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Expression of C. fimi genes from transposon Tn10 tet promoters Din, Neena 1989

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EXPRESSION OF C. FIMI GENES FROM TRANSPOSON Tn10 TET PROMOTERS. by NEENA DIN B .Sc , University college, London, 1984. P.G.C.E. , Institute of Education, London, 1985. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1989 © Neena Din, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of \ ^ K . \ o ' C L ^ ^ u c a G - ' V The University of British Columbia Vancouver, Canada D a t e 2_o\/3l^°\ DE-6 (2/88) ABSTRACT In this study, the promoterless genes cenA and cex (both obtained from E. coli expressing recombinant DNA of C. fimh were placed under the control of the divergent tet promoters from Tn10 and cloned into a broad host range plasmid, pJRD215. This system allowed us to investigate the expression and interaction of the gene products of these two genes (CenA and Cex) in four different Gram-negative host organisms: E. coli C600, an E. coli K12 derivative which is able to survive on cellobiose, R. capsulatus B10, and K. pneumoniae M5a1. The objective was to eventually find a Gram-negative host organism which would secrete the CenA and Cex proteins so that we could investigate whether it was now possible for that host organism to survive on cellulose. All four host organisms used in this study expressed the cenA and the cex genes with different efficiencies, however, none of the host organisms secreted CenA and Cex to the culture medium. ii TABLE OF CONTENTS Page A b s t r a c t ii List of Tables v List of Figures v i i Acknowledgements i x Introduction 1 Materials and methods 1. Bacterial strains and plasmids 8 2. DNA manipulations 9 3. Enzymatic studies 13 Results Section 1. Construction of the plasmid P N D 3 18 Sect ion 2. Quantitation and location of endoglucanase and exog lucanase activities in E. coli recombinant c lones 31 Sect ion 3. Mobil ization of P N D 3 from E. coli C600 to R. capsulatus B10 31 Sect ion 4. Mobi l izat ion of P N D 3 from E. coli C600 to K- pneumoniae M5a1 36 Section 5. Transformation of E. coli C e l + strains 40 Sect ion 6. Viscometr ic studies on E. coli C 6 O O / P N D 3 clone #11 46 iii Se c t i o n 7 . R e p r e s s i o n a n d induct ion of e n d o g l u c a n a s e a n d e x o g l u c a n a s e ac t i v i t i e s in E. co l i s t r a in s ca r r y i ng the plasmid P N D 3 50 Discussion 60 References 64 i v LIST OF TABLES TABLE TITLE PAGE 1. Quantitation of enzyme activities from crude cell extracts of E. coli clones #9 and #11 32 2. Location of enzyme activities in E. coli clone #11 33 3. Triparental mating efficiency when using R. capsulatus B10 as recipients 35 4. Quantitation of enzyme activities from R. capsulatus B10 clones #10 and #12 37 5. Location of enzyme activities in R. capsulatus B10 clone #10 38 6. Triparental mating efficiency when using K. pneumoniae as recipients 39 7. Quantitation of enzyme activities from K. pneumoniae clones 41 8. Location of enzyme activities in K. pneumoniae clones 42 9. Quantitation of enzyme activities from E. coli C e l + / p N D 3 clones..... 44 10. Location of enzyme activities in E. coli Cel+/pND3 clones 45 v 1. Quantitation of enzyme activities from E. coli JC10240/pND3 clone #12 vi LIST OF FIGURES F I G U R E T I T L E P A G E 1. The t ransposon Tn10 2 2. T h e enzyma t i c degradat ion of ce l l u lose 5 3. The const ruct ion of the p lasm id P N D 3 19 4. A u t o r a d i o g r a m fo l l ow ing the a t t a chmen t of # 3 2 P l abe l l ed E c o R I l i nkers onto p c E C 2 Dra11 D N A f r agmen t ca r r y i ng the cenAgene 20 5. P l a sm id p N D i d iges ted with B amH1 21 6. P l a sm id p N D 2 d iges ted with Apa1 23 7. Apa1 d iges t ion of c l ones #9 and #11 25 8. P lasmid P N D 3 d igest ions 27 9. P lasmid P N D 3 d igest ions 28 10. P lasmid P N D 3 d igest ions 29 11. P lasmid P N D 3 d igest ions 30 12. C annon - Fen s ke v iscometer 47 13 . S pe c i f i c f luidity aga i n s t rea l i n cuba t i on t ime 4 8 14. A S pe c i f i c f luidity aga in s t A r educ ing s uga r s 49 vii 15. G r ow th c u r v e s of E. co l i M 8 8 2 0 / p R 7 5 1 ::Tn10, P N D 3 cu l t u r e s 52 16. R e su l t s of D N S a s s a y s ca r r i ed out on ce l l ex t rac t s of 5m l s a m p l e s r e m o v e d f rom the g r ow ing cu l tu res of E. c o l i M8820/pR751 : :Tn10,pND 3 53 17. R e su l t s of p N P C a s s a y s ca r r i ed out on ce l l ex t rac t s of 5m l s a m p l e s r e m o v e d f rom the g row ing cu l t u re s of E. c o l i M8820/pR751 : :Tn10,pND 3 54 18. G row th cu r ve s of E. co l i J C 1 0 2 4 0 / p N D 3 cu l tu res 56 19 . R e su l t s of D N S a s s a y s ca r r i ed out on ce l l ex t rac t s of 5m l s a m p l e s r e m o v e d f rom the g r ow i ng cu l t u re s of E _ c _ , J C 1 0 2 4 0 / p N D 3 57 20 . R e su l t s of p N P C a s s a y s ca r r i ed out on ce l l ex t rac ts of 5m l s a m p l e s r e m o v e d f rom the g r ow i ng cu l t u re s of E. coli J C 1 0 2 4 0 / p N D 3 58 ( viii ACKNOWLEDGEMENTS I would like to thank Drs. R. Miller, D. Kilburn, and T. Warren for allowing me the opportunity to work on this project and for their instruction and encouragement during my research. I would also like to thank Dr. J . Smit for his useful advice and comments. Lastly, I thank all my friends in the Lab and Department for their help and support, especially Terry Bird for his never ending patience. ix INTRODUCTION DIVERGENT CONTROL REGIONS Sequences which al low divergent transcription from c losely spaced sites are widespread in Prokaryotes, Eukaryotes and their viruses. More than 60 examples have been found to date (1). The first sets of divergent promoters were identified in the genome of bacteriophage X (2,3) and in the bio operon on the E. coli genome (4). Subsequent ly, more than 20 cases of such divergent control regions have been identified on the E. coli chromosome. The widespread occurence of these divergent control regions implies that they play an important role in the regulation of gene express ion. Divergent control regions have also been identified on transposable e lements like Tn10 (5,6,7,8). Transposon Tn10 was originally isolated from nature as part of the conjugative plasmid R100, also known as R222 (9,10,11). The intact transposon is 9300bp in length and has 1400bp inverted repeats at its ends. The 6500bp of non-repeated material include the 2500bp tetracycl ine resistance determinant (see Figure 1). The 1400bp repeat sequences are c lose ly related but non-identical IS elements, IS10-Right and IS10-Left, which encode the functions responsib le for Tn10 t r a n s po s i t i o n . The tetracycl ine resistance determinant in the transposon Tn10 consists of two genes; the tetA res istance gene and the tetR repressor gene that are transcr ibed from divergent overlapping promoters (see Figure 1). The tetA gene encodes a 43.2 KDa membrane protein that appears to be both necessary and sufficient for res istance to tetracycl ine (12,13,14,15). The tetR gene encodes a 23.3 K D a protein that negatively regulates both its own synthesis and the synthesis of the TetA resistance protein (16,17). Both the resistance gene and the repressor are synthesized in the presence of tetracycl ine. Studies with purified Tet repressor have shown that in the absence of tetracycline the repressor binds to two adjacent operator sites, 0|_ and O R , and that tetracycline is able to induce transcription of both tetA and tetR by binding to this 1 F IGURE 1: THE T R A N S P O S O N Tn10. 1 'S/<9-L>- L 1 1 L_^jST^Rl -— tet  1 THE INTACT T R A N S P O S O N Tn10. -35 -10 PA O R SO TCC T A A T T T T TG T TGACJAC TC T A TCAljlTGA T AGAG TlTA T T T TACCAc!rcldCTATCA|GlT^ATAQA •AGGA TTAAAA AC A AC TQJTG AG A TAG TJAJACTA TC TCAlA. TA A A A TGG TG^GJG}3A T AG TjC_C_T A T_T SD PR1 -10 -35 PR2 -10 THE DIVERGENT CONTROL REGION OFTnIO 2 repressor and hence reducing its affinity for the operators (17,18,19). The operators overlap the promoters for tetA and tetR to different extents. Each operator is recognized and bound by a dimer of the tetR gene product (12,19). The tetracycline resistance genes of Tn10 are readily dist inguishable in both DNA sequence and phenotype from other types of tetracycl ine resistance genes, including those found on the plasmid pSC101 (and its derivatives pMB9 and pBR322) or the broad host range plasmids RP1 and RP4 (20,21). Binding sites for regulatory proteins have often been observed within the divergent control regions and these proteins may regulate transcr ipt ion in both direct ions. This character ist ic makes these divergent control regions very attractive when systems are required for the simultaneous expression of more than one gene. For example, it is possible to clone two genes of interest, one on either s ide of a divergent control region, and to then tightly regulate the expression of both genes in vivo. The fusion of divergent control regions to promoterless genes has also a l lowed the regulatory character ist ics of such divergent promoters to be ana lysed (22,23,24,25). Promoter-probe vectors containing the gene combinations lacZ/galK and lacZ/phoA were used to study the divergent control regions of the pBR322 tet gene and the tet genes of transposon Tn10 (24). Levels of express ion of the indicator genes a l lowed determination of promoter strengths. The induction kinetics of the divergent tet regulatory region from Tn10 were also studied. Express ion of the indicator genes was repressed if the tetR regulatory gene of Tn10 was provided in trans by a second plasmid within the host strain. Addition of an inducer (the non-inhibit ing Tc der ivat ive, 2-acetyl-2-decarboxyamide Tc.) resulted in induction of express ion from promoters tetP/\ and tetPR . In other words, upon induction, transcript ion started s imultaneously in both direct ions from the over lapping promoters of the tet control region. 