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The identification of a third recombinant DNA plasmid encoding a Cellulomonas fimi cellulase gene in… Wakarchuk, Warren William 1983

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IDENTIFICATION OF A THIRD RECOMBINANT DNA PLASMID ENCODING A CELLULOMONAS FIMI CELLULASE GENE IN ESCHERICHIA COLI by WARREN WILLIAM WAKARCHUK B.Sc., University of B r i t i s h Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Deparbment of Microbiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1983 © Warren William Wakarchuk, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of /WicrO K)>6l®jy The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 . 1 1 ABSTRACT A shotgun c l o n i n g o f C e l l u l o m o n a s f i m i DNA i n t o E s c h e r i c h i a c o l i y i e l d e d 2 t y p e s o f r e c o m b i n a n t p l a s m i d s e n c o d i n g c a r b o x y m e t h y l c e l l u l a s e (CMCase) a c t i v i t y ( 2 3 ) . These c l o n e s were d e s i g n a t e d p E C l and pEC2. A t h i r d t y p e o f r e c o m b i n a n t p l a s m i d c o n t a i n i n g a d i f f e r e n t C. f i m i c e l l u l a s e gene has now been i s o l a t e d and d e s i g n a t e d pEC3. The new c l o n e was d i f f e r e n t i a t e d from E. c o l i ( p E C l ) and E. c o l i (pEC2) by the f o l l o w i n g c h a r a c t e r i s t i c s : the s i z e o f the C. f i m i DNA i n s e r t ; i t s r e s t r i c t i o n d i g e s t i o n p a t t e r n and map; the l e v e l s o f CMCase i n c e l l f r e e e x t r a c t s ; h a l o f o r m a t i o n on CMC-Congo Red p l a t e s ; and the m i g r a t i o n o f CMCase a c t i v i t y i n n o n - d e n a t u r i n g p o l y a c r y l a m i d e g e l s . As w i t h E. c o l i ( p E C l ) and E. c o l i (pEC2), CMCase a c t i v i t y was d e t e c t e d An the p e r i p l a s m i c space o f E. c o l i <pEC3). i i i T A BLE OF CONTENTS Page A b s t r a c t . . . i i L i s t o f T a b l e s v L i s t o f F i g u r e s . . . . . . . . . . . v i A c k n o w l e d g e m e n t v i i I n t r o d u c t i o n 1 M a t e r i a l s a n d M e t h o d s A. B a c t e r i a l s t r a i n s a n d m e d i a . . . . . . . . . . 3 B. C o l o r i m e t r i c c e l l u l a s e a s s a y 4 C. DNA t e c h n i q u e s 4 D. N o n - d e n a t u r i n g p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . . . . . 5 E. L o c a l i z a t i o n o f c e l l u l a s e a c t i v i t y i n E. c o l i ( p E C ) s t r a i n s . . . 5 F. E n z y m e s a n d R e a g e n t s 6 iv Page Results A. Screening f o r c e l l u l a s e producing recombinant clones . . . 7 B. D i f f e r e n t i a t i o n of pEC3 from pECl and pEC2 . . . . . . . . 9 C. L o c a l i z a t i o n of c e l l u l a s e a c t i v i t y in E. c o l i (pEC) st r a i n s . . . . . . . 9 D. Determination of the r e s t r i c t i o n map of pEC3 . 9 Discussion A. The enzymes . 25 B. The genes 26 Lit e r a t u r e Cited . . . 28 V LIST OF TABLES Table T i t l e Page 1. Comparison of c e l l u l a s e a c t i v i t y from representative group 3 clones 8 2. C h a r a c t e r i s t i c s of c e l l u l a s e plasmids 19 3. S p e c i f i c a c t i v i t i e s of CMCase and marker enzymes in fracti o n a t e d E. c o l i (pEC) strains . 20 v i LIST OF FIGURES Figure T i t l e Page 1. Agarose-gel electrophoresis of Bam HI digested pEC plasmids 11 2. R e s t r i c t i o n endonuclease digests of pEC plasmids 13 3. Polyacrylamide gel electrophoresis analysis of c e l l u l a s e encoded by pEC plasmids 15 4. Zones of CMC hydrolysis produced by pEC strains and v i s u a l i z e d by Congo Red staining , . 17 5. Autoradiogram of pEC3 from a Smith and B i r n s t i e l r e s t r i c t i o n s i t e mapping experiment 21 6. R e s t r i c t i o n map of the plasmid pEC3 ; . . . 