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The molecular cloning of Cellulomonas fimi cellulase genes Whittle, Daniel Joseph 1982

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THE MOLECULAR CLONING OF CELLULOMONAS FIMI CELLULASE GENES by DANIEL JOSEPH WHITTLE - B . S c , Q u e e n ' s U n i v e r s i t y , 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( Depar tment o f M i c r o b i o l o g y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A u g u s t , 1982 0 D a n i e l J o s e p h W h i t t l e , 1982 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 for 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 available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of M i c r o b i o l o g y  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A u g u s t 5, 1982 D E - 6 C T / R - n i i ABSTRACT Recombinant DNA techniques were used to clone and isolate Cellulo-monas fimi cellulase genes. A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant plasmids carrying cellulase genes. The screening procedure is based on binding cellulases and other proteins released from lysed clones to CNBr-acti-vated paper. The paper is treated with anti-cellulase antibody and 125 the antigen-antibody complex is detected by autoradiography using I-labeled protein A from Staphylococcus aureus. This- immunoassay, was used to identify recombinant plasmids contai-ning strains, carrying at least two different cellulase genes. The enzymes present in extracts of E^. col i cellulase clones were active in catalysing the hydrolysis of carboxymethy1 eel 1ulose as indicated by the production of reducing sugars. Osmotic shock treatment of one E_. col? eel 1ulase clone revealed that the majority of the cellulase enzyme synthesized by this clone was transported to the periplasmic space. Cellulase encoding plasmids were characterized by the presence of either a 6.6 or a 5.0 kilobase C_. fimi DNA gene fragment. TABLE 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 ures v i Acknowledgement 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 and Methods A. B a c t e r i a l s t r a i n s and media ^ 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 C. P r e p a r a t i o n o f a n t i s e r a •.. 5 D. P r e p a r a t i o n o f C N B r - a c t i v a t e d paper 5 I O C E. P r e p a r a t i o n o f l - l a b e l e d p r o t e i n A .. 6 F. I m m o b i l i z a t i o n o f p r o t e i n s from c o l o n i e s l y s e d i n s i t u 6 G. D e t e c t i o n o f c e l 1 u 1 a s e - p r o d u c i n g c o l o n i e s 7 H. I s o l a t i o n and p u r i f i c a t i o n o f DNA — . 7 I. C l o n i n g o f BamHI fragments o f C. f i m i DNA i n t o E_. c o l i 8 J . S c r e e n i n g recombinant E_. c o l i c l o n e s f o r c e l l u l a s e a c t i v i t y ° 8 K. Enzymes and r e a g e n t s 9 i v Page R e s u l t s A. Development o f a s e n s i t i v e i m m u n o l o g i c a l s c r e e n i n g method t o d e t e c t e e l 1u1ase enzymes 10 B. C o n s t r u c t i o n and i d e n t i f i c a t i o n o f a c e l l u l a s e p r o d u c i n g recombinant c l o n e 13 C. E n z y m a t i c c h a r a c t e r i z a t i o n o f the recombinant pi asm i d 19 D. M o l e c u l a r c h a r a c t e r i z a t i o n o f the c e l l u l a s e -e n c o d i n g p l a s m i d ... ..... lh E. I s o l a t i o n o f o t h e r e e l 1ulase p r o d u c i n g recombinant c l o n e s 30 Di s c u s s i o n A. . A g e n e r a l i m m u n o l o g i c a l s c r e e n i n g method t o d e t e c t c l o n e d gene p r o d u c t s : 3^ B. C h a r a c t e r i z a t i o n s t u d i e s on c e l l u l a s e p r o d u c i n g c l o n e s 35 L i t e r a t u r e Ci t e d 38 V LIST OF TABLES T a b l e T i t l e Page 1 L o c a l i z a t i o n o f E_. c o l i pDWl c e l l u l a s e i n t h e p e r i p l a s m i c space 25 v i LIST OF FIGURES F i g u r e T i t l e Page 1 Immunoabsorption o f C_. f i m i c e l l u l a s e enzymes by a n t i - c e l l u l a s e a n t i b o d y bound t o a p r o t e i n A column . .. 11 2 A u t o r a d i o g r a m o f C. f i m i c o l o n i e s 14 3 A u t o r a d i o g r a m o f an E_. c o l i c o l o n y p r o d u c i n g c e l 1 u l a s e ... . 17 h C e l l u l a s e a c t i v i t y o f E_. c o l i c a r r y i n g pDW1 DNA 20 5 Immunoabsorption o f E. c o l i pDWI c e l l u l a s e enzymes by a n t i - c e l 1 u 1 a s e a n t i b o d y bound t o a p r o t e i n A column 22 6 A g a r o s e - g e l e l e c t r o p h o r e s i s o f BamHI d i g e s t e d p l a s m i d pDWT 26 7 R e s t r i c t i o n enzyme c h a r a c t e r i z a t i o n o f the p l a s m i d pDW1 28 8 A g a r o s e - g e l e l e c t r o p h o r e s i s o f BamHI d i g e s t e d p l a s m i d s pEC7 and pEC28 32 v i i ACKNOWLEDGEMENT I would l i k e t o thank Dr. R. M i l l e r f o r h i s encouragement, g u i d a n c e , i n s t r u c t i o n and s u p p o r t t h r o u g h o u t the c o u r s e o f my r e s e a r c h . I am v e r y g r a t e f u l t o Drs. D. K i l b u r n and T. Warren f o r t h e i r h e l p f u l comments and s u g g e s t i o n s . T h i s t h e s i s i s d e d i c a t e d t o my w i f e J e n n i f e r f o r a l l her u n d e r s t a n d i n g and moral s u p p o r t . 