3 CELLULASES Cel lu lose is a linear polymer composed of g lucose subunits linked by (3-1,4-glucosidic bonds. In the native state, ce l lu lose molecules form fibers which are composed of compact crystal l ine domains separated by more amorphous regions. Cel lu lose degradation by bacteria and fungi is carr ied out by complex mult ienzyme systems. Work in our lab is concerned with the enzyme system in one of these organisms, the Gram-posi t ive bacterium Ce l l umonas f imi. The convers ion of cel lulose to g lucose requires the activity of three types of enzymes . The cel lu lose is first co-operat ively attacked by two types of extracel lular ce l lu lases; the endoglucanases (1,4,f3-D-glucan glucanohydrolase, E C 3.2.1.4) and the exog lucanases (1,4,6-D-glucan cel lobiohydrolase, E C 3.2.1.91). The product of these enzymes, cel lobiose, is further hydrolysed by ce l lob iase (6-D-glucoside glucohydrolase, E C 3.2.1.21) to g lucose (see Figure 2). Two C. fimi cel lulase genes, cenA and cex, have been studied in detail (26,27). The cenA gene encodes a secreted glycosylated endoglucanase of 53.0 KDa and the cex gene encodes a secreted glycosylated exoglucanase of 49.3 KDa . The two structural genes have been c loned independently in E. coli on the vector pBR322 (26,27). The genes were expressed in E. coli to give non-g lycosy lated proteins retaining the specif ic i t ies of the native enzymes from C. fimi. Both genes have been sequenced. Both cenA and cex encode proenzymes in which the mature polypeptide is preceded by a leader peptide. Although both activities are found in the periplasm of E. coli cel ls, neither enzyme is secreted into the culture medium (26,27). 4 I I Crystalline region ' ' Amorphous region adsorption of cellutase «nzym«s FIGURE 2: THE ENZYMATIC DEGRADATION O F CELLULOSE . 5 THE BROAD HOST RANGE PLASMID PJRD215 The plasmid RSF1010 belongs to the incompatibility group Q (Inc Q) and has the very useful property of replicating in most, if not all, spec ies of Gram-negat ive bacteria. For this reason, it (and related or identical plasmids) is attractive as a cloning vector in Gram-negative organisms other than E. coli. RSF1010 is also of special interest due to its ability to be transferred between spec ies by conjugation in the presence of a variety of sel f-transmiss ib le plasmids including RK2 (28). However the s ize of RSF1010 (8.7kbp) and the fact that it contains few unique sites for c loning, makes it a far from ideal c loning vector. The cloning versatil ity of RSF1010 has been improved by changing the selective markers, inserting a cos site for in vitro packaging and by introducing a restriction site bank. This plasmid has been named pJRD215 (29). It retains the broad host range replicon and mobil ization functions of RSF1010 , which enables it to be used to carry novel genetic information to those bacter ia that cannot be transformed readily. OBJECTIVES An understanding of the mechanisms of cel lulose degradation by an organ ism requires the isolation and character izat ion of the individual components of the system. Once the relevant enzymes are known and purified, it should be possible to study further their role, for example by reconstituting mixtures and testing for synergy or by character iz ing mutants unable to synthesise def ined components. Hence a more detailed understanding of the mode of action of the CenA and the Cex proteins from C. fimi. whether they act individually or in concert, may be obtained if the cenA and the cex genes are c loned into non-cellolytic organisms and the express ion of these genes studied in these hosts. In this study, where cenA and cex (both obtained from E. coli express ing recombinant DNA of C. fimi) are placed under the control of the divergent tet promoters from Tn10, the interaction and 6 synergism of the gene products of these two genes (CenA and Cex) can be investigated in E. col i . P lacing the two cel lulase genes on either s ide of the divergent promoter region al lows the s imultaneous transcription of both genes . In addition, cloning these genes into a broad host range plasmid (pJRD215 (29)) al lows the transfer of these genes to other Gram-negat ive organisms by conjugation or by transformation. In this way it is poss ib le to study the interaction of the endoglucanase and the exoglucanase in other host organisms. The two Gram-negat ive organisms used in this study, other than E. coli were Rhodobac t e r capsulatus and K l ebs i e l l a  pneumon i ae . The photosynthetic bacterium R. capsu latus is of interest in this study because it possesses a B-glucosidase and K_. p n e u m o n i a e , a nitrogen-fixing bacterium, has the ability to grow on ce l lob iose (30). Ce l lu lose degradat ion by microorganisms is usual ly accompl i shed more efficiently if a supplementary source of nitrogen is avai lable (31,32). For these reasons various approaches have been suggested as ways to combine nitrogen fixation and cel lulolysis and hence one strategy would be to c lone cel lu lase genes into host organisms which are able to fix nitrogen. A second E. coli strain was also used which is a derivative of K12 and can survive on ce l lob iose. The main objective of constructing a broad host range plasmid which carries the cenA and cex genes (under the control of a divergent promoter region) is to eventual ly find a Gram-negat ive host organism which will secrete the CenA and the Cex proteins. In this way we can investigate whether it is now poss ib le for this host organism to survive on cel lu lose. 7 MATERIALS AND METHODS BACTERIAL STRAINS AND PLASMIDS E. coli C600/pEC1 and C600/pcEC2 were used as sources of C. fimi exog lucanase (cex) and endoglucanase (cenA) genes, respectively. The Tn10 tet promoter genes were provided on plasmid pCB168 . This plasmid was present in E. coli strain CB874 (thi, galK, rpsL, phoA8, rec A56::Tn10 site unknown (24). The broad host range plasmid p JRD215 (27) was carried in E. coli strain MM294. The two E. coli strains which can survive on ce l lob iose (Cel+), were derivatives of the K12 strain W4860 (33) The bacterial strains used in conjugative studies (see this section) were R. capsulatus strain B10 and K. pneumoniae strain M5a1. The 'helper' p lasmid, pRK2013 , (used to mobil ize pJRD215) was carr ied in E. coli strain HB101 (34). For repression and induction studies, the E. coli strains M8820/pR751 ::Tn10 and JC10240 (which has Tn10 integrated into the chromosome) were used. MEDIA All E. coli strains were grown in Luria broth (LB; 10g tryptone, 5g YE , 10g NaC l per litre). Appropriate antibiotics were added to a final concentrat ion as listed below: u,g/ml Abbreviations Ampicill in Kanamycin Streptomycin Trimethoprim 50 40 25 25 Amp Kan Sm Tm 8 R. capsulatus was grown on R C V medium: Solution ml/litre of medium 10% ( N H 4 ) 2 S 0 4 10% DL-Malate 1% EDTA 20% M g S 0 4 . 7 H 2 0 10 40 2.0 1.0 1.0 1.0 2.4 1.0 15 Trace elements 7.5% C a C l 2 . 2 H 2 0 0.5% F e S 0 4 . 