APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1986, p. 880-8870099-2240/86/100880-08$02.00/0Copyright C) 1986, American Society for Microbiology

Bacteroides pectinophilus sp. nov. and Bacteroides galacturonicussp. nov.: Two Pectinolytic Bacteria from the Human Intestinal Tract

NEIL S. JENSEN AND ERCOLE CANALE-PAROLA*Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003

Received 28 April 1986/Accepted 29 July 1986

Studies on the physiological characteristics of two obligately anaerobic, rod-shaped bacteria from the humanintestinal tract indicated that the organisms represented two previously undescribed species of Bacteroides, forwhich we propose the names Bacteroides pectinophilus (type strain, N3) and Bacteroides galacturonicus (typestrain, N6). Both strains were pectinophilic; that is, they utilized as fermentable substrates for growth onlypectin and a few related compounds. The two species differed significantly from each other in guanine pluscytosine content of the DNA, in substrate utilization patterns, and in other phenotypic characteristics. Bothspecies deesterified pectin by means of an extracellular pectinesterase (EC 3.1.1.11) activity. Polygalacturonate(the main component of deesterified pectin) was depolymerized extracellularly with formation of unsaturatedproducts by both species. The depolymerizing activity required Ca2+, functioned at a higher rate whenpolygalacturonate was the substrate as compared with pectin, and had an alkaline pH optimum. These data,as well as viscosity decrease studies and identification of products formed from polygalacturonate, indicatedthat the extracellular depolymerizing activity of either species was characteristic of an exopectate(exopolygalacturonate) lyase. The exopectate lyase activity had an unusual action pattern that resulted interminal cleavage of unsaturated trigalacturonic acid units from polygalacturonate. An unsaturated trimer wasthe major product that accumulated in cell-free reaction mixtures, where it was not cleaved further. Growingcells of both Bacteroides species released the exopectate lyase into the external environment by processes thatdid not involve cell lysis to any significant extent. In addition to the extracellular lyase activity, cell-associatedpectate lyase activity was detected in both species.

A significant portion of the food ingested by humansconsists of plant cell wall polysaccharides (dietary fiber)(31). Species of Bacteroides are among the most activefermenters of plant cell wall polysaccharides in the humancolon (9, 27, 31). Pectin, a plant cell wall polysaccharide, isnot degraded by human enzymes but is extensively brokendown in the human colon by bacteria (24), many of whichhave been identified as species of Bacteroides (1, 11, 31, 32).Pectin digestion by these and other intestinal bacteria affectsphysiological processes of the human body inasmuch as (i)end products of the bacterial fermentation of pectin areabsorbed from the intestine and may serve as energy sourcesfor the host (24) and (ii) pectin in the human diet decreasesserum cholesterol levels and increases the amount of lipidsexcreted (12, and references therein).

In the course of a recent study on intestinal pectin-degrading bacteria, we isolated 42 strains of pectinolyticbacteria from human feces (11). Two of the three isolatesthat exhibited the greatest pectinolytic activity were identi-fied as species of Bacteroides (11). However, the substrateutilization pattern of the two strains (N3 and N6) wasmarkedly different from that of recognized species of Bac-teroides, and it was suggested that they represented two newspecies of that genus. In the present article, we describe thegeneral physiological properties of strains N3 and N6, withemphasis on their pectinolytic enzymatic activities. As dis-cussed below, we propose the names Bacteroidespectinophilus and Bacteroides galacturonicus for strains N3and N6, respectively, and henceforth we refer to the strainsby the proposed names.

* Corresponding author.

(A preliminary report of part of this work has beenpresented [N. S. Jensen and E. Canale-Parola, Abstr. Annu.Meet. Am. Soc. Microbiol. 1985, K128, p. 193].)

MATERIALS AND METHODSBacterial strains and growth media. B. pectinophilus N3

and B. galacturonicus N6 were isolated from human feces(11). The strains were grown in medium PF, which had thefollowing composition (grams per 100 ml of distilled water):sodium polygalacturonate (grade II; Sigma Chemical Co.,St. Louis, Mo.), 0.4; Trypticase (BBL Microbiology Sys-tems, Cockeysville, Md.), 0.5; yeast extract (Difco Labora-tories, Detroit, Mich.), 0.25; MgSO4 7H20, 0.25;CaCl2. 2H20, 0.015; FeSO4 7H20, 0.002; (NH4)2SO4,0. 14; L-cysteine hydrochloride, 0.1; resazurin, 10- 4;NaHCO3, 0.2; and agar (when needed), 1.5. NaHCO3 wasfilter sterilized separately as a 10% (wt/vol) solution and thenwas added to the rest of the medium which had beenpreviously sterilized by autoclaving. In some experiments,B. galacturonicus was grown in a chemically defined me-dium (medium BGD) similar to medium PF except that theTrypticase and yeast extract were omitted and the followingadditional components were included (grams per 100 ml ofmedium): KH2PO4, 0.2; K2HPO4, 0.6; biotin, 5 x 10-;