1 INTRODUCTION M i c r o o r g a n i s m s c a p a b l e o f d e g r a d i n g c e l l u l o s e have a g r e a t e c o -nomic p o t e n t i a l f o r c o n v e r t i n g m u n i c i p a l and i n d u s t r i a l w a stes i n t o s u b s t r a t e s s u i t a b l e f o r f e r m e n t a t i o n . The c o n v e r s i o n o f c e l l u l o s e t o g l u c o s e i n v o l v e s a s e r i e s o f r e a c t i o n s m e d iated by enzymes known as c e l l u l a s e s w h i c h a r e s y n t h e s i z e d by p r o k a r y o t i c and e u k a r y o t i c m i c r o -o r g a n i s m s . Complete breakdown o f c e l l u l o s e t o g l u c o s e i s a c c o m p l i s h e d by t h r e e d i f f e r e n t t y p e s o f c e l l u l a s e enzymes: an endo-$'1 ,4-g 1 ucanase w h i c h c l e a v e s c e l l u l o s e i n t e r n a l l y ; an exo-6 1,^-glucanase w h i c h remo-ves d i s a c c h a r i d e u n i t s known as c e l l o b i o s e m o l e c u l e s from the e x t r e m i -t i e s o f the c e l l u l o s e c h a i n ; and a 3 - g l u c o s i d a s e w h i c h c l e a v e s c e l l o -b i o s e i n t o two g l u c o s e u n i t s . The i n s o l u b l e c r y s t a l l i n e n a t u r e o f c e l l u l o s e p r e s e n t s a major d i f f i c u l t y i n d i r e c t m i c r o b i a l c e l l u l o s e d e g r a d a t i o n . F u r t h e r m o r e , the a s s o c i a t i o n o f c e l l u l o s e w i t h o t h e r complex compounds such as l i g n i n make i t r e f r a c t o r y t o m i c r o b i a l ' i n v a s i o n v B a c t e r i a l i k e C e l 1 u - lomonas f i m i s e c r e t e c e l l u l a s e s t h a t p e n e t r a t e the wood m a t r i x and a t t a c k the c e l l u l o s e e x t r a c e l 1 u l a r l y . U s u a l l y , the amount o f c e l l u l a s e s e c r e t e d and the subsequent r a t e o f c e l l u l o s e d i g e s t i o n a r e low. One approach t o i n c r e a s i n g c e l l u l a s e p r o d u c t i o n by c e l l u l o l y t i c o r g a n i s m s would be t o i s o l a t e the genes c o d i n g f o r c e l l u l a s e s on r e - -combinant DNA p l a s m i d s and then t o m o d i f y the e x p r e s s i o n o f t h e s e genes by c u r r e n t m o l e c u l a r g e n e t i c t e c h n i q u e s . T h i s t h e s i s r e p o r t s the s u c -c e s s f u l m o l e c u l a r c l o n i n g o f c e l l u l a s e genes from C e l l u l o m o n a s f i m i on the p l a s m i d pBR322 i n E s c h e r i c h i a c o l i . The c l o n i n g o f s p e c i f i c 2 cellulase DNA framents in E. coli required a procedure for identi-fying those clones which had acquired recombinant plasmids containing the desired gene sequence. The development of a sensitive screening procedure for E_. col i carrying cellulase genes was the major problem addressed in this thesis. A common assay for the isolation of specific recombinants is based on the immunological detection of translation products produced by cloned foreign genes. These immunoassays provide an approach when it is not possible to use selection techniques that depend on nucleic acid hybridization or functional expression of the cloned genes (1). Immunoassays are capable of detecting incompletely translated products as well as proteins which have no easily detectable or selectable function (7). This immunological approach to screening cellulase producing clones was adopted because the desired cellulase genes encode proteins for which specific antibodies could be produced. One limita-tion of most immunoassay/ screening methods is that each desired pro-tein molecule must simultaneously bind two different antibody molecules (1,2,4,7,9). The screening procedure outlined in this thesis is based on binding cellulase from lysed colonies directly to CNBr-activated paper. The bound antigen is then treated with anti-cel1ulase serum. "The antigen-125 antibody complex is detected by autoradiography using I-labeled protein A from Staphylococcus aureus. The method demands binding of only one antibody molecule per antigen and therefore should allow the use of monoclonal antibodies which in general bind to only one deter-minant (9) • 3 In addition to the development of an immunological screening method, the construction and characterization of recombinant plasmids containing cellulase genes was also accomplished and is presented in this thesis. 4 MATERIALS AND METHODS A . B a c t e r i a l s t r a i n s and m e d i a . The b a c t e r i a s t r a i n s used w e r e : £ . f i m i ATCC 484; E_. c o l i SF8(pBR322) ; and E_. c o l i C600. C_. f i m i was grown i n b a s a l medium ( i g NaNO^, 1g ^ H P O ^ , 0.5g K C l , 0.5g M g S 0 i ( . 