Laminarinase (p8-glucanase) Activity inbacteroides from the

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1977, p Copyright 1977 American Society for Microbiology Vol. 33, No. 5 Printed in U.S.A. Laminarinase (p8-glucanase) Activity inbacteroides from the Human Colon A. A. SALYERS,* J. K. PALMER, AND T. D. WILKINS Anaerobe Laboratory and Department of Food Science and Technology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia Received for publication 13 December 1977 Laminarin, a,8(1-*3)-glucan similar to those found in plant cell walls, is fermented by some species of anaerobic bacteria from the human colon. Laminarinase (EC ) and,-glucosidase (EC ) activities were determined in strains representing Bacteroides thetaiotaomicron, Bacteroides distasonis, and an unnamed deoxyribonucleic acid homology group of Bacteroides fragilis. In all three species, laminarinase activity was inducible by laminarin and was predominantly cell bound. The products of laminarinase activity varied with each species. In the case ofb. thetaiotaomicron, the major product of laminarin hydrolysis was glucose (70 to 90%), and there were small amounts of laminaribiose (G2) and oligomers of glucose as high as G4. In the case of group '0061-1,' glucose (40 to 50%) and oligomers of glucose as high as G6 were found. The laminarinase of B. distasonis differed from the laminarinases of the other two species in that it mainly produced oligomers of glucose (G2-G5).,B-Glucosidase activity was also found in all three species. 8-Glucosidase was induced by glucose-containing disaccharides as well as by laminarin. The f3-glucosidases of the three Bacteroides species differed with respect to level of activity, induction pattern, and sensitivity to inhibition by D-glucono-1,5-lactone. Bacteroides species from the human colon can ferment a variety of polysaccharides (18). Since "dietary fiber" consists primarily of plant cell wall polysaccharides that are not digested in the stomach or small intestine, it is possible that dietary fiber components are an important source of carbohydrate for colon bacteria. Burkitt and others (3, 7) have suggested that dietary fiber may decrease the risk of colon cancer and other intestinal disorders by adsorbing harmful substances and speeding their passage through the colon. This hypothesis does not take into account the possibility that metabolism of dietary fiber polysaccharides by colon bacteria may reduce their concentration in the colon and may even alter their physical properties. To study the degradation of plant polysaccharides by colon bacteria, we chose the simple, well-characterized polysaccharide laminarin. Laminarin, a storage polysaccharide synthesized by brown algae, is a polymer consisting of glucose residues linked by 8-D-(1---3) glycosidic bonds. A small proportion (2 to 3%) of the laminarin chains are terminated by a mannitol rather than by a glucose residue (1). f3-d-(1-3)- glucans similar to laminarin in structure occur widely in plant and fungal cell walls. Frequently they form part of a heteropolysaccharide containing other linkages besides 83(1->3) and other sugars besides glucose (2). Thus, the process of laminarin utilization should give some indication of the way in which components of plant cell wall polysaccharides might be degraded by Bacteroides species. Laminarinase [,8(1-+3)-glucanohydrolase, EC ] is typically an enzyme complex that can involve as many as three types of activity: (i) /3-glucosidase (EC ), which hydrolyzes low-molecular-weight glucose-containing substrates such as di- and trisaccharides or p-nitrophenyl-,3-r-glucosides; (ii) an exoglucanase that cleaves single glucose units from the non-reducing end of laminarin; and (iii) an endoglucanase that releases laminaribiose (G2), laminaritriose (G3), or higher,8(1-3) oligomers of glucose from laminarin (5). The,8-glucosidase activity differs from the exoglucanase activity with respect to both substrate specificity and mechanism. f3-glucosidase is more sensitive than exoglucanase to inhibition by D-glucono-1,5-lactone (17). We have studied the f-d-(1-+3)-glucanase system in strains representing three species of Bacteroides that ferment laminarin: Bacteroides thetaiotaomicron, Bacteroides distasonis, and an unnamed deoxyribonucleic acid homology group (reference strain VPI ). B. thetaiotaomicron and B. distasonis were for- 1118

