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Characterization of cbg : a cloned gene encoding an extracellular [beta]-glucosidase from Cellulomonas… Bates, Nancy Carol 1987

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Characterization of cbg: a cloned gene encoding an extracellular B-glucosidase from Cellulomonas fimi By Nancy Carol Bates B.Sc. (Honours Program), The University of British Columbia, 1985 A thesis submitted in partial fulfillment of requirements for the degree of Master of Science in The Faculty of Graduate Studies (Department of Microbiology) We accept this thesis as conforming to the required standard September 1987 ©Nancy Carol Bates, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia 1956 Main Mall Vancouver, Canada Department V6T 1Y3 DE-6(3/81) (ii) ABSTRACT A group of Escherichia coli clones harbouring recombinant pBR322 plasmid, containing Cellulomonas fimi DNA inserts, that reacted with antiserum to C.fimi culture supernatant, was screened for their ability to hydrolyze carboxymethyl cellulose (CMC) and 4-methylumbeliferyll-B-D-cellobioside (MUC). A clone, pEC62, hydrolyzed MUC but did not hydrolyze CMC. The recombinant enzyme encoded by pEC62 was shown to be a B-glucosidase (cellobiase). C.fimii itself was shown to encode an extracellular B-glucosidase in C.fimi. This is the first report of an extracellular B-glucosidase from a bacterium. Deletion analysis localized the cloned gene (cbg)\o the tet promoter proximal region of the 7.0 kilobase insert of pEC62. Further analysis and sequence data showed a highly active derivative of pEC62 contained a translational gene fusion between lacZ of pUC13 and cbg. From this data, a location for the cbg start site was proposed. (iii) TABLE OF CONTENTS Page number I INTRODUCTION 1 I Cellulases 1 II 13-glucosidases 2 A. The role of 6-glucosidases in cellulose degradation 2 B. Regulation of R-glucosidase expression and the role of B-glucosidase in regulating cellulase production 3 C. The molecular cloning of genes encoding B-glucosidases 3 MATERIALS AND METHODS 5 I Bacterial strains, plasmids and phage 5 II Media 5 III Protein methodology 6 A. Preparation of crude cell extract as a source of recombinant B-glucosidase . . . 6 B. Preparation of native enzymes from C.fimi. 6 (1) Crude cell extracts 6 (2) Concentration of C.fimi culture supernatant 7 C. Determination of protein concentration 7 D. Enzymatic assays 7 (1) p-nitrophenyl-B-glucoside (pNPG) assay 7 (2) p-nitrophenyl-8-cellobioside assay (pNPC) assay 7 (3) Cellobiose assay 8 (4) Carboxymethyl cellulose assay 8 E. Non-denaturing polyacrylamide gel electrophoresis 8 F. Denaturing polyacrylamide gel electrophoresis 8 IV DNA methodology 9 A. Preparation of plasmid DNA 9 B. Restriction endonuclease digestions 9 C. Agarose gel electrophoresis 9 D. Isolation of DNA from agarose gels 10 E. Construction of deletions with nuclease BAL-31 10 F. Ligation conditions 11 G. Transformation 11 (iv) TABLE OF CONTENTS (continued! Page number H. DNA sequencing 11 (1) Chemical modification and cleavage method 11 (2) Enzymatic method 12 (3) Denaturing polyacrylamide gel electrophoresis 13 (4) Autoradiography 13 RESULTS 14 I Isolation and characterization of E.coli clones expressing cellulase genes from C.fimi... 14 II Preliminary characterization of the enzyme encoded by pEC62 14 III Identification of the corresponding native enzyme in C.fimi 18 IV Characterization of the cbg gene 18 A. Restriction mapping the pEC62 insert 18 B. Dependence of cbg expression on an external (E.coli) promoter 22 C. Delimiting the gene by restriction fragment deletion analysis 25 D. Delimiting the 5'-end of cbg by deletion using nuclease BAL-31 25 E. Sequencing the junctions of pUC13::A24,21,31 25 G. Creation of a reading frame shift in lacZ in pUC13::62A31 30 DISCUSSION 34 I Characterization of the recombinant enzyme encoded by pEC62 34 II Characterization of the cbg gene 36 REFERENCES 38 (v) LIST OF FIGURES Page number Figure 1: Screening of immunopositive clones by Pstl digestion of plasmid DNA 15 Figure 2: Hydrolysis of Cm-cellulose by recombinant enzymes in crude cell extracts 16 Figure 3: Non-denaturing polyacrylamide gel electrophoresis of crude cell extract from C.fimi, and E.colicells containing pEC62, pUC13::62 and pUC13::62A21 19 Figure 4: Denaturing polyacrylamide gel electrophoresis of crude cell extract from E.coli cells containing pUC13A21 and concentrated Avicel grown C.fimi culture supernatant 20 Figure 5: Autoradiogram of an agarose gel containing partially digested end-labelled DNA used to construct a restriction map 21 Figure 6: Schematic representation of linear restriction map of the 7.0 kb insert of pEC62 23 Figure 7: Schematic summarizy of restriction fragment deletions used to localize cbg . . 24 Figure 8: Outline of the strategy used to make deletions into the 5'-end of cbg 26 Figure 9A: Agarose gel of Pstl digested plasmid DNA showing deletions from the 5'-end of cbg 27 Figure 9B: Photograph of plates streaked with the deletion clones showing their relative MUGase activities 29 Figure 10A: Schematic summarizing the 5'-end deletions, their corresponding MUGase activities 30 Figure 10B: Sequencing map 30 Figure 11: Autoradiogram of a representative dideoxy sequencing gel 31 Figure 12: pUC13::62A21 sequence derived using chemical and dideoxy sequencing . . 32 Figure 13: Translation of pUC13::62A21 DNA seqeunce using the lacZ reading frame . . 33 (vi) LIST OF TABLES Page number Table 1: Preliminary characterization of the recombinant enzyme in crude cell extract from E.coli C600 containing pEC62 17 ABBREVIATIONS AND SYMBOLS AMP ampicillin BPB bromophenol blue BSA bovine serum albumin Ci curie CMC (ase) carboxymethyl cellulose(ase) dA deoxyadenosine dC deoxycytosine dG deoxyguanosine dNTP deoxynucleoside triphosphate dT deoxythymidine DEAE diethylaminoethyl A deletion in DNA DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid insertion of DNA IPTG isopropyl-B-D-thiogalactoside Kb 1000 base pairs Kd kilodalton lacZ E.coli B-galactosidase gene A bacteriophage lambda mA milliampere MOPS morpholinopropanesulfonic acid MUC (ase) 4-methyl-umbelliferyl-B-cellobioside (ase) MUG (ase) 4-methyl-umbelliferyl-B-glucoside (ase) NaOAc sodium acetate PEG polyethylene glycol 6000 PMSF phenylmethylsulfonyl fluoride pNP para-nitrophenol pNPGase para-nitrophenyl-6-D-glucosidase SDS . sodium dodecyl sulfate UV ultraviolet XGAL 5-bromo-4-chloro-3-indoyl-B-D-galactoside (viii) ACKNOWLEDGEMENTS I would like to thank Drs. R.C. Miller, R.A.J. Warren and D.G. Kilburn for their efforts in all aspects of this project. I would like to thank Drs. G. Spiegelman and S. Withers for their helpful suggestions during the course of this work. I would also like to thank my fellow members of the cellulase group, Cheryl Keen, Steve Wellington and Vera Webb for many helpful discussions. I would like to thank Patti Miller for her continual kindness and encouragement. Finally, I would especially like to thank my husband Fereydoun for his advice, his technical assistance when needed and his continual emotional support. 