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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., U n i v e r s i t y 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 M i c r o b i o l o g y )  We accept t h i s to  t h e s i s as conforming  the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA November, 1983 © Warren W i l l i a m Wakarchuk, 1983  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 a t 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 t h e s i s f o r s c h o l a r l y purposes may  be  department o r by h i s or her  granted by  the head o f  representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  Department o f  /WicrO  K)>6l®jy  The U n i v e r s i t y of B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3  Columbia  written  .  1  1  ABSTRACT  A shotgun c l o n i n g o f Cellulomonas y i e l d e d 2 types  of recombinant plasmids  (CMCase) a c t i v i t y third  type  (23).  gene h a s now b e e n i s o l a t e d f r o m E. c o l i  characteristics: digestion  and  p a t t e r n a n d map;  i n non-denaturing  E. c o l i  o f E. c o l i  (pEC2), <pEC3).  into Escherichia c o l i  encoding  carboxymethylcellulase  and d e s i g n a t e d (pECl)  pECl  containing a different pEC3.  a n d E. c o l i  t h e s i z e o f t h e C. f i m i  h a l o f o r m a t i o n on CMC-Congo activity  DNA  These c l o n e s were d e s i g n a t e d  of recombinant plasmid  differentiated  fimi  the l e v e l s  DNA  C. f i m i  The new c l o n e  A  cellulase  was  ( p E C 2 ) by t h e f o l l o w i n g insert;  i t s restriction  o f CMCase i n c e l l  Red p l a t e s ;  a n d pEC2.  free  extracts;  a n d t h e m i g r a t i o n o f CMCase  polyacrylamide  gels.  As w i t h E. c o l i  (pECl)  CMCase a c t i v i t y was d e t e c t e d An t h e p e r i p l a s m i c s p a c e  i i i T A B L E OF  CONTENTS  Page Abstract  . . .  List  of Tables  List  of Figures  i v . . . . . . . . . . .  v i  Acknowledgement  v i i  1  Introduction  Materials  and Methods  A.  Bacterial  B.  Colorimetric  C.  DNA  D.  Non-denaturing  E.  Localization  strains  and media  cellulase  . . . . . . .  . . .  assay  Enzymes  3 4  techniques  4 polyacrylamide  of cellulase  strains F.  i  and Reagents  g e l electrophoresis  activity . .  i n E. c o l i  5  . . . . .  (pEC) .  5 6  iv  Page Results A.  S c r e e n i n g f o r c e l l u l a s e producing recombinant  B.  Differentiation  C.  Localization (pEC)  D.  o f pEC3 from pECl and pEC2  of c e l l u l a s e  strains  clones  . . .  . . . . . . . .  7 9  a c t i v i t y i n E. c o l i . . . . . . .  9  D e t e r m i n a t i o n o f the r e s t r i c t i o n map o f pEC3  .  9  Discussion A.  The enzymes  B.  The genes  Literature  Cited  .  25 26  .  . .  28  V  LIST OF TABLES  Table 1.  Title Comparison  of c e l l u l a s e  activity  Page  from  representative  group 3 c l o n e s 2.  Characteristics  3.  Specific  8 of c e l l u l a s e plasmids  19  a c t i v i t i e s of CMCase and marker enzymes i n  fractionated  E. c o l i  (pEC) s t r a i n s  .  20  vi  LIST OF FIGURES  Figure 1.  Title  Agarose-gel electrophoresis  Page  o f Bam HI d i g e s t e d pEC  plasmids  11  2.  Restriction  endonuclease d i g e s t s  3.  Polyacrylamide g e l electrophoresis  of pEC plasmids analysis  13  of c e l l u l a s e  encoded by pEC plasmids 4.  Zones of CMC h y d r o l y s i s  produced by pEC s t r a i n s  15 and v i s u a l i z e d  by Congo Red s t a i n i n g 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  6.  17  R e s t r i c t i o n map of the plasmid pEC3  21 ;  . . .  23  VI  1.  