7 H 2 0 0.1% Thiamine. HCI 0.64 M K P 0 4 (add last) Deionised water to 1000mls K. pneumoniae was grown on M9 medium (35): 6g N a 2 H P 0 4 j 3g K H 2 P 0 4 , 0.5g NaC l , 1g N H 4 C I . This solution was autoclaved, cooled and then 2ml 1M M g S 0 4 and 0.1ml 1M CaC l2 were added. Agar plates contained 15g agar/litre. In case of carboxymethylce l lu lose plates, 11 g agar were added. DNA MANIPULATIONS PREPARATION AND PURIFICATION OF PLASMID DNA For smal l sca le isolation of plasmid DNA, 5 ml of LB, containing the appropriate antibiotic, were inoculated with a s ingle co lony from a plate and grown to stationary phase (overnight, 37°C). Cel ls were col lected by centrifugation and plasmid DNA was isolated by the alkal ine lysis method (36). P lasmid DNA was further purified on a NACS -P r epa c column, as descr ibed by the supplier, BRL . For large sca le isolation of plasmid DNA, 500 ml of LB plus the appropriate antibiotic, were inoculated with a s ingle co lony from a 9 plate and incubated overnight at 37°C. P lasmid DNA was isolated by alkal ine lysis and purif ied by ultracentrifugation in a CsCI-eth id ium bromide gradient (36). RESTRICTION ENDQNUCLEASE DIGESTIONS. Restrict ion enzyme reactions were carr ied out as descr ibed by Maniatis et al (36). All enzymes were used under the condit ions suggested by the manufacturer. AGAROSE GEL ELECTROPHORESIS OF DNA. Restrict ion fragments were separated on 0.8-1% agarose gels using T B E (89mM Tris Borate, 89mM Boric acid, 8mM EDTA, pH 8.0) buffer. Ge l s contained 1u.g ethidium bromide/ml of buffer. DNA bands were v isual ized by f luorescence using a UV transil luminator. Desired DNA fragments were recovered from agarose gels using NA-45 D E A E membrane (Schleicher and Schuel l) . A strip of NA-45 was placed in an incision just ahead of the desired DNA band. Elecrophoresis was cont inued (150V, 2-3 mins) until binding was complete, as judged by ethidium bromide f luorescence using long wave UV. The strip was freed of residual agarose by shaking in 500u.l NET buffer (0.15M NaCl , 0.1 mM EDTA, 200mM Tris pH 8.0). DNA was eluted off the NA-45 strip by addition of 250u1 high salt NET buffer (1.0 M NaC l , 0.1 mM EDTA, 20mM Tris pH 8.0) and incubating at 65°C for 45 mins with occas iona l mixing. Res idual ethidium bromide was removed by wash ing with water saturated n-butanol. The DNA was precipitated with 2.5 vo lumes of 95% ethanol. ATTACHMENT OF LINKERS TO DNA Blunt ended EcoR I linkers were attached to DNA fragments using the following procedure: (i) DNA was blunt-ended using the Klenow fragment of DNA polymerase. The reaction was carried out as descr ibed by Maniat is et al (36). (ii) A fraction of the linkers (1u.g out of 16u.g) to be used in the ligation to the DNA, was labelled 10 using tf-32p A T P : 2u,l of 0.5u.g/uJ blunt-ended EcoRI linkers were mixed with 0.1u.l of y- 3 2 P A T P (specif ic activity = 7000Ci/mMole) , 1uJ 10x linker kinase buffer (0.7M Tris. C l , pH 7.6, 0.1 M MgC^ , 50 mM DTT), 9u,l sterile distil led water ( sdH20) and 10 polynucleot ide kinase (Pharmacia). This mixture was incubated at 37°C for 15 mins, then 1u.l 10x l inker-kinase buffer, 1u.l 10mM ATP , 7u.l s d H 2 0 and 10 U polynucleotide kinase were added and the reaction incubated for a further 30 mins at 37°C. (iii) 1u.g of the radioactively label led linkers, 15u.g of unlabeled l inkers and 5|ig of DNA (to which the linkers were to be attached) were precipitated together and resuspended in 5jil 10x Apa1 assay buffer (60mM NaCl , 60mM 2-mercaptoethanol, 1 mg/ml BSA) , 5u.l 4mM ATP , 5LLI 10mM spermidine, 5LII 20mM DTT, 28u.l s d H 2 0 and 20 U T4 DNA ligase. This mixture was incubated overnight on ice. After extraction with phenol/chloroform, the DNA was precipitated with 2 vo lumes 95% ethanol before resuspension in 50jil TE (pH 8.0) . AUTORADIOGRAPHY. Attachment of l inkers to DNA was monitored by running an aliquot of the DNA on an agarose gel before and after digestion with E c oR I , transferring the gel to 3M Whatman filter paper and drying on a Biorad gel drier. The dried gel was placed in a Kodak X-Omatic film cassete with Kodak XRP-1 film and the film was exposed overnight at -70°C. LIGATION DNA fragments, at a molar ratio of insert to vector of 3:1, were precipitated together with ethanol and resuspended in 9uJ s d H 2 0 . One |il of ligation buffer (50mM Tris pH 7.4, 10mM MgCl2,; 10mM DTT, 1mM spermidine, 1mM ATP , 100jig/ml BSA) and T4 DNA ligase were added. For sticky ended ligations, 10 U of l igase were added, whereas for blunt ended ligations, 400 U were added. The ligation 11 mixture was incubated overnight at 16°C. TRANSFORMATION Competent cel ls were prepared as descr ibed by Maniatis (36). Up to 100ng of DNA were used to transform cel ls. Suitable volumes of transformation mixtures (50u.l, 1 OOLLI, 150u.l) were spread onto appropriate media to select for transformants. P lates were incubated overnight at the appropriate temperature. CONJUGATIVE C R O S S E S The broad host range plasmid, pJRD215, used in the construction of p N D 3 (see results section) is transferable between spec ies by conjugation in the presence of a mobilising plasmid (28). Hence, triparental matings were carr ied out using two donors (E. coli C600 / p N D 3 and E. coli HB101/pRK2013) and the recipient cel ls. Donor and recipient cel ls were grown in liquid media to log phase, mixed in a 1.5ml Eppendorf tube (ratio 1:1:1) and pelleted by 30 sec. centrifugation. The cel ls were careful ly resuspended in 1ml minimal medium (RCV in the case of R. capsulatus as recipient cells, and M9 in the case of K. pneumoniae as recipient cells), and 10u.l spotted onto a nitrocel lulose filter (0.7u.m pore size) on a prewarmed minimal medium plate. The plates were incubated at 30°C overnight. The filters were placed into 1.5ml Eppendorf tubes with 1ml minimal medium and the cel ls resuspended on a vortex mixer (5 mins). Samp les of the cell suspens ions (50u.l, 100u.l, 150jil) were then plated onto selective medium. In the case of R.  c ap su l a t u s as recipient cells, the selective media was R C V Sm and in the case of K. pneumoniae being the recipient cells, M9 Sm was used to select for transcongugants. The plates were incubated at 30°C for 24-48 hours. 12 ENZYMATIC STUDIES PLATE ASSAYS FOR THE DETECTION OF ENDOGLUCANASE AND  E X O G L U C A N A S E ACTIVITIES Endog lucanase activity of recombinant c lones was detected as fol lows: cel ls were grown overnight, at 37°C, on LB plates supplemented with 11 g C M C (Sigma low viscosity grade)/litre and the appropriate antibiotic. Co lon ies were washed off the agar surface and the plates were flooded with 0.2% congo red solution. After 15 minutes gentle agitation on a rotary shaker (Labline), the congo red solution was poured off and 1M NaC l was used to wash off excess dye (15-30 mins gentle agitation on the rotary shaker). Zones of clearing of the dye appeared at the sites of endoglucanase posit ive co lon ies. Exog lucanase activity of recombinant c lones was detected as fol lows: Methylumbel l i feryl ce l lobios ide (MUC) was used to detect any co lonies express ing exog lucanase activity. Cel ls were grown overnight at 37°C, on LB plates containing IOOLUTI M U C and the appropriate antibiotic. The exog lucanase c leaves the substrate to re lease methylumbell i ferone, which f luoresces under long wave U.V. i r r a d i a t i o n . PREPARATION OF CRUDE CELL EXTRACTS Single co lonies of recombinant c lones were inoculated into 25ml of medium and incubated overnight at 37°C. Cel ls were harvested by centrifugation (10 mins at 8K in a JA20 Beckman rotor) and resuspended in 5ml 50mM K P O 4 (pH 7.0), 0.02% N a N 3 . The suspens ions were sonicated using a Bronson sonifier with a microprobe (Intensity setting of two, 3x30secs.) . The suspens ions were then centrifuged again (12K, 40 mins) to give a c lear supernatant which was used in enzyme assays . 13 pNPCase A S S A Y The p-nitrophenylcel lobioside assay (as descr ibed by G i lkes et al (37)) g ives a measure of exog lucanase activity in cell extracts. The assay measures the hydrolysis of the agluconic bond of p N P C by following the re lease of p-nitrophenol (pNP). The reaction condit ions were as fol lows: 500u.l of prewarmed cel l extract were mixed with 500u.l of prewarmed p N P C (12.5mM in 100mM K P O 4 and 0.02% NaN3) for 30 mins at 37°C, when 500u.l of 1M N a 2 C 0 3 were added. Absorbance at 410nm was measured against a blank solution (carbonate added to the p N P C prior to enzyme). A standard curve using known dilutions of pNP was constructed. p N P C a s e activity was ca lcu lated using the following formula: O.D.410 x X = Units (u.mol/min/ml undiluted enzyme). Yt where Y= u.l undiluted enzyme in the reaction mix t= incubation time (30mins) 10 .D. 410 = X nmoles pNP/1.5ml CARBOXYMETHYLCELLULASE ASSAY The C M C a s e assay measures reducing sugars released from C M C by reaction of the reducing groups with dinitrosal icycl ic ac id (DNS) (36). D N S reagent was prepared by dissolv ing 10g dinitrosal icyl ic acid, 2g phenol, 0.5g sodium sulfite and 200g sodium potassium tartrate in 500ml of 2% NaOH and then diluting this solution with 500ml water. The assay condit ions were as fol lows: 250u.l of prewarmed cell extract and 500u.l of prewarmed 4% C M C (Sigma low viscosity; 50mM K P O 4 , pH 7.0; 0.2mg/ml BSA) were mixed together and incubated at 37°C for 30mins. The reaction was stopped with 800uJ DNS reagent. 50u.l of standard g lucose solution (1mg/ml) were added and the mixture steamed at 100°C for 15 minutes. It was then al lowed to cool and absorbance at 550nm was measured against a blank solution (DNS reagent was added to the C M C before 14 the enzyme mixture). A standard curve was constructed using dilutions of the standard g lucose solution, to give a value for the j imoles of glucose/0.75ml which gives 1 O.D.550 (X). C M C a s e activity was then calculated using the formula: AO.D . 