- Email: [email protected]
Cellulomonas flavigena: characterization of an endo-1,4-xylanase tightly induced by sugarcane bagasse
FEMS Microbiology Letters 214 (2002) 205^209
www.fems-microbiology.org
Cellulomonas £avigena: characterization of an endo-1,4-xylanase tightly induced by sugarcane bagasse Lino Mayorga-Reyes
a;b
, Yolanda Morales a , Luis M. Salgado c , Arturo Ortega d , Teresa Ponce-Noyola a;
a
c
Departamento de Biotecnolog|¤a y Bioingenier|¤a, Centro de Investigacio¤n y de Estudios Avanzados del Instituto Polite¤cnico Nacional, Apartado Postal 14-740, Mexico D.F. 07300, Mexico b Departamento de Sistemas Biolo¤gicos, Universidad Auto¤noma Metropolitana-Xochimilco, Xochimilco, Mexico Departamento de Bioqu|¤mica, Centro de Investigacio¤n y de Estudios Avanzados del Instituto Polite¤cnico Nacional, Apartado Postal 14-740, Mexico D.F. 07300, Mexico d Departamento de Gene¤tica y Biolog|¤a Molecular, Centro de Investigacio¤n y de Estudios Avanzados del Instituto Polite¤cnico Nacional, Apartado Postal 14-740, Mexico D.F. 07300, Mexico Received 20 February 2002; received in revised form 17 July 2002 ; accepted 18 July 2002 First published online 9 August 2002
Abstract Xylanases, an important group of enzymes for biomass degradation in the industry, are commonly found forming complex multienzyme systems. As a preliminary step to the construction of efficient xylanase producers using genetic engineering, we have characterized a gene encoding an endo-L-1,4 xylanase (xyncflA) from Cellulomonas flavigena. The xylanase activity and the xyncflA synthesis were higher when C. flavigena was grown on sugarcane bagasse. In this substrate, both activity and transcript increased with approximately the same rate during the culture period. When C. flavigena grew on glucose, low signal of mRNA was observed, suggesting that the xyncflA gene is regulated at the transcriptional level. 5 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Cellulomonas £avigena; Xylanase; Transcriptional regulation; Sugarcane bagasse
1. Introduction Hemicelluloses are widely distributed heteropolysaccharides. The enzymes that degrade them are ubiquitous and diverse. In nature, xylans have L-arabinose, acetyl, glucuronic, 4-O-methylglucuronic, and p-coumaric side chains, and ferulic acid cross-linkages. Intra-chain hydrogen bonding occurs through the O-3 position giving unsubstituted xylan a helical twist. Acetylation and substitution, however, disrupt and complicate that structure. Xylan is complexed with cellulose and pectin and is bound to lignin. As esteri¢cation and substitution increase, digestibility of the hemicellulose decreases [1].
* Corresponding author. Tel. : +52 (5) 747 7000; Fax : +52 (5) 5557 4770 02. E-mail address : [email protected] (T. Ponce-Noyola).
Microbial degradation of xylan requires the action of several enzymes such as L-1,4-xylanases, arabinofuranosidases, L-glucuronidases, esterases and L-xylosidases. Recently, xylanases are drawing increased attention because of their usefulness in facilitating the bleaching of kraft pulp, by increasing the extractability of lignin and chromophore release from pulp. Xylanases also improve the quality of dough and help bread to rise and can also be used in the bioconversion of lignocellulosic materials to fuels and chemicals [2]. These enzyme complexes are produced for some fungal and bacterial strains, being the best characterized fungal enzymes. The genes encoding for xylanases from di¡erent microorganisms have been cloned and expressed in Escherichia coli [3,4]. However, current understanding of the regulation of xylanase is mostly based on biochemical and physiological studies, while little information is available on the characterization of xylanase genes and the molecular mechanisms which govern their expression. In this regard, Cellulomonas £avigena is a potent cellulolytic and xylano-
0378-1097 / 02 / $22.00 5 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 7 6 - 5
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lytic bacterium for the production of L-xylosidase and L-xylanases [5]. Therefore in this work we describe the cloning, sequence and regulation of xync£A, a gene which encodes a L-1,4-xylanase from C. £avigena.