ACKNOWLEDGEMENT  I would l i k e  to thank Dr's. R. M i l l e r , D. K i l b u r n , and T. Warren f o r  t h e i r encouragement, grateful coli  i n s t r u c t i o n and p a t i e n c e d u r i n g my r e s e a r c h .  t o Dr. N. G i l k e s f o r p r o v i d i n g some data on E. c o l i  (pEC2).  I would e s p e c i a l l y l i k e  extreme p a t i e n c e and understanding w r i t i n g of this  thesis.  to thank Maureen  I am  (pECl) and E  Langsford  f o r her  d u r i n g my i l l n e s s proceeding the  1 INTRODUCTION  The  c o n v e r s i o n of c e l l u l o s i c waste to glucose  p r o d u c t i o n of s i n g l e c e l l  p r o t e i n has enormous economic p o t e n t i a l .  major drawback o f e n z y m a t i c a l l y degrading of h i g h l y a c t i v e ,  s t a b l e and i n e x p e n s i v e  systems should be done so t h a t t h e i r f u l l During  the l a s t  e l u c i d a t e d v a r i o u s enzymatic  The  classical  c e l l u l a s e complex  cellulose  The  c e l l u l o s i c m a t e r i a l s i s the l a c k enzymes.  A study o f c e l l u l a s e  p o t e n t i a l can be r e a l i z e d .  30 years r e s e a r c h on c e l l u l o l y t i c  has  synergistically:  f o r f e r m e n t a t i o n or  f u n g i and b a c t e r i a  components r e q u i r e d f o r c e l l u l o l y s i s . i s a consortium  endo-B-1,4-glucanase  o f 3 enzymes which a c t  (E.C.3.2.1.4) which c l e a v e s  randomly; exo-B-1,4-glucanase ( e i t h e r B - D - g l u c o s y l  gluco-  h y d r o l a s e or c e l l o b i o h y d r o l a s e E.C.3.2.1.91) -which removes g l u c o s e or c e l l o b i o s e u n i t s from c e l l u l o s e (B-D-glucoside  c h a i n ends; and B - g l u c o s i d a s e  g l u c o h y d r o l a s e , E.C.3.2.1.21) which produces g l u c o s e  from  c e l l o d e x t r i n s 4 g l u c o s e u n i t s long or s h o r t e r . One method of s t u d y i n g multi-enzyme complexes at the m o l e c u l a r is  to i s o l a t e  to manipulate  the v a r i o u s s t r u c t u r a l genes by m o l e c u l a r them by i_n v i t r o  approach i s best s u i t e d d i r e c t l y without necessary  with  c l o n i n g and then  recombinant DNA procedures.  to b a c t e r i a l genes:  level  T h i s type o f  a) the DNA may be  manipulated  the need to i s o l a t e mRNA and to make cDNA, as i s  f u n g a l genes because of i n t r a v e n i n g sequences; and b)  c l o n i n g v e c t o r s e x i s t with  regulatory signals  may allow i n c r e a s e d e x p r e s s i o n of cloned genes  f o r b a c t e r i a l hosts which (1,4).  The which  i s a gram p o s i t i v e ,  belongs to a genus comprised of s p e c i e s which  (7,27): It  bacterium Cellulomonas f i m i  I t i s easy to grow and  secretes c e l l u l a s e  i s capable of degrading m i c r o c r y s t a l l i n e  considered t r u l y c e l l u l o l y t i c . studied  (9).  identification In  The  degrade into  cellulose  this thesis  cellulose environment.  is therefore  enzyme system of C. f i m i has been  C. f i m i c e l l u l a s e  i n the  genes (23).  the i d e n t i f i c a t i o n of a t h i r d recombinant  encoding a C. f i m i c e l l u l a s e 3 genes i s g i v e n .  the  and  P r e v i o u s work i n t h i s l a b o r a t o r y r e s u l t e d of two  coryneform rod  gene i s r e p o r t e d ; a l s o ,  plasmid  a comparison  of these  3 MATERIALS AND METHODS  A.  Bacterial  s t r a i n s and media  The b a c t e r i a l (pBR322); E. c o l i (pEC3).  These  s t r a i n s used were: C600 ( p E C l ) ; E. c o l i  per l i t e r .  C600; E. c o l i  C600  C600 (pEC2); and E. c o l i C600  s t r a i n s were grown i n LB ( l O g t r y p t o n e ; 5 g y e a s t e x t r a c t ,  5 g NaCl, l g g l u c o s e , per l i t e r , (lOug/ml)  E. c o l i  pH 7.A), supplemented  and thymidine (5 ug/ml).  