molecular cloning, expression, andcharacterization of ... · endoglucanase activity and molecular...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, p. 359-3650099-2240/91/020359-07$02.00/0Copyright C 1991, American Society for Microbiology

Vol. 57, No. 2

Molecular Cloning, Expression, and Characterization ofEndoglucanase Genes from Fibrobacter succinogenes ARI

RICARDO CAVICCHIOLI* AND KENNETH WATSON

Department ofBiochemistry, Microbiology, and Nutrition, University ofNew England,Armidale 2351, New South Wales, Australia

Received 10 September 1990/Accepted 9 November 1990

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminalanaerobe Fibrobacter succinogenes ARL. Clones were screened on carboxymethyl cellulose, and 8 colonies thatproduced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blothybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, whichis representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and theresulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIIIinsert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 andpGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level ofendoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethylcellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%)was localized in the periplasm ofE. coli, with low but significant levels (16%) being detected in the extracellularmedium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimumtemperature of 39°C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.

Fibrobacter succinogenes is one of the most prolificcellulolytic microorganisms in the rumen, possessing ahighly active cellulase system (16, 46). However, difficulty inisolating F. succinogenes (43) has resulted in a large propor-tion of studies concentrating on strain S85, which wasoriginally isolated by Bryant and Doetsch in 1954 (7). Ex-tensive research has focused on the characterization ofvarious enzymes involved in the saccharification of lignocel-lulosic compounds. Enzyme activities associated with thebreakdown of cellulose and hemicellulose from S85 includeendoglucanase (30, 31), cellodextrinase (22), esterase (29),cellobiosidase (23), ,B-glucosidase (16), xylanase (41), andlichenase (10).

Recently, Bacteroides succinogenes was reclassified toFibrobacter succinogenes with two subspecies, F. succino-genes subsp. succinogenes and F. succinogenes subsp.elongata (33). Strain AR1 has not been extensively charac-terized; however, it appears to belong to F. succinogenessubsp. elongata (type strain HM2). Other strains belongingto F. succinogenes subsp. elongata include MM4 and MB4(1). Evidence that AR1 may belong to F. succinogenessubsp. elongata is suggested by the fact that genomic DNAisolated from strains MM4, MB4, and HM2 (42a), like AR1(7a), is not successfully digested with the restriction enzymeSau3Al, whose recognition sequence is GATC. BamHI(whose core recognition sequence is GATC) also fails todigest AR1 DNA (7a). By comparison, genomic DNAs fromstrains BL2 and S85 are able to be restricted by BamHI (12).This property may be helpful in distinguishing the twosubspecies.The cloning of separate genes or operons would facilitate

the analysis of individual cellulase enzymes, allowing abetter understanding of their role in lignocellulose break-down in the rumen. Elucidation of the mechanisms involvedin gene expression would allow an informed attempt at

* Corresponding author.

genetic manipulation of these bacteria. Furthermore, itwould provide important knowledge that is required beforethe release of genetically engineered microorganisms can beconsidered responsible. In this study, a gene library fromstrain AR1 was constructed to allow analysis of the geneticand enzymatic components responsible for the organism'sability to actively degrade lignocellulose. The isolation fromF. succinogenes AR1 of three distinct endoglucanase genes,and in particular, the characterization of one clone with ahigh level of activity, is reported. The nucleotide sequenceand novel aspects of gene control in the endoglucanase genefrom AR1 with a high level of activity are presented in aseparate report (7b).

MATERIALS AND METHODS

Bacterial strains and cloning vectors. F. succinogenes AR1(formerly B. succinogenes [33]) was provided by Tom Bau-chop. Escherichia coli HB101 and DH1 (17) were grown in2 x YT medium containing (per liter) 16 g of tryptone, 10 g ofyeast extract, and 5 g of sodium chloride. Incubations wereconducted at 37°C in flasks placed in an orbital shakeroperating at 200 opm (orbits per minute). Ampicillin wasadded at 100 ,ug/ml. For plate media, agar was added at 2%.Cosmid vector pHC79 (20) was used for cosmid cloning, andplasmids pGem-1, pGem-2, pGem7Zf+, and pGem7ZF(Promega, Sydney, Australia) were used for subcloning andexonuclease deletions.Gene library and screening for endoglucanase activity.