23 V I 1. ACKNOWLEDGEMENT I would l i k e to thank Dr's. R. M i l l e r , D. Kilburn, and T. Warren f o r th e i r encouragement, i n s t r u c t i o n and patience during my research. I am grat e f u l to Dr. N. Gilkes for providing some data on E. c o l i (pECl) and E c o l i (pEC2). I would e s p e c i a l l y l i k e to thank Maureen Langsford f or her extreme patience and understanding during my i l l n e s s proceeding the writing of this t h e s i s . 1 INTRODUCTION The conversion of c e l l u l o s i c waste to glucose for fermentation or production of single c e l l protein has enormous economic p o t e n t i a l . The major drawback of enzymatically degrading c e l l u l o s i c materials is the lack of highly active, stable and inexpensive enzymes. A study of c e l l u l a s e systems should be done so that t h e i r f u l l p o t e n t i a l can be r e a l i z e d . During the l a s t 30 years research on c e l l u l o l y t i c fungi and bacteria has elucidated various enzymatic components required for c e l l u l o l y s i s . The c l a s s i c a l c e l l u l a s e complex is a consortium of 3 enzymes which act s y n e r g i s t i c a l l y : endo-B-1,4-glucanase (E.C.3.2.1.4) which cleaves c e l l u l o s e randomly; exo-B-1,4-glucanase (either B-D-glucosyl gluco-hydrolase or cellobiohydrolase E.C.3.2.1.91) -which removes glucose or cellobiose units from c e l l u l o s e chain ends; and B-glucosidase (B-D-glucoside glucohydrolase , E.C.3.2.1.21) which produces glucose from ce l l o d e x t r i n s 4 glucose units long or shorter. One method of studying multi-enzyme complexes at the molecular l e v e l is to i s o l a t e the various s t r u c t u r a l genes by molecular cloning and then to manipulate them by i_n v i t r o recombinant DNA procedures. This type of approach is best suited to b a c t e r i a l genes: a) the DNA may be manipulated d i r e c t l y without the need to i s o l a t e mRNA and to make cDNA, as is necessary with fungal genes because of intravening sequences; and b) cloning vectors e x i s t with regulatory signals for b a c t e r i a l hosts which may allow increased expression of cloned genes (1,4). The bacterium Cellulomonas fimi is a gram p o s i t i v e , coryneform rod which belongs to a genus comprised of species which degrade c e l l u l o s e (7,27): It is easy to grow and secretes c e l l u l a s e into the environment. It is capable of degrading m i c r o c r y s t a l l i n e c e l l u l o s e and is therefore considered t r u l y c e l l u l o l y t i c . The enzyme system of C. fimi has been studied (9). Previous work in t h i s laboratory resulted in the i d e n t i f i c a t i o n of two C. fimi c e l l u l a s e genes (23). In t h i s thesis the i d e n t i f i c a t i o n of a t h i r d recombinant plasmid encoding a C. fimi c e l l u l a s e gene i s reported; also, a comparison of these 3 genes is given. 3 MATERIALS AND METHODS A. B a c t e r i a l s t r a i n s and media The b a c t e r i a l strains used were: E. c o l i C600; E. c o l i C600 (pBR322); E. c o l i C600 (pECl); E. c o l i C600 (pEC2); and E. c o l i C600 (pEC3). These str a i n s were grown in LB (lOg tryptone; 5 g yeast extract, 5 g NaCl, l g glucose, per l i t e r , pH 7.A), supplemented with thiamine (lOug/ml) and thymidine (5 ug/ml). S o l i d media contained 11 g agar per l i t e r . Strains with plasmids were grown with a m p i c i l l i n (50 ug/ml). The enzymatic a c t i v i t y of cer t a i n recombinant c e l l u l a s e gene products was v i s u a l i z e d by a modification pf the carboxymethylcellulose - Congo Red plate assay of leather and Wood (21). Minimal media, M9S (3) without glucose, was prepared with 1.0% high v i s c o s i t y carboxymethylcellulose (CMC), 1.0% Nobel agar, and a m p i c i l l i n (50 pg/ml). Bacteria were grown on the plates for 24 hours at 30°C, the colonies then were washed o f f the plate. The plates were flooded with Congo Red dye (1 mg/ml in d i s t i l l e d water) and shaken gently on an