7 H 2 0 , 0.5g y e a s t e x t r a c t , pe r l i t e r , pH 7.0) and e i t h e r 1g g l u c o s e o r Ig c a r b o x y m e t h y l e e l l u l o s e (CMC) pe r l i t e r as an e n e r g y s o u r c e (8). E_. c o l i s t r a i n s were grown i n LB (10g t r y p -t o n e , 5g y e a s t e x t r a c t , 5g N a C l , 1g g l u c o s e , pe r l i t e r , pH 7-4) o r M9S medium (3) s u p p l e m e n t e d w i t h u r i d i n e (200ug /ml ) , t h i a m i n e (20ug/ml) and t h y m i d i n e ( I 0 u g / m l ) . A m p i c i l l i n (50ug/ml) o r t e t r a c y c l i n e (20ug/ml) was used when g r o w i n g b a c t e r i a c o n t a i n i n g p l a s m i d s . S o l i d med ia c o n t a i n e d 11g a g a r pe r l i t e r . Top a g a r c o n t a i n e d 1Og t r y p t o n e , 5g NaCl and 7g a g a r pe r l i t e r , pH 7-4. 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 . C e l l u l a s e a c t i v i t y was a n a l y z e d by m e a s u r i n g the i n c r e a s e i n r e d u c i n g g r o u p s by t he h y d r o l y s i s o f a c a r b o x y m e t h y l e e l 1 u l o s e s u b -s t r a t e (15) . R e a c t i o n m i x t u r e s c o n t a i n e d 0.5 ml o f a p p r o p r i a t e l y d i l u t e d enzyme s o l u t i o n and 1.0 ml o f h% CMC i n 100 mM_ p h o s p h a t e b u f f e r , pH 7 . 0 . A f t e r 30 m i n u t e s a t 37°C, 1.5 ml d i n i t r o s a 1 i c y c l i c a c i d (DNS) r e a g e n t (12) were a d d e d , and t he t u b e s were p l a c e d i n b o i l i n g w a t e r f o r 15 m i n u t e s . The a b s o r b a n c e was read a t 550 nm a g a i n s t b l a n k s c o n t a i n i n g e q u i v a l e n t amounts o f b o i l e d enzyme. One u n i t o f enzyme r e l e a s e d 1ug o f g l u c o s e e q u i v a l e n t s pe r m i n u t e 5 by reference to a standard curve. Protein concentrations were der-termined by the Bio-Rad protein assay. C. Preparation of antisera. C_. fimi was grown to late log phase in CMC basal medium. The cells were removed by centrifugation, and the supernatant was con-centrated 100-fold by ultrafiltration (Amicon PM10 membrane) at 4°C. Residual cells were removed by passage of the concentrate' through a 0.45 um membrane (Mi 11ipore). One ml samples, containing approximately 1500 units of total cellulase activity, were mixed with 1 ml complete Freund's adjuvant and injected into mature white New Zealand rabbits. Booster injections of 1500 units of cellulase were given in the same manner four and six weeks later. The rabbits were sacrificed, and the sera were collected a week after the last i njection. D. Preparation of CNBr-act?vated paper. This paper was prepared for the covalent attachment of protein by a modification of a previous method (4). Twenty Whatman No. 40 paper disks were washed in water. The disks then were placed in 500 ml 2M Na2C0^  and mixed by occassional swirling. Ten ml of acetoni-trile solution of CNBr (2g of CNBr per ml of anhydrous acetonitrile) was added, and the mixture was swirled vigorously until the crystals disappeared. The reaction was stopped by decanting the CNBr solu-? tion, and the disks were washed with 0.1 NaHCO^ . The activated ? disks then were sequentially washed by suction with 0.1M NaHC0_ 6 water, 50% acetone in water and acetone. The disks were dried and stored over dessicant at h°C. 125 E. Preparation of l-labeled protein A. 125 l-labeled protein A was prepared with Bio-Rad enzymobead radioiodination:reagent. The first of two radioactive peaks from a Bio-Gel P10 column was pooled. This pooled fraction contained 125 6 l-labeled protein A with a specific activity of 5x10 cpm/ug protein. F. Immobilization of proteins from colonies lysed in situ. C_. fimi grown on plates containing CMC medium (induced) and plates containing glucose medium (un-induced) were lysed in situ by overlaying the agar with 2 ml top agar containing sodium dode-cyl sulfate (SDS) (0.25mg/ml) and lysozyme (0.5mg/ml) (7). The plates were covered, incubated at 30°C for 3 hr and then inverted over chloroform-saturated paper disks for 15 min at room tempera- :' ture. CNBr-activated paper disks were soaked in 25 mM_ phosphate," pH l.k, then placed on top of the lysed colonies. In some cases, the disks were used without prior activation. The plates were covered, and incubated at 30°C for 3 hr. The disks were removed and placed in 1 M g1ycine-phosphate buffer, pH 7.k at 30°C for 5 hr to inactivate residual protein binding sites, then stored overnight at h°C. 7 G. Detection of cel1ulase-producing colonies. After inactivating the residual protein binding sites with glycine, the disks were washed with phosphate buffered saline (PBS) then:.incubated with rabbit anti-cel1ulase serum (diluted 1/250-1/1000) for 3 hr at room temperature. The disks then were washed, 125 suction filtered with PBS, and incubated with l-labeled protein A (diluted to 5x10^  cpm/ml) for 3 hr at room temperature. Finally, the disks were washed, suction filtered, dried at k2°C for 1 hr and placed over Kodak XRP-1 film for 10-20 hr. All dilutions of anti-125 serum and l-labeled protein A were made in PBS containing \% bovine serum albumin. H. Isolation and purification of DNA. C_. fimi DNA was obtained by treating C_. fimi cel 1 s sequentially with lysozyme, RNase, SDS and pronase (11). It was purified by phenol extraction and banding