2. Materials and methods 2.1. Organisms, plasmids and culture conditions C. £avigena CDBB-531 was used as the source of chromosomal DNA. The strain was grown on mineral medium supplied with 1% glucose and yeast extract 0.02% [6]. For induction experiments, C. £avigena grown on glucose was washed twice with 0.85% NaCl before switching to growth on glycerol, sugarcane bagasse, xylan, cellobiose or glucose medium for 48 h at 37‡C on an orbital shaker at 150 rpm (New Brunswick Sci., Edison NJ, USA). To remove the residual insoluble carbon source, samples were ¢ltered through GD 120 glass ¢ber ¢lter discs and then centrifuged (5000Ug, 10 min, 10‡C). The supernatant £uids were recovered for xylanase activity assays and protein [7]. Cells grown on sugarcane bagasse were used to isolate total RNA. E. coli DH5K, used as the recipient strain for recombinant plasmids, was cultured in Luria^Bertani medium at 37‡C supplemented with 100 Wg ml31 of ampicillin. The pBluescript II SK plasmid (Stratagene, La Jolla, CA, USA) was used as the subcloning vector. 2.2. Enzyme activity assay Xylanase activity was determined by measuring the released reducing sugars as xylose by the dinitrosalicylic acid [8]. One milliliter of appropriately diluted enzyme was mixed with 1 ml citrate/phosphate bu¡er pH 7.0 and 1 ml 1% xylan (Larchwood, Sigma). The reaction mixture was incubated at 40‡C for 5 min. The reaction was stopped by addition of 3 ml dinitrosalicylic acid reagent. One unit of activity (U) is de¢ned as the amount (Wmol) of xylose formed per min under standard assay conditions. All determinations were made in triplicate. The maximum di¡erence among the three values was less than 5% of the mean. 2.3. Molecular techniques C. £avigena genomic DNA, as well as plasmid DNA were isolated according to standard protocols [9]. Restriction endonucleases and other DNA-modifying enzymes were used according to the manufacturer’s instructions (New England Biolabs, Boehringer, Gibco). Doublestranded DNA was sequenced by the dideoxy chain termination method, using the protocol recommended for the Sequenase DNA sequencing kit (US Biochemical Corp.). The standard primers used for sequencing were T3 and T7 (Gibco).
2.4. Primers and PCR conditions The following primers were used to amplify the sequence between map positions 398 and 1132 corresponding to the catalytic domain of xylanase D from Cellulomonas ¢mi [10]: 5P-ccgctggtcgagtactacatc-3P and 3P-gttggggtccgtcgtcgaa-5P. The conditions for the PCR reactions were as follows : C. £avigena DNA 2^5 ng Wl31 ; primers 20 pmol; NTPs 10 mM; MgCl2 1.5 mM; 1Ubu¡er and 2 units of Taq polymerase (Gibco) in a ¢nal volume of 50 Wl. The reaction conditions were hot start at 94‡C for 3 min one cycle, followed by 30 cycles of 1 min at 94‡C, 1 min at 62‡C and 1 min at 72‡C. The ampli¢ed DNA was cloned into E. coli Bluescript, and digested with PvuII. Ampli¢ed PCR product was analyzed by acrylamide gel electrophoresis and labeled with [K-32 P]dCTP (6000 Ci mmol31 ), according to the manufacturer’s instructions. Incorporation of label was monitored by liquid scintillation counting. 2.5. Southern blot analysis Genomic DNA from C. £avigena was completely digested with either EcoRI, Pst, BamHI, HindIII and Sau3AI restriction enzymes. The samples were then fractionated on 1.0% agarose gels and blotted onto nylon ¢lters (Hybond-N ; Amersham). The PCR-DNA fragment was [K-32 P]dCTP labeled by random priming method and used as a probe. Prehybridization was carried out at 42‡C for 2^4 h, and hybridization was carried out at 42‡C for 12^16 h. The nylon membrane was washed twice with 2USSC+0.2% SDS (w/v) at 42‡C for 15 min and then washed twice with 1USSC+0.1% SDS (w/v) at 50‡C for 20 min. The membrane was dried and exposed to high performance autoradiography ¢lm. 2.6. Isolation of RNA for Northern and dot-blot analysis Total RNA was isolated from a cell suspension of C. £avigena grown on sugarcane bagasse or glucose according to Oelmu«ller et al. [11]. The RNA concentration was determined by measuring the absorbance at 260 nm. For Northern blotting, 10 Wg RNA from cells grown on sugarcane bagasse medium were separated on 1.2% formaldehyde^agarose gels ; the integrity of the RNA was evaluated by ethidium bromide staining. Following electrophoretic separation, RNA was blotted onto nylon membranes (Hybond-NtTM , Amersham) and hybridized according to standard protocols [9] at 55‡C overnight. The size was estimated using RNA molecular mass markers (RNA leader 0.24^9.5 kb, Gibco BRL). For dot-blot hybridization, samples were taken at various time intervals. Approximately 10 Wg of RNA from each sample were denatured with formaldehyde and formamide, and then applied to Hybond-Nþ membrane using a dot-blotting apparatus (The Convertible Filtration Mani-
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xylan, cellobiose or glucose, in order to identify the best xylanolytic activity inducer. When the cells were grown on sugarcane bagasse, the xylanase activity was 13.6 U mg31 protein; in sharp contrast, when the cells were grown on xylan an activity of 5.6 U mg31 protein was present. In the rest of the substrates tested, the activity was about 10% of that found on sugarcane bagasse. Interestingly, when the cells were grown on glucose, no xylanase activity was detected (Fig. 1). These results point out that xylanase activity in C. £avigena was induced by some of the components of sugarcane bagasse, while xylanase expression was repressed by glucose. Fig. 1. E¡ect of di¡erent carbon sources on xylanase synthesis from C. £avigena.