o r b i t a l shaker for 15-30 minutes at. room temperature. Excess dye was poured o f f . The plates then were flooded with 1 M NaCl and gently shaken to wash out excess dye. The 1 M NaCl wash was repeated 2 or 3 times. Zones of cle a r i n g could be seen at th i s time; they appeared yellow while the plate was red. Plates could be stored by f i x i n g the dye with 10% acetic acid. 4 B. Colorimetric c e l l u l a s e assay Cel l u l a s e a c t i v i t y was analyzed by measuring the increase in reducing groups from the hydrolysis of CMC. C e l l - f r e e extracts were prepared as described by Whittle et a l . , (24); these were incubated with CMC at 37°C for varying lengths of time depending on which E. c o l i (pEC) s t r a i n was being analyzed. The reducing groups were quantitated by the d i n i t r o -s a l i c y c l i c acid method of M i l l e r (13). A standard curve was prepared using glucose. CMCase a c t i v i t y was expressed as ug glucose equivalents produced per minute per mg of protein. Protein was determined by the method of Lowry et. a l . , ( 1 0 ) . C. DNA techniques Plasmid DNA was i s o l a t e d using the rapid a l k a l i n e l y s i s technique (11) with the following modification: excess s a l t was removed by the mic r o d i a l y s i s technique of Marusyk and Sergeant (12). R e s t r i c t i o n endonuclease digestions were done according to the manufacturers s p e c i f i e d conditions (New England Biolabs). R e s t r i c t i o n fragments were separated on horizontal agarose gels using t r i s (hydroxymethyl) aminomethane/boric acid/ethylenediamine t e t r a c e t i c acid buffer pH 8.3 (11). Gels contained 1 ug/ml ethidium bromide. DNA was v i s u a l i z e d by fluorescence under short-wave u l t r a v i o l e t l i g h t . Gels were photographed with type 57 polaroid f i l m using an orange f i l t e r . R e s t r i c t i o n endonuclease s i t e mapping was done by a modification of the Smith and B i r n s t i e l method (19). Plasmid DNA (pEC3) was l i n e a r i z e d 32 with EcoRI and end l a b e l l e d with a- -P-dATP and Klenow DNA 5 polymerase (5). A 29 base pair fragment containing one end l a b e l was removed by digestion with Hind III followed by mi c r o d i a l y s i s . This l e f t a plasmid s e l e c t i v e l y end-labelled at the remaining EcoRI s i t e . This l a b e l l e d fragment of pEC3 then was p a r t i a l l y digested with various r e s t r i c t i o n endonucleases. The p a r t i a l digestion products were detected by autoradiography of gels dried onto Whatman 3 MM paper. D. Non-denaturing polyacrylamide gel electrophoresis C e l l - f r e e extracts were electrophoresed in 1.5 mm thick, 67o polyacrylamide gels using a tris(hydroxymethyl) aminomethane/glycine running buffer pH 8.3 (9) . CMCase a c t i v i t y was detected by s l i c i n g the gels into 2 mm fra c t i o n s and assaying the gel s l i c e s d i r e c t l y (see B above) . E. L o c a l i z a t i o n of c e l l u l a s e a c t i v i t y in E. c o l i (pEC) st r a i n s A periplasmic enzyme f r a c t i o n was obtained by the osmotic shock procedure of Nossal and Heppel (16). Cytoplasmic enzymes were released 2 from spheroplasts by french pressure (12,000 l b / i n ). Tot a l c e l l extracts were prepared by french pressure treatment of 25 f o l d concentrated cultures. C e l l debris was removed by cen t r i f u g a t i o n at 50,000 X g for 20 minutes at 4°C. The enzyme B-lactamase was used as a periplasmic marker; i t was assayed c o l o r i m e t r i c a l l y with n i t r o c e f i n as substrate (17). A unit of B-lactamase produced 1 nmole n i t r o c e f o i c acid per minute per mg protein. B-galactosidase was used as a cytoplasmic marker; i t was assayed with o-nitrophenyl-B-D-galactoside (ONPG) as substrate (15). A unit of 13-galactos idase produced 1 nmole o-nitrbphenol per minute per mg protein. CMCase a c t i v i t y