550 x X x100 = Units (Limol/min/ml undiluted enzyme). Yt where Y= u.l undiluted enzyme in 0.75ml reaction mix t= assay time. B -GALACTOSIDASE ACTIVITY (3-galactosidase was used as a cytoplasmic marker in E. coli cel ls (see enzyme localization studies) and was measured according to Miller (38). One unit of B-galactosidase is def ined as that amount of enzyme that re leases I j imole of ONP/min at 37°C. ALKALINE P H O S P H A T A S E ASSAY . Alkal ine phosphatase was used as a periplasmic marker in E. coli cel ls (see enzyme local ization studies) and was measured according to Garen and Levinthal, (39) in which the rate of release of p-nitrophenol from p-nitrophenol phosphate is determined by following absorbency changes at 410nm. One unit is that activity l iberating 1u.mole p-nitrophenol/min at 25°C. MALATE DEHYDROGENASE ASSAY Malate dehydrogenase was used as a cytoplasmic marker in FL c a p s u l a t u s cel ls in enzyme local ization experiments (see this sect ion). This enzyme cata lyses the following reaction: 15 L-malate + NAD^oxa l o a ce t a t e + N A D H 2 The activity was determined by a decrease in absorbency at 340nm owing to the oxidation of N A D H 2 . The following were p laced into a 3ml cuvette : 2.6ml phosphate buffer (0.1 M, pH 7.4), 0.2ml N A D H 2 , (3.75mM), 0.1ml cel l extract and finally 0.1ml 6mM oxaloacetate. Readings were taken at 15 second intervals for 2 minutes against a blank solution (no substrate added). Enzyme activity was calculated using the formula : Specif ic activity = A O P 340/min  6.2 x mg enzyme/ml reaction mix One unit of activity causes N A D H 2 to be oxidized at an initial rate of 1u.mole/min. under speci f ied condit ions at 25°C . PROTEIN CONCENTRATION Protein concentrat ions were determined using the B io-Rad assay (Bio-Rad labs) which is based on the differential color change of a dye in response to various concentrat ions of protein (40). Five u.l of cel l extract were diluted with 795 ul sdh^O and 200u.l of B io-Rad reagent were added. This mixture was incubated at room temperature for no less than 15 minutes. Absorbancy measurements were taken at 595nm against a blank solution (5u.l of 50mM K P O 4 subst ituted for cel l extract). Standard curves were constructed, using bovine p lasma albumin, to determine the u.g of protein/0.5ml that give 1 O.D. at 595nm (X). The protein concentration was calculated using the following formula : Q.D.595 x X x dilution = mg protein/ml (relative to 1000 bovine plasma albumin). 16 LOCATION O F ENZYMATIC ACTIVITY The osmotic shock procedure descr ibed by Nossa l and Heppel (41) was used to obtain per ip lasmic and cytoplasmic cell fractions. Briefly, 30ml of medium and antibiotic were inoculated with a single colony and incubated overnight at 30°C. Cel ls were harvested by centrifugation at 8K for 5 mins and resuspended in 10ml of a cold solution of Tris.CI (pH 7.1, 33mM), Sucrose (40%) and EDTA (1mM). The cel ls were sedimented and then resuspended in 10ml cold M g C I 2 solution (0.5mM). The cel ls were kept on ice (mixing gently) for 10 min, and then centrifuged again (8K, 5 min). The supernatant obtained was the per ip lasmic fraction. The cytoplasmic fraction was obtained by resuspending the sedimented material in 5ml K P O 4 buffer (0.1 M, pH 7.0) and sonicating the suspension (see Preparat ion of crude cel l extracts). After centrifugation (12K, 40min) the supernatant obtained was used as the cytoplasmic cell f ract ion. VISCOMETRIC A S S A Y S The rate of hydrolysis of 4% C M C (Sigma low viscosity grade) by crude cel l extracts was fol lowed by determining 0 (specif ic fluidity). 0 is an index of polymer length. The method is as descr ibed by Gi lkes et al (37) using a Cannon-Fenske viscometer (Fischer s c i e n t i f i c - # 1 3 - 6 1 6 E ) . 17 RESULTS SECTION ONE : CONSTRUCTION OF PLASMID pND 3 - A BROAD HOST RANGE PLASMID IN WHICH AN ENDOGLUCANASE AND EXOGLUCANASE FROM C. FIMI A R E PLACED UNDER THE CONTROL OF TRANSPOSON Tn10 TET PROMOTERS PART ONE : THE CONSTRUCTION OF pND-|. SEE FIGURE 3 P lasmid p cEC2 , carrying the endoglucanase gene (cenA) from C.  fimi was cut with Drall enzyme. The DNA fragment carrying the endoglucanase gene (1.7kbp) was isolated from a 0.8% agarose gel using D E A E membrane. The DNA was blunt-ended using a Klenow reaction, and blunt-ended EcoRI linkers were attached to the DNA. The DNA was then digested with an excess of EcoRI (see Figure 4). The plasmid pCB168, carrying the tet control region of transposon Tn10, was cut with E coR I and the 3.9kbp restriction fragment was isolated from a 0.8% agarose gel. This vector DNA and the DNA carrying the endoglucanase gene (insert DNA) were ligated together. The ligation mix was used to transform competent E. coli C600 cel ls. The cel ls were plated on LB Amp plates, and incubated overnight at 30°C. Of the 1171 transformants obtained on the LB Amp plates, 120 colonies were selected at random to be screened for endoglucanase activity by repl ica plating on LB Amp C M C plates (see materials and methods). Of these 120 colonies, four gave strong halos on the congo red stained C M C plates and two gave weaker halos. Hence all six c lones exhibited endog lucanase activity. The plasmid DNA from the six endoglucanase positive transformants was extracted by the alkal ine lysis method and digested with BamH1 to elucidate the orientation of the insert DNA in the constructs (see Figure 5). Two of the c lones gave 0.38kbp and 5.22kbp s i zed BamH1 restriction fragments suggest ing that the inserts were in the correct orientation. The plasmid that both these c lones carr ied was named pND-|. Two clones gave BamH1 restriction fragments of s izes 1.4kbp and 4.2kbp which suggested that the insert DNA was in the wrong orientation. Expression of the cenA 18 FIGURE 3: THE CONSTRUCTION O F PLASMID pND 3 . 19 F IGURE 4 : Auto radiogram following the attachment o fa"32p label led EcoRI linkers onto p cEC2 Dra11 DNA fragment carrying the cenA gene. Lane 1:loaded 1 jx) out of 50u.l of the p cEC2 DNA solution after the attachment of blunt ended EcoRI linkers. Lane 2: 45jil of the p c E C 2 DNA solution (after the ligation of linkers) was digested with EcoRI ( 4 0 units). After one hour, 1 JLLI was removed from this mix and loaded on the gel. Lane 3: one u.l of the digestion mix, after overnight digestion, was loaded on the gel. Lanes 2 and 3 show how the excess linkers were digested off the p cEC2 DNA, the majority being removed after one hour's digestion. 20 F IGURE 5: Plasmid pND-| digested with BamH1. Lane 1:Lamda DNA digested with Hind i 11. Lane 2:BamH1 digested pND-|, showing the 5.22kbp and the 0.38kbp fragments. 21 gene in these two c lones may have been from some unidentified vector promoter. The last two endoglucanase positive c lones gave restriction fragments whose s i zes suggested that two inserts had been cloned into the vector DNA. One of the two c lones (#5) in which the insert DNA was in the correct orientation, was selected for the second stage of the c on s t r u c t i o n . PART TWO:THE CONSTRUCTION OF pND2. SEE FIGURE 3. The broad host range plasmid, pJRD215, was digested with the enzymes EcoRI and Xba1 . P lasmid pND-|, isolated from clone #5 (see last stage), was then totally d igested with Xba1 and partially digested with E coR I (the DNA was digested 10-30 mins) to release a EcoRI -Xba1 fragment of 1.81kbp. This fragment was isolated from an agarose gel. The 1.81kbp p N D i DNA (insert DNA) was then ligated with the p JRD215 vector DNA and the ligation mix was used to transform competent E. coli C600 cells. The cel ls were plated on LB Kan plates and incubated overnight at 30°C. The transformants obtained on the LB Kan plates were replica plated onto LB Kan C M C plates to screen for endoglucanase activity. Of the 400 colonies screened, two were found to be endoglucanase positive. The plasmid DNA from these two c lones (designated p N D 2 ) was digested with Apa1 (see Figure 6). Both c lones gave Apa1 restriction fragments of s izes 10kbp and 1.84kbp. One clone (#1), was selected for the final stage in the construction of p N D 3 . 22 F IGURE 6: Plasmid p N D 2 digested with Apa1. Lane1 :pND 2 digested with Apa1, showing the 1.84kbp and the 10kbp fragments. Lane 2:Lamda DNA digested with Hind i 11. 