pantothenic acid, 10-4; and folic acid, 10-6. NaHCO3 wasadded to medium BGD to a final concentration of 0.1 g/100ml. The vitamins were filter sterilized as individual solutionsand then added to the autoclaved medium. The incubation ofcultures was at 37°C unless otherwise noted. Long termstorage of both strains was in liquid nitrogen.Media used in attempts to detect sporulation were the

cooked meat broth described by Holdeman et al. (8) (sup-plemented with 0.4 g of sodium polygalacturonate per 100 mlof medium) and PF broth to which 1.5% CaCO3 had been

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added and which contained 0.4% (wt/vol) D-galacturonate,D-glucuronate, or D-gluconate instead of polygalacturonate.

Microscopy. Equipment and methods for phase-contrastmicroscopy and photography (37) and for electron micros-copy (29) were similar to those described previously, exceptthat 2% (wt/vol) ammonium molybdate (pH 5) was used forthe negative stains. The method of Schaeffer-Fulton (6) wasused for spore stains.

Motility. Attempts to detect motility were made by ob-serving (i) cells in wet-mount preparations of PF and BGDbroth cultures with a phase-contrast microscope in air atmo-sphere, both at room temperature and at 37°C and (ii) cells inwet mounts prepared in an anaerobic chamber (Coy Labo-ratory Products, Ann Arbor, Mich.) containing an atmo-sphere of 10% C02, 10% H2, and 80% N2. Before theremoval of the preparations from the anaerobic chamber, thecover slips over the wet mounts were sealed at the edgeswith petrolatum. Then, cells in the preparations were ob-served at 37°C and at room temperature by phase-contrastmicroscopy. In addition, cells were placed in flat capillarytubes under anaerobic conditions as described previously (5)and observed by phase-contrast microscopy at 37°C. Finally,stab cultures of either strain in PF medium containing 0.07%(wt/vol) polygalacturonate and 0.4% (wt/vol) agar wereobserved to determine whether the cell population diffusedthrough the agar medium as a result of motility (14).The method of Leifson (6) was used for flagella stains.Phenotypic characteristics. For the catalase, gelatin hydro-

lysis, esculin hydrolysis, and bile tolerance tests, the cellswere grown in PF agar medium, PF broth supplemented with12% (wt/vol) gelatin (8, 34), PF agar medium supplementedwith both 0.01% (wt/vol) esculin and 0.05% (wt/vol) ferricammonium citrate (34), and PF broth supplemented with 2%dehydrated oxgall (wt/vol; Difco) (equivalent to 20% bile[vol/vol]) (34), respectively. Standard procedures (8, 34)were used for these tests. Cells used in the catalase test wereincubated for 30 min in air before adding H202 (8).Growth in the presence of penicillin-G (2 U/ml), tetracy-

cline (6 ,xg/ml), clindamycin (1.6 ,ug/ml), cephalothin (6,ug/ml), ampicillin (4 ,ug/ml), or chloramphenicol (12 ,ug/ml)was determined with the broth disk method of Wilkins andThiel (8). PF broth was used in these tests.

Enzymatic activities. Partially purified pectinolytic en-zymes were prepared from culture supernatant fluids byacetone precipitation and dialysis as described by Weber andCanale-Parola (38). Concentrated B. pectinophilus culturefluids that had been incubated in 1% (wt/vol, final concen-tration) sodium dodecyl sulfate (SDS) for 1 h at 23°C yieldedonly one band of polygalacturonate lyase activity whensubjected to nondenaturing polyacrylamide gel electropho-resis (unpublished data). The SDS treatment did not affectpolygalacturonate lyase activity.

Polygalacturonate (pectate) lyase or pectin lyase activitieswere determined by means of the thiobarbituric acid (TBA)and the Nelson-Somogyi reducing sugar assays, as previ-ously reported (38). Unless otherwise indicated, standardreaction mixtures contained (in a total volume of 0.2 ml)sodium polygalacturonate (1 mg); CaCI2 (0.1 ,umol); Trishydrochloride buffer (pH 8.5) (10 ,umol); and partially puri-fied enzyme preparation, cell extract, or culture supernatantfluid (0.1 ml). Reaction mixtures were incubated at 30°C foreither 30 or 60 min. In some reaction mixtures, polygalac-turonate was replaced by pectin (1 mg; 70% methylated). Inreaction mixtures used to measure the effect of pH onpolygalacturonate lyase activities, the buffer was either Trishydrochloride (pH 7.5 to 8.5) or glycine-NaOH (pH 8.5 to

10). As measured by the TBA assay, 1 U of lyase activitywas defined as the amount of activity at pH 8.5 and 30°C thatcaused an increase of 1.0 in optical density at 548 nm in 60min. A235 by unsaturated products formed from polygalac-turonate in lyase reactions (18) was measured as describedpreviously (38). Protein in enzyme preparations was deter-mined by means of the Bio-Rad assay (Bio-Rad Laborato-ries, Richmond, Calif.).