Genomic DNA was isolated essentially by the procedure ofAnderson and McKay (2). Genomic DNA was partiallydigested with HindlIl, and following separation on a 0.7%agarose gel, appropriately sized fractions were pooled andligated into the HindIII site of pHC79. Recombinant cosmidswere packaged and infected into E. coli DH1 by using thePromega Packagene kit. Cells were plated onto ampicillin(100 ,ug/ml)-containing plates, and 2,000 colonies werescreened onto plates containing (per liter) 2 g of carboxy-

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360 CAVICCHIOLI AND WATSON

methyl cellulose (CMC; low viscosity; Sigma Chemical Co.,St. Louis, Mo.), 5 g of yeast extract, the minimal salt ofGilardi (13), 100 mg of ampicillin, and 50 mg of cyclohexi-mide. Plates were incubated for 3 days at 37°C and thenflooded with 1 mg of Congo red per ml for 15 min. Plateswere drained and then washed with 1 M NaCl. Endogluca-nase-positive clones were identified by the clearing zonessurrounding the colonies (45). Thirty-three clones were

identified as endoglucanase positive, with 8 large clearingzones (LCZs; 3 cm in diameter after 2 days of growth) and 25small clearing zones (SCZs; 1 cm in diameter after 2 days ofgrowth). Recombinants pRC093 and pDA84 were chosen as

representatives of cosmid clones, producing large and smallclearing zones, respectively (see Table 1).

Subcloning and exonuclease deletions. Plasmid DNA wasisolated by the alkaline lysis procedure (5). Plasmid pRCEwas constructed by cloning an approximately 3.3-kb EcoRIfragment from pRC093 into pGem-1 (Table 1). A 2,075-bpEcoRI-HindIII fragment encoding FSendA (7b) was sub-cloned from pRCE into pGem-1, pGem-2, pGem7Zf+, andpGem7Zf-, generating pRCEH, pRCEH2, pRCZ+, andpRCZ-, respectively. The 1.55-kb HindIII-ApaI, 1.35-kbEcoRI-SphI, 1.38-kb EcoRI-Sau3AI, 1.62-kb EcoRI-XbaI,and 1.86-kb EcoRI-BglIll restriction enzyme fragments werecloned from pRCEH into pGem7Zf+. Exonuclease deletionsof pRCZ+ and pRCZ- were generated by using the Erase-a-base kit (Promega). Deletions from the HindIII end ofpRCZ+ were obtained by first digesting it with Sacl, toproduce a 3' overhang (to inhibit exonuclease III digestion),followed by digestion with HindIII to generate a 5' over-

hang. Nested sets of deletions were obtained by exonucleaseIII digestion for set time periods. Deletions from the EcoRIend were performed by first linearizing pRCZ- with XhoI (5'overhang), end filling with ot-phosphorothioate deoxynucle-oside triphosphates (39), restricting with EcoRI, and sequen-tially digesting with exonuclease III.

Southern hybridization. Cosmid clones were preparedfrom E. coli DH1 by using Qiagen plasmid preparationcolumns (Qiagen Inc., Studio City, Calif.). A small amountof E. coli genomic DNA was also extracted on the Qiagencolumns because of the size (>20 kb) of the cosmid recom-binants. DNA hybridization was performed essentially asdescribed by Southern (42). Cosmids and F. succinogenesAR1 genomic DNA were digested to completion withHindIII. These digests, together with an EcoRI-Hindlllfragment from pRCEH and an approximately 6-kb Hindlllfragment encoding cellulase activity from pDA84 (8), wereloaded onto 1% agarose gels and electrophoresed for 2 h at50 V. Lambda DNA Hindlll and EcoRI-HindIII digestswere included as molecular weight markers. Gels were

capillary blotted onto N+-nylon filters (Amersham, Sydney,Australia) according to the directions of the manufacturerand hybridized by using the insert from pRCEH which waslabeled with [32P]dATP by using a random priming kit(Bresa, Adelaide, Australia). High- and low-stringency hy-bridization and washing procedures were performed as out-lined in the Amersham blotting and hybridization protocolsfor Hybond membranes.