2 VOL.,V1-GLUCANASE 33, 1977 ACTIVITY IN BACTEROIDES 1119 merly classified as subspecies of Bacteroides fragilis (4). Strains of the deoxyribonucleic acid homology group '0061-1' were previously grouped with strains of B. fragilis subsp. thetaiotaomicron (J. L. Johnson, private communication). B. thetaiotaomicron and group '0061-1' are among the most numerous species in the colon. B. distasonis is found in the colon but at lower concentrations than the other two species (J. L. Johnson, private communication). Although laminarin utilization is found in a small proportion of strains from other Bacteroides species (18) and in strains of Clostridium ramosum from the human colon (A. A. Salyers, unpublished data), the three species chosen for this study probably account for most of the 8-i- (1--3)-glucanase activity in the colon. MATERIALS AND METHODS Chemicals. "Insoluble" laminarin was obtained from ICN Pharmaceuticals, Inc. (Cleveland, Ohio) and from Sigma Chemical Co. (St. Louis, Mo.). The composition of laminarin from both sources was checked by hydrolysis and chromatography of the component sugars as their alditol acetates (19). Glucose (96%), mannitol (3%), and traces of fucose and xylose were detected. Standards of laminaribiose (G2), laminaritriose (G3), and higher oligomers of glucose (G4-G7) were the kind gifts of E. Reese, U.S. Army Natick Development Center, Natick, Mass. Gentiobiose was purchased from Aldrich Chemical Co. (Milwaukee, Wis.). Cellobiose, lactose, and maltose were maintained from Difco Laboratories (Detroit, Mich.). All other chemicals were purchased from the Sigma Chemical Co. Bacterial strains. Bacterial strains of B. thetaiotaomicron (VPI 5008), deoxyribonucleic acid homology group '0061-1' (VPI ), and B. distasonis (VPI B1-20) were obtained from the culture collection of the Anaerobe Laboratory. These strains were classified according to species on the basis of deoxyribonucleic acid homology data (J. L. Johnson, private communication). Growth medium. The laminarin-containing growth medium was adapted from the minimal medium of Varel and Bryant (21). Phosphate buffer (0.1 M, ph 7.0) was used instead of carbonate buffer, and glucose (0.5%) was replaced by laminarin (0.5%). For induction experiments, minimal medium containing glucose, gentiobiose, maltose, salicin, cellobiose, or lactose instead of laminarin was used. Carbohydrates were autoclaved in the medium. Cultures were incubated at 37 C under a CO2 atmosphere. Growth rates. To compare the growth rate of laminarin-induced Bacteroides strains on laminarin with the growth rate of glucose-grown strains on glucose, a tube (30 ml) of minimal medium containing laminarin (0.5%) was inoculated with 3 ml of a logarithmic-phase culture grown on laminarin, and a tube (30 ml) of minimal medium containing glucose (0.5%) was inoculated with 3 ml of a logarithmic-phase culture grown on glucose. Each tube was stoppered with a rubber stopper fitted with a matched spectrophotometer tube (1-cm light path). Stoppers and spectrophotometer tubes were autoclaved and flushed with CO2 before being placed on the large tube containing the inoculated medium. Growth was measured periodically as absorbance at 650 nm by using a Bausch and Lomb Spectronic 20. Determination of enzyme activity. Cultures grown to maximum turbidity (16 h) were centrifuged at 10,000 x g for 15 min to harvest cells. The cell-free culture fluid was saved for assay. In some experiments, culture fluid was concentrated 20-fold by using a Minicon B15 concentrator (Amicon Corp., Lexington, Mass.). Cells were resuspended and washed once in citrate buffer (0.05 M, ph 6.8) and then resuspended in a final volume of buffer which produced a cell suspension with an optical density (at 650 nm) of approximately 6 to 7. Cells were disrupted by sonic treatment on a sonifier cell disrupter W185 (Ultrasonics, Inc., Plainview, N.Y.). Three minutes of sonic treatment was followed by a 2-min pause and then by a further 2 min of sonic treatment. During sonic treatment, the tube containing the cell