Cell extracts used to assay cell-associated lyase activitieswere prepared as follows. Cells were harvested by centrifu-gation at 4°C from 10 ml of a late-log-phase PF broth cultureand then washed with 10 mM Tris hydrochloride buffer (pH7). The washed cells were resuspended in 10 ml of the bufferand ruptured by passing them through a French pressure celltwice at 10,000 lb/in2. After removing the cell debris bycentrifugation (27,000 x g, 20 min, 4°C), the supernatantfluid was dialyzed against 1 mM CaCl2 for 10 h at 4°C beforebeing used in the TBA assay. The activity in dialyzed cellextracts was compared with that present in PF culturesupernatant fluid dialyzed against 1 mM CaCl2 for 10 h at40C.The production of extracellular polygalacturonate lyase

activity by cells grown on different fermentable substrateswas determined as follows. Cells were grown in PF broth(prepared without polygalacturonate) to which D-gluconicacid (Na salt), D-galacturonic acid, D-glucuronic acid, or

D-fructose had been added as filter-sterilized solutions to a

final concentration of 0.4% (wt/vol). The TBA assay was

used to measure polygalacturonate lyase activity in late-log-phase culture supernatant fluids after they were dialyzedagainst 10 mM glycine-NaOH buffer (pH 9) containing 0.5mM (final concentration) CaCl2 for 10 h.

Polygalacturonate lyase reaction products were separatedand identified by thin-layer chromatography (TLC) on cellu-lose TLC plates (Eastman Kodak Co., Rochester, N.Y.) andby descending paper chromatography. The reaction mixture(16-ml total volume) contained (final concentrations) sodiumpolygalacturonate, 1% (wt/vol); Tris hydrochloride buffer(pH 8.5), 0.05 M; CaCl2, 0.5 mM; and 1 ml of partiallypurified enzyme preparation (30 U of polygalacturonatelyase activity, TBA assay). Reaction mixtures were incu-bated at 300C. Samples were removed at time intervals andheated in a boiling water bath for 30 s. TLC plates were

developed twice in ethyl acetate-acetic acid-distilled water

(4:2:3 [vol/vol/vol]) (17). Paper chromatograms were devel-oped in ethyl acetate-pyridine-distilled water-acetic acid(5:5:3:1 [vol/voUvob'vol]) (36). The reaction products were

detected by spraying the TLC plates and paper chromato-grams with a p-anisidine solution (4) and then heating themat 100 to 105°C for 3 min (paper chromatograms) or 10 min(TLC plates). The products of polygalacturonate depolymer-ization appeared as brown to orange-brown spots. Theproducts were identified by measuring their RGaI values as

described by Stack et al. (36).Pectin methylesterase (EC 3.1.1.11) activity in culture

supernatant fluid was assayed by means of the hydroxamicacid reaction (23).The hydrolytic enzymes polygalacturonase (EC 3.2.1.15)

and exopolygalacturonase (EC 3.2.1.67) were assayed bymeasuring reducing sugar formation from polygalacturonateas previously described (38), except that 2 mM (final con-

centration) EDTA was added to the reaction mixture toinhibit lyase activity.

Correlation of viscosity decrease to percent polygalacturon-ate cleavage. The change in reaction mixture viscosity was

correlated with percent polygalacturonate degradation as a

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882 JENSEN AND CANALE-PAROLA

function of time (19) to determine the action pattern of thepolygalacturonate lyases. The reaction mixture was identicalto that used for the chromatography of polygalacturonatereaction products (see above), except that the total volumewas 7 ml. The procedure used was similar to that describedby Macmillan et al. (19). Viscosity was measured with aKimax viscometer (size 150). The initial flow time of thereaction mixture through the viscometer was 56.5 s (100%viscosity). Water had a flow time of 21.4 s (0% viscosity).Percent degradation of polygalacturonate in the reactionmixture was determined by measuring reducing sugar for-mation (38). Uronic acid concentrations were determined asdescribed by Bitter and Muir (2).

Fermentation products. Volatile and nonvolatile fatty acidsproduced by cells growing in PF broth were determined witha model 3700 gas chromatograph (Varian Instrument Divi-sion, Palo Alto, Calif.) equipped with a flame ionizationdetector, as described in a previous report (7). Formate wasmeasured colorimetrically (15). Ethanol was assayed withalcohol dehydrogenase from an ethyl alcohol reagent set(Worthington Diagnostics, Freehold, N.J.).