23 PART THREE- THE CONSTRUCTION OF pND 3 . S EE FIGURE 3. To make the final construct p N D 3 , p N D 2 (vector DNA) was partially digested with Apa1 and ligated with the Apa1 fragment from pEC1 (insert DNA) carrying the exoglucanase gene. Both the vector DNA (11.84kbp) and the insert DNA (1.53kbp) were isolated from an agarose gel . The ligation mix was then used to transform competent E. coli C600 cel ls. The cells were plated on LB Kan plates and incubated overnight at 30°C. Any transformants obtained were then screened for endog lucanase and exog lucanase activities by repl ica plating on LB Kan C M C and LB Kan M U C plates. This procedure was used twice but it failed to produce any endoglucanase/exog lucanase positive c lones. So the protocol was modif ied. Al l the Apa1 restriction fragments from a total digest ion of pEC1 and partial digestion of p N D 2 were used in a ligation reaction in the hope that the correct restriction fragments would ligate together to form the des i red construct. The ligation mix was then used to transform competent E. coli C600 cel ls and any transformants obtained were screened for endog lucanase and exog lucanase activities, as descr ibed earlier. Of the 150 transformants screened for endoglucanase/ exog lucanase activities, 14 were found to be exog lucanase positive and of these two were endoglucanase positive. Of the rest, 80 c lones were found to be endoglucanase positive but not exog lucanase positive, suggest ing that all these c lones had grown up from cel ls which had been transformed with religated vector DNA. The plasmid DNA from the 14 exoglucanase positive c lones was isolated and digested with Apa1 enzyme. Running the DNA from these c lones on an agarose gel showed that two of these released a 1.84kbp and 1.53kbp Apa1 fragment (corresponding to the endog lucanase and the exoglucanase inserts respectively) as well as a 10kbp fragment, corresponding to the vector DNA. These two c lones were identified as those which had been both endoglucanase and exog lucanase positive on indicator plates. One of the two endoglucanase/exog lucanase positive c lones (#9) also re leased an extra Apa1 fragment which corresponded to an Apa1 fragment from Apa1 digested pEC1 (see Figure 7). Hence, the clone which released the correct number and s ize fragments when its DNA was digested 24 F IGURE 7: Apa1 digestion of clones #9 and #11. Lane 1: C lone #9 digested with Apa1, showing the 10kbp vector band, the 1.84kbp and the 1.53kbp bands. An extra band of about 6kbp is also seen in this c lone. Lane 2: Undigested clone #9. Lane 3: C lone #11 digested with Apa1 , showing the three correct fragments of s i zes 10kbp, 1.84kbp and 1.53kbp. Lane 4: Undigested clone #11. Lane 5: Lamda DNA digested with H ind i 11. The DNA used in these digestions was isolated by a mini alkaline lysis procedure, and so the DNA is not being fully digested by the enzyme. 25 with Apa1.(#11) was used as a source of the constructed plasmid P N D 3 . The DNA from this clone was also digested with H ind i 11, Xba1 (Figures 8 and 9), Mlu1 (Figure 10) and Xba1 and Sea l enzymes (Figure11). 26 F IGURE 8: Plasmid P N D 3 digestions. Lane 1: P N D 3 digested with Hind111, showing the single band of approximately 13.4kbp. Lane 2 P N D 3 digested with Xba1, again showing the single band. Lane 3: P N D 3 undigested. Lane 4: Lamda DNA digested with Hind i 1 1 . 27 F IGURE 9 : Plasmid P N D 3 digestions. Lane 1: Lamda DNA digested with Hind i 1 1 . Lane 2: p N D 3 digested with Xba1, showing the single band of approximately 13.4kbp. Lane 3: p N D 3 undigested. Lane 4: p N D 3 digested with H ind i 11, again showing the single band. 28 F IGURE 10: Lane 1: Lamda DNA digested with Hindi 11. Lane 2: E. coli c lone #9 digested with Mlu1. Lane 3: E. coli c lone #11, carrying the plasmid P N D 3 , digested with Mlu1 and showing the bands of s izes 1.1 kbp and 12.2 kbp. From the s ize of these fragments released, the orientation of the cenA gene, in the plasmid P N D 3 , could be e luc ida ted . 29 F IGURE 11: Lane 1: Lamda DNA digested with Hindi 11. Lane 2: E. coli c lone #9 digested with Xba1 and Sea l enzymes. Lane 3: E. coli c lone #11, carrying the plasmid P N D 3 , digested with Xba1 and S e a l enzymes and showing the bands of s ize LOkbp and 12.3 kbp. From the s ize of these fragments released, the orientation of the cex gene, in the plasmid P N D 3 , could be elucidated 30 SECTION TWO QUANTITATION AND LOCATION OF ENDOGLUCANASE AND EXOGLUCANASE ACTIVITIES IN E. COLI RECOMBINANT CLONES. DNS and p N P C assays were carried out to quantitate the endog lucanase and exoglucanase activities present in crude cell extracts of the two E. coli recombinant c lones, #9 and #11 (see Table 1). Results of assays carried out by previous workers on E. coli C600/pcEC2 and C600/pEC1 are also listed as sources of reference (26,27). E. coli C600 cel ls alone do not express any endoglucanase or exoglucanase activities. The cenA and the c e x genes, in the plasmid P N D 3 , are lacking their promoter sequences . Presumably then, the expression of these two genes in the plasmid P N D 3 relies on the Tn10 tet promoters. Osmot ic shock was used to localise the endoglucanase and exog lucanase activities in c lone #11. The enzymes alkal ine phosphatase and G-galactosidase were used as periplasmic and cytop lasmic markers respect ively (see Table 2). The results show that both CenA and Cex are being exported to the periplasm in the E. coli cells, as was found by previous workers (26,27). This means that the leader sequences of the genes cenA and cex are being recognized and are functional in E. coli cel ls. No signif icant endog lucanase or exog lucanase activity was present in the culture medium. SECTION THREE: MOBILISATION OF P N D 3 FROM E. COLI C600 TO H C A P S U L A T U S B10. The broad host range plasmid, p JRD215 (29), which was used in the construct ion of P N D 3 , is transferable between gram-negat ive organisms by conjugation in the presence of a variety of self-transmiss ib le plasmids, including RK2 (29). These so cal led helper p lasmids provide trans-act ing fertility funct ions. The kanamyc in resistant helper plasmid, pRK2013 , cons ists of the RK2 transfer genes c loned onto a colE1 replicon (34). This plasmid (present in EL col i HB101) was used in triparental matings (E. coli HB101 /pRK2013 x E. coli C600/ pND3 x recipient R. capsulatus B10). 31 TABLE 1 :QUANTITATION OF ENZYME ACTIVITIES FROM C R U D E CELL EXTRACTS O F E. COLI C L O N E S #9 AND #11. C L O N E DNS pNPC U/mg protein U/mg protein #9 7.0 0.38 #11 19.0 3.80 pcEC2 15.2 pEC1 _ 1.43 U= units of activity: for the DNS assay, nmoles of g lucose equivalents released/min. and for the p N P C assay, nmoles of p-n i t rophenol re leased/min. 32 TABLE 2:L0CATI0N OF ENZYME ACTIVITIES IN E. COLI CLONE #11 ACTIVITY IN NMOLES PRODUCT/MIN/ML OF CULTURE ENZYMT"N PERTPLA^ IN"CELL % ASSAYED FRACTION FRACTION EXTRACT RECOVERY ENDO 5^ 70 757) ~2^92{29) 10 86 BO 0.57 (47.5) 0.44 (37) 1.2 85 ALK. PHOSPH. 0.64 (72) 0.27(30) 0.89 102 B-GAL. 0.93 (22) 3.49 (82) 4.26 104 Figures in brackets give % of total activity present in the fraction. Units of activity: for the endog lucanase activity, nmoles g lucose equiva lents released/min/ml culture; for the exog lucanase activity, nmoles p-nitrophenol released/min/ml culture; for the a lkal ine phospha tase activity, nmoles p-nitrophenol re leased/min/ml culture; and for the B-galactos idase activity, nmoles o-nitrophenol re leased/min/ml cu l ture . 33 Triparental matings were set up as descr ibed in the Materials and Methods sect ion. R. capsulatus has a doubling time of 2 hours and so a ratio of five vo lumes of recipient culture to one volume of donor cultures was used. R C V minimal medium supported the growth of the prototrophic R. capsu la tus recipient cel ls but se lected against the growth of the auxotrophic E. coli donor cells. A second triparental mating experiment was set up where the number of recipient ce l ls was increased (twice the volume used in the first mating experiment). The number of donor E. coli C 6 O O / P N D 3 and recipient cel ls used in each experiment was obtained by plating dilutions of samples , taken from the cultures used in the mating experiments, on appropriate media plates and counting colonies growing up overnight at 30°C. These values, in addition to the number of transconjugants obtained from each experiment, were used to calculate the eff iciency of the triparental matings (see Table 3). Using a greater number of recipient cel ls is shown to increase the number of transconjugants obtained. One hundred transconjugants were picked onto RCV C M C Sm (15u.g/ml) and R C V M U C Sm plates to screen for any endoglucanase and exoglucanase positive c lones. All 100 c lones were found to be posit ive for both activities, indicating that the p lasmid P N D 3 had been mobil ized to these cells. R. capsu latus cel ls alone did not score posit ive for either endog lucanase or exog lucanase activit ies. Sixteen endoglucanase/exoglucanase positive c lones were selected at random to test if any E. coli ce l ls were contaminat ing the colonies. This was done by restreaking the c lones onto R C V Kan Y E (0.1%) media. If any E. coli cel ls were present then they would grow up overnight on this media whereas R. capsulatus cel ls would not. E. col i and R. capsu latus cel ls are morphological ly distinct (R.  c ap su l a t u s colonies appear red with lighter coloring around them). Two of the 16 c lones showed contamination with E. coli co lonies, however upon further testing on LB M U C Kan plates, these E. coli were found to be neither endoglucanase or exoglucanase positive. They were probably HB101/pRK2013 cel ls, which had either not been d issoc iated from the recipient R. capsu la tus cell during the breaking of mating pairs, or had landed c lose to the recipient R.  c a p s u l a t u s cell during plating of the cel ls. 34 TABLE 3:TRIPARENTAL MATING EFFICIENCY WHEN USING R .CAPSULATUS B10 C E L L S A S RECIPIENTS . T R A N S C O N J U G A N T S / D O N O R C O N J U G A T I O N S 3x10-5 CONJUGAT ION #2 13x10 ' 5 Twice the vo lume of recipient cel ls used in conjugation #1 were used in conjugation #2. 