with thiamine  S o l i d media c o n t a i n e d 11 g agar  S t r a i n s w i t h plasmids were grown with a m p i c i l l i n  The enzymatic  a c t i v i t y o f c e r t a i n recombinant  (50 ug/ml).  c e l l u l a s e gene products  was v i s u a l i z e d by a m o d i f i c a t i o n p f the c a r b o x y m e t h y l c e l l u l o s e - Congo Red p l a t e assay o f l e a t h e r and Wood (21). Minimal media, M9S (3) w i t h o u t g l u c o s e , was prepared w i t h 1.0% high v i s c o s i t y c a r b o x y m e t h y l c e l l u l o s e (CMC), 1.0% Nobel agar, and a m p i c i l l i n on the p l a t e s f o r 24 hours at 30°C, the  plate.  (50 pg/ml).  B a c t e r i a were grown  the c o l o n i e s then were washed o f f  The p l a t e s were f l o o d e d w i t h Congo Red dye (1 mg/ml i n  d i s t i l l e d water) and shaken at. room temperature.  g e n t l y on an o r b i t a l  Excess dye was poured o f f .  f l o o d e d w i t h 1 M NaCl and g e n t l y shaken  shaker f o r 15-30 minutes The p l a t e s then were  t o wash out excess dye.  The 1 M  NaCl wash was r e p e a t e d 2 o r 3 times. Zones o f c l e a r i n g c o u l d be seen a t t h i s time; they appeared y e l l o w w h i l e the p l a t e was r e d . s t o r e d by f i x i n g  the dye w i t h 10% a c e t i c  acid.  P l a t e s c o u l d be  4 B.  Colorimetric cellulase  assay  C e l l u l a s e a c t i v i t y was  analyzed by measuring the i n c r e a s e i n r e d u c i n g  groups from the h y d r o l y s i s of CMC.  C e l l - f r e e e x t r a c t s were prepared  d e s c r i b e d by W h i t t l e e t a l . , (24); these were incubated w i t h CMC f o r v a r y i n g l e n g t h s of time depending on which E. c o l i being a n a l y z e d . salicyclic  r e d u c i n g groups were q u a n t i t a t e d by the  a c i d method of M i l l e r  using glucose. produced  The  (pEC)  (13).  CMCase a c t i v i t y was  per minute per mg  of p r o t e i n .  at 37°C  strain  was  dinitro-  A standard curve was  expressed  as  prepared  as ug g l u c o s e e q u i v a l e n t s  P r o t e i n 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 u s i n g the r a p i d a l k a l i n e l y s i s  (11) with the f o l l o w i n g m o d i f i c a t i o n : microdialysis endonuclease conditions  excess  s a l t was  technique of Marusyk and Sergeant  (12).  technique  removed by Restriction  d i g e s t i o n s were done a c c o r d i n g to the manufacturers (New  England  h o r i z o n t a l agarose  Biolabs).  Restriction  gels using t r i s  acid/ethylenediamine  tetracetic  ug/ml e t h i d i u m bromide. short-wave u l t r a v i o l e t  DNA light.  p o l a r o i d f i l m u s i n g an orange R e s t r i c t i o n endonuclease  (hydroxymethyl)  (11).  Gels c o n t a i n e d 1  Gels were photographed with type  57  filter. s i t e mapping was  l a b e l l e d with a-  32  done by a m o d i f i c a t i o n of  Plasmid DNA -P-dATP and  on  aminomethane/boric  v i s u a l i z e d by f l u o r e s c e n c e under  the Smith and B i r n s t i e l method (19). with EcoRI and end  specified  fragments were s e p a r a t e d  a c i d b u f f e r pH 8.3  was  the  (pEC3) was Klenow  DNA  linearized  5 polymerase  (5).  A 29 base p a i r fragment c o n t a i n i n g one end l a b e l was  removed by d i g e s t i o n with Hind plasmid  s e l e c t i v e l y e n d - l a b e l l e d at the remaining  l a b e l l e d fragment o f  by autoradiography  This l e f t a  EcoRI s i t e .  This  pEC3 then was p a r t i a l l y d i g e s t e d with v a r i o u s  r e s t r i c t i o n endonucleases.  D.  