Determination of DNA in culture supernatant fluid. Themethod of Burton (3) was used to determine DNA present inlate-log-phase culture fluids, in late-log-phase cells rupturedby being passed twice through a French pressure cell, and inuninoculated PF broth.DNA base composition. Cells grown in PF broth were

harvested by centrifugation, and the cell lysate was preparedas described by Miller and Wolin (26). DNA was isolated andpurified as described by Meyer and Schleifer (25). Theguanine plus cytosine (G + C) content of the DNA wasdetermined from its thermal denaturation temperature (21).Escherichia coli K-12 strain ATCC e23716 was used as acontrol (G + C content, 51.4 mol%).

RESULTS

Morphology and growth. The morphological characteris-tics of B. pectinophilus and B. galacturonicus cells (Fig. 1)have been described previously (11) and are summarizedbelow in the description of species.The cells of both species grew only anaerobically, reach-

ing densities of 1.6 x 109 cells per ml (B. pectinophilus) and2.3 x 109 cells per ml (B. galacturonicus) in PF broth at37°C. The cell population doubling time in PF broth at 37°Caveraged 1.2 h for B. pectinophilus and 1.0 h for B.galacturonicus. Highest growth yields occurred between 35and 40°C for both species, and no growth was observed at 20or 45°C.

B. galacturonicus grew abundantly (final yield, 4.9 x 108cells per ml) in BGD broth (see Materials and Methods),whereas B. pectinophilus did not grow in this defined me-dium nor in the defined medium described by Socransky etal. (35) from which pimelic acid, choline chloride, andDL-mevalonic acid lactone were omitted and to which 0.4 gof sodium polygalacturonate per 100 ml was added.

Spores were not detected by light microscopy examinationof either wet mount preparations or spore stains of cellsgrown in sporulation media or in any of the other mediaused. When inocula were pasteurized (3 min in boilingwater), cultures did not yield growth.

Motility. Negative stains of B. galacturonicus cellsshowed the presence of peritrichous flagella (6 to 8 per cell)(Fig. 2), whereas flagella were not seen on cells of B.pectinophilus. Similar observations were made when flagellastains were examined by light microscopy. However, motil-

FIG. 1. Phase-contrast photomicrographs ofB. pectinophilus (A)and B. galacturonicus (B) cells in PF broth cultures. Wet mountpreparations. Bar, 5 ,um.

ity of cells of either species was not observed under any ofthe experimental conditions described in Materials andMethods. Possibly, some component(s) of the media usedinhibited motility of B. galacturonicus, or the B. galacturon-icus strain isolated had nonfunctional flagella.

Nutrition. Both species were pectinophilic; that is, theyutilized as fermentable substrates for growth only pectin anda few related compounds (see species descriptions below)(11). Species of Bacteroides with such a specialized sub-strate utilization pattern have not been described previously.B. pectinophilus did not ferment D-galacturonate, the

major component unit of pectin. It is possible that D-galacturonate cannot enter cells of B. pectinophilus. Whenthis bacterium ferments pectin, oligomers derived from thedepolymerization of the polysaccharide by extracellular en-zymes may be transported into the cells and utilized asenergy and carbon sources. Indeed, both B. pectinophilusand B. galacturonicus grew in media containing, as the

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I'i<: I0 culture supernatant fluids, as well as in partially purified

preparations of extracellular enzymes.Both species possessed an extracellular enzymatic activity

that served to depolymerize polygalacturonate (the maincomponent of deesterified pectin) with the formation ofunsaturated products, as determined by assaying reactionmixtures by means of the TBA reaction. With polygalactu-ronate as the substrate, the increase in A548 per minute permilligram of protein was 0.9 for B. pectinophilus and 0.5 forJi B. galacturonicus. In addition, the products formed frompolygalacturonate by partially purified enzyme preparations

l of either species exhibited a UV absorption maximum at 235nm. An absorption maximum at this wavelength is charac-teristic of unsaturated products (18). The rate of productformation was greater when polygalacturonate was the sub-strate in reaction mixtures, as compared with the rateobserved when pectin was the substrate. The addition ofEDTA to the reaction mixture almost completely eliminatedthe polygalacturonate-depolymerizing activity (Table 1). Ac-tivity was restored by adding CaCl2 to the EDTA-containingreaction mixture, an indication that Ca2+ was required forpolygalacturonate depolymerization. For both organisms,the optimum polygalacturonate-depolymerizing activity inreaction mixtures was observed at alkaline pH values (Fig.