suspension was kept in an ice bath. After sonic treatment, the extent of disruption was checked by phase-contrast microscopy. To determine whether enzyme activity from disrupted cells was soluble or membrane bound, the sonically treated cells were first centrifuged at 10,000 x g for 10 min to remove undisrupted whole cells and then at 30,000 x g for 60 min to pellet the membranes. The 30,000 x g supernatant and the resuspended membrane pellet were assayed for activity. Protein content of the disrupted cell suspension was determined by using the method of Lowry et al. (12).,8-Glucosidase activity was measured by using the chromogenic substrate method of Ford et al. (8). Enzyme (0.1 to 0.2 ml) was added to p-nitrophenylf3-d-glucoside (1 mm in 0.05 M citrate buffer, ph 6.8) in a 1-cm path-length cuvette. The final volume of the assay mixture was 2.0 ml. Activity was measured by determining the increase in absorbance per min at 370 nm (at 37 C), using a Gilford recording spectrophotometer, and comparing it to a standard curve based on the absorbance of p-nitrophenol at 370 nm. One unit of 18-glucosidase activity was defined as 0.1,imol ofp-nitrophenyl-,f-d-glucoside hydrolyzed per min at 37 C. Laminarinase (exo- plus endoglucanase) activity was determined by adding 0.2 ml of enzyme to 2.3 ml of citrate buffer and 2.5 ml of laminarin (500 gtg/ml), which had been dissolved in 0.05 M citrate buffer (ph 6.8) by heating to 60 C and then brought to 37 C. The concentration of laminarin in the assay mixture (250 gg/ml) fell within the region in which increases of laminarin concentration did not significantly affect enzyme activity. Activity was measured by determining the increase in reducing sugar (as glucose) by the neocuproine method of Dygert et al. (6). In each experiment, replicate time zero measurements were compared with replicate measurements at two other times, usually 10 and 20 min. Enzyme activity varied linearly with time for at

3 1120 SALYERS, PALMER, AND WILKINS least 30 min. A boiled enzyme control was run in parallel. No increase in reducing sugar was observed in the boiled enzyme control. One unit of laminarinase activity was defined as 1,mnol of reducing sugar (as glucose) released per 10 min. All enzyme activities were obtained by averaging values obtained from at least two different experiments. A variation of less than 20% was obtained when replicate assays were compared. Inducibility of enzyme activity. To determine whether f-glucosidase activity and laminarinase activity were constitutive, enzyme activities were measured in disrupted cells of cultures grown on glucose (0.5%). To determine whether enzyme activities were inducible by disaccharides other than laminaribiose, activities were also measured in disrupted cells from cultures grown on cellobiose (0.5%), gentiobiose (0.5%), lactose (0.5%), maltose (0.5%), or salicin (0.5%). Group phase and enzyme activity. To compare enzyme activities at different phases of growth, 500 ml of laminarin-containing medium-was inoculated with 30 ml of laminarin-grown cells of B. thetatiotaiomicron. Cells were incubated, with stirring (100 rpm), in a Microferm fermenter vessel (New Brunswick Scientific Co., Inc., New Brunswick, N.J.) maintained at 370C. Samples of cells at various stages of growth were removed from the fermenter, washed, and resuspended to give a final optical density of at least 5. Resuspended cells were disrupted by sonic treatment. Disrupted cells and cell-free culture fluid were assayed to determine,3-glucosidase and laminarinase activity. To estimate the length of time required to induce f3-glucosidase and laminarinase activity, 60 ml of glucose-grown cells of B. thetaiotaomicron (5008), at maximum turbidity, was added to 1 liter of minimal medium containing laminarin (0.5%). Samples (150 ml) were withdrawn at 1-h intervals, and the optical density at 650 nm was determined. Cells were washed once in citrate buffer, resuspended in a final volume of 5 ml, and disrupted by sonic treatment. Glucosidase and laminarinase activities of the disrupted cell suspension were measured. f3- Gluconolactone inhibition. D-glucono-1,5-lactone inhibition of /8-glucosidase activity was measured by adding different