fold System, Gibco BRL). The membrane was baked, prehybridized, hybridized, and washed as previously described [9]. The ampli¢ed PCR fragment was [K-32 P]dCTP labeled by the random priming method and used as a probe for Northern and dot-blot analysis. Membranes were visualized by autoradiography after exposure at 370‡C. The ¢lms were scanned with a ZERO-Dscan 1.0 program (EagleEye II, Stratagene).
3. Results 3.1. E¡ect of carbon source over xylanase activity C. £avigena was grown on glycerol, sugarcane bagasse,
Fig. 2. (A) Primers used for ampli¢cation of the PCR fragment and (B) products of the restriction analysis. 1, PvuII; 2, molecular size markers; 3, PCR fragment not digested.
3.2. Cloning of an endo-L-xylanase gene fragment from C. £avigena In order to isolate the gene encoding an endo-L-xylanase from C. £avigena, we designed a set of primers based on the peptide sequences of the catalytic domain of several xylanases from di¡erent xylanolytic microorganisms. These oligonucleotides were used to amplify C. £avigena genomic DNA. A 781-bp fragment was obtained. The identity of this fragment was established, ¢rst with a restriction analysis with the appropriate enzymes according to the published xylanases sequences (Fig. 2) and ultimately by DNA sequencing. This sequence showed an extensive homology (81%) with the xynD from C. ¢mi [10]. Therefore, one can be con¢dent that the ampli¢ed product corresponds to one xylanase. The isolated gene was named xync£A and was registered in the GenBank date base under accession No. AF338352. The sequence analysis was using the Multalin program [12]. To evaluate whether the xync£A gene was reiterated throughout the Cellulomonas genome, C. £avigena genomic DNA was subjected to Southern hybridization using the PCR product as probe. As depicted in Fig. 3A, xync£A was present as a single copy within the C. £avigena genome.
Fig. 3. (A) Southern blot hybridization of xync£A with C. £avigena genomic DNA restricted with (1) EcoRI, (2) Pst, (3) BamHI, (4) HindIII, (5) Sau3AI, using the PCR fragment as probe. (B) Northern blot analysis of xync£A transcript. RNA was extracted from C. £avigena cultures grown on mineral medium supplemented with (1) sugarcane bagasse or (2) glucose. Riboprobe indicates the ribosomal control. Molecular size markers are indicated on the right.
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Fig. 4. (A) Time course of xylanase activity and relative amount of xync£A mRNA and (B) kinetics of appearance of xync£A mRNA by dot-blot analysis in C. £avigena grown on glucose and transferred to medium with sugarcane bagasse. 0 indicates the glucose-grown culture before replacement, 14, 18, 36 and 48 h.
3.3. Regulation by carbon source of the xync£A transcripts The e¡ect of sugarcane bagasse over the relative mRNA levels in C. £avigena was determined by Northern blot analysis. RNA samples were prepared from exponential phase grown cultures on sugarcane bagasse or glucose. The 781-bp PCR fragment probe hybridized to a species of C. £avigena RNA that was approximately 2200 bases long, isolated from sugarcane bagasse-grown cells (Fig. 3B, lane 1). However, signal was not detected on RNA samples isolated from glucose-grown cultures (Fig. 3B, lane 2). 3.4. Time course of xylanase activity and xync£A mRNA synthesis of C. £avigena growing on sugarcane bagasse C. £avigena was grown on sugarcane bagasse in order to follow, at di¡erent time intervals, the xylanase activity and expression levels of the xync£A gene. As shown in Fig. 4A,B, the xylanase activity and levels of the xync£A gene transcript were very low at 18 h. However, the xync£A transcript and the xylanase activity reach their maximum values at 48 h. On the other hand, the xync£A transcript could be detected at very low levels from cultures of C. £avigena grown on glucose as carbon source. Moreover, the expression of xync£A on sugarcane bagasse was more than 8-fold higher than that observed on cultures grown on glucose. These results point out that the carbon source provided during growth can regulate the levels of the xync£A gene transcript and the transcription is not completely repressed in the presence of glucose as substrate in C. £avigena.