_ ~~~~~3.It was concluded that the extracellular polygalacturonate-

depolymerizing activity of B. pectinophilus and B. galactu-ronicus was characteristic of a pectate (polygalacturonate)lyase (30), as indicated by the formation of unsaturatedproducts, the preference for polygalacturonate as a sub-strate, the requirement of Ca2 for activity (Table 1), and thealkaline pH optimum (Fig. 3).The action pattern of pectate lyases may be either of the

"endo" type (random cleavage of the polymer) or of theexo" type (terminal cleavage) (30). The change in reaction

FIG. 2. Electron micrograph of a negatively stained cell of B.galacturonicus. Peritrichously arranged flagella are visible. Bar, 1Ftm. ' '

E1

it.6-.carbon and energy source, a mixture of unsaturated oligo- /mers obtained by incubating polygalacturonate with a par- 1.4tially purified preparation of exopectate lyase. /

B. galacturonicus required as growth factors folic acid, w /pantothenic acid, and biotin. When any of these vitamins z I.2 1were omitted from BGD broth, the medium did not support /growth of this species.

Pectinolytic enzymatic activities. Growing cells of B. ° 1.0 /pectinophilus or B. galacturonicus deesterified pectin by mmeans of an extracellular pectinesterase (pectin methyles- . d/terase, EC 3.1.1.11) activity. This activity was detected in z 0.8 /

wU,< 0.6-w

TABLE 1. Ca2+ requirement of the extracellular lyase activities 0.4of B. pectinophilus and B. galacturonicus Z 0Ar

Added to the Reaction ratea for:reaction mixtureb B. pectinophilus B. galacturonicus

None 2.4 1.0EDTA 0.1 <0.1EDTA + CaC12 1.1 0.7

a Reaction rate is the increase in A5" over 60 min (TBA assay). A partiallypurified enzyme preparation was added to the reaction mixture, whichcontained 0.03 mg of protein.

b EDTA, 0.4 ,umol; CaC12, 1.0 Lmol.

pHFIG. 3. Lyase activity at different pH values as measured by the

TBA assay. - - -, B. pectinophilus; -, B. galacturonicus. Each B.pectinophilus reaction mixture contained 0.025 mg of partiallypurified enzyme preparation protein (2.4 U of lyase activity). B.galacturonicus reaction mixtures contained 0.03 mg of partiallypurified enzyme preparation protein (1.85 U of lyase activity).Reaction mixtures were incubated for 30 min.

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884 JENSEN AND CANALE-PAROLA

mixture viscosity was correlated with percent polygalactu-ronate degradation to determine the action pattern of thepectate lyases of the two Bacteroides species. At a 50%decrease in reaction mixture viscosity, B. pectinophiluspectate lyase activity had cleaved 8 to 9% of the bonds in thepolygalacturonate and B. galacturonicus lyase activity hadcleaved approximately 13%o (Fig. 4). These results indicatedthat the pectate lyases had an exo-type action patternbecause, at a 50% decrease in reaction mixture viscosity,endo-acting enzymes are expected to have cleaved only 2 to3% of the bonds in the polygalacturonate, whereas exo-acting enzymes would have cleaved up to 10% or more of thebonds (13, 19).The occurrence of an exo-type action pattern was con-

firmed by using TLC and paper chromatography to separateand identify the products formed from polygalacturonate bythe pectate lyase activities in reaction mixtures. Pectatelyase activity from either organism formed unsaturatedtrigalacturonate (trimer) as the initial product frompolygalacturonate (Table 2). Only after prolonged incubationof reaction mixtures were unsaturated di- and tetragalac-turonates detected (Table 2). It may be surmised thatpentamers, formed after prolonged terminal cleavage of thepolymer, were finally cleaved into dimers and trimers andthat oligomers smaller than pentamers were not cleaved bythe enzymatic activity. The major product accumulating inthe reaction mixture was unsaturated trigalacturonate,which apparently was not cleaved further. An endo-actinglyase would have produced oligomers of different lengthsearly in the incubation of the reaction mixture. In view of theresults reported above, we concluded that the extracellularpectin-depolymerizing activity of B. pectinophilus and B.galacturonicus was characteristic of an exopectate (exopoly-galacturonate) lyase that cleaves unsaturated trigalacturonicacid units from polygalacturonate. The formation of unsatur-ated di- and tetragalacturonate after prolonged incubation ofthe reaction mixtures is consistent with the possibility that

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TIME (min)FIG. 4. Relative viscosity changes and percent degradation of

polygalacturonate (PGA) by a partially purified pectate lyase prep-aration from B. galacturonicus. At a 50% decrease in relativeviscosity (A), approximately 13% of the polygalacturonate bondshad been cleaved (B).