in CsCl density gradients (6). Plasmid DNA was isolated by gently lysing chloramphenicol am-plified E_. col ? plasmid carrying strains with lysozyme and triton. Plasmids molecules in the cleared lysates were purified by banding in CsCl density gradients. A rapid microscale technique for isolating plasmid from pos i t i ve-ce 1 1 ul ase producing E_. col i was used.to initially charac-terize recombinant clones (10). After amplification with chloram-phenicol, cells were ruptured with lysozyme. Buffer-equilibrated • phenol was added, and the acqueous layer was removed. This volume was ethanol precipitated and the resulting pellet was dissolved 8 in water. I. Cloning of BamHI fragments of C. fimi DNA into E. coli. Purified pBR322 DNA was linearized with BamHI in BamHI buffer (l50mM'NaCl, 6mM Tris-HCl, pH 7-9, 6mM MgCl2, and 100ug gelatin/ml). C_. fimi chromosomal DNA was partially digested with BamHI under similar buffering conditions. Digestions were terminated by phenol extraction. The extent of digestion was monitored by horizontal gel electrophoresis. A lug sample of linearized pBR322 was mixed with 5ug of a BamHI partial digest of C_. fimi DNA; and the DNA was precipitated with ethanol. After adding 50 ul ligase buffer (66mM_ Tri s-HCl , pH 7-8, 10mM MgCl2, 20m^  dithiothreitol, 1mM ATP and 50ug gelatin/ml), the mixture was kept at 0°C for 3 hr. Th DNA ligase was ad'ded, and the mixture was held at k°C for 14 hr, at 14°C for 8 hr, and then used to transform E.coli C600 to ampicillin resistance (5). The transformed cells were stored at -20°C in LB containing k0% glycerol. The proportion of cells carrying recombinant plasmids was determi-ned by plating appropriately diluted samples on ampicillin containing medium ± tetracycline. J. Screening recombinant E. coli clones for cellulase activity. Colonies containing recombinant plasmids were picked onto LB plates supplemented with ampicillin and grown overnight at 30°C. The colonies were lysed and the proteins were immobilized as out-lined above. Induced C. fimi cells were spotted at defined posi-9 ions as markers and as positive controls. Screening was performed as outlined above. Potential cel lulase-producing E_. col i col on ies were picked either from replica plates or from the original lysed plates and re-streaked on LB plates. These colonies were screened a second t i me. K. Enzymes and reagents. Jk DNA ligase and the restriction enzymes were from New England Biolabs. Protein A was obtained from Pharmacia. DNS reagent contained 10g (0„N)„C.,H_-2-(OH)C0_H, 2.0g C,Hr0H, 0.5g L L D L L o p Na2S03> 200g KO^C^H^Na.kW£>, 10g NaOH, per liter. PBS contained 8g NaCl, 0.2g KC1 , 0.2g KH2P0/f, 2.17g Na2HP0/j.7H20, per liter, pH l.k. 10 RESULTS A. Development of a sensitive immunologj cal screening method to  detect eel 1ulase enzymes. The goal of this work was to clone the C_. fimi cellulase genes into the E_. col i plasmid pBR322. The initial step was the develop-ment of a sensitive and simple screening method for cellulase gene products. The approach adopted was immunological. Proteins from lysed colonies were transferred to CNBr-activated papers; which then were treated with-anti-cel1ulase antibody. Antibody bound to the 125 filters was detected by autoradiography using I-protein A which binds specifically to the portion of IgG molecules. Log-phase culture supernatants from C_. fumi growing on CMC medium contained 20 units of cellulase activity per ml. Concentr-ate's of such supernatants were injected into rabbits to elicit anti-body production (see MATERIALS AND METHODS). Log phase rather than stationary phase cultures were chosen to limit contamination with intracellular proteins released by cell death and lysis. These contaminating proteins would greatly increase the number of false-positive clones detected during screening. The presence of anti-eel 1ulase antibodies in the rabbit serum was demonstrated by using it to prepare an immunoadsorbent column that was subsequently shown to remove cellulase activity from C_. fimi culture supernatant (Figure 1). Protein A bound sepharose was used to bind anti-cellulase antibodies and these antibodies in turn bound to and removed cellulase from C. fimi culture 11 Figure 1.. Immunoabsorption of C. fimi cel1ulase enzymes by anti-cell ulase antibody bound to a protein A column. Sera from normal and cel1ulase-immunized rabbits were compared for their cellulase binding properties. Normal serum (1 ml) was added to a protein A-Sepharose CL-kB column (5 ml). After washing with PBS, 250 units of C_. fimi cellulase was added , and the column was eluted with PBS. One ml fractions were collected, and cellulase activity was analyzed by measuring the increase in reducing groups released by the hydrolysis of a CMC substrate (circles). The column was eluted with glycine-HCl buffer (pH 3-0) and re-equili-brated with PBS. The experiment then was repeated with serum from the cel1ulase-immunized rabbit added to the column, (squares). 12 Figure 1 . 