concentrations of D-glucono-1,5-lactone to the /8-glucosidase assay mixture and comparing this activity with the activity obtained in the absence of D-glucono-1,5-lactone. The concentration of D-glucono-1,5-lactone that gave a 50% inhibition of 3-glucosidase activity (I50) was determined graphically from a plot of percent inhibition versus concentration of D-glucono-1,5-lactone (,um). I50 values were obtained by using disrupted cell preparations from each of the three strains studied. Chromatography of laminarinase products. The relative amounts of endoglucanase and exoglucanase activity in each of the three Bacteroides strains were determined by incubating the enzyme preparation (50 to 70 mg of protein) with 50 ml of laminarin dissolved in 0.05 M citrate buffer (ph 6.8). The enzyme preparation consisted of the 30,000 x g supernatant of sonically disrupted cells. The final laminarin concentration was 10 mg/ml. Portions of the APPL. ENVIRON. MICItOBIOL. incubation mixture were removed at intervals and heated at 90 C for 5 min to stop the reaction. Three volumes of acetonitrile were added to 1- to 2-ml portions of the cooled reaction mixture to precipitate undigested laminarin. Acetonitrile at this concentration did not precipitate oligomers of glucose shorter than G7. Precipitation of higher oligomers could not be determined directly, because standards for oligomers longer than G7 were not available. The solutions were centrifuged (1,200 x g, 10 min) to sediment the precipitated laminarin. The supernatant fluid was filtered through a Fluoropore filter (2-,um pore diameter; Millipore Corp., Bedford, Mass.) utilizing a Swinnex-type syringe filter. Portions of this filtered solution were either analyzed undiluted or concentrated 10-fold at 800C under a stream of nitrogen. Sugars were determined in 20- to 80-,il portions by high-performance liquid chromatography (LC), as described by Palmer (15, 16), using a ", Bondapak Carbohydrate" column (Waters Associates, Inc., Milford, Mass.) with acetonitrilewater (75:25, vol/vol) as the eluant. Glucose (Gl), laminaribiose (G2), and the higher oligomers of glucose (G3-G6) were tentatively identified from the LC retention times determined with a standard mixture of Gl to G6. Identity was confirmed by paper chromatography of several of the concentrated samples. Samples were spotted on Whatman no. 1 paper and developed overnight (descending) with isopropyl alcohol-acetic acid-water (54:8:18) as described by Mandels and Reese (13). Sugars were located by spraying with p-anisidine hydrochloride in ethanol, followed by brief heating at 1000C (10). The Rf values obtained were identical to those of authentic samples. Quantitation of glucose (Gl), laminaribiose (G2), laminaritriose (G3), and higher oligomers (G4-G6) was obtained from LC peak area measurements relative to the peak area for glucose standards. In samples containing low concentrations of oligomers, glucose (normally the major component) was determined on both the concentrated and unconcentrated samples and used as an "internal standard" to calculate the oligomer values in the concentrated samples. This procedure corrected for any losses or errors in volume measurement during concentration. As a control of LC quantitation, the increase in reducing sugars, calculated by summing the concentrations of Gl through G6 obtained by LC, was compared with the increase in the reducing sugar concentration obtained from reducing sugar assays of the boiled samples prior to acetonitrile precipitation. In all cases, the reducing-sugar assay values agreed to within 10% with the values obtained from summing the LC peak area concentrations. RESULTS Cellular location of enzyme activity. a3-glucosidase and laminarinase activity were both detected in disrupted cells ofbacteroides grown on laminarin (Table 1). No f8-glucosidase activity was found in the cell-free culture fluid. Extracellular laminarinase activity was observed, but it was very close to the limit of detection for