1 INTRODUCTION  I Cellulases Cellulase is a complex of enzymes capable of degrading cellulose to cellobiose or glucose. Many different organisms ranging from bacteria to fungi to arthropods (Gascoigne and Gasciogne, 1960) are capable of producing cellulase. Potential applications of cellulase for the production of fuels from biomass and degradation of agricultural pollutants have intensified research in the enzymatic degradation of cellulose. However, cellulases also provide useful models for the study of 1) regulation of gene expression, 2) glycosylation, processing and secretion of extracellular proteins and 3) mechanisms of catalysis. Cellulose is a linear polymer of more than 1000 glucose residues joined by B-1,4 linkages. Cellulose polymers associate and form inter- and intra-molecular H bonds producing insoluble fibres (Alberts et al., 1983). Cellulose fibres contain highly ordered regions called crystalline areas and less well ordered regions called amorphous areas. These amorphous regions are subject to attack by cellulase enzymes. Degradation of cellulose to glucose requires the activities of three basic types of enzymes. Endo-B-1,4-glucanases (EC3.2.1.4) hydrolyze the internal 1,4-B-glucosidic linkages of cellulose creating new non-reducing ends which are attacked by exoglucanases that hydrolyze the B-1,4 linkages to effect the release of either cellobiose (1,4-B-D-cellobiohydrolase(EC3.2.1.91)) or glucose (1,4-B-D-glucan glucohydrolase (EC3.2.1.74)). Cellobiose and short cello-oligosaccharides are hydrolyzed by sequential removal of glucose from the non-reducing ends by B-glucosidase (EC3.2.1.21). Although B-glucosidases are involved in the complete degradation of cellulose, they are not considered cellulases because they do not attack cellulose polymers directly. Characterization of the components of different cellulase complexes has been in two main areas: 1) the roles and mechanisms of individual cellulase enzymes (purified native enzymes and cloned gene products) in cellulose degradation and 2) the regulation of gene expression and production of cellulases. In additition, much work has been devoted to the cloning of cellulase genes for the purpose of engineering high level production of the enzymes on an industrial scale. This report will focus on the study of B-glucosidases as it pertains to these areas. 2 II B-glucosidases A. The role of B-glucosidase in cellulose degradation Although B-glucosidases with a wide range of substrate specificities exist in non-cellulolytic organisms, it is those B-glucosidases from cellulolytic organisms that are of primary interest. Cellobiose and glucose are the major end-products of cellulose degradation by cellulases (Wood and M cCrae, 1978). Cellobiose is an inhibitor of endo-and exo-glucanase activity (Halliwell, 1975; Howell and Stuck, 1975; Wood and McCrae, 1975; Mandels et al., 1974) whereas glucose has less of an inhibitory effect (Shewale, 1984; Stoppok et al., 1982). The role of the B-glucosidase in the degradation process is thought to be release of end-product inhibition by hydrolysis of cellobiose into its constituent glucose residues. The synergistic effect of B-glucosidase on cellulose degradation by cellulase enzymes has been demonstrated (Sternberg, 1976; Sternberg et al., 1977). Srinivasan and Han (1969) showed that cellulase repression could be avoided if Cellulomonas was co-cultured with a B-glucosidase producing bacterium. This led to a fivefold increase in Cellulomonas cell mass. In fungi, B-glucosidases are extracellular or cell-asscociated or both, depending on the particular organism. Extracellular and cell-associated B-glucosidases usually have different substrate specificities and/or are regulated by different mechanisms (Woodward and Wiseman, 1982). In bacteria, B-glucosidases are located in the periplasmic space (in Gram positive organisms, the space between the cell membrane and the cell wall). A model for cellulose degradation by some prokaryotes has been proposed (Lamed et al., 1983) in which, cellulosomes containing the cellulases are in contact with cellulose and are associated with the bacterial cell. Cellobiose diffuses through the cell wall into the periplasmic space where it is converted by B-glucosidases to glucose which is metabolized inside the cell. Although Saddler and Khan (1980) reported a B-glucosidase activity in the culture supernatant from Acetivibrio celluloyticus, the inability of the organism to utilize extracellular glucose (Patel et al., 1980) suggests that its presence in supernatants results from cell lysis. This is further supported by the observation that the activity does not appear extracellularly until the cells reach the stationary phase of growth. This thesis is the first report of a demonstrated extracellular 6-glucosidase from a bacterium. 3 B. Regulation of B-glucosidase expression and the role of B-glucosidase in regulation of cellulase production Glucose is generally an inhibitor of B-glucosidase activity. It can also effect repression of B-glucosidase biosynthesis. Inducers of B-glucosidase biosynthesis are less well characterized; vary depending on the particular type of B-glucosidase and on the organism producing the enzyme. For example, aryl-B-glucosidase and cellobiase from Neurospora crassa are induced by 1 mM cellobiose (Ebehart and Beck, 1973) whereas little B-glucosidase is produced by a Basidiomycetes species grown on cellobiose (Shewale and Sadana, 1978). Induction of the latter enzyme required growth of the organism on cellulose. Optimal 8-glucosidase biosynthesis in Schizophyllum commune is dependent on the K 2 H P 0 4 concentration in the medium (Desrochers et al., 1981). In Trichoderma reseei a complex induction pathway for the biosynthesis of extracellular B-glucosidase has been proposed (Woodward and Wiseman, 1982) in which a constitutive, cell-associated B-glucosidase converts cellobiose to sophorose which induces the biosynthesis of the extracellular B-glucosidase. In Cellulomonas uda, B-glucosidase production appears to be constitutive and uninducible (Stoppok et al., 1982). The mechanisms of regulation of B-glucosidase synthesis are not known for most organisms, but its biosynthesis in Mucor racemosus has been shown to be sensitive to catabolite repression (Borgia and Sypherd, 1972). A 6-glucosidase is implicated in the regulation of synthesis of the cellulases otT.reesei (Kubicek, 1987). A constitutive B-glucosidase in the plasma membrane converts cellobiose to sophorose, a powerful inducer of the cellulases and the extracellular B-glucosidase. The importance of B-glucosidase in the cellulolytic process has been recognized and has led to gene cloning and characterization of B-glucosidases from fungi and bacteria. Q. The molecular cloning of genes encoding B-glucosidases B-glucosidase genes have been cloned from the fungi, Aspergillus niger (Penttila et al., 1984), Candida pelliculosa (Kohchi and Toh-e, 1986) and Kluyveromyces fragilis (Raynal and Guerineau, 1984) and the bacteria, Erwinia carotovora (Barras et al., 1984), Clostridium thermocellum (Schwarz et al., 1985), Clostridium acetobutylicum (Zappe et al., 1986), Agrobacterium ATCC21400 (Wakarchuk el a\., 1986), Cellulomonas uda (Nakamura et al., 1986), Escherichia adecarboxylata (Armentrout and Brown, 1981) and 4 Caldocellum saccharolytium (Love and Streiff, 1987) for a variety of reasons. Although cloning of the genes allows isolation and amplification of specific gene products for characterization, there is particular interest in cloning B-glucosidase genes for the purpose of high level production of enzymes with desirable kinetic properties (low K m for cellobiose and high Kj for glucose) for use in the industrial scale degradation of cellulose (eg. A.niger and Agrobacterium). This thesis describes the characterization of cbg, a cloned gene encoding a B-glucosidase (Cbg) from Cellulomonas fimi. Current research in our laboratory is focussed on the cloning and expression of genes encoding the cellulase complex of C. fimi in Escherichia coli for the purpose of industrial application and for further study of the interesting intrinsic properties of the enzymes. Although a B-glucosidase from Agrobacterium was cloned for the purpose of enhancing the degradation of cellulose by the recombinant C.fimi cellulase complex, the recombinant 6-glucosidase from C.fimi may prove to be more valuable in allowing optimal synergism between the enzymes. Perhaps of greater interest is the fact that cbg encodes the first bacterial extracellular B-glucosidase to be described. 