100 80 2 4 6 8 10 Fract ion no. 13 supernatants. A control column prepared using normal rabbit serum did not absorb the enzyme activity. Experiments with phage 11 and anti-T7 serum showed that antigen could be bound directly to CNBr-activated paper and that less than 1 ng of bound antigen could be detected with antibody 125 and l-labeled protein A. To determine the specificity and sensitivity of the immuno-logical screening method, induced and un-induced C_. fimi colonies as well as E_. col i C 6 0 0 colonies were screened as outlined in MATERIALS AND METHODS. It was shown that E_. col i colonies gave a negative response, un-induced C_. fimi a weak positive, and induced C^. fimi colonies a strong positive response to the anti-cellulase serum when screened in the same manner (Figure 2 ) . Some cellulases have a high affinity for cellulose. In-duced colonies of C_. fimi still gave a strong positive response by this screening method when the filter paper was used without CNBr-activation. However, since it was not known if all C_. fimi cellulases would bind strongly to native paper, the CNBr-activ-ated paper:was used routinely in the present work. B. Construction and identification of a cellulase producing  recombinant clone. On the assumption that cellulase genes might contain inter-nal BamH I restriction sites, a partial BamH I digest of C_. fimi DNA was prepared and used for the construction of recombinant plasmids from pBR322 . A higher concentration (5 times more) Figure 2. Autoradiogram of C_. fimi colonies. This autoradiogram represents the results of an experiment designed to demonstrate the specificity and sensitivity of the immunological screening method described in MATERIALS AND METHODS. Uninduced C_. fimi and E_. col i colonies were grown on LB plates and screened using (A) CNBr-activated paper and (B) normal paper. The location of E_. col i colonies is indicated by circles. Induced — f' m' colonies were grown on CMC-plates and screened using (C) CNBr-activated paper and (D) normal paper. 15 F i g u r e 2. 16 of C_. fimi DNA to pBR322 DNA was used in attempts to increase the proportion of transformants containing plasmids with C_. fimi i nserts. Cloning restriction fragments into the BamHI restriction site in the plasmid pBR322 leads to inactivation of the tetracycline resistance gene in the native plasmids. Transformants containing recombinant plasmids were selected initially by their ability to grow on media containing ampicillin and their inability to propagate on media supplemented with tetracycline. Some k0% of the E_. coli ampici 11 in transformants obtained were also tetra-cycline sensitive, an indication of the presence of inserted DNA. These ampicillin resistant, tetracycline sensitive colonies were picked on LB-plates supplemented with ampicillin and grown overnight at 30°C. Screening for cellulase activity was performed as described in MATERIALS AND METHODS. A trial experiment showed that with an anti-eel 1ulase serum dilution of 1/250, sites where E. coli C600 colonies had been lysed appeared clear against a darker background. The darker background was assumed to be caused by the presence of contaminating anti-yeast antibodies binding to components of the yeast-extract present in the medium. The back-ground was eliminated by diluting the antiserum 1/1000 or by excluding yeast extract from the medium. One positive clone was obtained in the initial screening of 1000 clones (Figure 3). Upon re-streaking and repeated screening, it was confirmed as an immunoreactive, recombinant clone. Phage typing with bacteriophage T7 confirmed that the bacterium was 17 Figure 3- Autorad i ogram of an E_. col i colony producing cellulase. E_. coli containing recombinant plasmids were spotted on LB-plates supplemented with ampicillin and incubated at 37°C for 14 hr. Colonies then were screened as outlined in MATERIALS AND METHODS at a serum dilution of 1/250. Two C. fimi colonies were used as references and positive controls. One E_. col i colony was detected which carried a recombinant plasmid pDW1 encoding C. fimi cellulase. 18 19 col' • The plasmid it contained was given the designation pDW1. C. Enzymatic characterization of the recombinant plasmid The doubly-screened, positive recombinant clone was grown overnight in LB supplemented with ampicillin. Cells were concent-rated 100-fold in 100 mM phosphate buffer pH 7.0. DNase (25 ug/ml) was added, and the cells were ruptured by passing them twice 2 through a French Press (12000 lb/in ). The extract was cent-rifuged at 25,000 rpm in a Beekman type 40 rotor for 60 min. Supernatants were collected and assayed for cellulase activity as outlined previously. Cell-free extracts of E_. col i pDW1 contained 5 units of cellu-lase per ml of original culture volume. The activity was approx-imately linear with respect to amounts of extract added over the range of 1.5 mg to 6.0 mg protein (10 ul - hO ul of extract) (Figure 4). No activity was detectable in an extract of E_. col i pBR322 over an identical range of added protein. As was the case with C_. fimi culture supernatants, the cellulase activity in the E_. coli pDW1 could be absorbed onto immobilized anti-C_. fimi cellulase antibodies (Figure 5) . To determine whether the cytoplasm or the perip.lasmic space was the major site of cellulase accumulation in E_. col ? pDW1 , cell cultures were subjected to osmotic shock. E_. col ? pDW1 cells were first suspended in a concentrated solution of sucrose ( 1 3 ) . The suspension then was treated with ethylene-diaminetetraacetate (EDTA) and pelleted by centrifugation. 