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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.Sc,  Queen's  A T H E S I S SUBMITTED  University,  1980  IN P A R T I A L FULFILMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES ( Department of  We a c c e p t to  this  the  Microbiology  t h e s i s as  required  conforming  standard  THE UNIVERSITY OF B R I T I S H August,  0  Daniel  )  COLUMBIA  1982  Joseph W h i t t l e ,  1982  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by  the head of  representatives.  understood t h a t copying or p u b l i c a t i o n of t h i s f o r f i n a n c i a l gain  Department of  Microbiology  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  CT/R-n  August  5,  1982  my  It is thesis  s h a l l not be allowed without my  permission.  thesis  written  ii  ABSTRACT  Recombinant DNA techniques were used to clone and isolate Cellulomonas 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-activated 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 containing 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 Abstract  i i  L i s t of Tables  v  Li s t o f Fi gures  vi  Acknowledgement  v i i  Introduction  1  M a t e r i a l s and M e t h o d s A.  Bacterial  strains  and media  ^  B.  C o l o r i m e t r i c c e l l u l a s e assay  C.  Preparation of antisera  D.  Preparation of CNBr-activated  •..  5  paper  5  I O C  E.  Preparation of  F.  Immobilization  l-labeled  p r o t e i n A ..  o f p r o t e i n s from c o l o n i e s  6 lysed  in s i tu  6  G.  Detection  of cel1u1ase-producing  H.  I s o l a t i o n and p u r i f i c a t i o n  I.  Cloning  colonies  o f DNA  o f BamHI f r a g m e n t s o f C. f i m i  7 —  DNA  .  7  into  E_. c o l i J.  Screening  8 r e c o m b i n a n t E_. c o l i c l o n e s  activity K.  Enzymes a n d r e a g e n t s  for cellulase °  8 9  iv  Page  Results A.  Development o f a s e n s i t i v e  immunological  screening  method t o d e t e c t e e l 1u1ase enzymes B.  C.  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  of a  10 cellulase  producing  recombinant clone  13  Enzymatic  c h a r a c t e r i z a t i o n o f the recombinant  19  p i asm i d D.  Molecular 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 encoding  E.  plasmid  ...  .....  I s o l a t i o n o f o t h e r e e l 1ulase  producing  lh recombinant  clones  30  Di s c u s s i o n A.  . A general cloned  B.  immunological  gene p r o d u c t s  s c r e e n i n g method t o d e t e c t :  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 clones  Li t e r a t u r e Ci t e d  3^ producing 35  38  V  L I S T OF TABLES  Table 1  Title Localization periplasmic  o f E_. c o l i pDWl space  Page cellulase  i n the 25  vi  L I S T OF FIGURES  Figure 1  Title  Page  I m m u n o a b s o r p t i o n o f C_. f i m i  cellulase  by a n t i - c e l l u l a s e a n t i b o d y bound A column  enzymes  to a protein . ..  2  Autoradiogram  o f C. f i m i  3  Autoradiogram  o f a n E_. c o l i  11  colonies  14  colony  producing  c e l 1 u l a s e ... . h  17  Cellulase activity  o f E_. c o l i  carrying  pDW1  20  DNA 5  I m m u n o a b s o r p t i o n o f E. c o l i  pDWI  cellulase  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 protein 6  Agarose-gel plasmid  7  plasmids  26 enzyme c h a r a c t e r i z a t i o n o f t h e  pDW1  Agarose-gel  22  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  pDWT  Restriction plasmid  8  A column  28 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  pEC7  and  pEC28  32  vi i  ACKNOWLEDGEMENT  I would guidance,  like  t o t h a n k D r . R. M i l l e r  i n s t r u c t i o n and s u p p o r t  f o r h i s encouragement,  throughout  the course  o f my  research. I am v e r y their  helpful This  grateful  D. K i l b u r n  and T. W a r r e n f o r  comments and s u g g e s t i o n s .  thesis  understanding  t o Drs.  i sdedicated  and m o r a l  t o my w i f e J e n n i f e r  support.  for a l lher  1  INTRODUCTION  Microorganisms capable of nomic p o t e n t i a l  for converting municipal  substrates suitable glucose  involves  for fermentation.  a series  c e l l u l a s e s which are organisms. by  types of  disaccharide units  biose  the  cellulose  i n t o two The  known as  of  a t t a c k the  approach to  c o m b i n a n t DNA by  on  the  to  to  as  micro-  is accomplished  endo-$'1 ,4-g 1 u c a n a s e  m o l e c u l e s from the  to m i c r o b i a l ' i n v a s i o n v  increasing  remo-  extremicello-  presents a major Furthermore,  p l a s m i d s and  of  Usually,  the  cellulase  like  amount o f are  p r o d u c t i o n by  cellulolytic  e x p r e s s i o n of  This thesis  The  cloning  and  low.  on  r e p o r t s the  of  re--  t h e s e genes  c e l l u l a s e genes from C e l l u l o m o n a s coli.  