I I I f o l l o w e d by m i c r o d i a l y s i s .  The p a r t i a l d i g e s t i o n products were d e t e c t e d  o f g e l s d r i e d onto Whatman 3 MM paper.  Non-denaturing p o l y a c r y l a m i d e  gel electrophoresis  C e l l - f r e e e x t r a c t s were e l e c t r o p h o r e s e d i n 1.5 mm t h i c k , 67o polyacrylamide  gels using a tris(hydroxymethyl)  running b u f f e r pH 8.3  (9).  aminomethane/glycine  CMCase a c t i v i t y was d e t e c t e d by s l i c i n g the  g e l s i n t o 2 mm f r a c t i o n s and a s s a y i n g the g e l 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  i n E. c o l i  (pEC)  strains  A p e r i p l a s m i c enzyme f r a c t i o n was o b t a i n e d by the osmotic procedure  of Nossal  and Heppel ( 1 6 ) .  Cytoplasmic  shock  enzymes were r e l e a s e d 2  from s p h e r o p l a s t s by f r e n c h p r e s s u r e e x t r a c t s were prepared concentrated  cultures.  (12,000 l b / i n  by f r e n c h p r e s s u r e treatment  ). T o t a l  cell  o f 25 f o l d  C e l l d e b r i s was removed by c e n t r i f u g a t i o n a t  50,000 X g f o r 20 minutes at 4°C. The assayed  enzyme B-lactamase was used as a p e r i p l a s m i c marker; i t was 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  B-lactamase produced 1 nmole n i t r o c e f o i c  as s u b s t r a t e ( 1 7 ) . acid  A unit of  per minute per mg p r o t e i n .  B - g a l a c t o s i d a s e was used as a c y t o p l a s m i c marker; i t was assayed  with  o-nitrophenyl-B-D-galactoside  (ONPG) as s u b s t r a t e  (15).  A u n i t of  13-galactos idase produced 1 nmole o - n i t r b p h e n o l per minute per mg CMCase a c t i v i t y was  F.  determined as d e s c r i b e d  protein.  i n s e c t i o n B above.  Enzymes and reagents All  restriction  B i o l a b s ; Klenow DNA  endonucleases were purchased from New polymerase  England  from Bethesda Research Labs; CMC  and ONPG  32 from Sigma Chemical Co; aN i t r o c e f i n was  a gift  -P-dATP from New  England N u c l e a r ;  from Dr. Margaret Mathew of Glaxo  Laboratories;  7 RESULTS  A.  S c r e e n i n g f o r c e l l u l a s e producing  recombinant  A shotgun c l o n i n g of Cellulamonas  f i m i DNA  pBR322 (23, 24) y i e l d e d 64 immunopositive measurable c e l l u l a s e 3 groups. kb  Clones  l e v e l s of CMCase.  i n t o the E. c o l i  clones.  plasmid  The c l o n e s showing  (CMCase) a c t i v i t y were c h a r a c t e r i z e d and d i v i d e d  of the f i r s t  i n s e r t of C. f i m i  clones  into  group (pECl) contained a p l a s m i d with a 6.6  DNA, were s t r o n g l y a n t i g e n i c , and c o n t a i n e d moderate  Clones  of the second  group (pEC2) c o n t a i n e d a p l a s m i d  with a 5.0 kb i n s e r t of C. f i m i DNA, were weakly a n t i g e n i c , and c o n t a i n e d high l e v e l s of CMCase a c t i v i t y .  Those i n the t h i r d  a n t i g e n i c but c o n t a i n e d o n l y low l e v e l s of CMCase. plasmids  these c l o n e s had i d e n t i c a l  present  (data not shown).  5.6 kb i n s e r t s .  i n i n d i v i d u a l c l o n e s which  group 3 plasmids  i s shown i n T a b l e 1.  (pEC3).  The plasmids  i n 3 of  Q u a n t i t a t i o n of the CMCase  were r e p r e s e n t a t i v e of the v a r i o u s S t r a i n s with the most  c o n t a i n e d a 5.6 kb Bam HI fragment of C. f i m i DNA. E. c o l i  An a n a l y s i s of the  i n 10 of the 25 c l o n e s i n the t h i r d group showed t h e r e were  v a r i o u s types o f plasmids  activity  group were a l s o weakly  CMCase  These were d e s i g n a t e d  8 Table  1.  