20 Figure k. Cellulase activity of E_. col ? carrying pDW1 DNA. E_. col i carrying recombinant plasmid pDW1 DNA was grown to g 5x10 /ml in L-broth. The cells were collected by centrifugation, lysed in a French press and assayed for cellulase activity by the colorimetric assay outlined in MATERIALS AND METHODS (circles). The experiment was repeated with E. coli pBR322 (squares). c 22 Figure 5- Immune-adsorption of E. coli pDW1 cellulase enzymes by anti-cellulase antibody "bound to a protein A column. Normal serum was added to a protein A-Sepharose CL-4B column. After washing with PBS, 350 units of E_. col ? pDW1 cellulase were added to the column. Fractions of 1.5 ml were collected and assayed colorimetrically for cellulase activity (circles). The column was eluted with glycine-HCl buffer (pH 3.0) and re-equi-librated with PBS. The experiment was repeated using anti-cellu-lase serum instead of normal serum (squares). 23 2k The pellet was rapidly mixed with a medium of low osmotic strength (water) and the supernatant known as the cold water wash, was col-lected. The remaining cell pellet was resuspended in phosphate buffer, and the cells were ruptured using the French Press to ob-tain a cell-free extract. The cold water wash contained periplas-mic proteins, and the cytoplasmic proteins were found in the cell extract. The cell extract and the cold water wash were assayed and compared for cellulase activity (Table l). It was discovered that the majority of the cellulase produced by E_. col i pDW1 was localized in the periplasmic space. D• Molecular characterization of the cel1u1ase-encoding plasmid The plasmid from E_. col ? pDW1 was isolated using the rapid microscale technique outlined in MATERIALS AND METHODS. The plasmid was digested with BamHI and analyzed by agarose-gel electrophoresis (Figure 6). It contained four components: one, of k.k kilobases, corresponded to linear pBR322; the other three, of 1.6, 6.6 and 12 kilobases, presumably were C_. fimi DNA. The guanine and cytosine content in C^. fimi DNA is close to 73% (16). Assuming that the Z_. fimi insert in the plasmid pDW1 contained base sequences characteristic of C_. fimi DNA, it would be possible to predict the extent of insert digestion using various restriction enzymes. The plasmid pDW1 was initially digested with BamHI followed by separate digestions with five other restriction enzymes (Figure 7)- As predicted, those restriction enzymes having 25 Tab 1e 1. Localization of E. coli pDWl cellulase in the periplasmic space. cytoplasm (cell extract) peripiasmic space .(cold Water wash) Total protein 3-25mg/ml 0.36mg/ml 3-lactamase activity 1x Cellulase activi ty 6 units/ml S.k units/ml Cellulase/mg protein 1.8 uni ts/mg 26 units/ml The cell extract and the cold water wash from osmotically shocked cells were compared for cellulase activity. Protein concentrations were determined using the Bio-Rad protein assay. To monitor the effectiveness of the osmotic shock procedure, the activity of the periplasmic enzyme 3-lactamase was determined using nitrocefin. Cellulase activity was measured colorimetrically as outlined in MATERIALS AND METHODS. 26 Figure 6. Agarose-gel electrophoresis of BamHI digested plasmid pDWl. Purified pDWl plasmid was isolated and digested with the restriction enzyme BamHI. The digested pDW1 plasmid was electro-phoresed on a 0.6% agarose gel (B). Plasmid pBR322 linearized with BamHI was also electrophoresed in the gel (A). Ethidium bromide was used to stain the gel. 27 28 Fi gure 7- Restriction enzyme characterization of the plasmid pDW1. The plasmid pDWl was first digested with BamHI restriction enzyme (A). Following dialysis of separate aliquots of pDW1 BamHI cut DNA in appropriate restriction enzyme buffers, the following restriction enzymes were used to digest the DNA; BglI I (B) , PstI (C), Sa_N (D), Xhol (E), and Hindi I I (F). Digested DNA was elec-trophoresed through a 0.6% agarose gel. Ethidium bromide was used to stain the gel. 2S F i g u r e 7. 