5 MATERIALS AND METHODS I Bacterial strains, plasmids and phage BamHI digested chromosomal DNA from Cellulomonas fimi ATCC484 was cloned into the unique BamHI site of pBR322 and the resulting plasmids were used to transform Eschericiha coll C600 (F", thi-1, thr-1, leuB6, lacY1, tonA21, supE44, ') (Gilkes et al., 1984). One clone, pEC62, contained a 7 Kb BamHI fragment and encoded B-glucosidase activity. pBR322, pEC62 and all other pEC62 derivatives were maintained in E.coli C600. The BamHI insert from pEC62 was also cloned into pUC13 (Messing, 1983) to give the plasmid pUC13::62 (see Results Section IV B.). pUCl3, pUC13::62 and all other pUCl3::62 derivatives were maintained in JM101(Yanisch-Perron et al., 1985). The sequencing plasmid pTZ18U (Vieira, 1985) was also maintained in JM101. M13K07 helper phage used in sequencing was described previously (Vieira, 1985). C. fimi ATCC484 served as a source of native cellulase enzymes. II Media All strains of E.coli were grown in either LB (10 g tryptone, 5 g yeast extract and 5 g NaCI per litre), 2YT (10 g tryptone, 16 g yeast extract and 10 g NaCI per litre), B Broth (10 g tryptone, 8g NaCI, 10mg thiamine and 5.0 mg thymidine per litre ) or M9 (Champe and Benzer, 1962) supplemented with thiamine (10 ug/ml) and thymidine (5 ng/ml). When required, media contained the following at the given final concentrations: Ampicillin (Amp) 100ug/ml XGAL 40 ug/ml IPTG 100 u.M MUG 100 uM MUC 100 uM Agar 15g/L C.fimi was grown in basal salts medium (Whittle et al.,1982) supplemented with 0.2% carbon source (glucose, cellobiose or Avicel). Solid media contained 11 g agar per litre. Yeast extract, tryptone and agar were obtained from DIFCO. All other compounds were 6 obtained from Sigma Chemicals. Release of 4-methylumbelliferone by hydrolysis of MUC or MUG was monitored by fluorescence under UV (unhydrolyzed substrate does not fluoresce). Glass plates were used for solid medium containing MUC or MUG in order to minimize the background fluorescence created when plastic plates are used. Ill Protein methodology A. Preparation of crude cell extract as a source of recombinant B-glucosidase Crude cell extracts prepared from the clones pEC62 and pUC13::62A21 served as sources of recombinant B-glucosidase. A 2 ml overnight culture was used to inoculate 50 ml of LBAmp (pEC62) or M9Amp (pUC13::62A21). pEC62 was grown overnight at 37°C with vigorous shaking and pUC13::62A21 was grown at 37°C to an OD 6 0 0 =0.2 at which time IPTG (100 u.M) was added (to induce expression of the B-glucosidase) and incubation was continued overnight. The corresponding pBR322 and pUCl3 controls were grown under the same conditions as their recombinant derivatives. Cells were harvested by centrifugation (10 minutes at 8K in a Beckman JA20 rotor) at 4 0 C . The cell pellet was resuspended in 2 ml of ice cold 50 mM sodium phosphate buffer (pH7.0). The cell suspension was sonicated using a Branson sonifier with a microprobe set an intensity of 2 for three 15 second bursts. Cell debris was pelleted by centrifugation (15 minutes in an Eppendorf microfuge) at 4 ° C . Cleared cell extract was then assayed directly. B. Preparation of native enzvmes from C.fimi (1) Crude cell extracts A single growing colony of C.fimi was taken from a with cellobiose as a carbon source and inoculated into a 2L flask containing 500 ml of basal salts medium with glucose or cellobiose as a carbon source. Cultures were grown for 2 days at 300C with vigorous shaking. Cells were harvested by centrifugation (20 minutes at 6K in a Beckman JA10 rotor). The wet weight of the pellet was determined before it was frozen at -200Q The frozen pellet was transferred to an ice cold mortar and alumina (Sigma chemicals) (2.5 X wet weight of cell pellet) was added. The cells and alumina were ground to a paste before cells were broken by the addition of ice cold 50 mM phosphate buffer (pH 7.0). NaN3 (0.02%) and PMSF (20 u.g/ml) were added to the cell extract before it was cleared 7 by centrifugation (30 minutes at 18K in a Beckman JA20 rotor). Cleared cell extract was assayed directly. (2) Concentration of C.fimi cell culture supernatant 10 ml overnight cultures of C.fimi were used to inoculate 500 ml of basal salts medium supplemented with 0.2% Avicel as the sole carbon source. Cultures were incubated for three days at 3 0 ° C with vigorous shaking. Avicel and cells were removed by first allowing the culture to settle out at 4 ° C for several hours. The supernatant was removed and was further cleared by repeated centrifugation. NaN3 (0.02%) and PMSF (20 ug/ml) were added to the cleared supernatant which was then concentrated 100X on an Amicon apparatus using a PM10 Diaflo ultrafilter (Amicon). Concentrated supernatants were then assayed directly. C. Determination of protein concentration Protein concentration was determined by the method of Lowry et al. (1951) using BSA Fraction V (Sigma chemicals) as a standard. Assays were always performed in duplicate. D. Enzymatic assays (1) p-nitrophenvl-B-D-olucosidfl toNPG^ assay pNPG is an analogue of cellobiose and was used to quantify B-glucosidase activity. 100 JJ.I of suitably diluted enzyme were added to 500 u.1 of 50 mM sodium phosphate buffer (pH 7.0) prewarmed to the incubation temperature (37nC) containing 1.2 mM pNPG. Reactions were stopped by the addition of 600 u.l of 1 M Na 2C03, m i x e d and kept on ice. The release of pNP was quantified by measuring the absorbance at 400 nm. Calculation of enzyme activity has been described previously (Stoppok et al., 1982). 1 unit of pNPGase activity is defined as that amount of enzyme releasing 1u.mol pNP from pNPG in 1 minute at 37 0C. (2) p-nitrophenvl-B-D-cellobioside (pNPC^ assay pNPC is an analogue of cellotriose. The ability of enzyme to hydrolyze pNPC was assayed as follows. 100 uJ of suitably diluted enzyme were added to 900 ul of 50 mM sodium phosphate buffer prewarmed to the incubation temperature (37nC) containing 0.56 mM pNPC . Reactions were stopped with 1 ml 1 M Na2C03, mixed and kept on ice. pNP release was monitored as for the pNPG assay. 1 unit of pNPCase activity is defined 8 as that amount of enzyme required to release 1 umol of pNP from pNPC in 1 minute at 370 C. (3) Cellobiose assay 100 u.l of suitably diluted enzyme were added to 500 ul of 50 mM sodium phosphate buffer (pH7.0) containing 41.7 mM cellobiose (Sigma chemicals) prewarmed to the incubation temperature (37°C). Reactions were stopped by the addition of D-gluconic acid lactone at a final concentration of 5 mM. Glucose release was measured using a Sigma glucose detection kit (Hexokinase/glucose-6-phosphate dehydrogenase). (4) Carboxvmethvl cellulose (Cm-cellulose^ assay Cm-cellulose hydrolysis was followed by determination of specific fluidity ( 0 ) , an index of polymer length, as described by Gilkes et al. (1984). E. Non-denaturing polvacrvlamide gel electrophoresis Recombinant and native enzyme preparations were electrophoresed through non-dissociating discontinous polyacrylamide gels (Jovin et al., 1964). Proteins were suspended in loading dye (62.5 mM Tris (pH 6.8), 10% glycerol and 0.002% BPB) and samples were stacked (16 mA constant current) in a 1.5 mm thick 3% polyacrylamide gel (acrylamide to methylene bis-acrylamide 45:1.2 in 125 mM Tris HCI (pH 6.8)). Proteins were separated (45 mA constant current) on a 1.5 mm thick 10% polyacrylamide (45:1.2 acrylamide to methylene bis-acylamide) gel dissolved in 375 mM Tris HCI (pH 8.8) and 75 mM NaCI. Gels were run in buffer ( 28.8 g glycine and 6.0 g of Tris base in 2 L of distilled water) until the bromophenol blue reached the bottom. To visualize enzymatically active bands, the gels were soaked in warm 50 mM phosphate buffer (pH 7.0) for 15 minutes before being transferred to a warm solution of 100 u.M MUG (dissolved in 50 mM phosphate buffer). Gels were incubated in substrate until a fluorescent band was visible under UV illumination. The positions of bands were marked with pin holes before staining for protein with Coomassie blue (Schleif and Wensink, 1981). F. Denaturing polvcrvlamide oel electrophoresis Denaturing polyacrylamide gel electrophoresis was performed as for the non-denaturing PAGE except