4 VOL. 33, GLUCANASE ACTIVITY IN BACTEROIDES 1121 TABLE 1. Extracellular and cell-bound 3- glucosidase and laminarinase activities of colon Bacteroides strains grown on laminarin /3-Glucosi- Laminarinase oase(ua/mg (Ubl/mg of protein) Bacteroides sp. protin (VPI strain no.) ExtracExtr- Cell Extra- Cell Cellau lar bound cellular bound B. thetaiotaomi < cron (5008) Bacteroides group < '0061-1' (0061-1) B. distasonis (BR < ) a One unit = 0.1,mol of substrate hydrolyzed per min. b One unit = 1 umol of reducing sugar (as glucose) released in 10 min. the laminarinase assay (ca U/mg of protein) and accounted for less than 1% of the total laminarinase activity. Neither the /8-glucosidase nor the laminarinase activity appeared to be membrane associated in any of the three strains tested. When the disrupted cell suspensions were centrifuged at 30,000 x g for 60 min to pellet the membranes, approximately 90% of both the /3-glucosidase and laminarinase activity was found in the supernatant. The remaining activity was found in the resuspended pellet. Undisrupted whole cells exhibited both laminarinase and 8-glucosidase activity. With B. distasonis (B1-20), the enzyme activity found with whole cells was the same as the activity found when the cells were disrupted. In the other two species, disruption increased activity. Disrupted cells of B. thetaiotaomicron (5008) had twice the,b-glucosidase and laminarinase activity of undisrupted cells. Disrupted cells of group '0061-1' (0061-1) had a 20% higher activity than undisrupted cells. Differences in the enzyme activity found in disrupted cells from the three different species were not due to differences in the extent of disruption of the cells. Microscopic examination of cells after sonic treatment indicated that nearly all cells had been disrupted. Moreover, concentrations of protein released by sonic treatment were comparable for all three strains. Inducibility of enzyme activity. Neither 38- glucosidase nor laminarinase activity was constitutive in any of the Bacteroides strains tested. No enzyme activity was detected in cells grown on glucose.,8-glucosidase, but not laminarinase, was inducible by several glucose-containing disaccharides (Table 2). The specific activity of /3-glucosidase, which was induced, varied considerably with different disaccharides. These activities also differed from the j3-glucosidase activities induced by laminarin (Table 1). In /8. distasonis (B1-20), the highest 3-glucosidase activities were obtained by induction with gentiobiose, salicin, and cellobiose. None of these activities was as high as the activity obtained by induction with laminarin. The best inducers of /3-glucosidase in group '0061-1' (0061-1) were cellobiose and salicin. These disaccharides were better inducers of /8-glucosidase than laminarin. In B. thetaiotaomicron (5008), none of the disaccharides induced /3-glucosidase activities comparable to those found in cells grown on laminarin. Although B. thetaiotaomicron (5008) grew much more slowly and achieved lower turbidity on cellobiose than on maltose, lactose, or gentiobiose, cellobiose induced the highest 83-glucosidase activity in this strain. In all three Bacteroides species, the lowest levels of (-glucosidase were found in cells grown on maltose and lactose. Laminarinase activity was found only in cells grown on laminarin. Gluconolactone inhibition. In all three species, 83-glucosidase activity was inhibited by D- glucono-1,5-lactone, although the concentration of D-glucono-1,5-lactone required for 50% inhibition varied considerably from species to species. B. distasonis (B1-20), with an IO of 50,M, was much less sensitive to D-glucono-1,5- lactone inhibition than either B. thetaiotaomicron (5008) with an I50 of 2,uM, or group '0061-1' (0061-1), with an I50 of 1 MiM. Growth rates and variation of enzyme activity during growth. Laminarin-induced cells grew as rapidly on laminarin as on glucose. All three strains achieved an optical density of 1.2 to 1.4 within 12 h. A sample growth curve is shown in Fig. 1 for laminarin-grown cells of B. TABLE 2. /&Glucosidase activity in Bacteroides cells grown on various glucose-containing disaccharides /3-Glucosidase activity (Ua/mg of protein) of: Inducer B. thetaio- Bacteroides B. distataomicron group'0061- sonis (B1- (5008) 1' (0061-1) 20) Cellobiose Gentiobiose Lactose < Maltose < Salicin NGb a One unit = 0.1 ALmol of substrate hydrolyzed per min. b NG, No growth.