Cellular fractionation. Cells in the late logarithmic phasewere harvested at 11,000 x g for 10 min, and the supernatantwas taken as the extracellular phase. Periplasmic enzymes

were prepared by the osmotic shock procedure (19), withmodifications. Cells were washed in 10 mM Tris hydrochlo-ride (pH 8), resuspended in 25% sucrose-10mM Tris hydro-chloride (pH 8), and shaken gently at 22°C for 10 min. Cellswere pelleted (11,000 x g for 10 min) and resuspended in ice

water with further gentle shaking at 4°C for 10 min. Cellswere pelleted, and the supernatant that was recovered wasused as the periplasmic fraction. Cytoplasmic fractionationwas performed by sonicating the remaining cell pellet (resus-pended in 50 mM sodium citrate buffer [pH 5.7]) with aBranson sonifier fitted with a B-30 microtip at 70% maximumpower and 50% duty cycle. The cytoplasmic fraction was thesupernatant after centrifugation at 16,000 x g for 1 h at 40C.,B-Galactosidase and alkaline phosphatase were assayed todetermine the activities of these respective cytoplasmic (36)and periplasmic (19) marker enzymes. Activity was deter-mined by a spectrophotometric method (34) by using para-nitrophenyl (pNP) derivatives.Endoglucanase activity and molecular weight determina-

tion. Endoglucanase activity was assayed by measuring therelease of reducing sugars (35) on 0.5% CMC, phosphoricacid-swollen cellulose (Avicel PH101 prepared by themethod of Wood [48]), oatspelt xylan (batch no. X-0376;Sigma), lichenan (batch no. 124F-0106; Sigma), laminarin(batch no. 78F-3798; Sigma), ball-milled cellulose (Whatmanno. 1; Whatman, Maidstone, England), and 5% AvicelPH101 (38-,um average particle size; Honeywill and Stein,Poole, United Kingdom), filter paper (Whatman No. 1), andsliva cotton buffered in 50 mM citrate phosphate buffer (pH6). Activity was measured as (i) pNP liberated from pNP-glucoside, pNP-xyloside, pNP-lactoside, and pNP-cellobio-side (34) and (ii) glucose liberated from maltose, cellobiose,lactose, sucrose, melibiose, and melezitose with a glucoseoxidase-peroxidase kit (Sigma). For evaluating the effect ofpH, HCI-KCI (100 mM) was used for pH 1.0 to 2.2, citrate-phosphate buffer (various concentrations of citrate and phos-phate were used, depending on the pH) was used for pH 2.6to 7.0, 50 mM Tris hydrochloride was used for pH 7.2 to 9.0,and 50 mM glycine-NaOH was used for pH 9.0 to 12.5 (15).The pH of the final endoglucanase assay mixture (includingadded enzyme fractions) was measured directly, as thebuffering capacities of final solutions were found to varymarkedly. One unit of activity was defined as that whichreleased 1 ,umol of glucose equivalent as reducing sugar or 1,umol of pNP per min at 39°C. Protein concentration wasdetermined by the Lowry procedure (28). Thermal inactiva-tion of endoglucanase activity was determined by measuringreducing sugar from CMC at 390C, after preincubation in 50mM citrate phosphate (pH 6) at 4, 39, and 50°C. The mode ofcellulase action was determined in an Ubbelohde viscometerby quantitating the decrease in viscosity with time caused byenzyme action in a solution of 1% CMC-50 mM citratephosphate (pH 6). The molecular weight of the endogluca-nase was determined from periplasmic extracts by gel filtra-tion (3) by using Sephadex G100 (Pharmacia, Sydney, Aus-tralia). The column was calibrated with bovine serumalbumin, ovalbumin, pepsin, and lysozyme, and the voidvolume was calculated with blue dextran (Sigma). Thecolumn was eluted with 50 mM citrate buffer (pH 6), andfractions were assayed for endoglucanase activity by mea-suring the release of reducing sugar by the para-hydroxy-benzoic acid hydrazide method (26), with CMC used as thesubstrate.