35 DNS and p N P C assays were carried out on cell extracts of two randomly picked R. capsulatus clones, #10 and #12. The results of these assays are shown in Table 4. The exoglucanase activities obtained from these c lones were comparable to those obtained from E. coli c lone #11, however the endog lucanase activities were lower (see d iscuss ion). Further experiments to local ise the enzyme activit ies (see Tab le 5), indicated that endog lucanase activity could not be detected in the periplasmic fractions whereas exog lucanase activity could. This could mean that the cenA leader sequence is not being recognized in the R. capsu latus cells and so is not functional in exporting the protein to the per ip lasm. Malate dehydrogenase was used as a cytoplasmic marker. A suitable per iplasmic marker, which is easi ly assayed, is not avai lable in R.  c a p su l a t u s cel ls . SECTION FOUR: MOBILIZATION OF p N D 3 FROM E. COLI C600 TO KLEBSIELLA PNEUMONIAE M5A1. Triparental mating experiments, similar to those used with R.  c a p s u l a t u s B10 as recipient cel ls, were set up to mobil ize plasmid p N D 3 into K. pneumoniae M5A1 cel ls. The plasmid pRK2013 (carried in E. coli HB101) was used again to help mobilize the plasmid p N D 3 into the recipient cel ls. Both E. coli strains C600 and HB101 are a u x o t r o p h i c whereas the K. pneumoniae strain used is prototrophic. This meant that transconjugants from the triparental mating exper iments could be se lected on minimal-medium plates (M9 medium without addition of amino acids). Hence, the mating pair mixture was plated on M9 Sm (15u.g/ml) plates. Two conjugation experiments were set up as before, where the second experiment used a larger volume of recipient cel ls. The efficiency of the conjugation experiments was calculated, as descr ibed for the R.  c ap su l a t u s matings (see mobilization into R. capsu latus B10) and the results are shown in Table 6. Again the results show that using a larger number of recipient cel ls increases the eff iciency of the conjugat ions. Sixteen random transconjugants were picked onto M9 M U C Sm and 36 TABLE 4:QUANTITATI0N OF ENZYME ACTIVITIES FROM R. CAPSULATUS B10 C L O N E S #10 AND #12 C L O N E DNS pNPC U/mg protein U/mg protein #10 8.33 3.28 #12 6.24 2.37 U= units of activity. For the DNS assay, nmoles g lucose equivalents released/min. and for the p N P C assay, nmoles p-nitrophenol released/min. 37 TABLE 5 1 0 C A T I 0 N OF ENZYME ACTIVITIES IN R. C A P S U L A T U S B10 C L O N E #10. ACTIVITY IN nMOLES PRODUCT/MIN/ML O F C U L T U R E ENZYMEE TN PERIPLASMIC~ IN CYTOPLASMIC IN C E L L % A S S A Y E D FRACTION FRACTION EXTRACT R E C O V E R Y ENDO 0 3.50"(79) 4.4 ~ ~79 EXO 0.036 (22.5) 0 .115(72) 0.16 94.5 MALATEDEHY. 0.027 (1.3) 1.58 (76) 2.06 77 F igures in brackets give % of total activity present in the fraction. Units of activity: for the endoglucanase, nmoles of g lucose equiva lents released/min/ml culture; for the exog lucanase activity, nmoles p-nitrophenol released/min/ml culture and for the malate deydrogenase activity, nmoles NAD released/min/ml culture. 38 TABLE 6:TRIPARENTAL MATING EFFICIENCY W H E N USING K.PNEUMONIAE A S RECIPIENT CELLS . T R A N S C O N J U G A N T S / D O N O R CONJUGAT ION #1 2.6x10-4 CONJUGAT ION #2 4.2 x10-4 Twice the volume of recipient cel ls used in conjugation #1 were used in conjugation #2. 39 M9 C M C Sm plates. All the clones were endoglucanase and exoglucanase positive. To test that the K. pneumoniae transconjugants were not contaminated with E. coli cel ls, samples of all 16 were examined microscopical ly after treating with Indian ink. K. pneumoniae cells possess a capsule and this is seen clearly using the indian ink. Three of the 16 clones seemed to be contaminated with E. coli cells, but the other 13 appeared E. coli free, even upon restreaking on LB Kan plates. Two of the endoglucanase/exoglucanase positive c lones were selected at random and pNPC and DNS assays were carried out on their cel l extracts (see Table 7). The values obtained for both the assays were much lower than those obtained from the E. coli c l one #11. Carrying out osmotic shock procedures on the two c lones was not very successful (see Table 8) as the percentage recovery obtained was very low. This may have been because the osmotic shock procedure was not working well with these cel ls or due to the very low O.D. readings obtained from the assays, which may have been beyond the sensit ive range of the spectrophotometer. SECTION FIVE: TRANSFORMATION OF E i_QQJJ CEL+ STRAINS. The genes for cel lobiose utilization are normally cryptic in E.  co l i . but recently the eel gene cluster for ce l lob iose util ization has been identified (33). The eel cluster is located at 37.8 min. on the E. coli map. It is not expressed in wild type strains of E. coli . Act ivat ion of the eel cluster by spontaneous mutation permits util ization of ce l lobiose, arbutin and sal ic in. Two derivatives of the E. coli strain W4680 (33) were used in this study (TW34 and TW35). The two E. coli strains were transformed with the plasmid PND3 and the transformants obtained (selected on LB Kan plates) were screened for endoglucanase and exoglucanase activities on LB Kan C M C and LB Kan M U C plates. Of the 120 transformants screened, 60 from each transformation, all were positive for endog lucanase and exog lucanase act iv i t ies. 40 TABLE 7:QUANTITATI0N OF ENZYME ACTIVITIES FROM K. PNEUMONIAE C L O N E S C L O N E DNS pNPC U/mg protein U/mg protein #1 2.78 0.83 #2 3.70 0.74 Units for the DNS assay, nmoles glucose equivalents released/min. and for the p N P C assay, nmoles p-nitrophenol released/min. 41 TABLE 8:L0CATI0N OF ENZYME ACTIVITIES IN K. PNEUMONIAE CLONES ACTIVITY IN nMOLES PRODUCT/MIN/ML O F C U L T U R E ENZYME IN PERIPLASMIC IN CYTOPLASMIC IN CEL L % ASSAYED FRACTION FRACTION EXTRACT R E C O V E R Y C LONE #1 ENDO 0.03 (5.6) 0.144 (27) 0.53 33 D O 0.003 (3.6) 0.03 (36) 0.083 40 C L O N E #2 ENDO 0 0.04 (29) 0.14 29 EXO 0.003 (3.6) 0.03 (50) 0.063 51 Figures in brackets give % of total activity present in the fraction. Units for the endoglucanase, nmoles g lucose equivalents released/ min/ml and for the exoglucanase, nmoles p-nitrophenol r e l e a s e d / m i n / m l . 42 Two c lones were selected at random, one from each transformation and the CenA and Cex activities were quantitated (see Table 9). The activities were also local ized, (see Table 10) and again no CenA or Cex activity could be detected in the culture supernatants. Ten c lones were selected at random to test if they could still grow up on medium containing cel lobiose as the carbon source. The c lones were grown in 5ml M9 media (where the 0.2% glucose had been replaced with 1% cel lobiose solution) overnight at 30°C. All ten c lones grew up in this medium. In the next stage these c lones were tested for growth on C M C containing medium (M9 medium, with 0.1% C M C as the carbon source replacing glucose). The original host strain, E. coli TW34, was also tested for growth on this medium. All eleven cultures were streaked heavily onto the M9 0.1% C M C Kan plates and incubated overnight at 30°C . None of the eleven strains grew up on the C M C medium, even after seven days incubation at 30°C. It was then decided to try growing the ten c lones and the control strain on M9 media containing 0.1% C M C and 0.05% cel lobiose to help start the growth of the E. coli cel ls. However, after a week's incubation on this medium at 30°C, no significant growth of any of the strains was observed. It is known that secretion of the ce l lu lases is essent ia l for their degradative action on cel lulose. The fact that CenA and Cex are not being exported to the cel ls ' exterior in these E. coli C e l + / p N D 3 strains probably helps explain why these cel ls are unable to grow on medium containing C M C (see discussion). 