4. Discussion The commercial importance of xylanases has resulted in an increased interest in the elucidation of the structure and catalytic mechanisms of microbial xylanases. At this stage, either further engineering to improve the enzyme properties or the characterization of xylanases from other sources
should be undertaken. Therefore, in the present work, we decided to initiate the characterization of a xylanase gene from C. £avigena. The xylanolytic complex was induced when C. £avigena was grown on sugarcane bagasse. It has been observed that complex substrates such as rice straw, corn stalk, corn cob and sugarcane bagasse are better inducers of xylanases or cellulases than pure substrates (xylan or cellulose) [13]. Cross-induction of xylanase activity by cellulosic substrates has been documented for Cellulomonas IIbc [14], C. ¢mi [15], C. £avigena [5] and Bacillus strain BP-7 [16]. Besides, many glucanases such as cellulases also harbor xylanolytic activity [4]. Our ¢rst approach was to make an alignment of the published sequences of bacterial xylanases in order to amplify via PCR part of a xylanase gene. The ampli¢ed fragment from C. £avigena as part of a xylanase gene showed high identity and homology with xynD from C. ¢mi [10]. Therefore one can be con¢dent that xync£A encodes an endo-L-1,4-xylanase. The most abundant xync£A transcripts were detected by Northern blot and dot-blot analysis from cells grown on sugarcane bagasse, a complex substrate known to induce both cellulases and xylanases in C. £avigena [5]. The low signal of RNA observed from cells grown on glucose suggest that the xync£A gene is regulated at the transcriptional level, but its constitutive expression is small. Similar results have been observed for xylanase and L-xylosidase from Bacillus subtilis [17] and xynA of Bacillus stearothermophilus [18]. The function of the constitutively expressed enzymes is presumably to generate low-molecular-mass xylan-speci¢c degradation products, which can act as true inducers once a suitable substrate is encountered [19]. This could explain why xync£A mRNA was detected in RNA prepared from glucose-grown cultures of C. £avigena. The xync£A gene was transcribed as a monocistronic mRNA of about 2200 nucleotides in length. This is the expected size for a mRNA encoding a xylanase D from C. ¢mi [10]. In summary, we have cloned a fragment of a xylanase gene from C. £avigena that is tightly regulated by the
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carbon source. Work currently in progress in our lab is aimed at the characterization of the promoter of this gene.
Acknowledgements This work was supported by CONACYT-Me¤xico (3528P-B9607 and 28784-B). L.M.-R. was supported by a CONACYT-Me¤xico Ph.D. scholarship.
References [1] Kulkarni, N., Shendye, A. and Rao, M. (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol. Rev. 23, 411^ 456. [2] Je¡ries, T.W. (1996) Biochemistry and genetics of microbial xylanases. Curr. Opin. Biotechnol. 7, 337^342. [3] Bhalerao, J., Patki, A.H., Bhave, M., Khurana, I. and Deobagkar, D.N. (1990) Molecular cloning and expression of a xylanase gene from Cellulomonas sp. into Escherichia coli. Appl. Microbiol. Biotechnol. 34, 71^76. [4] Chaudhary, P. and Deobagkar, D.N. (1997) Characterization of cloned endoxylanase from Cellulomonas sp. NCIM 2353 expressed in Escherichia coli. Curr. Microbiol. 34, 273^279. [5] Pe¤rez-Avalos, O., Ponce-Noyola, T., Magan‹a-Plaza, I. and de la Torre, M. (1996) Induction of xylanase and L-xylosidase in Cellulomonas £avigena growing on di¡erent carbon sources. Appl. Microbiol. Biotechnol. 46, 405^409. [6] Ponce-Noyola, T. and de la Torre, M. (1995) Isolation of a highspeci¢c-growth-rate mutant of Cellulomonas £avigena on sugar cane bagasse. Appl. Microbiol. Biotechnol. 42, 709^712.