was determined as described in section B above. F. Enzymes and reagents A l l r e s t r i c t i o n endonucleases were purchased from New England Biolabs; Klenow DNA polymerase from Bethesda Research Labs; CMC and ONPG 32 from Sigma Chemical Co; a- -P-dATP from New England Nuclear; N i t r o c e f i n was a g i f t from Dr. Margaret Mathew of Glaxo Laboratories; 7 RESULTS A. Screening f o r c e l l u l a s e producing recombinant clones A shotgun cloning of Cellulamonas fimi DNA into the E. c o l i plasmid pBR322 (23, 24) yielded 64 immunopositive clones. The clones showing measurable c e l l u l a s e (CMCase) a c t i v i t y were characterized and divided into 3 groups. Clones of the f i r s t group (pECl) contained a plasmid with a 6.6 kb in s e r t of C. fimi DNA, were strongly antigenic, and contained moderate le v e l s of CMCase. Clones of the second group (pEC2) contained a plasmid with a 5.0 kb insert of C. fimi DNA, were weakly antigenic, and contained high l e v e l s of CMCase a c t i v i t y . Those in the t h i r d group were also weakly antigenic but contained only low l e v e l s of CMCase. An analysis of the plasmids in 10 of the 25 clones in the t h i r d group showed there were various types of plasmids present (data not shown). The plasmids in 3 of these clones had i d e n t i c a l 5.6 kb i n s e r t s . Quantitation of the CMCase a c t i v i t y in i n d i v i d u a l clones which were representative of the various group 3 plasmids is shown in Table 1. Strains with the most CMCase contained a 5.6 kb Bam HI fragment of C. fimi DNA. These were designated E. c o l i (pEC3). Table 1. Comparison of c e l l u l a s e a c t i v i t y from representative group 3 clones. 8 Clone designation CMCase (U/mg) % Relative to pECl pECl 3.97 100 pEC 3A 0 0 pEC 3B 0.66 17 pEC 3C 0.59 15 pEC 3D 1.30 33 The c e l l extracts of various group 3 clones were compared for c e l l u l a s e a c t i v i t y . Extracts were obtained by french pressure; protein and carboxymethylcellulase (CMCase) were determined as described in MATERIALS AND METHODS. 9 B. D i f f e r e n t i a t i o n of pEC3 from pECl and pEC2 The sizes of the C. fimi DNA inserts in the pEC plasmids was determined by digestion with Bam HI (Fig. 1). These inserts gave d i f f e r e n t r e s t r i c t i o n fragment patterns with the enzymes Sal I and Pst I (Fig. 2). C e l l free extracts from c e l l s carrying these plasmids gave single bands of CMCase a c t i v i t y in non-denaturing polyacrylamide gels (Fig. 3). Colonies of E. c o l i (pEC2) and E. c o l i (pEC3) produced halos on CMC-containing plates which were stained with Congo-Red (Fig. 4). E. c o l i (pECl) did not produce a halo on these plates even though i t s CMCase a c t i v i t y was higher than that of E. c o l i (pEC3). C l e a r l y the enzymatic a c t i v i t y of E. c o l i (pECl) was q u a l i t a t i v e l y d i f f e r e n t from those of E. c o l i (pEC2) and E. c o l i (pEC3). These data are summarized in Table 2. C. L o c a l i z a t i o n of c e l l u l a s e a c t i v i t y in E. c o l i (pEC) s t r a i n s L c o l i (pEC) strains contained s i g n i f i c a n t amounts of CMCase a c t i v i t y in the periplasmic space (Table 3). The very low l e v e l s of 8-galactosidase from the periplasmic f r a c t i o n was consistent with i t s known cytoplasmic l o c a t i o n in the c e l l and indicated that there was no s i g n i f i c a n t contribution of cytoplasmic enzymes to the a c t i v i t i e s measured in the periplasmic f r a c t i o n . D. Determination of the r e s t r i c t i o n map of pEC3 Re s t r i c t i o n s i t e s were determined r e l a t i v e to the l a b e l l e d 5* EcoRI terminus by the p a r t i a l digestion method of Smith and B i r n s t i e l (19) see 10 M a t e r i a l s and Methods. S e v e r a l enzymes were used: S a l I ; P s t I ; Pvu I I ; Sma I and Kpn I . An example o f a mapping g e l i s shown i n F i g u r e 5. The map d i s t a n c e s were c o n f i r m e d by an a n a l y s i s o f r e s t r i c t i o n f r a g m e n t s on agarose g e l s . The r e s t r i c t i o n s i t e map i s shown i n F i g u r e 6. 