TABLE 2. B. pectinophilus exopectate lyase reaction productsseparated by TLC

ProductsaTime(h) Monomerb Unsaturated Unsaturated Unsaturated Oligomers larger

dimer trimer tetramer than tetramer

00.25 - - (+) - -0.75 - - + _ _1.5 - - ++ (+) -3.0 - (+) +++ + -

11 - ++ ++++ ++ -

a-, No product seen; (+), very weak spot; + + + +, very intense spot.Saturated dimers, trimers, and tetrarners were not detected.

bIncludes both saturated and unsaturated galacturonic acid. The TLCplates were sprayed with a 2% (wt/vol) solution of o-phenylenediaminedihydrochloride in 80% (vol/vol) ethanol (16) to detect unsaturatedgalacturonic acid.

the pectate lyases act on the nonreducing ends of thepolygalacturonate chains (33).The exopectate lyase activity was present in culture

supernatant fluids of both Bacteroides species. The bulk ofthe enzymatic activity in culture supernatant fluids did notoriginate from the release of cell-associated protein by lysingcells. Light microscopy examination of broth cultures atvarious growth stages did not show alterations in cell mor-phology known to occur during lysis. The amount of lysisthat occurred in broth cultures was estimated by measuringthe amounts of DNA in late-log-phase PF culture superna-tants, in cultures lysed by passage through a French pressurecell, and in uninoculated PF medium. On the basis of thesemeasurements, it was calculated that less than 5% of thecells had lysed in late-log-phase cultures. Although less than5% of the cells had lysed, 50% of the total lyase activity incultures was in the supernatant fluid, and the remaining 50%was cell associated. This indicated that the exopectate lyase,after being synthesized by the cells, was released into theexternal environment by processes that did not involve celllysis to any significant extent.

Cells of B. pectinophilus grown in media containing fer-mentable substrates other than polygalacturonate producedless than 10% of the extracellular exopectate lyase activityfound in supernatant fluids of polygalacturonate-containingcultures (Table 3), an indication that the lyase activity wasinducible. In contrast, the level of extracellular lyase activityof B. galacturonicus remained approximately constantwhether the organism was grown with polygalacturonate,galacturonate, or glucuronate as the fermentable substrate.The exopectate lyases of the two species differed with

TABLE 3. Extracellular polygalacturonate lyase activityproduced by cells growing in media containing different

fermentable substrates

Reaction ratea:Substrate

B. pectinophilus B. galacturonicus

Polygalacturonate 0.96 0.17D-Galacturonate NTb 0.18D-Glucuronate NT 0.16D-Fructose 0.04 NTD-Gluconate 0.06 NT

a Reaction rate is defined in Table 1. All supernatant fluids used in the TBAassay reaction mixtures were from cultures with a cell density of 109 cells perml.bNT, Not tested (not used as fermentable substrate).

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TABLE 4. Products of polygalacturonate fermentation formed bygrowing cells

Amta produced by:Product

B. pectinophilusb B. galacturonicus

Acetate 2.0 (1.0) 2.3Formate 1.0 (0.36) 1.3Ethanol <0.1 (0.1) NDcLactate <0.1 (0.4) 0.1

a Expressed as millimoles per 100 ml of culture. Cultures were incubated for24 h at 37°C. Products tested for but not detected: pyruvate, oxaloacetate,fumarate, succinate, propionate, n-valerate, n-butyrate, n-caproate. Produc-tion of CO2 or H2 was not determined.

b Numbers within parentheses are the amounts of end products formedwhen D-gluconate (0.4% [wt/vol]) was the fermentable substrate.

c ND, Not detected.

respect to their pH optima. B. pectinophilus pectate lyaseactivity had a pH optimum of 9.0 to 9.5, whereas B.galacturonicus lyase activity peaked at pH 8.2 to 8.5 (Fig. 3).

Hydrolytic enzyme activities (e.g., exopolygalacturon-ases, endopolygalacturonases) were not detected in culturesupernatant fluids or in partially purified preparations ofextracellular enzymes of either Bacteroides species.

Fermentation products. Acetate and formate were themajor nongaseous products of polygalacturonate fermenta-tion by either species (Table 4). Acetate and formate pro-duced from polygalacturonate were present approximatelyin a 2:1 molar ratio in the culture fluid of both species (Table4), an observation suggesting that CO2 was also produced.Cells of B. pectinophilus growing in PF broth lackingpolygalacturonate and containing gluconate as the ferment-able substrate produced more lactate and less acetate andformate than did cells fermenting polygalacturonate (Table4).

Other phenotypic characteristics. The results of antibioticsensitivity and other tests are summarized in Table 5.G + C content ofDNA. The mol% G + C of the DNA was 45

+ 1 for B. pectinophilus and 36 + 1 for B. galacturonicus.