5 1122 SALYERS, PALMER, AND WILKINS thetaiotaomicron (5008) inoculated into laminarin-containing minimal medium. The specific activities of 8-glucosidase and laminarinase at various stages of growth are also given in Fig. 1 for disrupted whole cells of this strain. The specific activities of both 8-glucosidase and laminarinase were highest during the logarithmic phase of growth. The specific activity of laminarinase in the cell-free culture fluid (not shown) was constant throughout growth. When uninduced glucose-grown cells of B. thetaiotaomicron (5008) were inoculated into laminarin-containing medium, there was a lag period of approximately 4 to 6 h during which no growth and no enzyme activity were detected. Once the cells began to grow, the rate of growth was identical with that shown in Fig. 1. The specific activity of both 8-glucosidase and laminarinase increased rapidly during the logarithmic phase of growth and then decreased when the cells entered the stationary phase. Products of laminarinase activity. Incubation of disrupted cells with laminarin produced glucose and varying proportions of higher oligomers of glucose (G2-G6). A typical LC chromatogram is shown in Fig. 2. Distributions of the hydrolysis products are given in Table 3. The laminarinase of B. thetaiotaomicron (5008) produced mainly glucose, with small amounts of G2-G4. G3 and G4 appeared only at longer incubation times. The laminarinase of group '0061-1' (0061-1) produced a lower percentage of glucose than that of B. thetaiotaomicron (5008) and oligomers as high as G6. G6 was detected only after prolonged incubation times. The laminarinase of B. distasonis (B1-20) produced the 20 r 1.0t -i 4 I. c TIME (HRS) FIG. 1. Cell-bound laminarinase and /3-glucosidase activities in B. thetaiotaomicron (5008) at different stages of growth. 25 z a. 0 Ua a. w C') z 0- w0 w I) -,,. APPL. ENVIRON. MICROBIOL. o MINUTES FIG. 2. Separation of the products of laminarin hydrolysis by the laminarinase of Bacteroides group '0061-1' (0061-1) using high-performance LC. TABLE 3. Distribution of hydrolysis products of laminarinases from sonically disrupted cells of three Bacteroides species % of Gla and higher oli- Concn of Bacteroides Time of gomers detected hydrolysp. and incuba- sis prodstrain no. tion (h) Gl G2 G3 G4 G5 G6 ucts (mg/ ml)" B. thetaio taomi cron (5008) Bacteroides group '0061-1' (0061-1) B. dista sonis (B1-20) 22 Tc agl, Glucose G2, laminaribiose; etc. b Sum of concentrations of Gl to G6 determined by LC, with an initial laminarin concentration of 10 mg/ml. c T, Below the level of quantitation (ca mg/ml). lowest percentage of glucose and had the lowest activity of the three strains tested. At longer incubation periods, higher oligomers of glucose appeared, and at 22 h, glucose itself had dropped below the level of detection. When cell-free culture fluid was incubated

6 VOL. V,-GLUCANASE 33, 1977 ACTIVITY IN BACTEROIDES 1123 with laminarin, we detected traces of substances tentatively identified as G2 and G3. Concentrations were very close to the limit of detection (ca mg/ml). No glucose was detected. DISCUSSION The 8-D-(1-.-3)-glucanase systems of Bacteroides from the human colon differed substantially from f3-d-(1---+3)-glucanase systems reported for other bacteria and fungi. First, the /8- D4(1-3)-glucanases of all three of the Bacteroides species studied were predominantly cell bound, whereas in previously studied microorganisms, the,8-d-(1--+3)-glucanase activity was extracellular (5). Second, the enzyme activity in Bacteroides was found only when cells were grown on laminarin. Previously described laminarinases have been constitutive (5) or semiconstitutive (11, 20). The fact that B3-i- (1-3)-glucanases from colon Bacteroides were cell bound and inducible may reflect the requirements imposed by the colonic environment. In the colon, each species must compete for energy sources with a large number of other organisms existing in close proximity (9, 14). It is likely