Comparison of c e l l u l a s e  activity  from  r e p r e s e n t a t i v e group 3 c l o n e s .  Clone d e s i g n a t i o n  CMCase  % R e l a t i v e to pECl  (U/mg)  pECl  3.97  pEC 3A  0  pEC 3B  0.66  17  pEC 3C  0.59  15  pEC 3D  1.30  33  The cellulase and  cell  100 0  e x t r a c t s of v a r i o u s group 3 c l o n e s were compared f o r  activity.  E x t r a c t s were o b t a i n e d by f r e n c h p r e s s u r e ; p r o t e i n  carboxymethylcellulase  MATERIALS AND METHODS.  (CMCase) were determined as d e s c r i b e d i n  9  B.  D i f f e r e n t i a t i o n o f pEC3 from pECl and pEC2 The  s i z e s of the C. f i m i DNA  determined different (Fig.  2).  by d i g e s t i o n with Bam restriction Cell  fragment  HI  i n the pEC plasmids  ( F i g . 1).  was  These i n s e r t s gave  p a t t e r n s w i t h the enzymes S a l I and Pst I  f r e e e x t r a c t s from c e l l s  s i n g l e bands of CMCase a c t i v i t y (Fig.  inserts  carrying  these plasmids  i n non-denaturing  gave  polyacrylamide gels  3). C o l o n i e s o f E. c o l i  (pEC2) and E. c o l i  (pEC3) produced  h a l o s on  c o n t a i n i n g p l a t e s which were s t a i n e d with Congo-Red ( F i g . 4 ) . (pECl) d i d not produce a c t i v i t y was  (pECl) was  (pEC2) and E. c o l i  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  activity  coli  (pEC)  (pEC3).  Clearly  qualitatively different  coli  L  coli  a h a l o on these p l a t e s even though i t s CMCase  h i g h e r than t h a t of E. c o l i  a c t i v i t y of E. c o l i  E.  CMC-  (pEC3).  i n E. c o l i  from those of E.  (Table 3 ) .  i n the c e l l  and  (pEC)  2.  strains  amounts of CMCase  The very low l e v e l s  8 - g a l a c t o s i d a s e from the p e r i p l a s m i c f r a c t i o n was known c y t o p l a s m i c l o c a t i o n  enzymatic  These d a t a are summarized i n T a b l e  strains contained s i g n i f i c a n t  i n the p e r i p l a s m i c space  the  of  consistent with i t s  i n d i c a t e d that t h e r e was  no  s i g n i f i c a n t c o n t r i b u t i o n of c y t o p l a s m i c enzymes to the a c t i v i t i e s measured in the p e r i p l a s m i c f r a c t i o n .  D.  D e t e r m i n a t i o n of the r e s t r i c t i o n map Restriction  terminus  s i t e s were determined  of pEC3  relative  to the l a b e l l e d  by the p a r t i a l d i g e s t i o n method of Smith  and B i r n s t i e l  5*  EcoRI  (19)  see  10 Materials  and M e t h o d s .  Several  enzymes w e r e u s e d :  S a l I ; P s t I ; Pvu I I ;  Sma  I a n d Kpn I .  map  d i s t a n c e s w e r e 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  agarose g e l s .  An e x a m p l e o f a m a p p i n g g e l i s shown  The r e s t r i c t i o n  s i t e map  i s shown  i n Figure  i n Figure  5.  fragments 6.  The on  11  F i g u r e 1.  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 Bam HI d i g e s t e d pEC p l a s m i d s .  Plasmid DNA was  isolated  from E. c o l i  C600(pEC) s t r a i n s ,  digested  with Bam HI and s e p a r a t e d on a 0.7% agarose g e l c o n t a i n i n g 1 ug/ml e t h i d i u m bromide. p E C l , Lane C:  Lane A:  pEC2, Lane D:  Hind I I I c l e a v e d fragments of \, Lane B: pEC3.  The arrow i n d i c a t e s l i n e a r pBR322  DNA.  12  13  F i g u r e 2.  R e s t r i c t i o n endonuclease d i g e s t s o f pEC  P u r i f i e d pEC plasmids were d i g e s t e d with endonucleases S a l I and P s t I . agarose g e l c o n t a i n i n g  the r e s t r i c t i o n  The fragments were separated  1 ug/ml e t h i d i u m  bromide.  d i g e s t s of pECl, pEC2 and pEC3 r e s p e c t i v e l y . phage DNA as a m o l e c u l a r  plasmids.  weight marker.  