Cel1u-  cellulase  genes c o d i n g f o r c e l l u l a s e s  pBR322 i n E s c h e r i c h i a  as  wood m a t r i x  cellulose digestion  then to m o d i f y the  of  Bacteria  t h a t p e n e t r a t e the  i s o l a t e the  molecular cloning plasmid  eukaryotic  to g l u c o s e  cellulose  current molecular genetic techniques.  cessful  and  c e l l u l o s e w i t h o t h e r c o m p l e x compounds s u c h  subsequent rate  o r g a n i s m s w o u l d be  enzymes known  cellulose degradation.  secrete cellulases  the  cellulose  3 -glucosidase which cleaves  cellulose extracel1ularly.  s e c r e t e d and One  cellobiose a  into  exo-6 1,^-glucanase which  c r y s t a l l i n e nature of  l i g n i n make i t r e f r a c t o r y lomonas f i m i  m e d i a t e d by  cellulose  eco-  units.  in d i r e c t microbial  association  conversion of  prokaryotic  i n t e r n a l l y ; an  have a g r e a t  i n d u s t r i a l wastes  c e l l u l a s e e n z y m e s : an  c h a i n ; and  glucose  insoluble  difficulty the  by  and  The  reactions  Complete breakdown o f  three d i f f e r e n t  t i e s of  of  synthesized  which cleaves c e l l u l o s e ves  degrading c e l l u l o s e  suc-  fimi  specific  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 limitation of most immunoassay/ screening methods is that each desired protein 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 antigen125  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 determinant (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.  Bacterial  strains  The b a c t e r i a  and m e d i a .  strains  SF8(pBR322) ; and E_. c o l i C_. f i m i KCl,  0.5g  either  i (  source  i n b a s a l medium  2  (3)  yeast extract,  (8).  5g N a C l ,  (I0ug/ml).  media c o n t a i n e d  5g NaCl and 7g a g a r p e r  strate  (15).  buffer,  pH 7 . 0 .  After  (DNS) r e a g e n t  boiling  water  against  blanks  One u n i t o f  for  (12)  pH 7.0)  (CMC) p e r  liter,  (200ug/ml), or  containing  and  liter  i n LB (10g  as tryp-  pH 7-4)  thiamine  or (20ug/ml)  tetracycline plasmids.  Top a g a r c o n t a i n e d  1Og  Solid tryptone,  7-4.  was a n a l y z e d by m e a s u r i n g t h e  and 1.0  of  ml o f at  ml o f  h% CMC i n 37°C,  w e r e a d d e d , and t h e  15 m i n u t e s .  increase  a carboxymethyleel 1ulose  c o n t a i n e d 0.5  30 m i n u t e s  containing  0.5g  assay.  Reaction mixtures  enzyme s o l u t i o n  acid  pH  by t h e h y d r o l y s i s  diluted  E_. c o l i  1g ^ H P O ^ ,  liter,  (50ug/ml)  bacteria  liter.  liter,  cellulase  Cellulase activity reducing groups  per  1g g l u c o s e , p e r  Ampicillin  11g a g a r p e r  Colorimetric  NaNO^,  E_. c o l i s t r a i n s w e r e grown  ( 2 0 u g / m l ) was u s e d when g r o w i n g  B.  (ig  supplemented w i t h u r i d i n e  and t h y m i d i n e  ATCC 484;  Ig c a r b o x y m e t h y l e e l l u l o s e  5g y e a s t e x t r a c t ,  M9S medium  fimi  C600.  M g S 0 . 7 H 0 , 0.5g  1g g l u c o s e o r  an e n e r g y tone,  was grown  used w e r e : £ .  1.5  100 mM_ p h o s p h a t e ml  dinitrosa1icyclic  tubes were p l a c e d  enzyme r e l e a s e d 1ug o f  amounts o f  sub-  appropriately  The a b s o r b a n c e was r e a d a t  equivalent  in  boiled  glucose equivalents  in  550 nm  enzyme. per  minute  5  by reference to a standard curve. Protein concentrations were dertermined 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 concentrated 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 acetonitrile 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 solution, 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 125  l-labeled protein A.  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 dodecyl 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 ture.  paper disks for 15 min at room tempera- :'  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 1/2501/1000) 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. the disks were washed, suction filtered, dried at  k2°C  Finally,  for 1 hr and  placed over Kodak XRP-1 film for 10-20 hr. All dilutions of anti125 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 amplified 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 characterize recombinant clones (10). After amplification with chloramphenicol, 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 MgCl , and 100ug gelatin/ml). 2  C_. fimi chromosomal DNA was partially digested with BamHI under similar buffering conditions. extraction.  