DISCUSSIONPectin depolymerization by B. pectinophilus and B.

galacturonicus is accomplished through the action of at leasttwo enzymes. One of these enzymes is a pectinesterase (EC3.1.1.11) that serves to remove methyl ester groups frompectin; the other is an exopectate lyase which releasesunsaturated trimers from the demethylated polymer. Exo-acting pectate lyases that release unsaturated trimers arequite uncommon and have only been detected in the soilbacterium Streptomyces nitrosporeus (33) and in the ruminalbacterium Butyrivibrio fibrisolvens (40).The Nomenclature Committee of the International Union

of Biochemistry has recommended (28) that EC number4.2.2.9 be used to designate exopectate (exopolygalacturon-ate) lyases that release unsaturated dimers from poly-galacturonate (20). No recommendation was made fortrimer-releasing exopectate lyases. Wojciechowicz et al. (40)used EC number 4.2.2.9 for the trimer-releasing exopectatelyase of Butyrivibrio fibrisolvens, whereas Sato and Kaji(33), who described the trimer-releasing exopectate lyase ofS. nitrosporeus, concluded that the latter was a new type ofexopectate lyase and did not assign to it an EC number.Evidently, dimer- and trimer-releasing exopectate lyases aretwo distinct enzymes, as indicated by their dissimilar modeof action, and probably should be given different EC num-bers.

The properties of the cell-associated pectate lyase activitywere not studied. Thus, it is not known whether the cell-associated and the extracellular lyase activities differ fromeach other. A cell-associated pectate lyase present in thecolon bacterium Bacteroides thetaiotaomicron has beenreported to produce mainly unsaturated dimers frompolygalacturonate (22).The degree of nutritional specialization exhibited by the

pectinophilic intestinal Bacteroides species suggests thatthese bacteria occupy a rather narrow ecological niche in thehuman colon. The pectinesterases and exopectate lyases,which they release into the external environment, convertthe esterified polygalacturonate chains in the pectin mole-cules to unsaturated trimers and oligomers that are used bythe pectinophiles as fermentable substrates and that mayalso be utilized by commensal intestinal bacteria lackingpectinolytic enzymes. Furthermore, pectin fermentation endproducts such as acetate are absorbed from the human largeintestine and may contribute to the nutrition of the host (24,31, 41).According to current taxonomic criteria (10), the two

pectinolytic bacteria described in this paper (strains N3 andN6) are appropriately assigned to the genus Bacteroidesinasmuch as they are gram-negative, obligately anaerobic,nonsporeforming, chemoorganotrophic rods that produceacetate and formate as major fermentation end products.Strain N3 has no flagella and is not motile, whereas strain N6has peritrichous flagella but does not exhibit motility in thegrowth media used in this study.The substrate fermentation patterns of strains N3 and N6

are markedly different from those of all other known speciesof Bacteroides. Strains N3 and N6 ferment only pectin and afew related compounds (see species descriptions below),whereas pectinolytic strains of Bacteroides previously de-scribed (1, 10, 32, 39) utilize, in addition to pectin, a broadspectrum of pentoses, hexoses, and disaccharides as fer-mentable substrates for growth.Even though strains N3 and N6 are similarly specialized

with respect to their carbon and energy sources, they differsignificantly from each other in the G +C content of theirDNA; in flagellation; in the ability to utilize galacturonate,glucuronate, and gluconate (see species descriptions below);and in other phenotypic characteristics (Table 5). Theyutilize different exopectate lyases to cleave polygalacturon-ate, as indicated by the dissimilarity of the pH optima of thetwo lyase activities (Fig. 3).

In view of the considerations outlined above, we concludethat strains N3 and N6 represent two previously undescribedspecies of Bacteroides. We propose the name Bacteroides

TABLE 5. Phenotypic characteristics

Test B. pectinophilus B. galacturonicus

Catalase produced no noGelatin digested yes noEsculin hydrolyzed no noGrowth in 20% bile yes yesGrowth with antibioticsa

Penicillin-G + +Tetracycline - -ClindamycinCephalothinAmpicillin + +Chloramphenicol - +

a +, Growth; -, no growth. Antibiotic concentrations are listed in Materialsand Methods.

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886 JENSEN AND CANALE-PAROLA

pectinophilus (pectin-loving) for strain N3 to denote itsextreme nutritional specialization and the name Bacteroidesgalacturonicus (galacturonate-utilizing) for strain N6 to in-dicate its ability to utilize galacturonate or polygalacturonateas an exogenous carbon and energy source.

It should be noted, however, that even though the avail-able information indicates that the two pectinolytic isolatesdescribed in this paper are appropriately classified in thegenus Bacteroides, it is possible that further work involvingribosomal RNA oligonucleotide cataloging or DNA homol-ogy studies will show that these species are sufficientlydifferent from other Bacteroides species to justify proposinga new genus to which the two isolates would be assigned.Description of Bacteroides pectinophilus sp. nov.

pec.ti.no'phil.us. M.L. n. pectinum pectin; Gr. adj. philusloving; M.L. adj. pectinophilus pectin-loving.

Cells grown in broth containing polygalacturonate asfermentable substrate (PF broth) are rod shaped, 0.5 by2.5-5 Am, arranged singly or in pairs, and rarely in shortchains. Nonsporeforming, gram-negative, nonmotile.