that the running buffer, loading dye and gel contained 0.2% SDS. 9 IV DNA methodology A. Preparation of plasmid DNA For small scale isolation of plasmid, 2-3 ml of LB containing ampicillin (LBAmp)(pBR322 derivatives) or M9Amp (pUC13 derivatives) were inoculated with a single colony from a plate and grown overnight at 37 OC on a tube roller. Plasmid DNA was isolated by the alkaline lysis method (Maniatis et al., 1982). DNA prepared using this method is referred to in the text as mini-prep DNA. When required, plasmid DNA was further purified on a NACS Prepac column (BRL) by the procedure described by the manufacturer. For large scale isolation of plasmid, 500 ml of M9Amp were inoculated with a single colony from a plate and grown overnight at 37 n C with vigorous shaking. Plasmid was isolated by the alkaline lysis method and purified on a CsCI gradient (Maniatis et al., 1982). DNA from the gradient was extracted with water saturated n-butanol to remove ethidium bromide and dialyzed against three 1L changes of 10 mM TrisHCI and 1 mM EDTA (pH8.0) or precipitated from CsCI directly with two volumes of 95% ethanol and reprecipitated from 0.3M NaOAc (pH5.2). Quantity and purity of the DNA were determined by measuring the absorbance at 260 nm and 280 nm on a Perkin Elmer Lambda 3 UV/Vis spectrophotomter (1 OD 26o u n i t = 5 0 M5 double stranded DNA). The relative amount of covalently closed circular (ccc) DNA was determined by mini agarose gel electrophoresis (see Section III C.) B- Restriction endonuclease digestions Analytical and preparative restriction endonuclease digestions were performed according to the manufacturers' specifications. Partial restriction endonuclease digestions for analytical (restriction site mapping) and preparative (restriction fragment deletions) purposes were carried out as for complete digests except that aliquots of the digests were taken at short time intervals and digestion stopped in EDTA (250 mM). All digests were monitored by agarose gel electrophoresis (see next section). C. Agarose gel electrophoresis 0.7-1.2% (w/v) agarose (BIO RAD ultra pure DNA grade) gels were prepared in TBE (89 mM Tris borate, 89 mM boric acid, 8 mM EDTA) or TAE (90 mM Tris base, 20 mM NaOAc, 2 mM EDTA) and contained ethidium bromide at 1ug/ml. Gels were run in TBE or 10 TAE at 80V constant voltage for 1-2 hours as required for sufficient separation of fragments. Migration of DNA was estimated by tracking dye in the sample (40% sucrose, 25 mM EDTA and 0.025% bromophenol blue). DNA was visualized by fluorescence using a UV transilluminator (Ultraviolet products limited). DNA fragments labelled with 32p were visualized by autoradiography (see Section IIG (3)). D. Isolation of DNA from agarose gels DNA was electrophoresed through an agarose gel until the desired fragments had separated sufficiently, then the desired fragment was electrophoresed (150 V for 3-5 minutes) onto a NA-45 DEAE membrane (Schleicher and Schuell). DNA was eluted from the membrane in 300 u.l of 2M NaCI in TE (pH7.5) at 60°C for 1 hour and precipitated directly with 2 volumes of 95% ethanol (when required DNA was reprecipitated from 0.3M NaOAc (pH5.2)). Migration of DNA onto the membranes and its elution from the membranes was monitored by the presence and abscence of fluorescence on the membrane under UV illumination, respectively. Fragment recovery was estimated by mini agarose gel electrophoresis. E. Construction of deletions with nuclease BAL-31 Thirty-five u.g of linearized plasmid DNA were resuspended in 60 u.l of BAL-31 buffer (600 mM NaCI, 12 mM MgCI2,12 mM CaCI 2, 1 mM EDTA, 10 mM TrisHCI (pH8.0)). 1 unit (1 unit releases 1 ug of acid-soluble nucleotide from denatured calf thymus DNA in one minute at 370C)of BAL-31 (BRL) was added at time 0 and the mixture incubated at 300C. 15 ul aliquots were taken at 15 minute intervals and added to an equal volume of 0.5 M EDTA (pH8.0) and set on ice to stop further digestion. The extent of nuclease digestion was monitored by agarose gel electrophoresis as described previously. The volume of each aliquot was increased to 50 u.l with sterile distilled water before extraction of the DNA with 50 ul of phenol:CHCI3 and precipitation from 0.3 M NaOAc (pH5.2). Although nuclease BAL-31 can create blunt ends, some overhangs are still present in the DNA. To increase the proportion of blunt ended DNA molecules, 5' overhangs were filled in using DNA polymerase 1 (Klenow fragment) and dNTPs (Maniatis et al., 1982). DNA was then cut with EcoR1 and cloned into pUC13 digested with Hindll (Boehringer Mannheim) and EcoRI (see next section for ligation conditions). 11 F. Ligation conditions Unless otherwise noted, DNA fragments were resuspended in 20ul of ligation buffer (50 mM Tris, pH 7.4,10 mM MgCI 2,10 mM DTT, 1mM spermidine, 1mM ATP, 100 ng BSA/ml) with a molar ratio of vector to insert.of 3:1. Fragments were ligated with 10 units of T 4 DNA ligase (NEB) for "sticky end" ligations and with 400 units of T 4 DNA ligase (NEB) for blunt end ligations. All ligation reactions were carried out overnight at 160C. G. Transformation Single growing colonies of E.coli C600 (for transformation with pBR322 derived plasmids) orJM101 (for pUC 13 derived plasmids) were inoculated into 50 ml of 2YT. Cultures were grown to an O D 6 5 0 =0.2 (C600) or 0.5 (JM101). The cells were made competent by the method of Dagert and Ehrlich (1979) except that 50 mM CaCI 2 was substituted for 100 mM CaCI 2. DNA (0.1-1 .Ong) was used to transform 100 u.l of competent cells by incubating the mixture on ice for 10 minutes followed by heat shocking for 5 minutes at 370C. Cells were diluted with 2 ml of 2YT and incubated one hour at 37°C on a tube roller. Then samples of 10, 50 and 100 u.l were plated on the appropriate media and incubated at 37 °C. H. DNA sequencing (1) Chemical modification and cleavage method DNA (pUC13::62A21,24,31) to be sequenced by the chemical method was first linearized by cleavage at the unique Hindlll site. 5' terminal phosphates were removed from the linearized DNA (65 u.g=20 pmole 5'ends) with calf intestinal alkaline phosphatase (CIAP) (Boehringer special quality for molecular biology) and replaced with 32 p transferred from [tf-32p]dATP by T 4 polynucleotide kinase (Boehringer) using the procedures described in Maniatis (1982). A total of 8 units of CIAP was used to dephosphorylate the DNA and 400uCi of [«- 3 2 P]dATP (ICN crude) with a specific activity of 7000Ci/mmole was used to label the 5' ends of the DNA. Labelled DNA was precipitated, digested with BstEII and the appropriate labelled fragment was isolated from an agarose gel run in TAE. The DNA fragment was then sequenced by the method described by Maxam and Gilbert (1980). Fragmented DNA was resuspended in formamide dye (90% formamide, 0.025 M EDTA, 0.02% XC and 0.02% 12 BPB) and analyzed by denaturing polyacrylamide gel electrophoresis (see Section H(3)). (2) Enzvmatic method Single stranded template DNA was used for dideoxy sequencing as described by Sanger et al. (1977) using the universal (forward) primer (P-L Biochemicals). DNA fragments to be sequenced were cloned into the sequencing vector pTZ18U . To obtain single stranded template, the following procedure modified from the Pharmacia protocol for pTZ18R was used. Cells containing recombinant pTZ18U plasmid were grown in 25 ml of 2YTAmp at 37 nC to an O D 6 0 0 ~ 0 . 