that simple sugars are seldom encountered by these bacteria. Rather, their carbon and energy sources probably consist mainly of undigested food materials (primarily plant cell walls) and mucins that contain a wide variety of polysaccharide types. In this situation, the ability to produce the appropriate polysaccharidase in response to changes in the nature of the available energy source would confer a definite growth advantage. Although laminarinase activity was found in intact cells, the activity did not appear to be closely associated with membranes. When disrupted cells were centrifuged to pellet membranes, the activity remained in the soluble supernatant. Possibly the laminarinase activity is periplasmic or loosely associated with membranes. The fact that mechanical disruption increased activity in two of the three Bacteroides species indicates that there may be a permeability barrier between substrate and enzyme. The three Bacteroides species differed from one another in the level of laminarinase activity and in the products of hydrolysis. The laminarinase from B. distasonis had a lower activity than laminarinases from the other two species and appeared to be mainly an endoglucanase. The fact that glucose (Gl) concentrations resulting from laminarin hydrolysis by this strain initially increased and then decreased over longer incubation periods (>8 h) indicates that further metabolism of glucose (e.g., repolymerization) may have occurred. The laminarinases ofb. thetaiotaomicron and group'0061-1' were more active than the B. distasonis enzyme, and their activity was predominantly of the exoglucanase type. B. thetaiotaomicron produced mainly glucose, whereas group '0061-1' produced a small proportion of glucose and a higher proportion of glucose oligomers as high as G6. The glucose oligomers produced by the B. thetaiotaomicron laminarinase may have resulted from the accumulation of portions of laminarin chains that had not been completely degraded rather than from endoglucanase activity. The three species ofbacteroides also differed somewhat with respect to /8-glucosidase activity. The group '0061-1' strain had a lower,3- glucosidase activity than did strains of the other two species when grown on laminarin. However, /8-glucosidase activity comparable to that of the other two Bacteroides species was obtained when strain '0061-1' was grown on cellobiose and salicin. f3-glucosidase activities in B. thetaiotaomicron and B. distasonis were higher in cells grown on laminarin than in cells grown on any of the disaccharides tested. Thus, the f8-glucosidases of B. thetaiotaomicron and B. distasonis were induced more effectively by a f3(1-3) glycosidic linkage, whereas the group '0061-1' strain appeared to be induced most effectively by a f8(1-*4) linkage or by a /3-glucoside such as salicin. 83-Glucosidases of all three Bacteroides species were inhibited by concentrations of D-glucono-1,5-lactone comparable to concentrations required for inhibition of 18-glucosidases from other microorganisms and from plants. However, the 83-glucosidase of B. distasonis was 25 to 50 times less sensitive to inhibition than were the 83-glucosidases of the other two Bacteroides species. Thus, strains of three Bacteroides species from the colon have cell-bound, inducible enzyme activities which permit them to obtain carbohydrate from /3(1-*3)-glucans in vitro. In the colon, the utilization of /3-glucans is probably limited by a number of factors, such as accessibility of the 3-glucan polymer within the plant cell wall complex. Moreover, the presence of other types of fermentable complex carbohydrate (e.g., mucin) may affect the induction of,8-glucanase activity in these organisms. ACKNOWLEDGMENTS This research has been supported by Public Health Service contract no. NO1-CP55685 from the National Cancer Institute. We would like to thank J. R. Vercellotti (Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University) for his help and advice on this project.