Digestions were terminated by phenol  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 MgCl , 20m^ dithiothreitol, 1mM ATP and 50ug gelatin/ml), 2  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 determined  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 outlined 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,H 0H, 0.5g r  L  Na S0 2  3>  L D L  200g KO^C^H^Na.kW£>,  8g  NaCl,  pH  l.k.  10g  L  o p  NaOH, per liter.  0.2g KC1 , 0.2g KH P0 , 2.17g Na HP0 .7H 0, 2  /f  2  /j  2  PBS contained per liter,  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 development 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 binds specifically to the  I-protein A which  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 antibody 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 falsepositive 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 anticell ulase antibody bound to a protein A column. Sera from normal and cel1ulase-immunized rabbits were compared for their cellulase binding properties. added to a protein A-Sepharose  CL-kB  Normal serum (1 ml) was  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-equilibrated 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  Fraction  6 no.  8  10  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  C600  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 anticellulase 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-activated 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' ' colonies were grown on CMC-plates and screened using (C) m  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 tetracycline 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 background 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 LBplates 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 rifuged at  25,000  (12000  lb/in ). The extract was cent-  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 cellulase per ml of original culture volume. The activity was approximately 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-equilibrated 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 collected.  The remaining cell pellet was resuspended in phosphate  buffer, and the cells were ruptured using the French Press to obtain a cell-free extract. The cold water wash contained periplasmic 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.  Total protein  cytoplasm  peripiasmic space  (cell extract)  .(cold Water wash)  3-25mg/ml  3-lactamase activity  0.36mg/ml  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 electrophoresed 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 electrophoresed 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 immunogenic 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 (Figure  8).  kilobase  pBR322  vector plasmid  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 restriction 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  Figure  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 successfully 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 applicable for the screening of any recombinant clones which express a peptide 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 affinity 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 cellulase. 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 provides a simple way to monitor the movement of antigens on polyacrylamide 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 identified.  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 aniinoterminal 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 cleavage of C_. fimi  DNA in the original recombinant DNA formation, it  is not possible'to say whether or not the insert represents a contiguous 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:immunogenecity,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 inducibi1i ty of eel 1 u lase genes; stabi1ity of eel 1 ul ase in E_. col i ; and, transcriptional or translational differences between Grampositive 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~fluorouracil; 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,  5-  PP.  436-442.  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.: Isolation 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 radioimmunoassay for detecting products translated from cloned DNA fragments. Cel1.  8.  13(1978) 681-689.  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 Escherichia coli by osmotic shock and during the formation of spheroplasts. 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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