9 , at which time the culture was infected with 0.4 ml of M13K07 helper phage (1011/ml) and incubated at 37°C with vigorous shaking for 1 hour. Kanamycin (75 ug/ml) was added to the culture and cells were incubated for a further 18 hours at 370C with vigorous shaking. To recover phage, cells were removed by centrifugation (2X 10 minutes at 10K rpm in a Sorvall SS34 rotor) and 5 ml of 27% PEG (6000) in 3.3 M NaCI was added to 20 ml of the supernatant. Phage was precipitated for 1 hour on ice and pelleted by centrifugation (20 minutes at 12K rpm in a Beckman JA20 rotor). Phage pellets were resuspended in 0.5 ml of T E (pH 8.0) and loaded onto a 4 ml step gradient made up of 1 ml amounts of 5,10,15 and 20% sucrose dissolved in 0.8 M NaCI and 0.2M NaOH. Gradients were centrifuged for 16 hours in a Beckman SW50.1 rotor at 25K rpm. 200 u,l fractions were collected dropwise from the gradient and fractions containing DNA template were identified and quantified by absorbance measured at 260 nm and 280 nm (10D26o unit= 33 ug single stranded DNA). DNA was precipitated directly from the pooled fractions and then reprecipitated from 0.3 M NaOAc (pH 5.2) and resuspended in 15-20 nl T E (pH 8.0). Sequencing reactions contained 4 ul template DNA (4-6 ug), 3 u.l sterile distilled water, 2 ul primer (2 ng) and 1 ul 10X H buffer (100 mM TrisHCI (pH 7.9), 600 mM NaCI, and 66 mM MgCl2). The mixture was incubated at 60°C for 5 minutes, room temperature for 5 minutes and then placed on ice at which time 1 u.l of 0.1 M DTT, 1 \i\ 0.015 mM dATP, 1 |xl (10uCi)[<*-- 32pjdATP (NEN 3000 Ci/mmole) and 3 units of DNA polymerasel (pol I) (Klenow fragment) (BRL) were added. 3 u1 aliquots of the mixture were dispensed into four tubes each containing 2 ui of one of the dideoxynucleoside triphosphate/deoxynucleoside triphosphate mixes made up as follows: A mix: 63 u.M 7-deaza dGTP (Boehringer), dCTP, dTTP, 77 uM ddATP C mix: 83 uM 7-deaza dGTP, dTTP, 8 uM dCTP, 25 u.M ddCTP 13 T mix: 83 uM 7-deaza dGTP, dCTP, 8 u M dTTP, 250 uM ddTTP G mix: 83 uM dCTP, dTTP, 8 u M 7-deaza dGTP, 33 u.M ddGTP All mixes were made up in 1X H buffer. Each tube was incubated at 30°C for 15 minutes at which point 1 uJ of cold chase (0.5 mM dNTPs) was added. Reactions were incubated for a further 10 minutes and then stopped by the addition of 7 u.1 of formamide dye. Reactions were then electrophoresed through a denaturing polyacrylamide gel as described in the next section. (3) Denaturing polvacrvlamide gel electrophoresis Sequencing samples were boiled for 2 minutes then immediately placed on ice before being loaded onto the gel. Sequencing samples were electrophoresed through a 6,8 or 20% polyacrylamide (29:1 acrylamide to methylene bis-acrylamide) gel containing 7 M urea. Gels were run in TBE at 1500-2000 Volts (constant voltage). Gels were transferred to 3M Whatman filter paper and dried on a BIORAD gel drier. (4) Autoradiography Dried gels were placed in a Kodak X-Omatic film cassete with Kodak XRP-1 film. Film was exposed overnight at room temperature or overnight at -70 °C if an intensifying screen was required. 14 RESULTS I Isolation and characterization of E.coli'clones expressing cellulase genes from C.fimi. BamH1 digested chromosomal DNA from C.fimi was cloned into pBR322 and E.coli C600 cells were transformed with the resulting plasmids. Transformants were screened for reactivity to antiserum against C.fimi culture supernatant and for cellulase activity (Cm-cellulose hydrolysis) (Gilkes et al., 1984b). Twenty-three out of 61 immunopositive clones were screened by Pstl digestions of plasmid DNA (Gilkes et al., 1984b). Three different Pstl restriction patterns were identified (pEC1, pEC2 and pEC3) and these clones have been further characterized (Gilkes et al., 1984a). The remaining unidentified clones were screened by Pstl digestion of plasmid DNA (Figure 1). Clones were also screened for CMCase by the Congo red assay (Gilkes et al., 1984b) and MUCase activity on plates(see Section II in Materials and Methods). A fourth Pstl restriction pattern was identified in the clones pEC41, pEC56, pEC57 and pEC62. These clones did not contain any CMCase activity but did contain MUCase activity. pEC62 was chosen for further characterization. II Preliminary characterization of the enzvme encoded bv pEC62 To characterize the activity of the recombinant enzyme, cell extracts were prepared (Section III A in Materials and Methods) and incubated with 4 different diagnostic substrates (Figure 2 and Table 1). Lack of CMCase activity on plates suggested pEC62 did not encode an endoglucanase. CMCase activity could not be detected by the more sensitive viscometric assay for CMC hydrolysis (Gilkes et al .,1984a)(Figure 2) confirming that pEC62 did not encode an endoglucanase activity. The extracts hydrolyzed pNPG, pNPC and cellobiose, indicating that the enzyme was a 13-glucosidase (cellobiase) (Table 1 A). Because all assays were performed using a phosphate buffer, it was possible that pEC62 encoded a phosphate-dependent cellobiose phosphorylase known to be present in Cellulomonas strains (Schimz et al., 1983). To test this, cell extracts were dialyzed against 50mM MOPS buffer to remove cellular inorganic phosphate and assayed for pNPGase and cellobiase activity in the presence and absence of 15 L A N E : 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 FIGURE 1: Screening of immunopositive clones by Pstl digestion of plasmid DNA. Mini-prep plasmid DNA was digested with Pstl and electrophoresed through a 0.8% agarose gel. Lanes 1 and 18: * DNA digested with Hindlll (standards) Lane 2: pBR322 Lane 10: pEC8 Lane 3: pEC1 Lane11:pEC19 Lane 4: pEC2 Lane 12: pEC35 Lane 5: pEC3 Lane 13: pEC41 Lane 6: B2 Lane 14: pEC52 Lane 7: B5 Lane 15: pEC56 Lane 8: B7 Lane 16: pEC57 Lane 9: B21 Lane 17: pEC62 16 FIGURE 2: Hydrolysis of Cm-cellulose by recombinant enzymes in crude cell extract. Graph showing (specific viscosity)"1 of Cm-cellulose versus incubation time of Cm-cellulose with crude cell extract from the positive control pEC2 (encoding an endoglucanase) and pEC62 containing 3.2 aand 1.0 mUnits of pNPCase activity, respectively. 17 TABLE 1: Preliminary characterization of the recombinant enzyme in crude cell extract from E.coli C600 containing pEC62 A. Hydrolysis of pNPG, pNPC and cellobiose using pEC62 cell extract8  Substrate mUnitsb/ma protein0 pNPC 4.9 pNPG 40.7 Cellobiiose 4.8 B. Hydrolysis of pNPG and cellobiose using pEC62 cell extract dialysed against 50 mM MOPS Substrate mUnits/mg protein + 50 mM phosphate buffer d -50 mM phosphate buffer8 pNPG 60 46 Cellobiose 3.9 2.8 a All assays were performed using the same cell extract b 1 unit is defined as that amount of enzyme required to release 1 umole of product pNP (from pNPG or pNPC) or glucose (from cellobiose) in 1 minute at 3 7 ° C c Total protein was assayed in all cell extracts d Dialyzed extract was added to reaction mixes containing substrate dissolved in 50 mM phosphate buffer (pH 7.0) e Dialyzed extract was added to reaction mixes containing substrate dissolved in 50 mM MOPS buffer (pH 7.0) 18 inorganic phosphate. The enyzme activity was not dependent on inorganic phosphate and therefore the enzyme was not a cellobiose phosphorylase (Table 1B). Accordingly, the recombinant gene product was designated Cbg {C^fimi J3-glucosidase) and the corresponding gene was designated cbg. III Identification of the corresponding native enzvme in C.fimi Initial studies on the B-glucosidases of C.fimi (Wakarchuk et al., 1984) suggested that they were localized within the cell and that cell culture supernatants were devoid of such activities. Although pEC62 had been isolated using antibody to C.fimi culture supernatants, it was thought that pEC62, which was weakly immunopositive, encoded a cellular B-glucosidase that was cross-reacting with antibody to supernatant cellulase enzymes . To identify the corresponding C.fimi B-glucosidase, cell extracts of pEC62 and C.fimi were examined by non-denaturing polyacrylamide gel electrophoresis followed by staining for MUGase activity (Figure 3). It appeared that pEC62 did not encode a recombinant C.fimi cellular B-glucosidase (see Discussion). Cell extracts from pUC13::A21 and 100X concentrated supernatant from C.fimi cultures grown on 0.2% Avicel for 3 days were compared by SDS-PAGE followed by staining for MUGase activity . The C.fimi supernatant contained a MUGase activity believed to be the native Cbg (Figure 4). IV Characterization of the cba aene A. Restriction mapping of the pEC62 insert A partial restriction map of the BamHI insert in pEC62 was created using the method of Smith and Bernstiel (1976). The plasmid was linearized by digestion at the unique Hindlll site, then end-labelled using the Klenow fragment of DNA pol I and [<*.