11 Figure 1. Agarose-gel electrophoresis of Bam HI digested pEC plasmids. Plasmid DNA was i s o l a t e d from E. c o l i C600(pEC) s t r a i n s , digested with Bam HI and separated on a 0.7% agarose gel containing 1 ug/ml ethidium bromide. Lane A: Hind III cleaved fragments of \, Lane B: pECl, Lane C: pEC2, Lane D: pEC3. The arrow indicates l i n e a r pBR322 DNA. 12 13 Figure 2. R e s t r i c t i o n endonuclease digests of pEC plasmids. P u r i f i e d pEC plasmids were digested with the r e s t r i c t i o n endonucleases Sal I and Pst I. The fragments were separated on a 1.1% agarose gel containing 1 ug/ml ethidium bromide. Lanes A - C are Sal I digests of pECl, pEC2 and pEC3 respectively. Lane D is Hpa I digested T7 phage DNA as a molecular weight marker. Lanes E-G are Pst I digests of pECl, pEC2 and pEC3, respectively. 1 4 15 Figure 3. Polyacrylamide gel electrophoresis analysis of c e l l u l a s e s encoded by pEC plasmids. Whole c e l l extracts of E. c o l i C600(pEC) strains were run on 6% non-denaturing polyacrylamide gels. The gels were s l i c e d in 2 mm f r a c t i o n s , these s l i c e s were placed d i r e c t l y into a CMCase assay mixture and incubated for 18 - 20 hours at 37°C. The reactions were terminated, and reducing sugars were determined c o l o r i m e t r i c a l l y as outlined in MATERIALS AND METHODS. Panel A shows the a c t i v i t y p r o f i l e s of pECl, Panel B shows pEC2 and Panel C shows pEC3. N.B. Only the top part of the gel is shown here; since no a c t i v i t y was seen in the remainder of the g e l , the top part i s graphed in an expanded form . 16 0-2 0 4 6 c O m m Q o 0 2 • 0 2 PERCENT 20 40 LENGTH OF G E L 17 Figure 4. Zones of CMC hydrolysis produced by pEC strains and v i s u a l i z e d by Congo Red st a i n i n g . Cultures of the E. c o l i (pEC) strains were grown to stationary phase and then plated for single colonies. Single colonies were transferred to CMC containing plates which were incubated 24 hours at 30°C. Zones of hydrolysis then were v i s u a l i z e d with Congo Red as outlined in MATERIALS AND METHODS. A l l of the colonies were approximately the same s i z e . (A) denotes where colonies of E. c o l i (pBR322) were; (B) where colonies of E. c o l i (pECl) were; (c) where colonies of E. c o l i (pEC2) were; (D) where colonies of E. c o l i (pEC3) were. I O O v 9 w Table 2. Ch a r a c t e r i s t i c s of Cell u l a s e Plasmids 19 Plasmid Insert Size A n t i g e n i c i t y CMCase U/mg Zone of Clearing on (kb) in E. c o l i CMC-Congo Red PECl 6.6 strong 3.6 pEC2 5.0 weak 39.0 + + pEC3 5.6 v. weak 1.2 + The size of C. fimi DNA inserts was determined from Bam HI r e s t r i c t i o n endonuclease digestion fragments separated on an agarose g e l . An t i g e n i c i t y was based on the in t e n s i t y of the colony on the screening autoradiograms. CMCase and protein were determined as described in MATERIALS AND METHODS. Zones of CMC hydrolysis were v i s u a l i z e d with Congo. Red staining as outlined in MATERIALS AND METHODS (Fig. 4). 20 Table 3. S p e c i f i c a c t i v i t i e s of CMCase and marker enzymes Enzyme C e l l Fraction Periplasmic Cytoplasmic C e l l extract pECl CMCase fi-galactos idase B-lactamase 26 160 14,480 0.4 2,722 72.9 3.6 1,932 2,200 PEC2 CMCase B-galactosidase B-lactamase 95 325 9,372 29 4,048 63 39 2,738 2,604 PEC3 CMCase B-galactosidase B-lactamase 10. 584 67 ,653 ,3 0.36 4,909 2,556 1.2 2,892 6,390 CMCase = yg glucose eqv/min/mg protein B-Galactosidase = nmole p-nitrophenol/min/mg protein B-Lactamase = nmole n i t r o c e f o i c acid produced/min/mg protein The E. c o l i (pEC) strains were fractionated by the osmotic shock, procedure of Nossal and Heppel. The periplasmic marker enzyme was B-lactamase, and the cytoplasmic marker was B-galactosidase. A l l three enzymes were assayed as outlined in MATERIALS AND METHODS. 