RESULTS

Isolation of endoglucanase genes from F. succinogenes AR1genomic DNA. Two thousand cosmid clones were screenedin E. coli DH1 for endoglucanase activity. Eight clonesexhibited LCZs on CMC plates, and 25 clones exhibitedSCZs. Plasmid pRC093 (Table 1), which was representative

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F. SUCCINOGENES ENDOGLUCANASE GENES 361

TABLE 1. Recombinant plasmids and cosmids used in this study

Plasmid orVector and construction Endoglucanase

cosmid activitypRC093 pHC79 cosmid gene bank LCZCosmids 2 to 8 pHC79 cosmid gene bank LCZpDA84 pHC79 cosmid gene bank SCZpRCE pGEM-1, approx. 3.3-kb Hindlll insert from pRC093 LCZPRCEH pGEM-1, 2.1-kb EcoRI-HindIII insert from pRC093 LCZpRCEH2 pGEM-2, 2.1-kb EcoRI-Hindlll insert from pRC093 LCZpRCZ+ pGEM7Zf+, 2.1-kb EcoRI-HindIII insert from pRC093 LCZpRCZ- pGEM7Zf-, 2.1-kb EcoRI-HindIII insert from pRC093 LCZpRCZHA+ pGEM7Zf+, 1.55-kb HindIII-ApaI insert from pRCEHa hpRCZES+ pGEM7Zf+, 1.35-kb EcoRI-SphI insert from pRCEHpRCZEM+ pGEM7Zf+, 1.38-kb EcoRI-Sau3AI insert from pRCEHpRCZEX+ pGEM7Zf+, 1.62-kb EcoRI-XbaI insert from pRCEHpRCZEB+ pGEM7Zf+, 1.86-kb EcoRI-BgIII insert from pRCEH LCZ

Exonuclease deletionspRCZ-1 pRCZ-, deletion from HindIll terminus, 1.97-kb insert LCZpRCZ-2 pRCZ-, deletion from HindIll terminus, 1.74-kb insertpRCZ+1 pRCZ+, deletion from EcoRI terminus, 1.93-kb insert LCZpRCZ+2 pRCZ+, deletion from EcoRI terminus, 1.58-kb insert

a Restriction enzyme sites and exonuclease deletion sites have been mapped by DNA sequence analysis (7b).b-, No clearing zone and no detectable activity by reducing sugar assay.