7 1124 SALYERS, PALMER, AND WILKINS We would also like to acknowledge the excellent technical assistance of Roger Van Tassell and Eva Hudlicky. LITERATURE CITED 1. Black, W. H., and E. T. Dewar Laminarin, p In R. Whistler (ed.), Industrial Gums, 2nd ed. Academic Press Inc., New York. 2. Bull, A. T., and C. G. C. Chesters The biochemistry of laminarin and the nature of laminarinase. Adv. Enzymol. 28: Burkitt, D. P., A. R. P. Walker, and N. S. Painter Dietary fiber and disease. J. Am. Med. Assoc. 229: Cato, E. P., and J. L. Johnson Reinstatement of species rank for Bacteroides fragilis, B. ovatus, B. distasonis, B. thetaiotaomicron, and B. vulgatus: designation of neotype strains for Bacteroides fragilis (Veillon and Zuber) Castellani and Chalmers and Bacteroides thetaiotaomicron (Distaso) Castellani and Chalmers. Int. J. Syst. Bacteriol. 26: Chesters, C. G. C., and A. T. Bull The enzymic degradation of laminarin. I. The distribution of laminarinase among micro-organisms. Biochem. J. 86: Dygert, S., L. H. Li, D. Florida, and J. A. Thoma Determination of reducing sugar with improved precision. Anal. Biochem. 13: Eastwood, M. A Vegetable dietary fiber-potent pith. J. R. Soc. Health 95: Ford, J. R., J. A. Nunley, Y.-T. Li, R. P. Chambers, and W. Cohen A continuously monitored spectrophotometric assay of glycosidases with nitrophenyl glycosides. Anal. Biochem. 54: Holdeman, L. V., I. J. Good, and W. E. C. Moore Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Appl. Environ. Microbiol. 31: Hough, L., J. K. N. Jones, and W. H. Wadman APPL. ENVIRON. MICROBIOL. Quantitative analysis of mixtures of sugars by the method of partition chromatography. V. Improved methods for the separation and detection of their methylated derivatives on the paper chromatogram. J. Chem. Soc., p Lilley, G., and A. T. Bull The production of,3-1,3 glucanase by a thermophilic species of streptomyces. J. Gen. Microbiol. 83: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Mandels, M., and E. T. Reese Induction of cellulase in fungi by cellobiose. J. Bacteriol. 79: Moore, W. E. C., and L. V. Holdeman Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl. Microbiol. 27: Palmer, J. K A versatile system for sugar analysis via liquid chromatography. Anal. Lett. 8: Palmer, J. K Liquid chromatography for monitoring the conversion of cellulosic wastes to sugars. Appl. Polym. Symp. 28: Reese, E. T., and F. W. Parrish Norjirimycin and D-glucono-1,5-lactone as inhibitors of carbohydrases. Carbohydr. Res. 18: Salyers, A. A., J. R. Vercellotti, S. E. Wedt, and T. D. Wilkins Fermentation of mucin and plant polysaccharides by strains ofbacteroides from the human colon. Appl. Environ. Microbiol. 33: Sawardeker, J. S., J. H. Sloneker, and A. Jeanes Quantitative determination of monosaccharides as their alditol acetates by gas-liquid chromatography. Anal. Chem. 37: Tanka, H., and H. J. Pfaff Enzymatic hydrolysis of yeast cell walls. I. Isolation of wall-decomposing organisms and separation and purification of lytic enzymes. J. Bacteriol. 89: Varel, V. H., and M. P. Bryant Nutritional features ofbacteroides fragilis subsp. fragilis. Appl. Microbiol. 28: Downloaded from on March 22, 2019 by guest