21 Figure 5. Autoradiogram of pEC3 DNA from a Smith and B i r n s t i e l r e s t r i c t i o n s i t e mapping experiment. P u r i f i e d pEC3 plasmid DNA was end-labelled s e l e c t i v e l y and then digested with various r e s t r i c t i o n endonucleases as outlined in MATERIALS AND METHODS. Lane A is a molecular weight marker. Lanes B-E are Sal I time points. Lanes F-J are Pst I time points. Lanes K-0 are Pvu II time points. Sal I time points were 3, 15, 30 and 120 minutes of digestion. The Pst I and Pvu II time points were 1, 3, 15, 30 and 120 minutes of digestion. 22 23 Figure 6. R e s t r i c t i o n map of the plasmid pEC3. Using the r e s t r i c t i o n mapping technique outlined in MATERIALS AND METHODS the s i t e s for 5 d i f f e r e n t r e s t r i c t i o n endonucleases were determined. A l l distances were determined r e l a t i v e to the unique Eco RI (E) s i t e in the vector pBR322. The crosshatched area represents the inserted C. fimi DNA. A shows the Sal I (S) s i t e s . B shows the Pvu II (P) s i t e s . G shows the Pst I (Ps) s i t e s . D shows both Sma I (Sm) and Kpn I (K) s i t e s . The Bam HI s i t e s (B) are also shown. o o CD r- o - ro - * — cn o CT Q tn 9 I T3 O — cn — CD -m - - c o to 3 CO 3 00 m —03 -m 09 " D T I to " T I i n 07 •m =-0) -m T I • n 03 -m CO •to -co -co CD m 25 DISCUSSION A. The enzymes The components of the c e l l u l a s e system of Cellulomonas fimi are being elucidated by the molecular cloning of the s t r u c t u r a l genes in Escherichia  c o l i . To date 3 genes encoding c e l l u l a s e (CMCase) a c t i v i t y have been i s o l a t e d and p a r t i a l l y characterized. Each gene is present on a si n g l e , d i f f e r e n t , Bam HI r e s t r i c t i o n fragment. The r e s t r i c t i o n endonuclease digestion patterns are also d i s t i n c t . In addition, each clone appears to encode a single c e l l u l a s e enzyme. This conclusion is based on enzyme migration in non-denaturing polyacrylamide gels. The enzymatic a c t i v i t i e s of the 3 C. fimi DNA recombinant clones appear not only q u a n t i t a t i v e l y d i f f e r e n t , but q u a l i t a t i v e l y d i f f e r e n t as well. The standard c e l l u l a s e assay (CMCase) would not d i s t i n g u i s h endoglucanase from exoglucanase a c t i v i t y ; however, one might expect an exoglucanase to generate fewer reducing sugar equivalents because of i t s s e n s i t i v i t y to the substituted glucose residues (8). The Congo Red plate assay is l i k e l y to detect only endoglucanase a c t i v i t y . The dye binds only to c e l l o d e x t r i n s 6 glucose residues long or longer (25). Halos then are produced by cleaving long CMC chains into short oligomers which do not bind dye. A l t e r n a t e l y longer oligomers produced by infrequent cleavage may be washed out during the staining procedure again producing a halo. From this rationale then, E. c o l i (pECl) l i k e l y encodes an exoglucanase, while E. c o l i (pEC2) and E. c o l i (pEC3) encode endoglucanases of d i f f e r e n t s p e c i f i c a c t i v i t i e s . This hypothesis w i l l have to be confirmed by checking the c l a s s i c a l ertdo-26 glucanase property of v i s c o s i t y reduction and the c l a s s i c a l exoglucanase property of cellobiohydrolase action on c r y s t a l l i n e substrates (8). Because there are d i f f e r e n t enzymatic a c t i v i t i e s present in C. fimi culture supernatants the use of a screening method l i k e Congo Red-CMC plates for recombinant DNA clones is quite l i m i t e d . Congo Red screening has been used to detect endoglucanase from recombinant DNA clones of Clostridium thermocellum (2). The immunological screening