p E C l , pEC2 and pEC3, r e s p e c t i v e l y .  on a 1.1%  Lanes A - C are S a l I  Lane D i s Hpa I d i g e s t e d T7  Lanes E-G are P s t I d i g e s t s o f  1 4  15  F i g u r e 3.  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 a n a l y s i s of c e l l u l a s e s  encoded by pEC p l a s m i d s .  Whole c e l l non-denaturing fractions, and  e x t r a c t s o f E. c o l i polyacrylamide gels.  C600(pEC) s t r a i n s were run on 6% The g e l s were s l i c e d  these s l i c e s were p l a c e d d i r e c t l y  incubated f o r 18 - 20 hours  at 37°C.  and r e d u c i n g sugars were determined MATERIALS AND METHODS.  part  i s graphed  mixture  The r e a c t i o n s were t e r m i n a t e d ,  Panel A shows the a c t i v i t y  shown here; s i n c e no a c t i v i t y was seen top  i n t o a CMCase assay  colorimetrically  B shows pEC2 and Panel C shows pEC3.  i n 2 mm  as o u t l i n e d i n  p r o f i l e s of p E C l , P a n e l  N.B. Only the top p a r t o f the g e l i s i n the remainder  i n an expanded form .  of the g e l , the  16  0-2  0 4 6  c  O  m m  Q  0 2 •  o 0 2  20 PERCENT  LENGTH  40 OF  GEL  17  F i g u r e 4.  Zones o f CMC h y d r o l y s i s produced by pEC s t r a i n s and v i s u a l i z e d  by Congo Red s t a i n i n g .  C u l t u r e s of the E. c o l i and  (pEC) s t r a i n s were grown to s t a t i o n a r y phase  then p l a t e d f o r s i n g l e c o l o n i e s .  CMC c o n t a i n i n g  S i n g l e c o l o n i e s were t r a n s f e r r e d to  p l a t e s which were incubated 24 hours at 30°C.  Zones of  h y d r o l y s i s then were v i s u a l i z e d with Congo Red as o u t l i n e d i n MATERIALS AND METHODS.  A l l of the c o l o n i e s were approximately the same s i z e .  denotes where c o l o n i e s of E. c o l i coli  (pBR322) were; (B) where c o l o n i e s of E.  (pECl) were; (c) where c o l o n i e s of E. c o l i  c o l o n i e s of E. c o l i  (pEC3) were.  (A)  (pEC2) were; (D) where  I  O O v  9  w  19 T a b l e 2.  Plasmid  Insert  Size  Characteristics  of C e l l u l a s e  Antigenicity  CMCase U/mg  (kb)  6.6  strong  pEC2  5.0  weak  pEC3  5.6  v. weak  CMC-Congo Red  3.6 39.0  ++  1.2  +  s i z e o f C. f i m i DNA i n s e r t s was determined from Bam HI  restriction Antigenicity  endonuclease d i g e s t i o n  CMCase and p r o t e i n  MATERIALS AND METHODS. staining  fragments separated on an agarose g e l .  was based on the i n t e n s i t y of the colony on the s c r e e n i n g  autoradiograms.  Red  Zone o f C l e a r i n g on  i n E. c o l i  PECl  The  Plasmids  as o u t l i n e d  were determined as d e s c r i b e d i n  Zones of CMC h y d r o l y s i s  were v i s u a l i z e d with Congo.  i n MATERIALS AND METHODS ( F i g . 4 ) .  20 Table  3.  Specific  activities  of CMCase and marker enzymes  Enzyme  Cell Fraction Periplasmic  Cytoplasmic  C e l l extract  pECl CMCase fi-galactos idase B-lactamase  26 160 14,480  CMCase B-galactosidase B-lactamase  95 325 9,372  0.4 2,722 72.9  3.6 1,932 2,200  29 4,048 63  39 2,738 2,604  PEC2  PEC3 CMCase B-galactosidase B-lactamase  10.,3 584 67 ,653  0.36 4,909 2,556  1.2 2,892 6,390  CMCase = yg glucose eqv/min/mg p r o t e i n B-Galactosidase  = nmole p-nitrophenol/min/mg p r o t e i n  B-Lactamase = nmole n i t r o c e f o i c  The E. c o l i  a c i d produced/min/mg p r o t e i n  (pEC) s t r a i n s were f r a c t i o n a t e d by the osmotic shock,  procedure o f N o s s a l  and Heppel.  B-lactamase, and the c y t o p l a s m i c  The p e r i p l a s m i c marker enzyme was marker was B - g a l a c t o s i d a s e .  enzymes were assayed as o u t l i n e d i n MATERIALS AND METHODS.  A l l three  21  Figure  5.  