30 predominantly guanine and cytosine in their recognition sequences (Pst I, Sal I and Xhol) were able to extensively digest the C_. fimi insert. On the other hand, those restriction enzymes having mainly adenine and thymine in their recognition sequence (Bg1 I I and H i nd I I I) were unable to cut the C_. fimi insert. E. Isolation of other eel 1ulase producing recombinant clones Following the isolation and characterization of E_. col ? pDW1, approximately 5,000 additional recombinant plasmid containing colonies were screened immunologically as outlined in MATERIALS AND METHODS. Two distinct positive-cellulase producing colonies were identified. These clones were classified as E_. col i pEC7 and IE. col i pEC28. E_. col i pEC7 and E_. col i pEC28 were initially classified as distinct because they produced different intensities on the autoradiograms. E_. col i pEC7 produced an intense dark spot similar to that produced by the control or marker C_. fimi location. E_. col i : pEC28 however produced a spot on the autoradiogram that was approximately half as intense as that observed in E_. col i pEC7. From these results, E_. col i pEC7 was classified as a high immuno-genic clone, while E_. col i pEC28 was considered a low immunogenic clone. E_. col i pDWl was also classified as a high immunogenic clone because the intensity that it produced on the autoradiogram was similar to that produced by E_. col ? pEC7. Cellulase assays indicated that cell-free extracts of E_. coli pEC7 contained approximately 5 units of cellulase per ml of origi-31 nal culture volume, while E_. col i pEC28 cell-free extracts contai-ned 35 units of cellulase per ml of original culture volume. BamH I digestion of the plasmid isolated from E_. col i pEC7 followed by agarose-gel electrophoresis revealed a 6.6 kilobase gene fragment in addition to the k.k kilobase pBR322 vector plasmid (Figure 8 ) . Recombinant plasmid isolated from E_. col i pEC28 were characterized by a 5 . 0 kilobase gene fragment insert. 32 Figure 8. Agarose-gel electrophoresis of BamHI digested plasmids pEC7 andpEC28. Purified plasmid DNA was isolated and digested with the rest-riction enzyme BamHI. Digested pEC7 (A) and pEC28 (B) were elecr trophoresed on a 0.6% agarose gel. Plasmid pBR322 linearized with BamHI was also electrophoresed in the gel (C). Ethidium bromide was used to stain the gel. 33 F i g u r e 8 . A B C 34 DISCUSSION .' A. A general immunological screening method to detect cloned gene  products. The direct immunological screening method described in this thesis represents a very useful and sensitive general method for defecting •.clones containing defined genes. The assay has been suc-cessfully used in the identification of recombinant plasmids carrying cellulase genes from C_. f imi. This screening method has several advantages. First, the use of CNBr-activated paper to bind proteins released from lysed colonies renders the technique generally applica-ble for the screening of any recombinant clones which express a pep-tide for which an antibody is available. The use of l-labeled protein A to detect bound antibody makes possible the use of one labelled probe for many different screenings. A similar technique has recently been developed independently (9). In the present studies, CNBr-activated paper was used routinely, although because of the affi-nity of cellulases for their substrate, the use of activated paper can be omitted. This simplifies the procedure when applied in genetic engineering experiments designed to increase yield or to develop export of the cellulase encoded by pDW1. The sensitivity of the technique was estimated to be in the nanogram range, certainly adequate to detect the production of cel-lulase. in un-induced C_. f im? . In addition, the technique is responr' sive to the amount of cellulase in induced C_. fimi , which represents an increase of at least two orders of magnitude compared to the un-35 induced state. This will facilitate experiments designed to;increase levels of cellulase production. Another advantage of the procedure described here is the ability to recover live cells from:the bottom of colonies on the original lysed plate. This eliminates the necessity of replica plating (7). Furthermore, with minor variations, this immunoassay technique pro-vides a simple way to monitor the movement of antigens on polyacryla-mide gels. The direct screening method reported here has been shown to be sensitive, quantitative and reproducible, and should allow the use of monoclonal antibodies. The assay could be adapted to identify specific translated products produced by desired cloned foreign genes. B. Characterization studies on cellulase producing clones. Having developed a sensitive cellulase screening method, stan-dard recombinant DNA techniques were used to cut and 1 igate C_. fimi DNA fragments into the plasmid pBR322. Two important procedures were implemented to increase the probability of obtaining a cloned cellulase gene. First, a partial BamHI digest of £. fimi DNA was used in the construction of recombinant plasmids to insure that cellulase genes with internal BamHI sites would be cloned and iden-tified. Second, in an effort to increase the number of recombinant clones, a higher concentration of £. fimi DNA to pBR322 was used in the ligation reaction. It is encouraging that the cellulase made by E. coli pDW1 36 appeared to be transported to the periplasmic space. This suggests that a leader sequence, which is commonly associated with the aniino-terminal of excreted proteins, has also been cloned (14). This enzyme excretion mechanism will allow for the use of relatively simple and easy methods for cellulase purification. It is interesting that E_. coli pDWl carried pBR322 with a 20.2 kilobase insert without any apparent abnormalities in the growth of the bacteria or the plasmid. Since we used a partial BamHI clea-vage of C_. fimi DNA in the original recombinant DNA formation, it is not possible'to say whether or not the insert represents a conti-guous portion of the C_. fimi DNA. A number of similarities exist between E_. col i pDW1 and E_. col i pEC7 which indicate that they contain the same cellulase gene. Both clones produce the same amount of cellulase and they both have high immuna>genecity as indicated by the intensity that they produce on the autoradiogram. In addition, plasmids pDW1 and pEC7 are charac-terized by the presence of a 6 . 