procedure does not d i s t i n g u i s h between enzymatic a c t i v i t i e s and hence can be used to detect clones encoding a l l of the C. fimi c e l l u l a s e s present in c u l t u r e supernatents. Also, i f C. f imi's c e l l u l a s e requires peptides which in themselves are not c e l l u l o l y t i c but instead are necessary for synergy or substrate binding, they too could be detected as immunoreactive clones. The u t i l i t y of such peptides could be shown in reconstruction experiments. B. The genes The commercial u t i l i z a t i o n of c e l l u l a s e for degradation of c e l l u l o s i c wastes i s not yet economically f e a s i b l e . This is because large quantities of highly a c t i v e , stable c e l l u l a s e are not yet a v a i l a b l e . Increased production of c e l l u l a s e w i l l be the f i r s t step in making them less expensive. Molecular cloning o f f e r s an approach to increase production of the necessary enzymes by iri v i t r o recombinant DNA manipulations of cloned genes. For example, this very type of approach has been successful with human interferon (18) and a b a c t e r i a l a-amylase enzyme (22). The f i r s t step in these projects is to define the exact length of cloned DNA which makes a functional enzyme. The second is to determine the 27 nucleotide sequence of the gene and f i n d any possible regulatory sequences which may or may not function in various hosts. The t h i r d step is to couple the gene to known regulatory sequences to have ei t h e r an inducible or c o n s t i t u t i v e l y produced gene product which is released from the b a c t e r i a l c e l l s into the environment. This would either allow extra-c e l l u l a r conversion of substrate or provide an i n i t i a l p u r i f i c a t i o n away from the c e l l u l a r components. Currently cloned C. fimi genes are expressed in E. c o l i , and i t i s l i k e l y that strong E. c o l i regulating sequences could be used to increase t h e i r expression. In C. fimi the c e l l u l a s e genes are regulated (9); therefore, sequences from C. fimi must ex i s t which could possibly be exploited as c o n t r o l l i n g elements. Also c e l l u l a s e s from pEC plasmids are exported to the periplasmic space of E. c o l i . This indicates that the leaders or signal polypeptides for transport to the environment are present. The excretion of the pEC c e l l u l a s e s is then possible and in fac t has been achieved with a mutant E. c o l i (pEC) which s e l e c t i v e l y releases periplasmic enzymes into the culture medium (6). 28 LITERATURE CITED 1. Bernard, H.M., Remant, E., Hershfield, M.V., Das, H.K., H e l i n s k i , D.R., Yanofsky, C. and Franklin, N.: Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda P L promoter. Gene 5:59-69, 1979. 2. Cornet, P., Tronik, D., M i l l e t , J . , and Aubert, J . : Cloning and expression in Escherichia c o l i of Clostridium thermocellum genes coding f o r amino acid synthesis and c e l l u l o s e h y d r o l y s i s . FEMS Mic r o b i o l . Letters 16:137-141, 1983. 3. Champe, S.P. and Benzer, S. : Reversal of mutant phenotypes by 5- f l u o r o u r a c i l ; An approach to nucleotide sequences in messenger-RNA. Proc. Natl. Acad. S c i . USA 48:532-546, 1962. 4. de Boer, H.A., Comstock, L.J., Yansura, D.G., and Heynecker, H.L.: Construction of a tandem t r p - l a c promoter and a hybrid t r p - l a c promoter for e f f i c i e n t and co n t r o l l e d expression of the human growth hormone in Escherichia c o l i . In Promoter structure and function, Rodriquez, R.L. and Chamberlain, M.J. (Eds.), Praegar Publishers, New York. 1982. 5. Drovin, J . : Cloning of human mitochondrial DNA in Escherichia c o l i . J. Mol. B i o l . 140:15-22. 1980. 