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f LCZ, was isolated and completely digested with HindlIl, tralia) and PBS- (Stratagene, Sydney, Australia) were fre--?coRI, or BamHI. Digests were ligated into appropriately quently unstable. pGem7Zf+ and pGem7Zf- were mostestricted pGem-1, transformed into E. coli HB101, and frequently used for subcloning and exonuclease deletiongcreened for endoglucanase activity on CMC plates. A analysis because of the large number of suitable restrictionlumber of transformants derived from each digest gave enzyme sites in the multiple cloning site. Endoglucanase'MC-positive clones, indicating that all three restriction activity from the 2.1-kb fragment in pRCEH and pREH2 was'nzyme sites were absent from the functional endoglucanase independent of orientation, indicating that the gene was;ene. The EcoRI-derived clone pRCE harbored an insert of expressed from its own promoter in E. coli. Deletion analy-pproximately 3.3 kb. HindlIl digestion of this insert re- sis (Table 1) indicated that the functional gene was encodedulted in a 2.1-kb EcoRI-HindIII fragment which was cloned within a fragment of approximately 1.7-kb delimited at then pGem-1, generating pRCEH. Restriction map analysis of EcoRI end by pRCZ+1 and at the HindlIl end by pRCZ-1.he pRCEH insert (Fig. 1) indicated an absence of sites for Southern blot analysis. Seven of the recombinant cosmidslatII, NsiI, PstI, Sacl, Sall, SmaI, and XhoI. Fragments producing LCZ, pRC093, and cosmids 2 to 7 (see Table 1 forrom digestion with Sau3AI, which cut three times, and description) showed equivalent bands of approximately 3.4tpaI, BglII, SphI, and XbaI, which cut only once each, were kb when they were hybridized with 32P-labeled probe syn-igated into pGem7Zf', transformed into E. coli HB101, and thesized from the pRCEH insert (Fig. 2). By contrast,,creened onto CMC plates. Only clone pRCEB' (EcoRI- cosmid 8 (LCZ) and pDA84 (SCZ) revealed an absence of3glII insert) exhibited cellulase activity. However, activity hybridization under both high- and low-stringency condi-vas not stably maintained in cells harboring this recombi-iant, and the 2.1-kb EcoRI-HindIII fragment was considered tions of hybridization and washing. Moreover, when the

o etemniu-ze retito.nym rgetta filter was stripped and then reprobed with a 6-kb HindIIIloubende. edogluc activiy. Iwasmnoted fragment (cellulase encoding) derived from pDA84, only thehat cloning of DNA derived from F. succinogenes ARi in corresponding 6-kb band in lanes B and N (Fig. 2a) indicatedhGem-, pGem7Zf+n pGem7Zfr pHC79, and pKK232-8 (6) hybridization (data not shown). These observations indi-Gem1pGem7Zf'pGem7Zf.pHC79'and pKK2328 6 cated that three distinct genes are encoded by (i) pRC093 andvas generally successful, whereas cloning of recombinants cosmids 2 to 7, which produce LCZs, (ii) cosmid 8 (LCZ),.onstructed in pTZ18U and pTZ19U (Biorad, Sydney, Aus- and (iii) pDA84 (SCZ). Southern blot analysis of the EcoRI

HindlIl fragment from pRCEH hybridized to F. succino-EV A V Shl mx M B H genes AR1 genomic DNA digested with Hindlll showed a

single band of hybridization under high- and low-stringencyconditions. This indicated that F. succinogenes AR1 proba-bly encodes only a single copy of the gene and that there isan absence of other genes showing a high degree of similarity

500 bp to it. It was noted that the genomic band (Fig. 2a, lane L)

FIG..

Restrictionmapofthe21kbenoglucanasenappeared to be slightly smaller than the correspondingFIG. 1. Restriction map of the 2.1-kb endoglucanase-encoding cosmid bands (Fig. 2a, lanes D through J). This may havensert from pRCEH. Restriction enzyme abbreviations: E, EcoRI, . 'X, ApaI; V, AvaI; S, SphI; M, Sau3Al; X, XbaI; B, BglII; H, been due to a gel artifact, perhaps caused by overloading.HindIII. The insert was restricted with HindIll and ApaI to generate Cellular localization of endoglucanase in E. coli. Determi-)RCZHA' and with EcoRI combined with SphI, Sau3AI, XbaI, and nation of the cellular distribution of endoglucanase activity,gglIl to generate pRCZES', pRCZEM+, pRCZEX+, and pRC- as estimated by reducing sugar analysis by using the dinitro-ZEB respectively. salicylic acid method (32) or the procedure of Nelson (35)

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a bABC DE F GH I J K L MN ABC DE F G HI J K L MN