-32P]dATP. The linearized labelled plasmid was digested with EcoRI to give two end-labelled fragments: one fragment contained the C.fimi DNA insert and the other fragment contained the vector. Partial digests of the DNA were electrophoresed through an agarose gel. The gel was dried and subjected to autoradiography (Figure 5). Other restriction sites were mapped using single and double diagnostic digests. The schematic representation of the derived linear map is 19 L A N E : 1 2 3 4 5 6 7 8 9 10 11 12 FIGURE 3: Non-denaturing polyacrylamide gel electrophoresis of crude cell extract from C.fimi and from E.coli cells containing pEC62, pUCl3::62, pUCl3::62A21. Crude cell extract from cells containing pUC13 grown with IPTG (Lane 5) and without IPTG (Lane 3) served as negative controls for pUC13::62A21 cells grown with IPTG (Lane 6) and without IPTG (Lane 4) and for pUC13::62 grown wtihout IPTG (Lane 2). pBR322 (Lane 7) served as a negative control for pEC62 (Lane 8). Cell extracts from C.fimi grown in 0.2% cellobiose and 0.2% glucose were loaded into Lanes 11 and 12, respectively. Gels were stained for activity by incubation with 100 L I M MUG. 20 FIGURE4: Denaturing polyacrylamide gel electrophoresis of crude cell extract from E.coli cells containing p U C l 3 : : 6 2 A 2 1 and of concentrated Avicel-grown C.fimi culture supernatant. pUC13 cells induced with IPTG (Lane 2) served as a negative control for pUC13::62A21 cells induced with IPTG. 2 mUnits of pNPGase activity were loaded into Lane 3. Cell free culture supernatant from a C.fimi culture grown for 3 days on 0.2% Avicel was concentrated 100X and 1 mUnit of pNPGase activity was loaded onto the gel (Lane 4). Lane 1 contained molecular weight standards. Gels were stained by incubation in 100 u.M MUG (active bands are indicated by the arrows on the right) 21 LANE: 1 2 3 4 5 6 7 8 . • - - -1 FIGURE5: Autoradiogram of an agarose gel containing partially digested end-labelled DNA used to construct a restriction map. End-labelled pEC62 DNA was partially digested with Nrul (Lane 3), Pvull (Lane 4), Sail (Lane 5), Smal (Lane 6) and Xhol (Lane 7). Undigested DNA (Lanes 2 and 8) was used to mark labelled vector DNA (indicated by arrow). The size of the DNA fragments was estimated using labelled Hindlll digested A DNA. DNA was electrophoresed through an 0.8% agarose gel. The dried gel was subjected to autoradiography overnight at -70°C with an intensifying screen. 22 shown in Figure 6. B. Determination of the dependency of cbg expression on an external (E. coli) promoter To determine the dependency of cbg gene expression on an E.coli promoter, the BamHI insert from pEC62 was subcloned into pUCl3 in both orientations. pl_IC13::62 contained the BamHI insert in the same orientation (relative to the lacZ promoter) as it was in pBR322 (relative to the tet gene promoter) and pUC13::62(R) contained the insert in the opposite orientation. E.coli JM83 (Yanisch-Perron et al., 1983) transformed with these plasmids were plated onto BBrothAmp plates containing IPTG and MUG. None of the recombinant cells had MUGase activity and all recombinant cells carried the plasmid pUC13::62(R). This suggested that cbg required an external promoter and that induced high level expression of the gene was lethal to the cells. To test this, E.coli JM101 carrying the laclq mutation (overexpression of the lac repressor) was transformed with the same plasmids and plated onto BBrothAmp plates. Recombinant clones were then picked onto BBrothAmp plates containing IPTG and MUG. Two types of clones were evident: weakly growing clones that were MUGase positive and strongly growing clones that were MUGase negative. The active clones contained pUC13::62 and the negative clones contained pUC13::62(R), confirming that cbg gene expression required an external promoter. C. Delimiting the oene bv restriction fragment deletion analysis Three restriction fragment deletions were constructed and screened for MUGase activity. A schematic representation of the deletions made and their corresponding MUGase activity is given in Figure 7. The first deletion was constructed by partially digesting pEC62 DNA with Sail, religating it and transforming E.coli C600. Transformants were screened for activity on MUG plates and active deletions were screened by Sail digestion. All active clones contained either parental plasmids or plasmids with a 1Kb deletion at the tet promoter distal end of the insert (600 bp of C.fimi insert and 400 bp of pBR322). The second deletion was constructed by digesting pUC13::62 DNA to completion with BstEII, religating it and transforming E.coli JM101. Transformants were screened for activity on MUG plates and by BstEII digestion. Clones missing the 1.8Kb BstEII fragment were devoid of MUGase activity. 23 T Sm B l _ _Pstl 1 — Sm Pstl 500 bp 250 bp Bst Pstl Pstl Ism V N i s m X Sm FIGURE 6: Schematic representation of linear restriction map of the 7.0 Kb insert of pEC62. Nrul(N), Pvull(P), Smal(Sm), (Xhol) and Sal(S) sites were mapped by partial digestion of end-labelled DNA. Loss of a single Pstl site by the deletion of a 600 bp Sail fragment (Figure 7) allowed mapping of the remaining Pstl sites. BstEII sites were mapped by double digestion with Pstl. 24 5-* 3' BamHI BstEII BstEII Sail BamHI . I I I I 1 ( + ) (") ( + ) -> ? cbg = 500 bp FIGURE7: Schematic summary of restriction fragment deletions used to localize c b g . The presence (+) or absence (-) of MUGase activity is indicated for each deletion construct. 25 Forced cloning of the 2.7Kb promoter proximal BamHI-Pstl fragment into pUC13 digested with BamHI and Pstl showed that this fragment was insufficient for MUGase activity. However, to determine if DNA contained in this 2.7 Kb fragment was necessary for enzyme expression, pUC13::62 was partially digested with BstEII, completely digested with BamHI and then filled in using DNA poll (Klenow fragment) to create blunt-ends before being religated. Deletion of the BamHI -BstEII fragment showed that all or part of this fragment was required for cbg expression. The properties of the deletions localized cbg to the promoter proximal end of the insert, which spanned the promoter proximal BstEII site in both directions (Figure 7). The actual size and the precise location of the gene could not be determined. D. Delimiting the 5'end of cba bv deletion using nuclease BAL-31 To further delimit cbg, deletions into the 5'-end of the gene were obtained using the strategy outlined in Figure 8. Figure 9A is an agarose gel showing the deletions series constructed and Figure 9B shows the MUG plates containing the various deletions. Figure 10A is a schematic representation and summary of the results shown in Figure 9. Figure 10B is a map showing the direction, length and method of sequencing a section of the pUC13::62 insert. E. Sequencing the junctions of pUC13::A24.21.31 Deletions 24,21 and 31 were sequenced chemically (Figure 10). The sequence between nucleotides 138-143 and between 161-167 fitted the consensus sequence determined for cellulase gene promoters in C.fimi (Miller et al.,1987). Lack of overlapping deletions in this region dictated that the shortest deletion be cloned into the vector pTZ18U and sequenced by the dideoxy method in order to locate a downstream ATG. F. Sequencing pTZ18U::A21Hindlll-BstEII pUC13::62A21 was digested to completion with BstEII and the 3'-recessed ends were filled in using the Klenow fragment of DNA pol I and all four nucleotides. The DNA was then digested with Hindlll and cloned into pTZ18U digested to completion with Hindi (blunt cutter) 26 Screen A ' s by Pstl digestion FIGURE8: Outline of the strategy used to make deletions into the 5'end of c b g . The largest deletion obtained by this method was pUCl3::62A21. Further deletions into the 5'-end of the gene were made by the strategy outlined beginning with this clone. 