6 kilobase C_. fimi gene sequence. It is' interesting that even though E_. col i pEC28 hasMowl:immuno-genecity,this clone produces 7 times more cellulase then the high immunogenic clones E_. col i pDWl and E_. col i pEC7. It is possible that a comparitively small amount of cellulase is synthesized by E_. col i pEC28 but that this cellulase enzyme is much more active in catalysing the hydrolysis of a cellulose substrate then are the cellulases produced by E_. col i pDW1 and E_. col 1 pEC7. It is also possible that the same quantity of cellulase protein is produced by each positive clone but that the antibody titer for the cellulase 37 produced by E_. col i pDW1 and E_. col I pEC7 is higher then the anti-body titer of E_. col i pEC28 cellulase. Improper post-translational modification could alter antigen-antibody binding efficiencies which might account for the unexpected low immunogenecity observed in IE. col? pEC28. A number of factors could influence the amount of cellulase production in the E_. col ? eel 1 ul ase clones. The most apparent factors include: cellulase gene copy numbers; regulation or indu-cibi1i ty of eel 1 u lase genes; stabi1ity of eel 1 ul ase in E_. col i ; and, transcriptional or translational differences between Gram-positive and Gram-negative systems. Any one or a combination of the above factors could influence the level of cellulase present in the E. coli cellulase clones. 38 LITERATURE CITED 1. Anderson, D., Shapiro, L. and Skalka, A.M.: In situ immunoassay for translation products, in Wu, R. (Ed.), Methods in Enzymology, Vol. 68, Academic Press, Toronto. 1979, pp. 428-436. 2. Broom, S. and Gilbert, W.: Immunological screening method to detect specific translation products. Proc. Natl. Acad. Sci. USA. 75 (1978) 2746-2749. 3. Champe, S.P.,and Benzer, S.: Reversal of mutant phenotypes by 5~flu-orouracil; An approach to nucleotide sequences in messenger-RNA. Proc. Natl. Acad. Sci. USA. 48(1962) 5 3 2 - 5 4 6 . 4. Clarke, L., Hitzman, R. and Carbon, J.: Selection of specific clones from colony banks by screening with radioactive antibody, in Wu, R. (Ed.), Methods in Enzymology, Vol. 68, Academic Press, Toronto. 1979, PP. 4 3 6 - 4 4 2 . 5- Cohen, S.N. and Chang, A.C.Y.: Recircularization and autonomous replication of a sheared R-factor DNA segment in Escherichia  coli transformants. Proc. Natl. Acad. Sci. USA. 70(1973) 1293" 1297. 6. Dunn, R., Delaney, A.D., Gilliam, I.C., Hayashi, S., Tener, G.M., Grigliatti, T., Misra, V., Taylor, D.M. and Miller, R.M.: Iso-lation and characterization of recombinant DNA plasmids carrying Drosophila tRNA genes. Gene. 7(1979) 197-215. 7- Erlich, H.A., Cohen, S.N. and McDevitt, H.O.: A sensitive radioimmu-noassay for detecting products translated from cloned DNA fragments. Cel1. 13(1978) 681-689. 8. Hitchner, E.V. and Leatherwood, J.M.: Use of a cel1ulase-derepressed mutant of Cel1ulomonas in the production of a single-cell protein product from cellulose. Appl. Environ. Microbiol. 39(1980) 382-386. 9- Kemp, D.J. and Cowman, A.F.: Direct immunoassay for detecting Escherichia coli colonies that contain polypeptides encoded by cloned DNA segments. Proc. Natl. Acad. Sci. USA. 78(1981) 4520-4524. 10. Klein, D. , Seising, E. and Wells, R.D.: A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis. Plasmid. 3(1980) 88-91 -11. Lovett, P.S. and Keggins, R.M.: Baci11 us subti1?s as a host for molecular cloning, in Wu, R. (Ed.), Methods in Enzymology. Vol. 68, Academic Press, Toronto, 1979, pp. 342-357-39 Miller, G.L.: Use of dinitrosalicyclic reagent for determination of reducing sugars. Anal. Chem. 31(1959) 426-428. Neu, H.C. and Heppel, L.A.: The release of enzymes from Escheri- chia coli by osmotic shock and during the formation of sphero-plasts. J. Bio. Chem. 240(1965) 3685- 3692. Palva, I ., Petterson, R., Kalkkinen, N., Lehtovaara, P., Sarva, M., Soderlund, H., Takkinen, K. and Kaariainen, K.: Nucleotide sequence of the promoter and NH2_terminal signal peptide region of the amylase gene from Bacillus amyloliquefaciens. Gene. 15 (1981) 43-51. Stewart,.B..J. and Leatherwood, J.M. Derepressed synthesis of Cellulomonas. J. Bacteriol. 128(1976) 609-615. Yamada, J. and Komagata, L.: Genus Cel1ulomonas, 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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