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[7] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265^275. [8] Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426^428. [9] Sambrook, J.E., Fritsch, F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [10] Millward-Sadler, S.J., Poole, D.M., Henrissat, B., Hazlewood, G.P., Clarke, J.H. and Gilbert, H.J. (1994) Evidence for a general role for high-a⁄nity non-catalytic cellulose binding domains in microbial plant cell wall hydrolases. Mol. Microbiol. 11, 375^382. [11] Oelmu“ller, U., Kru“ger, A., Steinbu“chel, A. and Friedrich, C.G. (1990) Isolation of prokaryotic RNA and detection of speci¢c mRNA with biotinylated probes. J. Microbiol. Methods 11, 73^84. [12] Corpet, F. (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 16, 10881^10890. [13] Haltrich, D., Nidetzky, B., Kulbe, K.D., Steiner, W. and Zupancic, S. (1996) Production of fungal xylanases. Biores. Technol. 58, 137^ 161. [14] Rodriguez, H. and Alea, F. (1992) Regulation activity in Cellulomonas sp. IIbc. Acta Biotechnol. 12, 337^343. [15] Khanna, S. and Gauri (1993) Regulation, puri¢cation, and properties of xylanase from Cellulomonas ¢mi. Enzyme Microbiol. Technol. 15, 990^995. [16] Lopez, C., Blanco, A. and Javier-Pastor, F.I. (1998) Xylanase production by a new alkali-tolerant isolate of Bacillus. Biotechnol. Lett. 20, 243^246. [17] Lindner, C., Stulke, J. and Hecker, M. (1994) Regulation of xylanolytic enzymes in Bacillus subtilis. Microbiology 140, 753^757. [18] Cho, S.G. and Choi, Y.J. (1999) Catabolite repression of the xylanase gene (xynA) expression in Bacillus stearothermophilus No. 236 and B. subtilis. Biosci. Biotechnol. Biochem. 63, 2053^2058. [19] Priest, F.G. (1977) Extracellular enzyme synthesis in the genus Bacillus. Bacteriol. Rev. 41, 711^753.
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Cellulomonas £avigena: characterization of an endo-1,4-xylanase tightly induced by sugarcane bagasse Lino Mayorga-Reyes
a;b
, Yolanda Morales a , Luis M. Salgado c , Arturo Ortega d , Teresa Ponce-Noyola a;
a
c
Departamento de Biotecnolog|¤a y Bioingenier|¤a, Centro de Investigacio¤n y de Estudios Avanzados del Instituto Polite¤cnico Nacional, Apartado Postal 14-740, Mexico D.F. 07300, Mexico b Departamento de Sistemas Biolo¤gicos, Universidad Auto¤noma Metropolitana-Xochimilco, Xochimilco, Mexico Departamento de Bioqu|¤mica, Centro de Investigacio¤n y de Estudios Avanzados del Instituto Polite¤cnico Nacional, Apartado Postal 14-740, Mexico D.F. 07300, Mexico d Departamento de Gene¤tica y Biolog|¤a Molecular, Centro de Investigacio¤n y de Estudios Avanzados del Instituto Polite¤cnico Nacional, Apartado Postal 14-740, Mexico D.F. 07300, Mexico Received 20 February 2002; received in revised form 17 July 2002 ; accepted 18 July 2002 First published online 9 August 2002
Abstract Xylanases, an important group of enzymes for biomass degradation in the industry, are commonly found forming complex multienzyme systems. As a preliminary step to the construction of efficient xylanase producers using genetic engineering, we have characterized a gene encoding an endo-L-1,4 xylanase (xyncflA) from Cellulomonas flavigena. The xylanase activity and the xyncflA synthesis were higher when C. flavigena was grown on sugarcane bagasse. In this substrate, both activity and transcript increased with approximately the same rate during the culture period. When C. flavigena grew on glucose, low signal of mRNA was observed, suggesting that the xyncflA gene is regulated at the transcriptional level. 5 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Cellulomonas £avigena; Xylanase; Transcriptional regulation; Sugarcane bagasse
1. Introduction Hemicelluloses are widely distributed heteropolysaccharides. The enzymes that degrade them are ubiquitous and diverse. In nature, xylans have L-arabinose, acetyl, glucuronic, 4-O-methylglucuronic, and p-coumaric side chains, and ferulic acid cross-linkages. Intra-chain hydrogen bonding occurs through the O-3 position giving unsubstituted xylan a helical twist. Acetylation and substitution, however, disrupt and complicate that structure. Xylan is complexed with cellulose and pectin and is bound to lignin. As esteri¢cation and substitution increase, digestibility of the hemicellulose decreases [1].
* Corresponding author. Tel. : +52 (5) 747 7000; Fax : +52 (5) 5557 4770 02. E-mail address : [email protected] (T. Ponce-Noyola).