Autoradiogram o f pEC3 DNA from a Smith and B i r n s t i e l  restriction  s i t e mapping experiment.  Purified digested  pEC3 plasmid DNA was e n d - l a b e l l e d  with various  restriction  selectively  endonucleases as o u t l i n e d i n MATERIALS  AND METHODS.  Lane A i s a m o l e c u l a r weight marker.  time p o i n t s .  Lanes  points.  and then  F - J are P s t I time p o i n t s .  Lanes B-E are S a l I  Lanes  K-0 are Pvu I I time  S a l I time p o i n t s were 3, 15, 30 and 120 minutes o f d i g e s t i o n .  The P s t I and Pvu I I time p o i n t s were 1, 3, 15, 30 and 120 minutes of digestion.  22  23  F i g u r e 6.  Restriction  map o f the plasmid pEC3.  Using the r e s t r i c t i o n METHODS the s i t e s determined. (E) s i t e  for 5 different  restriction  A l l d i s t a n c e s were determined  i n the v e c t o r pBR322.  i n s e r t e d C. f i m i sites.  mapping technique o u t l i n e d  DNA.  endonucleases  relative  A shows the S a l I (S) s i t e s .  The Bam HI s i t e s  AND  were  t o the unique  Eco RI  The c r o s s h a t c h e d area r e p r e s e n t s the  G shows the P s t I (Ps) s i t e s .  (K) s i t e s .  i n MATERIALS  B shows the Pvu I I (P)  D shows both Sma I (Sm) and Kpn I  (B) are a l s o shown.  o  r- o  -m  o -m  CD -m  09  - ro  -*  --co  —03  =-0) CO TI  — cn  "D  • n  •to  o  CT Q  tn 9 I  T3 O  — cn  to 3  TI  to  -co CO  3  "TI in  — CD  -co  00  07  03  CD  m  •m  -m  m  25 DISCUSSION  A.  The  enzymes  The components of the c e l l u l a s e  system of Cellulomonas f i m i  are b e i n g  e l u c i d a t e d by the m o l e c u l a r c l o n i n g of the s t r u c t u r a l genes i n E s c h e r i c h i a coli.  To date 3 genes encoding c e l l u l a s e  i s o l a t e d and p a r t i a l l y d i f f e r e n t , Bam  characterized.  HI r e s t r i c t i o n  encode a s i n g l e c e l l u l a s e enzyme. migration  The  restriction  endonuclease  In a d d i t i o n , each c l o n e appears to  This conclusion  i s based on enzyme  i n non-denaturing p o l y a c r y l a m i d e g e l s .  The enzymatic  activities  of the 3 C. f i m i DNA  appear not o n l y q u a n t i t a t i v e l y d i f f e r e n t , well.  have been  Each gene i s p r e s e n t on a s i n g l e ,  fragment.  d i g e s t i o n p a t t e r n s are a l s o d i s t i n c t .  (CMCase) a c t i v i t y  The  standard c e l l u l a s e  endoglucanase  but q u a l i t a t i v e l y  assay (CMCase) would  from exoglucanase a c t i v i t y ;  recombinant c l o n e s  not  different  as  distinguish  however, one might  expect an  exoglucanase to generate fewer r e d u c i n g sugar e q u i v a l e n t s because of i t s sensitivity  to the s u b s t i t u t e d  assay i s l i k e l y  to d e t e c t only endoglucanase  to c e l l o d e x t r i n s  Alternately  (8).  The Congo Red  activity.  6 g l u c o s e r e s i d u e s long or l o n g e r (25).  produced by c l e a v i n g long CMC dye.  glucose residues  The dye binds only Halos then are  c h a i n s i n t o s h o r t o l i g o m e r s which  l o n g e r oligomers produced by  do not b i n d  i n f r e q u e n t c l e a v a g e may  washed out d u r i n g the s t a i n i n g procedure again producing a h a l o . r a t i o n a l e then, E. c o l i  (pECl) l i k e l y  coli  (pEC3) encode endoglucanases  (pEC2) and E. c o l i  activities. classical  This hypothesis w i l l  ertdo-  encodes  plate  be  From t h i s  an exoglucanase, w h i l e E. of d i f f e r e n t  specific  have to be c o n f i r m e d by c h e c k i n g the  26 glucanase  p r o p e r t y of v i s c o s i t y r e d u c t i o n and  the c l a s s i c a l  p r o p e r t y of c e l l o b i o h y d r o l a s e a c t i o n on c r y s t a l l i n e Because t h e r e are d i f f e r e n t enzymatic c u l t u r e supernatants  substrates (8).  