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FIG. 2. Southern blot analysis. (a) Hybridization of a 32P-labeled probe (2.1-kb EcoRI-Hindlll fragment from pRCEH) to Hindlll-digestedcosmid clones (lanes B through J), F. succinogenes ARi genomic DNA (lane L), and 2.1- and 6-kb cellulase-encoding fragments from pRCO93(lane M) and pDA84 (lane N), respectively. X Hindlll-EcoRl (lane A) and X Hindlll (lane K) were used as molecular weight markers. Sizesof bands marked by arrows for X Hindlll-EcoRI are as follows, from the top: 21.2, 4.3, 2.0, 1.4, and 0.95 kb. (b) Ethidium bromide-stainedagarose gel of the gel used in panel a.

(see Materials and Methods), led to some inconsistencies inactivity determinations, particularly in the extracellular frac-tion, in which high and variable blank values were obtained.The Nelson method (35), however, provided the most con-sistent results and was used throughout this study. Becauseof its relative purity, the periplasmic fraction was used for allsubsequent enzyme activity determinations, including sub-strate utilization studies (see below). The cellular distribu-tion for each fraction was confirmed by comparing the ratesof fluidity change in an Ubbelohde viscometer. This methodallowed estimation of the enzyme distribution by assessingendoglucanase activity by a purely physical method ratherthan by a chemical assay. Samples were appropriatelydiluted until a constant rate of change in fluidity was ob-tained for each fraction. E. coli cells harboring plasmids witheither a >20-kb insert in pRC093 or a 2.1-kb insert inpRCEH produced clearing zones of equivalent sizes onCMC plates, and this was reflected by the endoglucanaseactivity (Table 2). The cellular distribution of endoglucanasefor E. coli harboring either recombinant was mainly periplas-mic (approximately 80%). A low but significant proportion(16%) was, however, found in the extracellular medium.Marker enzymes indicated the integrity of each fraction.Extracellular activity was also monitored, and endogluca-

TABLE 2. Cellular distribution of endoglucanase in E. coliHB101 harboring pRCEH and E. coli DH1 harboring pRC093

Activity (mU/ml) of original culture

Fraction Endoglucanase" pNP- pNP-alkaline

pRC093 pRCEH galactosidase phosphatase

Extracellular 6 (16)b 7 (17) NDc NDPeriplasmic 30 (78) 32 (78) ND 0.6 (100)Cytoplasmic 2 (6) 2 (5) 0.4 (100) NDTotal 38 (100) 41 (100) 0.4 (100) 0.6 (100)

a Endoglucanase activity was determined by the method of Nelson (35), andcellular distribution was confirmed by a viscometric assay.

b Values in parentheses are percentages of total activity.' ND, Not detectable (0.1 mU/ml or less).

nase levels were found to increase proportionally withrespect to the cell growth phase (data not shown). Theactivities of the cell-bound fractions (pelleted cellular mem-branes following sonication) were less than 1% (data notshown).

Physiochemical properties of endoglucanase activity. It wasnoteworthy that expression of endoglucanase activity of E.coli clones was observed under aerobic or anaerobic growthconditions. Cellobiose or glucose (100 mM) had no effect onthe expression of endoglucanase activity, as determined byclearing zone size on plates containing CMC and glucose orcellobiose. Endoglucanase pH profiles were constructed,and activity was high (>50% of maximum activity) betweenpH 4.5 and 8.0, peaking at about pH 5.0 for E. coli HB101harboring pRCEH (Fig. 3). Endoglucanase activity showed asharp decrease below pH 4.5 and a more gradual decrease atalkaline pH values. Endoglucanase activity was maximum at

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FIG. 3. Effect of pH on the endoglucanase activity of E. coliHB101 harboring pRCEH.



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TIME (HR)FIG. 4. Effect of preincubation at 4°C (A), 39°C (0), and 50°C

(A) on the endoglucanase activity of E. coli HB101 harboringpRCEH.