6. Gilkes, N.R., Kilburn, D.G., M i l l e r , R.C., J r . and Warren, R.A.J.: Is o l a t i o n of a mutant of Escherichia c o l i which leaks c e l l u l a s e a c t i v i t y encoded by cloned c e l l u l a s e genes from Cellulomonas f i m i . (Submitted to Nature.New Biotechnology) 1983. 7. Hagget, K.D., Gray, P.P., and Dunn, N.W. : C r y s t a l l i n e c e l l u l o s e degradation by a s t r a i n of Cellulomonas and i t s mutant d e r i v a t i v e s . Eur. J. Appl. M i c r o b i o l . Biotechnol. 8:183-190, 1979. 8. King, K.W., and Vessal, M.I.: Enzymes of the c e l l u l a s e complex, in Gould, R.F. (Ed.), Cellulases and Their Applications, Advances in Chemistry series 95. 1969, pp. 17-25. 9. Langsford, M.L., Gilkes, N.R., Wakarchuk, W.W., Kilburn, D.G., M i l l e r , R.C., J r . and Warren, R.A.J.: The c e l l u l a s e system of Cellulomonas f i m i . Submitted to J. Gen. Micro b i o l . , 1983. 10. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, J . : Protein measurement with the F o l i n phenol reagent. J. B i o l . Chem. 193:265-273, 1951. 11. Maniatis, T., F r i t s c h , E.F. and Sambrook, J . : Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory. 1982. 29 12. Marusyk, R. and Seargeant, A.: A simple method for d i a l y s i s of small-volume samples. Anal. Biochem. 105:403-404, 1980. 13. M i l l e r , G.L.: Use of d i n i t r o s a l i c y c l i c reagent for determination of reducing sugars. Anal. Chem. 31:426-428. 1959. 14. M i l l e r , G.L., Blum, R., Glennon, W.E. and Burton, A.L.: Measurement of carboxymethylcellulase a c t i v i t y . Anal. Biochem. 2:127-132. 1960. 15. M i l l e r , J.H;: Experiment in molecular genetics. Cold Spring Harbor Laboratory, pp. vxi + 466. 1972. 16. Nossal, N.G. & Heppel, L.A.: The release of enzymes by osmotic shock from Escherichia c o l i in exponential phase. J. B i o l . Chem. 241:3055-3062. 1966. 17. O'Callaghan, C.H., Morris, A., Kirby, S.M. and Shingler, A.H.: Novel method for detection of B-lactamase by using a chromogenic cephalosporin substrate. Antimicrobial Agents and Chemotherapy 1:283-2388. 1972. 18. Slocombe, P., Easton, A., Boseley, P., and Burke, D.C: High l e v e l expression of an interferon a2 gene cloned in phage M13mp7 and subsequent p u r i f i c a t i o n with a monoclonal antibody. Proc. Natl. Acad. S c i . USA 79:5455-5459, 1982. 19. Smith, H.O. and B i r n s t i e l , M.L.: A simple method for DNA r e s t r i c t i o n s i t e mapping. Nucleic Acids Research 3(9):2387-2398, 1976. 20. Stoppok, W., Rapp, P. and Wagner, F.: Formation, l o c a t i o n and regulation of endo-1, 4-B-glucanases and B-glucbsidases from Cellulomonas uda. Appl. Environ. M i c r o b i o l . 44:44-53, 1982. 21. Teather, R.M. and Wood, P.J.: Use of Congo red-polysaccharide i n t e r -actions in enumeration and characterization of c e l l u l o l y t i c b a cteria from bovine rumen. Appl. Environ. M i c r o b i o l . 43:777-780, 1982. 22. Willemot, K. and Cornelis, P.: Growth defects of Escherichia c o l i c e l l s which contain the gene of an oc-amylase from B a c i l l u s coagulans on a multicopy plasmid. J. Gen. Micro b i o l . 129:311-319, 1983. 23. Whittle, D.J., Master's Thesis, University of B r i t i s h Columbia, 1982. 24. Whittle, D.J., Kilburn, D.G., Warren, R.A.J, and M i l l e r , R.C., J r . : Molecular cloning of a Cellulomonas fimi c e l l u l a s e gene in Escherichia c o l i . Gene 17.: 139-145 , 1982. 25. Wood, P.J.: S p e c i f i c i t y in the int e r a c t i o n of d i r e c t dyes with polysaccharides. Carbohydr. Res. 85:271-287, 1980. 30 26. Wood, T.M., and McCrae, S.I.-: P u r i f i c a t i o n and some properties of the e x t r a c e l l u l a r 13-D-glucosidase of the c e l l u l o l y t i c fungus Trichoderma  k o n i n g i i • J. Gen. Mi c r o b i o l . 128:2973-2982, 1982. 27. Yamada, J. and Komagata, L.: Genus Cellulomonas, in Holt, J. (Ed.), Bergey's Manual of Determinative Bacteriology, 8th Ed., Williams and Wilkins Co., Baltimore, 1977, pp. 232-233. 

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