activities  present  i n C.  fimi  the use of a s c r e e n i n g method l i k e Congo Red-CMC  p l a t e s f o r recombinant DNA  clones  is quite limited.  Congo Red  has been used to d e t e c t endoglucanase from recombinant DNA C l o s t r i d i u m thermocellum ( 2 ) .  The  not d i s t i n g u i s h between enzymatic d e t e c t c l o n e s encoding supernatents.  exoglucanase  immunological activities  a l l of the C.  screening  c l o n e s of  s c r e e n i n g procedure  does  and hence can be used to  f i m i c e l l u l a s e s present  in c u l t u r e  A l s o , i f C. f imi's c e l l u l a s e r e q u i r e s p e p t i d e s which i n  themselves are not c e l l u l o l y t i c  but  i n s t e a d are necessary  f o r synergy  or  s u b s t r a t e b i n d i n g , they too c o u l d be d e t e c t e d as immunoreactive c l o n e s . The  B.  utility  of such  p e p t i d e s c o u l d be  The  genes  The  commercial u t i l i z a t i o n  wastes i s not yet e c o n o m i c a l l y of  highly active,  shown i n r e c o n s t r u c t i o n  of c e l l u l a s e feasible.  enzymes by  first  c l o n e d DNA  Increased  step i n making them l e s s  iri v i t r o recombinant DNA  manipulations  For example, t h i s very type of approach has  human i n t e r f e r o n The  cellulosic  M o l e c u l a r c l o n i n g o f f e r s an approach to i n c r e a s e p r o d u c t i o n of  the n e c e s s a ry genes.  of  T h i s i s because l a r g e q u a n t i t i e s  s t a b l e c e l l u l a s e are not yet a v a i l a b l e .  p r o d u c t i o n of c e l l u l a s e w i l l be the f i r s t expensive.  f o r degradation  experiments.  (18)  and  of c l o n e d  been s u c c e s s f u l with  a b a c t e r i a l a-amylase enzyme (22).  step i n these p r o j e c t s i s to d e f i n e the exact  which makes a f u n c t i o n a l enzyme.  The  l e n g t h of  second i s to determine  the  27 nucleotide  sequence of the gene and f i n d any p o s s i b l e r e g u l a t o r y  which may or may not f u n c t i o n  in various  couple the gene to known r e g u l a t o r y  hosts.  The t h i r d  step  sequences i s to  sequences to have e i t h e r an i n d u c i b l e  or c o n s t i t u t i v e l y produced gene product which i s r e l e a s e d from the bacterial cells  i n t o the environment.  c e l l u l a r conversion from the c e l l u l a r  of substrate  that strong  t h e i r expression.  or p r o v i d e  an i n i t i a l  extra-  p u r i f i c a t i o n away  components.  Currently cloned likely  T h i s would e i t h e r a l l o w  C. f i m i genes are expressed i n E. c o l i , and i t i s  E. c o l i  r e g u l a t i n g sequences c o u l d be used t o i n c r e a s e  In C. f i m i  the c e l l u l a s e genes are r e g u l a t e d ( 9 ) ;  t h e r e f o r e , sequences from C. f i m i must e x i s t which c o u l d p o s s i b l y be e x p l o i t e d as c o n t r o l l i n g exported leaders present.  elements.  to the p e r i p l a s m i c  A l s o c e l l u l a s e s from pEC p l a s m i d s are  space o f E. c o l i .  or s i g n a l p o l y p e p t i d e s  f o r transport  This  i n d i c a t e s t h a t the  to the environment are  The e x c r e t i o n of the pEC c e l l u l a s e s i s then p o s s i b l e and i n f a c t  has been achieved periplasmic  with  a mutant  E. c o l i  (pEC) which s e l e c t i v e l y  enzymes i n t o the c u l t u r e medium ( 6 ) .  releases  28 LITERATURE CITED 1.  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