Microbial degradation of xylan requires the action of several enzymes such as L-1,4-xylanases, arabinofuranosidases, L-glucuronidases, esterases and L-xylosidases. Recently, xylanases are drawing increased attention because of their usefulness in facilitating the bleaching of kraft pulp, by increasing the extractability of lignin and chromophore release from pulp. Xylanases also improve the quality of dough and help bread to rise and can also be used in the bioconversion of lignocellulosic materials to fuels and chemicals [2]. These enzyme complexes are produced for some fungal and bacterial strains, being the best characterized fungal enzymes. The genes encoding for xylanases from di¡erent microorganisms have been cloned and expressed in Escherichia coli [3,4]. However, current understanding of the regulation of xylanase is mostly based on biochemical and physiological studies, while little information is available on the characterization of xylanase genes and the molecular mechanisms which govern their expression. In this regard, Cellulomonas £avigena is a potent cellulolytic and xylano-
0378-1097 / 02 / $22.00 5 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 7 6 - 5
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lytic bacterium for the production of L-xylosidase and L-xylanases [5]. Therefore in this work we describe the cloning, sequence and regulation of xync£A, a gene which encodes a L-1,4-xylanase from C. £avigena.
2. Materials and methods 2.1. Organisms, plasmids and culture conditions C. £avigena CDBB-531 was used as the source of chromosomal DNA. The strain was grown on mineral medium supplied with 1% glucose and yeast extract 0.02% [6]. For induction experiments, C. £avigena grown on glucose was washed twice with 0.85% NaCl before switching to growth on glycerol, sugarcane bagasse, xylan, cellobiose or glucose medium for 48 h at 37‡C on an orbital shaker at 150 rpm (New Brunswick Sci., Edison NJ, USA). To remove the residual insoluble carbon source, samples were ¢ltered through GD 120 glass ¢ber ¢lter discs and then centrifuged (5000Ug, 10 min, 10‡C). The supernatant £uids were recovered for xylanase activity assays and protein [7]. Cells grown on sugarcane bagasse were used to isolate total RNA. E. coli DH5K, used as the recipient strain for recombinant plasmids, was cultured in Luria^Bertani medium at 37‡C supplemented with 100 Wg ml31 of ampicillin. The pBluescript II SK plasmid (Stratagene, La Jolla, CA, USA) was used as the subcloning vector. 2.2. Enzyme activity assay Xylanase activity was determined by measuring the released reducing sugars as xylose by the dinitrosalicylic acid [8]. One milliliter of appropriately diluted enzyme was mixed with 1 ml citrate/phosphate bu¡er pH 7.0 and 1 ml 1% xylan (Larchwood, Sigma). The reaction mixture was incubated at 40‡C for 5 min. The reaction was stopped by addition of 3 ml dinitrosalicylic acid reagent. One unit of activity (U) is de¢ned as the amount (Wmol) of xylose formed per min under standard assay conditions. All determinations were made in triplicate. The maximum di¡erence among the three values was less than 5% of the mean. 2.3. Molecular techniques C. £avigena genomic DNA, as well as plasmid DNA were isolated according to standard protocols [9]. Restriction endonucleases and other DNA-modifying enzymes were used according to the manufacturer’s instructions (New England Biolabs, Boehringer, Gibco). Doublestranded DNA was sequenced by the dideoxy chain termination method, using the protocol recommended for the Sequenase DNA sequencing kit (US Biochemical Corp.). The standard primers used for sequencing were T3 and T7 (Gibco).