37 to 39°C, decreasing markedly at elevated temperatures.This was reflected in thermal stability at 39°C compared withthat at 50°C (Fig. 4). At 39°C, the activities of the enzyme

fractions, which were buffered in 50 mM citrate phosphate(pH 6), remained high (75 to 100%) for 8 h, whereasendoglucanase was completely lost after 3 h when it was

stored at 50°C. By comparison, endoglucanase activity was

retained for several months at 0 to 4°C. The molecularweight of the endoglucanase was estimated by gel filtrationto be approximately 46,500.Mode of action and substrate specificity. As measured with

an Ubbelohde viscometer, a rapid increase in CMC fluiditywas observed with periplasmic enzymes from E. coli harbor-ing pRCEH, and this was indicative of an endo-actingenzyme. Activity on natural substrate (cellobiose) or substi-tuted substrate (pNP-glucoside) was not detectable. Thesesubstrates are diagnostic for P-glucosidase activity. Lowactivity on pNP-cellobioside, 0.72 nmol/min/mg of protein,indicated that exoglucanase activity was also minimal. Sim-ilarly, activity was not detected on other natural or substi-tuted short-chain glycans (maltose, lactose, sucrose, melibi-ose, melezitose, pNP-xyloside, pNP-lactoside, and pNP-glucoside). It was noted that trehalose was activelyhydrolyzed by periplasmic enzymes from E. coli HB101 andDH1 (7a).

Activity on recalcitrant substrates such as filter paper,

cotton, and Avicel was significant (Table 3). Less crystallinecelluloses were more extensively hydrolyzed, with the high-est activity detected on acid-swollen cellulose. It was notedthat hydrolysis of CMC resulted in the rapid production ofreducing sugar, with >70% maximum production observedafter 1 h and essentially 100% observed by 4 h. Specificactivity on CMC was about 70 nmol/min/mg of protein(30-min assay). Activity on lichenan (1,3-1,4-4-D-glucan),but not on laminarin (1,3-3-D-glucan), indicated that it wasprobably the 1,4-p-D-glucan component of lichenan that wasbeing hydrolyzed. It was noteworthy that the endoglucanasewas also active on xylan.

TABLE 3. Hydrolysis of glycan substratesa after 24 h ofincubation with periplasmic enzymes from E. coli

HB101 harboring pRCEH

Substrate Reducing sugar % Activity(p.g of glucose) relative to CMC

CMC 26.8b 100Avicel PH101 10.9 41Filter paper 8.5 32Cotton 2.4 9Ball-milled cellulose 46.2 173Acid-swollen cellulose 165.4 618Laminarin NDc NDLichenan 13.4 50Xylan 15.8 59

' Reducing sugars were determined by the method of Nelson (35) afterincubation of periplasmic enzymes in 50 mM sodium citrate buffer (pH 6) for24 h. Each substrate was 5 mg/ml, except for Avicel, filter paper, and cotton,which were 50 mg/ml.

b Production of reducing sugar on CMC at 1 h was 72% of the level at 24 hand was essentially 100% by 4 h.

ND, Not detectable (1 jLg or less in 24 h).

DISCUSSION

Genomic DNA from F. succinogenes AR1 was success-fully cloned into E. coli by using cosmid pHC79, and anumber of recombinants expressed endoglucanase activity.Distinct LCZs and SCZs on CMC plates were features ofendoglucanase expression in E. coli. A similar phenomenonhas been reported by other authors during the constructionof gene libraries of Cellulomonas (14), Clostridium thermo-cellum (40), Ruminococcus albus (21), and F. succinogenes(8, 9, 24). In all cases a correlation between clearing zonediameter and endoglucanase activity was observed. South-ern blot analysis revealed the isolation of three separategenes encoding endoglucanase (Fig. 2). It is possible thatother unique endoglucanase genes are represented in thefamily of 25 clones that produced SCZs. The fact that twoseparate genes were found for the eight clones that producedLCZs indicates that this possibility is likely. Multiple en-doglucanase genes have been identified for other cellulolyticbacteria, and in C. thermocellum, for example, 15 distinctendoglucanase genes have been isolated (18). In F. succino-genes S85, a number of endoglucanases have been identified,and three have been characterized in particular (30, 31). Itwill thus be useful to isolate and characterize all the genesencoding endoglucanases from our gene bank, because thiswill lead to a comprehensive understanding of the endoglu-canase requirements for this microorganism.