27 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 FIGURE 9A: Agarose gel of Pstl digested plasmid DNA showing deletions from the 5'-end of cbg. Mini-prep plasmid DNA was digested with Pstl and electrophoresed through a 0.8% agarose gel. Lanes 1 and 13:xDNA digested with Hindlll Lanes 2 and 12: ADNA digested with Hindlll and EcoRI Lane 3: pUC13 Lane4:pUC13::62 Lane 5: pUC13::62A27 Lane 6: pUC13::62A17 Lane 7: pUC13::62A24 Lane 8: pUC13::62A31 Lane 9: pUC13::62A21 Lane 10: pUC13::62A21-4 Lane11: pUC13::62A21-16 28 FIGURE 9B: Photograph of plates streaked with the deletion clones showing their relative MUGase activities. The deletion clones, pUC13 (negative control) and pUC13::62 (postive control) were streaked onto BBrothAmp plates containing MUG (100 u.M) and IPTG (100 u.M). The deletion numbers are indicated on the plates. A 2 1 - 1 6 — B pUC13: :62 A 2 4 Chemical Dideoxy 200bp FIGURE: 10A: Schematic summarizing the 5' end deletions and their corresponding MUGase activities. The diagram shows the /acZ-proximal 2.7 kb Pstl fragment containing the 5'-end of cbg. Relative MUGase activitites of the deletions (+ and - signs) were assessed visually using the plate assay. 10B: Sequencing map. A schematic showing the direction, length and method of the sequencing performed on the pUC13::62 insert. 30 and Hindlll. Single-stranded DNA template was then sequenced by extension from the universal primer. A representative autoradiogram is shown in Figurel 1. Sequence derived by the dideoxy method is shown in Figure 12. A start codon was not found within 170 nucleotides of the putative promoter sequence. Given that the maximum distance from a start codon to the -10 region found to date in C.fimi is about 100 nucleotides (Greenberg, personal communication), it seemed unlikely that the promoter like sequence identified was actually the cbg promoter. At this point two possible explanations for the deletion data were considered. The first possibility was that the promoter and start codon were further downstream of the determined sequence and deletion of that sequence in pUC13::62A21-4 created a lacZ-cbg translational fusion with decreased activity. The second possible explanation was that the promoter and start codon of cbg were upstream of the determined sequence and that active clones, pUC13::62A21 and A31, were lacZ-cbg in-frame translational fusions whereas the inactive clones, A24 and A28, were out-of-frame fusions with lacZ. To test the second possibility, a frameshift was created in the lacZ coding seqeunce in pUC13::62A31. G Creation of a reading frame shift in lacZ in pUC13::A21 To determine whether or not pUC13::A21 was a lacZ-cbotranslational fusion, plasmid was linearized at the unique Hindlll site in the coding sequence of lacZ and the 3' recessed ends were filled in using DNA poll Klenow fragment, creating a 4 bp insertion at this site and a reading frame shift in the religated plasmid. E.coli JM101 cells were transformed with this plasmid and screened for MUGase activity and loss of the unique Hindlll site. It was found that plasmids containing the frameshift were unable to confer MUGase activity on E.coli JM101. This result showed that pUC13::62A31 contained a lacZ-cogtranslational fusion. Translation of sequence from pUC13::62 in the lacZ reading frame is given in Figure 13. 31 FIGURE 11: Autoradiogram of a representative dideoxy sequencing gel. This gel is the sequence of pTZ18U::A21 Hindlll-BstEII obtained using the universal primer. Sequencing reactions were run on a 6% denaturing polyacrylamide gel and the dried gel was subjected to autoradiography overnight at room temperature without an intensifying screen. The sequence thought to be a promoter sequence is indicated. 32 10 20 30 40 50 GCTGCAGGTC GGTTCGGCTA CTCGCTGTTC GCGCTCGAGG TCTACGGCAC |A24 |A31 60 70 80 90 100 GCCGGGCGCG GTGGCGACGG CGTTCGGGAC GAGCGGTGTC CGGGTGCCGG |A21 110 120 130 140 150 CGGGCCAGAA CGCGCAGGTG CCGGTCGTCC TCGCGGCTCC GGTGGCGCAG 1G0 170 180 190 200 GACACGACCG TGCGGGTCGC GTCGACGGGC GGCACGGCGG TGCCGGGACG 210 220 230 240 250 ACTTCACCGC GGTCGACGAG ACGCTCACGT TCCCGGCCGG CGCGACGACG 260 270 280 290 300 GCCACGGTCG ACGTGTGAGA CGGACCACGG CCCGCTGGCC CGGTCCGGAC 310 320 330 340 GTCGTGCTGG AGCTGACGAG CCGGGCGACG CCTGTCTGGC pUC13 sequence |A number=junction with pUC13 for that deletion Underlined sequence fitted the consensus sequence for a C.fimi cellulase promoter FIGURE 12: pUCl3::62A24 sequence derived using chemical and dideoxy sequencing. Nucleotides upto position 170 were sequenced using both methods. The sequence from position 170 to the end was determined by the dideoxy method. Sequence from positions 1 to 10 is part of the multiple cloning site of pUC13. Sequence translated using nucleotides 3,4 and 5 as the first codon is in-frame with the lacZ coding sequence (Figure 13). 33 LacZ cqs-arq-ser-va I - tyr-g I y- thr—pro-g! y-a I a-va I -a I a- thr-a 1 a-phe-q 1 y- thr—ser-g I y-va I -arq-ua 1 -pro-a 1 a-q I y-q 1 n-asn-a I a-g I n-ua I -pro-ua I -va I -1 eu-a 1 a-a I a-pro-ya i -a I a-q I n-asp- thr~ thr-va 1 -arq-'va I -a I a-ser- thi—g I y-q I y- tht—a I a-va I -pro-g i y-arq-1 eu-h i s-arq-g I y-arg-ar-g-asp-a I a-h i s-va I -pro-g I y-ar-g-arg-asp-asp-g! y-h i s-g I y-at-g-arg-va I -ar^g-arq- thr- thr-a I a-arq- trp-pro-g I y-pro-asp-va i - va 1 -! eu-q I u-1 «su- thr-ssr-ar q-a I a- thr-pro-val-trp-FIGURE 13: Translation of pUCl3::62A31 DNA sequence using the/acZ reading frame. The predicted amino acid sequence of the N-terminus of the lacZ-cbg gene fusion protein was derived using the Macintosh DNA Inspector II software. 34 DISCUSSION I Characterizaton of the recombinant enzvme encoded bv pEC62 pEC62 is a member of the group of immunopositive, non-cellulolytic clones described by Gilkes et al.(1984a). The inability of the recombinant enzyme to hydrolyze CMC using the viscometric assay demonstrates that pEC62 does not encode an endoglucanase. However, because the release of reducing sugar could not be measured during the assay (due to high background), the possibility that pEC62 encoded an exoglucanase could not be excluded. Although the substrate specificity for the enzyme encoded by pEC62 could not be determined, it was identified as a 6-glucosidase by its ability of the enzyme to hydrolyze pNPG and its sensitivity to inhibition by D-glucono-i>1,5 lactone (Schimz et al., 1983). pEC1 encodes an exoglucanase (Exg) that releases cellobiose from the cellotriose analogue pNPC but does not hydrolyze the cellobiose analogue pNPG. The ability of pEC62 cell extract to hydrolyze both substrates identifies the recombinant enzyme as a B-glucosidase. Although there are B-glucosidases which only hydrolyze aryl-glucosides (aryl-B-glucosidases), the enzyme encoded by pEC62 is also a cellobiase since it hydrolyzes cellobiose. Identification of cbg as a true cellobiase (k c a t highest for cellobiose as a substrate) requires pure recombinant enzyme. Dialysis of pEC62 cell extract against MOPS buffer and subsequent assay for pNPGase and cellobiase activity in the presence and absence of inorganic phosphate, indicated that the recombinant enzyme is not a cellobiose phosphorylase. The reduced activity seen in assays without the addition of exogenous phosphate may be due inhibition by MOPS. It was shown that Tris buffers severely inhibited recombinant enzyme activity (data not shown). The native cbg gene product was identified as a C.fimi extracellular enzyme. Non-denaturing PAGE of pUC13::62 A21 cell extracts andC.fimi cell extracts showed no co-migration of recombinant and native enzymatically active bands. When pUC13::62A21 cell extract and C.fimi culture supernatant were electrophoresesd through an SDS gel (Figure 4), the recombinant enzyme, which had remained at the interface of the stacking and running gel in