2.4. Primers and PCR conditions The following primers were used to amplify the sequence between map positions 398 and 1132 corresponding to the catalytic domain of xylanase D from Cellulomonas ¢mi [10]: 5P-ccgctggtcgagtactacatc-3P and 3P-gttggggtccgtcgtcgaa-5P. The conditions for the PCR reactions were as follows : C. £avigena DNA 2^5 ng Wl31 ; primers 20 pmol; NTPs 10 mM; MgCl2 1.5 mM; 1Ubu¡er and 2 units of Taq polymerase (Gibco) in a ¢nal volume of 50 Wl. The reaction conditions were hot start at 94‡C for 3 min one cycle, followed by 30 cycles of 1 min at 94‡C, 1 min at 62‡C and 1 min at 72‡C. The ampli¢ed DNA was cloned into E. coli Bluescript, and digested with PvuII. Ampli¢ed PCR product was analyzed by acrylamide gel electrophoresis and labeled with [K-32 P]dCTP (6000 Ci mmol31 ), according to the manufacturer’s instructions. Incorporation of label was monitored by liquid scintillation counting. 2.5. Southern blot analysis Genomic DNA from C. £avigena was completely digested with either EcoRI, Pst, BamHI, HindIII and Sau3AI restriction enzymes. The samples were then fractionated on 1.0% agarose gels and blotted onto nylon ¢lters (Hybond-N ; Amersham). The PCR-DNA fragment was [K-32 P]dCTP labeled by random priming method and used as a probe. Prehybridization was carried out at 42‡C for 2^4 h, and hybridization was carried out at 42‡C for 12^16 h. The nylon membrane was washed twice with 2USSC+0.2% SDS (w/v) at 42‡C for 15 min and then washed twice with 1USSC+0.1% SDS (w/v) at 50‡C for 20 min. The membrane was dried and exposed to high performance autoradiography ¢lm. 2.6. Isolation of RNA for Northern and dot-blot analysis Total RNA was isolated from a cell suspension of C. £avigena grown on sugarcane bagasse or glucose according to Oelmu«ller et al. [11]. The RNA concentration was determined by measuring the absorbance at 260 nm. For Northern blotting, 10 Wg RNA from cells grown on sugarcane bagasse medium were separated on 1.2% formaldehyde^agarose gels ; the integrity of the RNA was evaluated by ethidium bromide staining. Following electrophoretic separation, RNA was blotted onto nylon membranes (Hybond-NtTM , Amersham) and hybridized according to standard protocols [9] at 55‡C overnight. The size was estimated using RNA molecular mass markers (RNA leader 0.24^9.5 kb, Gibco BRL). For dot-blot hybridization, samples were taken at various time intervals. Approximately 10 Wg of RNA from each sample were denatured with formaldehyde and formamide, and then applied to Hybond-Nþ membrane using a dot-blotting apparatus (The Convertible Filtration Mani-
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xylan, cellobiose or glucose, in order to identify the best xylanolytic activity inducer. When the cells were grown on sugarcane bagasse, the xylanase activity was 13.6 U mg31 protein; in sharp contrast, when the cells were grown on xylan an activity of 5.6 U mg31 protein was present. In the rest of the substrates tested, the activity was about 10% of that found on sugarcane bagasse. Interestingly, when the cells were grown on glucose, no xylanase activity was detected (Fig. 1). These results point out that xylanase activity in C. £avigena was induced by some of the components of sugarcane bagasse, while xylanase expression was repressed by glucose. Fig. 1. E¡ect of di¡erent carbon sources on xylanase synthesis from C. £avigena.
fold System, Gibco BRL). The membrane was baked, prehybridized, hybridized, and washed as previously described [9]. The ampli¢ed PCR fragment was [K-32 P]dCTP labeled by the random priming method and used as a probe for Northern and dot-blot analysis. Membranes were visualized by autoradiography after exposure at 370‡C. The ¢lms were scanned with a ZERO-Dscan 1.0 program (EagleEye II, Stratagene).
3. Results 3.1. E¡ect of carbon source over xylanase activity C. £avigena was grown on glycerol, sugarcane bagasse,
Fig. 2. (A) Primers used for ampli¢cation of the PCR fragment and (B) products of the restriction analysis. 1, PvuII; 2, molecular size markers; 3, PCR fragment not digested.
3.2. Cloning of an endo-L-xylanase gene fragment from C. £avigena In order to isolate the gene encoding an endo-L-xylanase from C. £avigena, we designed a set of primers based on the peptide sequences of the catalytic domain of several xylanases from di¡erent xylanolytic microorganisms. These oligonucleotides were used to amplify C. £avigena genomic DNA. A 781-bp fragment was obtained. The identity of this fragment was established, ¢rst with a restriction analysis with the appropriate enzymes according to the published xylanases sequences (Fig. 2) and ultimately by DNA sequencing. This sequence showed an extensive homology (81%) with the xynD from C. ¢mi [10]. Therefore, one can be con¢dent that the ampli¢ed product corresponds to one xylanase. The isolated gene was named xync£A and was registered in the GenBank date base under accession No. AF338352. The sequence analysis was using the Multalin program [12]. To evaluate whether the xync£A gene was reiterated throughout the Cellulomonas genome, C. £avigena genomic DNA was subjected to Southern hybridization using the PCR product as probe. As depicted in Fig. 3A, xync£A was present as a single copy within the C. £avigena genome.
