to XynR, probably through its unique N-terminal input domain, resulting in positive control of xylanase gene transcription via the C-terminal DNA-binding domain of XynR. Direct recognition of xylo-oligosaccharides by the XynR N-terminal domain cannot be ruled out, but seems less likely, as this would imply that one end of the regulatory protein could be in contact with large oligosaccharides at the same time as the other end is binding to DNA.
The present results confirm that not only xynA and xynB transcription, but also transcription of the unlinked xynC gene, is induced by WS-X. It is not known whether xynC is also under control of the xynR regulator, or of another regulator that responds to the same induction signal.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cotta, M. A. (1993). Utilization of xylooligosaccharides by selected ruminal bacteria. Appl Environ Microbiol 59, 35573563.[Abstract]
Cotta, M. A., Wheeler, M. B. & Whitehead, T. R. (1994). Cyclic AMP in ruminal and other anaerobic bacteria. FEMS Microbiol Lett 124, 355360.[CrossRef][Medline]
Fields, M. W. & Russell, J. B. (2001). The glucomannokinase of Prevotella bryantii B14 and its potential role in regulating -glucanase expression. Microbiology 147, 10351043.[Medline]
Flint, H. J., McPherson, C. A. & Martin, J. C. (1991). Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13. J Gen Microbiol 137, 123129.[Medline]
Flint, H. J., Whitehead, T. R., Martin, J. C. & Gasparic, A. (1997). Interrupted catalytic domain structures in xylanases from two distantly related strains of Prevotella ruminicola. Biochem Biophys Acta 1337, 161165.[Medline]
Flint, H. J., Aurilia, V., Kirby, J., Miyazaki, K., Rincon-Torres, M. T., McCrae, S. I. & Martin, J. C. (1998). Organization of plant cell wall degrading enzymes in the ruminal anaerobic bacteria Ruminococcus flavefaciens and Prevotella bryantii. In Genetics, Biochemistry and Ecology of Cellulose Degradation, pp. 511520. Edited by K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita & T. Kimura. Tokyo: Uni Publishers.
Garcia-Campayo, V., McCrae, V., Zhang, J.-X., Flint, H. J. & Wood, T. M. (1993). Mode of action, kinetic properties and physicochemical characterisation of two different domains of a bifunctional (1-4)--D-xylanase from Ruminococcus flavefaciens expressed separately in Escherichia coli. Biochem J 269, 235243.
Gardner, R. G., Wells, J. E., Russell, J. B. & Wilson, D. B. (1995). The cellular location of the Prevotella ruminicola -1,4-D-endoglucanase and its occurrence in other strains of ruminal bacteria. Appl Environ Microbiol 61, 32883292.[Abstract]
Gasparic, A., Martin, J. C., Daniel, A. S. & Flint, H. J. (1995). A xylan hydrolase gene cluster in Prevotella ruminicola B14: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and -(1,4)-xylosidase activities. Appl Environ Microbiol 61, 29582964.[Abstract]
Gouka, R. J., Hessing, J. G., Punt, P. J., Stam, H., Musters, W. & Van den Hondel, C. A. (1996). An expression system based on the promoter region of the Aspergillus awamori 1,4-beta-endoxylanase A gene. Appl Microbiol Biotechnol 46, 2835.[CrossRef][Medline]
Henkin, T. M. (1996). The role of the CcpA transcriptional regulator in carbon metabolism in Bacillus subtilis. FEMS Microbiol Lett 135, 915.[CrossRef][Medline]
Heuck, C. J., Kraus, A., Schmiedel, D. & Hillen, W. (1995). Cloning, expression and functional analyses of the catabolite control protein CcpA from Bacillus megaterium. Mol Microbiol 16, 855864.[CrossRef][Medline]
Kristensen, H. H., Valentin-Hansen, P. & Sogaard-Andersen, L. (1997). Design of CytR regulated, cAMP-CRP dependent class II promoters in Escherichia coli: RNA polymerase-promoter interactions modulate the efficiency of CytR repression. J Mol Biol 266, 866876.[CrossRef][Medline]
Mach, R. L., Strauss, J., Zeilinger, S., Schindler, M. C. & Kubicek, P. (1996). Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei. Mol Microbiol 21, 12731281.[CrossRef][Medline]
Martin, S. A. & Russell, J. B. (1986). Phosphoenolpyruvate-dependent phosphorylation of hexoses by rumen bacteria: evidence for the phosphotransferase system of transport. Appl Environ Microbiol 52, 13481352.[Medline]
Miyazaki, K., Martin, J. C., Marinsek-Logar, R. & Flint, H. J. (1997). Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii B14. Anaerobe 3, 373381.[CrossRef]
Miyazaki, K., Miyamoto, H., Mercer, D. K., Hirase, T., Martin, J. C., Kojima, Y. & Flint, H. J. (2003). Involvement of the two component regulatory protein XynR in positive control of xylanase gene expression in the ruminal anaerobe Prevotella bryantii B14. J Bacteriol 185, 22192226.
Monedero, V., Gosalbes, M. J. & Perez-Martinez, G. (1997). Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA. J Bacteriol 179, 66576664.
Nelson, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153, 375380.
Orejas, M., MacCabe, A. P., Perez Gonzalez, J. A., Kumar, S. & Ramon, D. (1999). Carbon catabolite repression of the Aspergillus nidulans xlnA gene. Mol Microbiol 31, 177184.[CrossRef][Medline]
Pedersen, H., Dall, J., Dandanell, G. & Valentin-Hansen, P. (1995). Gene-regulatory modules in Escherichia coli: nucleoprotein complexes formed by cAMP-CRP and CytR at the nupG promoter. Mol Microbiol 17, 843853.[CrossRef][Medline]
Postma, P. W., Lengeler, J. W. & Jacobson, G. R. (1993). Phosphoenol-pyruvate : carbohydrate phosphotransferase systems of bacteria. Microbiol Rev 57, 543594.[Medline]
Schneider, W. C. (1957). Determination of nucleic acids in tissues by pentose analysis. Methods Enzymol 3, 680684.
Zeilinger, S., Mach, R. L., Schindler, M., Herzog, P. & Kubicek, C. P. (1996). Different inducibility of expression of the two xylanase genes xyn1 and xyn2 in Trichoderma reesei. J Biol Chem 271, 2562425629.
Zhang, J.-X. & Flint, H. J. (1992). A bifunctional xylanase encoded by the xynA gene of the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 comprises two dissimilar domains linked by an asparagine/glutamine rich sequence. Mol Microbiol 6, 10131023.[Medline]
Received 15 June 2005;
revised 30 August 2005;
accepted 13 September 2005.
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