the non-denaturing gel system, migrated into the gel to a position corresponding to a molecular weight of 120 Kd. However, the enzyme was only partially denatured since it retained some enzymatic activity. A strong active band from the C.fimi supernatant remained 35 at the interface of the stacking and running gel. Evidence suggests that this band contains the native C.fimi enzyme corresponding to the recombinant Cbg. pEC62 encodes a 13-glucosidase; it carries the only fragment with B-glucosidase activity identified using antibody directed against C.fimi culture supernatant. C.fimi cell culture supernatant contains a single band of B-glucosidase activity (although the band may contain more than one protein, it is unlikely that it contains more than one B-glucosidase activity) suggesting that the antibody reacting with pEC62 was directed to this extracellular activity. The fact that the C.fimi enzyme migrated slightly differently and retained more activity in the S D S gel can be explained. All C.fimi extracellular enzymes studied to date are glycosylated to some degree (Langsford, personal communication) and this glycosylation could reduce S D S binding (Hames, 1981) such that the protein runs as if it were in a non-denaturing system. Cloned gene products are not glycosylated in E.coli and may be more readily denatured by SDS. Assuming that glycosylation has little effect on the charge to mass ratios of large proteins, one would expect the native and recombinant Cbgs to comigrate in a non-denaturing gel. Recombinant Cbg remains at the interface of the stacking and running gel in a non-denaturing system (Figure 3), as does the MUGase activity from C.fimi culture supernatants (data not shown). Therefore, it is concluded that cbg encodes an extracellular B-glucosidase in C.fimi. This is the first description of a bacterial extracellular B-glucosidase. A B-glucosidase from A. cellulolyticus can be found in culture supernatants (Saddler and Khan, 1980). However, the inability of these cells to utilize extracellular glucose suggests that the enzyme is actually an intracellular enzyme released into the medium by cell lysis. Although some extracellular glucosidases have been identified from Bacillus licheniformis (Thirunavukkarasu and Priest, 1984), they appear to be identical to known cell-associated enzymes suggesting that their presence in the supernatant is a result of leakage from the periplasmic space or of cell lysis. This is further supported by the enzymes not appearing in the medium until the cells reach stationary phase. The B-glucosidase identified in C.fimi culture supernatants differs from the intracellular B-glucosidases on denaturing and non-denaturing PAGE. Besides different migration patterns, the enzymes show a marked difference in their sensitivity to SDS. The cellular 6-glucosidases are completely inactive in 0.2% SDS whereas the extracellular 6-glucosidase remains fully active. 36 II Characterization of the cbg gene The first steps in characterizing cbg were to determine its direction of transcription and to determine if it was contained in its entirety within the 7.0 Kb insert. Because only one orientation of the BamHI insert in pBR322 had been isolated (pEC62) and expression of the gene product was required for immunoreactivity, it was thought that cbg gene expression was dependent on a heterologous promoter (tet in pBR322). This was tested by cloning the entire 7.0 Kb insert into both orientations relative to the lacZ promoter in pUC13. This experiment not only showed that an external promoter is required for gene expression but that the recombinant enzyme is not a gene fusion product (since the BamHI site is in a different reading frame in the lacZ gene in pUC13) and that therefore the entire gene must be present in the 7.0 Kb insert. To ensure pEC62 encoded a single enzyme activity (i.e. to ensure MUGase and MUCase activity were from single protein), the next step was to subclone the gene. Deletion of three restriction fragments provided some information about the general location of cbg but gave no indication of the size or precise translational start or stop sites of the gene. Analysis of the derived restriction map (Figure 6) showed no convenient restriction sites for a force cloning strategy based on the restriction fragment deletion information. The reasons for constructing a deletion series into the 5'-end of cog were three-fold. Firstly, the deletion series would be used to delimit the 5'-end of the gene and would provide clones from which probes for S1 mapping could be made. High expression of the cbg gene in cells carrying pUC13::62 induced with IPTG seemed to be deleterious to the cells but it seems possible that this was due to production of some other toxic protein such as a large lacZ fusion peptide (because of the high G/C content (76%) of C.fimi DNA ,stop codons are very rare). Therefore a clone with increased expression of the cbg gene was required to be able to more easily purify the recombinant enzyme. The deletion strategy described in Figure 8 yielded six clones (A17, 24, 27, 28, 21 and 31). The MUGase activity pattern was consistent with several hypotheses about the location of the 5'-end of the gene. Because there was a very small difference in the sizes of the deletions in A24 and A31 and there was a very large difference in the relative MUGase activities, the 5'-ends of these two deletions were carried out to determine if there was any kind of inhibitory 37 sequence removed from A31 that caused the sudden increase in activity. A24 contained 25 more nucleotides at its 5'-end than did A31 but the sequence did not appear to contain any regions of potential secondary structure or known transcription terminators (Figure 12). However, A24 and A31 were in different reading frames suggesting that they were out-of-frame and in-frame fusions with lacZ, respectively. The sequence from A21 revealed a putative promoter sequence (Miller et al., 1987), consistent with the hypothesis that A31 and A21 were transcriptional fusions with optimal spacing of the /acZtranscriptional start site and the cbg RBS and start codon (ATG or GTG) leading to an increased cbg translation efficiency. When sequence 200 bp downstream of the potential promoter sequence failed to yield a potential RBS and start codon, the translational fusion hypothesis was tested. Creation of a reading frame shift in lacZ and concurrent loss of detectable Cbg expression demonstrated that the increased expression in A21 and A31 was due to creation of a lacZ-cbg fusion peptide. The above results in conjunction with the activity patterns of the 3 deletions (A21-4, A21-15 and A21-16) constructed from A21 using the strategy shown in Figure 8, are consistent with the theory that the RBS and ATG of cbg lie in the DNA sequence present in A17 and absent in A28 (Figure 10). Since it is known that the recombinant enzyme in the pUC13::62 clone is translated from its own RBS and ATG (it is not a fusion) and that the level of MUGase activity in A27 and A17 is the same as for that of the parental clone, it is likely that expression of cbg is still dependent on the C.fimi RBS and ATG. Deletion of this sequence from C.fimi DNA in A28 and A24, coupled with the out-of-frame fusion of cbg with the reading frame of lacZ prevents the expression of cop. Fusion of lacZ with cbg in A21 andA31 allows efficient translation of cbg with minimal changes to the N-terminus of the enzyme. Further deletion of translated sequence in A21-4, A21-15 and A21-16 increasingly affects the N-terminus of the protein leading to decreased or no enzymatic activity. However, location of the RBS and start codon is required to verify any hypothesis. After the start of the gene has been sequenced and the size of the transcript has been determined by Northern blot, research should be directed in the area of purification of native and recombinant Cbg. 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