43 TABLE 9:QUANTITATI0N OF ENZYME ACTIVITIES FROM E. COLI CEL+/PND3 C L O N E S CLONE DNS pNPC U/mg protein U/mg protein #1 9.30 1.2 #2 10.5 1.3 Units for the DNS assay, nmoles g lucose equivalents released/min. and for the p N P C assay, nmoles p-nitrophenol released/min. 44 TABLE 10:LOCATION OF ENZYME ACTIVITIES IN E. COLI CEL+/PND3 C L O N E S ACTIVITY IN nMOLES PRODUCT/MIN/ML O F CULTURE E N Z Y M E IN PERIPLASMIC IN CYTOPLASMIC IN C E L L % A S S A Y E D FRACTION FRACTION EXTRACT R E C O V E R Y ENDO.CLONE#1 2.90 (47) 2.48 (40) 6.20 87 C L O N E #2 2.90 (44) 2.78 (41) 6.80 85 EXO.CLONE #1 0.47 (52) 0.32 (36) 0.90 88 C L O N E #2 0.45 (48) 0.36 (37) 0.98 85 ALK. P H O S P H J 1 0.55 (77) 0.14 (20) 0.71 97 C L O N E #2 0.61 (73) 0.18 (22) 0.83 95 B - G A L C L O N E #1 0.47 (15) 2.30 (74) 3.10 89 CLONE#2 0.51 (19) 2.14 (74) 2.90 93 Figures in brackets give % of total activity present in the fraction. Units of activity:the endog lucanase activity, nmoles g lucose equiva lents released/min/ml culture; for the exog lucanase activity, nmoles p-nitrophenol released/min/ml culture; for the a lkal ine phosphatase activity, nmoles p-nitrophenol re leased/min/ml culture; and for the B-galactos idase activity, nmoles o-nitrophenol re leased/min/ml cu l ture . 45 SECTION SIX: VISCOMETRIC STUDIES ON E. COLI C600 p N D 3 C LONE #11. Cel l extracts of E. coli C6OO/PND3 c lone #11, C600/pcEC2 and C600/pEC1 were made from 30ml cultures grown overnight at 30°C. The rates of hydrolysis of 4ml 4% C M C by 1ml of cell extract was then assayed using a Cannon-Fenske viscometer (see Figure 12). Spec i f i c fluidity was calculated using the formula: 1_ = _L -1 S P t 0 where t = time taken for the liquid meniscus to pass mark B. The real incubation time = t x (time at start of viscosity measurement + t ( viscosity) 2 t 0 = time for the solvent, without any enzyme or C M C added, to pass through the markers. Specif ic fluidity , 0 S p = 1 1 Sp See Figures 13 and 14 for the results of the viscometric assays . The graphs show how the cell extract from the E. coli C600 c lone #11 behaves more like the cell extract from the cel ls harboring the cenA gene alone (E. coli C600/pcEC2) . 46 F IGURE 12: Cannon-Fenske viscometer. At time zero, 12ml 4% C M C (Sigma low viscosity grade) in 50mM K phosphate pH7.0 and 0.02% N a N 3 were mixed with 3ml of enzyme or crude cel l extract. Five ml of this mix was then transferred immediately to the v iscometer and the liquid was drawn up to past mark C (into the bulb B) by attaching a pipette pump onto A. At 10 minutes, the time (t) was taken for the liquid meniscus to pass mark E. S imi lar readings were taken every 10 minutes for 60 minutes. At each t ime point, 0.75ml were removed from the remaining mix to determine the reducing sugars present (using the DNS assay). 47 FIGURE 13: SPECIFIC FLUIDITY AGAINST R E A L INCUBATION TIME. 600 1200 1800 2400 3000 -3600 420 REAL INCUBATION TIME (SECONDS) The curves represent results of viscometr ic assays carr ied out on cell extracts of E. coli C600 cel ls containing the plasmid p N D 3 ('pND3') curve , the plasmid p cEC2 ('Endo' curve) or the plasmid pEC1 ('Exo' curve). 48 FIGURE 14: A SPECIFIC FLUIDITY AGAINST A REDUCING SUGARS EXO 0.1 0.2 A REDUCING SUGARS 0.3 The curves represent results of viscometric assays carried out on cell extracts of E. coli C600 cells containing the plasmid p N D 3 CPND3') curve , the plasmid pcEC2 ('Endo' curve) or the plasmid pEC1 ('Exo' curve). 49 SECTION SEVEN: REPRESSION AND INDUCTION OF ENDOGLUCANASE AND EXOGLUCANASE ACTIVITIES IN E. COLI STRAINS CARRYING THE PLASMID p N D 3 . In order to test that the cenA and the cex genes are in fact under the control of the tet promoters from the transposon Tn10, the £ . co l i strain M8820/pR751 ::Tn10, which has Tn10 inserted into the plasmid pR751, was transformed with the plasmid p N D 3 . The trans-acting tetR gene product from the Tn10 would then, hopefully, recognize the operator sites, OL and OR, on the Tn10 tet region of p lasmid p N D 3 and hence repress the transcription of both the cenA and the cex genes. The addition of tetracycline or the non-inhibit ing tetracyc l ine der ivat ive, 2 -acety l -2 -decarboxyamide Tc, would then induce the transcription of these genes by binding to the repressor molecules and reducing their affinity for the operator sites. The plasmid pR751 carries resistance to trimethoprim (Tm) and so the E. coli M8820/pR751 transformants were selected for on LB Kan Tm (25u.g/ml) plates. Transformants were checked for endoglucanase and exoglucanase activities on LB Kan Tm C M C and LB Kan Tm M U C plates respectively. Of the 150 transformants screened, all were positive for both activities. When the levels of the endog lucanase and the exog lucanase activities were quantitated using DNS and p N P C assays, it was found that the activities were lower compared to those levels obtained from E. coli C 6 0 0 / p N D 3 . However it is probably not valid to compare the activities obtained from these two different host strains (see d iscuss ion) . One clone was selected at random and grown up in 5ml of LB Kan Tm overnight at 30°C. Two 1 litre f lasks containing 500ml LB Kan Tm were inoculated with 0.1ml of the overnight culture and the time of addition and O.D.600 readings, after addition of the inoculum, were noted. The flasks were then placed in a shaking water bath at 30°C. At regular time intervals, 6 ml of culture were removed from each flask . One ml of this was used to take O.D.600 readings and the rest was used for making cell extracts. At a given 50 time point during the exponential phase of growth (see Figure 15) 2u.g/ml of the inducer (2-acetyl 2-decarboxyamide Tc) was added to one of the flasks. Six ml samples, for both O.D.600 readings and cell extracts were removed as before but this time at shorter time intervals, up to 50 mins after the addition of the inducer. DNS and p N P C assays were carried out on the cell extracts to measure the levels endoglucanase and exog lucanase activities at each time point (see Figures 16 and 17). The results obtained indicated that the addition of the inducer, did not seem to have any effect on the levels of the enzyme activities compared to the levels obtained from the control culture. There were several possib le reasons. The inducer itself may not have been functioning; it is a l ight-sensitive compound and so could easi ly have been inactivated before use. Another possibil ity is that there was no repression of the endoglucanase and exoglucanase activities in the original E. coli M8820/pR751 ::Tn10 transformants and that the express ion of the cenA and the cex genes was simply low in this host strain compared to the levels obtained from the E. coli C600 clone #11. The lack of repression could be because Tn10 was carried on a low copy number plasmid (pR751) so that the levels of repressor molecules produced were insufficient to repress the transcription of the cenA and the cex genes on plasmid PND3. The E. coli strain JC10240 (42), with Tn10 on its chromosome, was used in a second set of repression/induction experiments. The E. coli cel ls were transformed with pND3, and transformants were selected on LB Kan plates. One hundred transformants were screened for endoglucanase/exoglucanase activities, and of the 100 sc reened all were double positives. One clone (#2) was selected at random and 0.1ml of an overnight culture was used to inoculate two one litre f lasks containing 500ml of LB Kan. The time of the inoculation and the O.D.600 readings after the addition of the inoculum were noted from both f lasks. The f lasks were then placed in a shaking water bath at 30°C. A s in the first experiment, six ml samples were removed from each flask at regular time intervals for O.D.600 readings and for preparing cell extracts. At a given time point during the exponential phase (see Figure 18), 2u.g/ml of tetracycline was added to one of the f lasks. Six ml samples were removed as before from each flask at five minute intervals, for up to 40 minutes after the addition of the inducer. Carrying out p N P C 51 F IGURE 15: Growth curves of E. coli M8820/pR751 ::Tn10, PND3 cultures. The plus (+) and minus (-) signs indicate whether the inducer (2-acetyl 2-decarboxyamide Tc) was added to the growing culture or not. The arrow indicates the time of addition of the inducer (2-acetyl 2 decarboxyamide Tc) to the plus (+) culture. 52 DNS ASSAY-SPECIFIC ACTIVITY AGAINST TIME 5.0-INDUCER I I I I I I 10 20 30 40 50 60 70 80 90 TIME (MINUTES) F I G U R E 16: Resu l t s of D N S a s s a y s c a r r i e d out on ce l l ex t rac ts of 5m l s a m p l e s r emoved f rom the g row ing cu l tu res of E. co l i M 8 8 2 0 / p R 7 5 1 : : T n 1 0 , p N D 3 . T h e ar row ind i ca tes the t ime of add i t ion of the i nduce r (2-acety l 2-d e c a r bo x yam i de Tc) to the p lus (+) cu l ture . 