Surface colonies on PF agar medium are cream colored,circular, flat, opaque, and smooth and measure 2 to 3 mm indiameter. Final growth yield in PF broth approximates 1.6 x109 cells per ml, and the cell population doubling time is 1.2h. Final pH of PF broth cultures ranges from 5.0 to 5.5.Optimum growth is between 35 and 40°C, with no growth at20 or 45°C.

Obligate anaerobes. Pectin, polygalacturonate, and D-gluconate are fermented. Occasionally, D-fructose is fer-mented after a lag of 1 to 3 days. L-arabinose, D-ribose,D-xylose, D-galactose, D-glucose, D-mannose, cellobiose,lactose, maltose, sucrose, L-rhamnose, D-galacturonate, D-glucuronate, pyruvate, glycerol, methanol, amino acids,alginate, amylose, arabinogalactan, carboxymethyl cellu-lose, cellulose, chondroitin sulfate, dextran, hyaluronic acid,inulin, starch, and xylan are not fermented. Catalase is notproduced. Gelatin is digested. Esculin is not hydrolyzed.Excellent growth in PF broth containing 20% bile.Acetate and formate are major end products of

polygalacturonate or pectin fermentation. Lactate andethanol are formed in minor amounts. In addition, pectinfermentation yields methanol. Gluconate fermentation yieldsacetate, formate, lactate, and a small amount of ethanol.

Pectin is depolymerized extracellularly by the cumulativeaction of pectinesterase (EC 3.1.1.11) and of an exopoly-galacturonate lyase activity that releases unsaturatedtrigalacturonate molecules from polygalacturonate and thathas a pH optimum of 9 to 9.5.

Isolated from human feces.The mol% G + C of the DNA is 45 as determined by

thermal denaturation.The type strain is N3; this strain has been deposited with

the American Type Culture Collection under the numberATCC 43243.Description of Bacteroides galacturonicus sp. nov.

ga.lac.tu.ro'ni.cus M.L. adj. galacturonicus pertaining togalacturonate, with reference to the ability to fermerit thiscompound or polygalacturonate.

Cells grown in broth containing polygalacturonate as thefermentable substrate (PF broth) are rod shaped, 0.6 by3.5-6 ,m, arranged singly or in pairs, and rarely in shortchains. Cells up to 12 4m in length are present in cultures.Nonsporeforming, gram-negative. Six to eight peritrichousflagella per cell are present. However, motility has not beenobserved under the cultural conditions used.

Surface colonies on PF agar medium are white with a tan

tinge, irregularly shaped with uneven margins, flat, opaque,and rough and measure 4 to 5 mm in diameter. Final growthyield in PF broth approximates 2.3 x 109 cells per ml, andthe cell population doubling time is 1 h. Final pH ofPF brothcultures ranges from 5.0 to 5.5. Optimum growth is between35 and 40°C, with no growth at 20 or 45°C.

Obligate anaerobes. Pectin, polygalacturonate, D-galac-turonate, and D-glucuronate are fermented. L-arabinose,D-ribose, D-xylose, D-fructose, D-galactose, D-glucose, D-mannose, cellobiose, lactose, maltose, sucrose, L-rhamnose,D-gluconate, pyruvate, glycerol, methanol, amino acids,alginate, amylose, arabinogalactan, carboxymethyl cellu-lose, cellulose, chondroitin sulfate, dextran, hyaluronic acid,inulin, starch, and xylan are not fermented. Folic acid,pantothenic acid, and biotin are required as growth factors.Neither hemin nor vitamin K is required for growth. Cata-lase is not produced. Gelatin and esculin are not hydrolyzed.Excellent growth in PF broth containing 20% bile.

Acetate, formate, and a small amount of lactate are endproducts of polygalacturonate or pectin fermentation. Inaddition, pectin fermentation yields methanol.

Pectin is depolymerized extracellularly by the cumulativeaction of pectinesterase (EC 3.1.1.11) and of an exopoly-galacturonate lyase activity that releases unsaturatedtrigalacturonate molecules from polygalacturonate and thathas a pH optimum of 8.2 to 8.5.

Isolated from human feces.The mol% G + C of the DNA is 36 as determined by

thermal denaturation. The type strain is N6; this strain hasbeen deposited with the American Type Culture Collectionas ATCC 43244.

ACKNOWLEDGMENTS

We are grateful to B. Himelbloom for valuable contributions tothe substrate fermentation studies, to F. H. Weber for his assistancein the determination of guanine plus cytosine values, and to T. L.Miller and M. J. Wolin for the use of their laboratory facilities. Wethank S. A. Fowler for carrying out the paper chromatography workand E. Musante for the electron microscopy.

This research was supported by Public Health Service grantAI-20620 from the National Institute of Allergy and InfectiousDiseases.

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