Fig. 3. (A) Southern blot hybridization of xync£A with C. £avigena genomic DNA restricted with (1) EcoRI, (2) Pst, (3) BamHI, (4) HindIII, (5) Sau3AI, using the PCR fragment as probe. (B) Northern blot analysis of xync£A transcript. RNA was extracted from C. £avigena cultures grown on mineral medium supplemented with (1) sugarcane bagasse or (2) glucose. Riboprobe indicates the ribosomal control. Molecular size markers are indicated on the right.
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Fig. 4. (A) Time course of xylanase activity and relative amount of xync£A mRNA and (B) kinetics of appearance of xync£A mRNA by dot-blot analysis in C. £avigena grown on glucose and transferred to medium with sugarcane bagasse. 0 indicates the glucose-grown culture before replacement, 14, 18, 36 and 48 h.
3.3. Regulation by carbon source of the xync£A transcripts The e¡ect of sugarcane bagasse over the relative mRNA levels in C. £avigena was determined by Northern blot analysis. RNA samples were prepared from exponential phase grown cultures on sugarcane bagasse or glucose. The 781-bp PCR fragment probe hybridized to a species of C. £avigena RNA that was approximately 2200 bases long, isolated from sugarcane bagasse-grown cells (Fig. 3B, lane 1). However, signal was not detected on RNA samples isolated from glucose-grown cultures (Fig. 3B, lane 2). 3.4. Time course of xylanase activity and xync£A mRNA synthesis of C. £avigena growing on sugarcane bagasse C. £avigena was grown on sugarcane bagasse in order to follow, at di¡erent time intervals, the xylanase activity and expression levels of the xync£A gene. As shown in Fig. 4A,B, the xylanase activity and levels of the xync£A gene transcript were very low at 18 h. However, the xync£A transcript and the xylanase activity reach their maximum values at 48 h. On the other hand, the xync£A transcript could be detected at very low levels from cultures of C. £avigena grown on glucose as carbon source. Moreover, the expression of xync£A on sugarcane bagasse was more than 8-fold higher than that observed on cultures grown on glucose. These results point out that the carbon source provided during growth can regulate the levels of the xync£A gene transcript and the transcription is not completely repressed in the presence of glucose as substrate in C. £avigena.
4. Discussion The commercial importance of xylanases has resulted in an increased interest in the elucidation of the structure and catalytic mechanisms of microbial xylanases. At this stage, either further engineering to improve the enzyme properties or the characterization of xylanases from other sources
should be undertaken. Therefore, in the present work, we decided to initiate the characterization of a xylanase gene from C. £avigena. The xylanolytic complex was induced when C. £avigena was grown on sugarcane bagasse. It has been observed that complex substrates such as rice straw, corn stalk, corn cob and sugarcane bagasse are better inducers of xylanases or cellulases than pure substrates (xylan or cellulose) [13]. Cross-induction of xylanase activity by cellulosic substrates has been documented for Cellulomonas IIbc [14], C. ¢mi [15], C. £avigena [5] and Bacillus strain BP-7 [16]. Besides, many glucanases such as cellulases also harbor xylanolytic activity [4]. Our ¢rst approach was to make an alignment of the published sequences of bacterial xylanases in order to amplify via PCR part of a xylanase gene. The ampli¢ed fragment from C. £avigena as part of a xylanase gene showed high identity and homology with xynD from C. ¢mi [10]. Therefore one can be con¢dent that xync£A encodes an endo-L-1,4-xylanase. The most abundant xync£A transcripts were detected by Northern blot and dot-blot analysis from cells grown on sugarcane bagasse, a complex substrate known to induce both cellulases and xylanases in C. £avigena [5]. The low signal of RNA observed from cells grown on glucose suggest that the xync£A gene is regulated at the transcriptional level, but its constitutive expression is small. Similar results have been observed for xylanase and L-xylosidase from Bacillus subtilis [17] and xynA of Bacillus stearothermophilus [18]. The function of the constitutively expressed enzymes is presumably to generate low-molecular-mass xylan-speci¢c degradation products, which can act as true inducers once a suitable substrate is encountered [19]. This could explain why xync£A mRNA was detected in RNA prepared from glucose-grown cultures of C. £avigena. The xync£A gene was transcribed as a monocistronic mRNA of about 2200 nucleotides in length. This is the expected size for a mRNA encoding a xylanase D from C. ¢mi [10]. In summary, we have cloned a fragment of a xylanase gene from C. £avigena that is tightly regulated by the
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carbon source. Work currently in progress in our lab is aimed at the characterization of the promoter of this gene.
Acknowledgements This work was supported by CONACYT-Me¤xico (3528P-B9607 and 28784-B). L.M.-R. was supported by a CONACYT-Me¤xico Ph.D. scholarship.
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