53 PNPC ASSAY-SPECIFIC ACTIVITY AGAINST TIME F IGURE 17: Results of p N P C assays carr ied out on cell extracts of 5ml samp les removed from the growing cultures of E. col i M8820/pR751: :Tn10,pND3. The arrow indicates the time of addition of the inducer (2-acetyl 2 decarboxyamide Tc) to the plus (+) culture. 54 and DNS assays on the crude cell extracts showed that induction of both endoglucanase and exoglucanase activity occurred 5 minutes after the addition of the tetracycline (see Figures 19 and 20). As additional ev idence that induction of enzyme activities was occurr ing after the addition of the tetracycl ine, two f lasks containing 100ml of LB Kan were inoculated with a single colony of c lone #2, and 2u.g/ml of tetracycline was added to one of the f lasks. The cultures were then incubated overnight in a shaking water bath at 30°C and cell extracts of the two cultures were then prepared and used in DNS and pNPC assays. The results of these assays are given in Table 11. The results showed that the cenA and the cex genes were indeed under the control of the tet promoters from Tn10 (see d i s c u s s i o n ) . 55 TIME (HOURS) F IGURE 18: Growth curves of E. coli JC10240/pND3 cultures. The plus (+) and minus (-) s igns indicate whether the inducer (tetracycline) was added to the growing culture or not. The arrow indicates the time of addition of the inducer to the plus (+) culture. 56 DNS ASSAY- SPECIFIC ACTIVITY AGAINST TIME TIME (MINUTES) FIGURE 19: Results of DNS assays carried out on cel l extracts of 5ml samples removed from the growing cultures of E. col i JC10240 /pND3 . The arrow indicates the time of addition of the inducer (tetracycline) to the plus (+) culture. 57 PNPC ASSAY- SPECIFIC ACTIVITY AGAINST TIME 10 20 30 40 50 60 7 0 80 TIME (MINUTES) F IGURE 20: Results of p N P C assays carried out on cel l extracts of 5ml samples removed from the growing cultures of E. col i JC10240 /pND3 . The arrow indicates the time of addit ion of the inducer (tetracycline) to the plus (+) culture. 58 TABLE 11 :QUANTITATION OF ENZYME ACTIVITIES FROM E. COLI JC10240/PND3 C L O N E #2 . DNS pNPC U/mg protein U/mg protein + TETRACYCL INE 5.2 0.64 - T E R A C Y C L I N E 3.0 0.36 Units for the DNS assay, nmoles g lucose equivalents released/min. and for the p N P C assay, nmoles p-nitrophenol released/min. 59 DISCUSSION The broad host range plasmid p N D 3 constructed in this project has been successfu l ly maintained in four different Gram-negat ive organisms and has al lowed the simultaneous express ion of the cenA and the cex genes in these host organisms. Placing the C .  f imi genes in non-cellulolytic organisms has also al lowed us to examine the effects of heterologous expression on these genes. The levels of expression of the cenA and the cex genes in these four organisms varies, as might be expected. Severa l factors could be influencing the accumulation of the CenA and Cex proteins. These include transcr ipt ional and translat ional eff ic iency, growth rate of the cel ls, and the sensitivity of the products to proteases present in the different host organisms. The levels of enzyme activity from E. coli C600 clone #11 were comparable to those obtained when cenA and cex were introduced separately into E. coli C600 cells on plasmid pBR322 (26,27). The levels of endoglucanase and exoglucanase activities obtained from the E. coli Ce l+/pND3 clones were lower than those obtained for the E. coli C600 clone #11 by a factor of two, but approximately the same percentage of each activity was present in the periplasm of the E. coli Ce l+/pND3 clones as was found in the periplasm of the E^  col i C600 clone #11. The levels of CenA and Cex enzyme activities were much lower in the K. pneumoniae host strain used, suggesting that the expression of the cenA and the cex genes were more strongly affected by one or more of the factors d iscussed above. The R. capsulatus recombinant c lones gave exoglucanase activities which were comparable to those obtained from E. coli C600 c lone #11, but the endoglucanase activity was lower. In addition CenA was not exported to the periplasm of the cel ls, implying that the cenA leader sequence was not recognized in the R. capsu latus cells, or that the protein was degraded by proteases present in the periplasm of the cells. The Cex leader peptide is 10 amino acids longer than that of CenA, and has more positive charge at its N-terminus. These properties may play a role in exporting Cex more efficiently in this organ ism. 60 R. capsulatus has been widely used in various biochemical and genetic studies but little is known about the factors that affect gene expression in this spec ies. Broad host range plasmid vectors were constructed for expression of heterologous genes in R.  capsu l a tus . These utilized an RK2 derived replicon and the rxcA promoter to obtain transcription of genes within appropriately posit ioned DNA fragments (43). The expression vectors were used to obtain the individual synthesis of CenA and Cex proteins. The ce l lu lase genes were expressed either from their native translation signals or from the rxcA B8703 gene translat ion initiation s igna ls to form a hybrid protein. R. capsu la tus cultures containing the expressed cel lulase genes could not be grown using C M C as a sole carbon source, though signif icant amounts of ce l lu lase activity were found in extracts of such cel ls (43). None of the four host organisms used in the present study secreted CenA and Cex to the culture medium. Presumably in these Gram-negat ive hosts, the outer membrane acts as a permeabil ity barrier to the release of proteins to the external medium. The excretion of the ce l lu lases is essent ia l for their degradat ive action on cel lulose, hence mutants of these organisms which leak the cel lu lases would be desirable. Leaky mutants have defects in the outer membrane, thereby allowing the diffusion of exported polypeptides from the periplasm into the surrounding medium. A leaky mutant strain of E. coli which leaked ce l lu lases was previously isolated in this lab (44). Before leaky mutants are isolated, it might be necessary to investigate whether the host organisms are in fact able to survive on cel lu lose which has been pretreated to break it down into smal ler subunits. Pretreatment could include using ac id swol len ce l lu lose or even enzymatical ly c leaved cel lu lose (i.e. treated with CenA and Cex proteins). The results of the viscometric assays indicate that the cell extracts from E. coli C600 clone #11 (carrying the plasmid PND3), gave C M C hydrolysis rates which were similar, if not identical, to those obtained from extracts of cel ls carrying the cenA gene alone (on plasmid pcEC2) . This is difficult to explain, but one possibil ity is that, in the construct pND3, the cenA gene is under the control of the stronger promoter (tetP/\) in the tet control region, 61 whereas the cex gene is under the control of the weaker promoter (tetPpi). Under maximally inducing condit ions, the tet A promoter (tetPA), has been reported to be 7-11 times more active than the tet R promoter, whereas under repressing condit ions, the basal level of transcription from tet PR is more nearly equal to that from tet Pj\ (45). Further v iscometr ic exper iments on the effect of mixing CenA and Cex in varying ratios, are required before firm conclus ions can be made from the viscometric assays carried out on the cel ls carrying the plasmid PND3. The results of the repression/induction exper iments carr ied out on the E. coli JC10240/pND3 clone #2 showed that the cenA and the cex genes are indeed under the control of the tet promoters from Tn10. The levels of induction of CenA and Cex activities were not very high, being about a 30% and 50% increase for the CenA and Cex activities respectively. There could be a number of reasons for this. A major one being that the intact Tn10 was present in the EL col i J C10240 /pND3 clone #2, thus the tetracycl ine resistance protein, TetA, would have been present and functioning in the host cel ls. Tn10 is thought to mediate resistance to tetracycl ine by caus ing active efflux of the antibiotic. Hence, once the resistance mechanism starts to function we would expect a decrease in the concentrat ion of tetracycl ine inside the cel ls, which could explain why the induction of both CenA and Cex decreases 5 minutes after the addition of the tetracycl ine. The modest level of induction might also be a result of the low level of repression in E. coli JC10240 which carries tetR on the chromosome as a Tn10 insertion. We may have obtained greater levels of repression if the TetR was on a high copy number plasmid and would then perhaps have seen greater levels of induction of the two proteins on addition of the tetracycl ine. Therefore a better repression and induction system may be to have the tetR gene alone carried on a high copy number plasmid and to use the non-inhibitory tetracycl ine derivative 2-acetyl 2-decarboxyamide Tc. 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