In view of the recent reclassification of Fibrobacter as agenus distinct from the genus Bacteroides and, furthermore,to the suggestion that it may form a new family distinct fromthe family Bacteroidaceae (33), it was significant that theendoglucanase genes in pRCEH and pRCEH2 were ex-pressed in E. coli, indicating that promoters from F. succi-nogenes may be recognized by E. coli RNA polymerase.Data from DNA sequencing and primer extension analysis tomap transcription start sites would be useful to confirm thepresence of functional promoters within the cloned insert.

Delimitation of the boundaries of the functional gene inpRCEH was undertaken by subcloning restriction fragmentsin pGem7Zf+ and constructing nested deletions from pRCZ+and pRCZ-. As a result, a 1.7-kb fragment was identified as

being important for the expression of endoglucanase. Thisindicated that the possible molecular weight of the encodedprotein could be up to 62,000. However, preliminary data

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indicated that the molecular weight of the periplasmic en-

doglucanase was 46,500, which suggested that regions inaddition to the coding sequence are required for activity.

In F. succinogenus ARI, 80% of endoglucanase activitywas found in the extracellular medium (7a), and a similarlevel was found in strain S85 (16). In E. coli harboringpRCEH and pRC093, most activity (approximately 80%)was localized in the periplasm (Table 2). E. coli is not notedfor its ability to secrete cloned products into the extracellularmedium; however, secretion to the periplasm is normallyefficient (38). Most cloned cellulases are secreted to theperiplasm, and DNA sequence analysis has revealed that allcellulases to date have signal peptides predicted for theiramino acid sequences (4).Maximum endoglucanase activity was at pH 5.0, which is

around the range (pH 5.5 to 7.0) reported for cloned endoglu-canases from F. succinogenes (44), R. albus (25, 37, 47), andBacteroides ruminicola (48). Maximum activity of extracel-lular endoglucanase from F. succinogenes ARI is observedat pH 5.5 (7a), which is similar to that for the single gene

product cloned in E. coli.F. succinogenes exhibits a diverse range of enzyme activ-

ities with the ability to solubilize lignocellulosic materials(23). Some of these properties were reflected in the broadrange of substrates hydrolyzed by the gene product ofpRCEH (Table 3). Most endoglucanases that have beencloned from ruminal bacteria show broad-spectrum activityon cellulosic compounds. Kinetic studies were not under-taken; however, it was noteworthy that activities on acid-swollen cellulose and ball-milled cellulose were higher thanthat on CMC. Initial production of reducing sugars on CMCwas, in fact, high, but within 4 h, hydrolytic action essen-

tially ceased. This can be explained by an endoglucanaseattack on the portions of CMC that have at least three or

more consecutive unsubstituted glucose units, resulting inonly about 2% of CMC that can be readily hydrolyzed (11,27).

ACKNOWLEDGMENTS

We are grateful to Peter East, John Pemberton, and coworkersKeith Gregg, Anil Lachke, Philip Vercoe, and Cheryl Ware forvaluable advice and discussion; Tom Bauchop for providing culturesof F. succinogenes AR1; Michelle Deegenaars for technical exper-

tise; and Jean Hansford for typing the manuscript. We also acknowl-edge in particular Ronald Teather, Geoff Hazlewood, Bryan White,and David Wilson for preprints and recent publications and DavidStahl and Chuzhao Lin for communication of unpublished data.

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