JOURNAL OF BACTERIOLOGY, Aug. 1987, p. 3792-3800 Vol. 169, No. 8 0021-9193/87/083792-09$02.00/0 Copyright © 1987, American Society for Microbiology Specialized Cell Surface Structures in Cellulolytic RAPHAEL LAMED,'* JENNY NAIMARK,' ELY MORGENSTERN,' AND EDWARD A. BAYER2 Center for Biotechnology, George S. Wise Faculty ofLife Sciences, Tel Aviv University, Ramat Aviv,1 and Department of Biophysics, The Weizmann Institute of Science, Rehovot,2 Israel Received 30 March 1987/Accepted 22 May 1987

The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characterisitic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular -containing polycellulosomes of thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of .

We have recently demonstrated by various means that MATERIALS AND METHODS cellulolytic enzymes of the gram-positive, thermophilic anaerobe Clostridium thermocellum are organized into a Organisms and culture conditions. The bacterial strains distinct multisubunit complex which we have called the used in this study are listed in Table 1. All strains of C. "cellulosome" (2, 16, 17). The cellulosome in this organism thermocellum were cultivated under anaerobic conditions at appears both in an extracellular and in a cell-associated 60°C in cellobiose-containing medium as described in refer- form. The latter is considered to comprise a discrete cell ence 2. Clostridium cellulovorans was also grown in the surface organelle which is responsible both for efficient same medium but at 37°C. cellulolysis and for the adhesion of the bacterium to its Bacteroides cellulosolvens (SPB25) NRC 2944 was cul- insoluble substrate. tured anaerobically at 37°C according to Murray et al. (20) on The ultrastructural localization of the cellulosome on the modified AC medium (pH 7.6) supplemented with 3-(N- cell surface of C. thermocellum has previously been studied morpholino)-2-hydroxypropane sulfonic acid (5 g/liter) and by transmission electron microscopy (TEM). In that study using cellobiose as a substrate. Acetivibrio cellulolyticus was (4), a multitude of novel protuberant surface structures were grown on the identical medium brought to pH 6.8. visualized both by specific immunolabeling and by a general Cellulomonas sp. was grown on medium (pH 6.5) contain- staining procedure using cationized ferritin (CF). In subse- ing 5 g each of peptone, tryptone, yeast extract, and either quent studies (3, 15), the fate of these structures upon glucose or cellobiose (31). Cells were grown aerobically at binding of the bacterial cell to cellulose was traced using an 32°C with reciprocal shaking at 150 rpm. In some cases, this immunocytochemical technique. In this manner, some of the strain was grown anaerobically on the cellobiose-containing polycellulosomal protuberances were shown to be trans- medium used for C. thermocellum. formed to yield an amorphous or fibrous network which Ruminococcus albus and Clostridium cellobioparum were appears to connect the cell to cellulosome clusters which grown at 37°C on medium containing the following additives coat the surface of the cellulose substrate. per 1 liter of distilled water: 10% (vol/vol) goat rumen, 1% The occurrence of cell-associated cellulolytic enzymes has (wt/vol) beef extract, 30 g of peptone, 5 g of yeast extract, been suggested in several anaerobic bacteria (6, 9, 10, 21, 24, 2.5 g of KH2PO4, 2.5 g of K2HPO4, 4 g of glucose, 1 g of 32). It was the purpose of this work to determine whether maltose, 1 g of starch, 0.1 g of CaC12, 3 g of NaCl, 1 g of our previous findings regarding the protuberant structures (NH4)2SO4, 0.1 g of MgSO4, 0.1 g of NaHCO3, 10 ml of observed on the surface of C. thermocellum would be vitamin solution (1), 1 mg of resazurin, and 0.5 g of cysteine relevant to other cellulolytic bacteria. hydrochloride. Cellobiose (3 g/liter) was added as a carbon We demonstrate here by scanning electron microscopy source, and the pH was adjusted to 7.3. As in all anaerobic (SEM) the occurrence of distinct protuberant structures on procedures, the culture medium was treated with a stream of the cell surface in a variety of both gram-negative and nitrogen gas, and the bottles were sealed with butyl rubber gram-positive cellulolytic bacterial species. The immuno- septum-type stoppers. The bacteria were cultured at 37°C. chemical properties of the cellulolytic bacteria examined in Thermoanaerobium brockii and Clostridium thermohydro- this study were compared with those of C. thermocellum by sulfuricum were grown under anaerobic conditions at 60°C using a specific anticellulosome antibody preparation. The on tryptone-yeast extract-glucose (TYEG) medium accord- results indicate that the cellulosome concept may be a more ing to Lamed and Zeikus (18). In some experiments, these general feature of cellulolytic microorganisms. strains were grown on the cellobiose-containing medium used for C. thermocellum. Escherichia coli and Serratia marcescens were grown at 37°C in nutrient broth (Difco Laboratories, Detroit, Mich.) * Corresponding author. either on agar plates or in liquid medium. 3792 VOL. 169, 1987 SURFACE STRUCTURES OF CELLULOLYTIC BACTERIA 3793

TABLE 1. Bacterial strains used in this study magnification). Controls (no lectin) showed no autoagglu- tination. Strains Referenceor source It is interesting that only one of the above lectins, G. simplicifolia GS-I, and only one of its homotypic isolectins, Cellulolytic B4, caused agglutination of C. thermocellum. p-Galactose Acetivibrio cellulolyticus ATCC 33288 ...... 22 final concentration) inhibited the agglutination. Thus, Bacteroides cellulosolvens NRC 2944...... 20 (1% was further in Cellulomonas sp. ATCC 21399 ...... 11 GS-I employed fluorescence-labeling experi- Clostridium cellobioparum ATCC 15832 ...... 13 ments described below. Clostridium cellulovorans ATCC 35296 ...... 27 Lectin-mediated surface labeling. A 10-ml culture of the Clostridium thermocellum NCIB 10682 (ATCC 27405).. 30 desired bacterial strain, grown to mid-exponential phase on Clostridium thermocellum YS . 2 the desired medium and under the desired conditions, was Clostridium thermocellum AD2' ...... 2 washed once with PBS by centrifugation (7,000 x g, 10 min, Clostridium thermocellum LQRI ...... 18 25°C), and the cells were suspended to 1 optical density unit Clostridium thermocellum Jl ...... 2 (400 nm). A 50-plI sample was combined with 10 pul of Ruminococcus albus DSM 20455 ...... 25 Noncellulolytic strains fluorescein isothiocyanate-labeled GSI-B4 (0.4 mg/ml; After 15 min at the cells were washed once, Clostridium thermohydrosulfuricum DSM 567 ...... 14 Sigma). 25°C, Escherichia coli B ...... TAU' suspended to the same volume with PBS, and mounted on a Serratia marcescens ...... TAUb microscope slide for fluorescence analysis under an Thermoanaerobium brockii ATCC 33075 ...... 33 Olympus BH2 fluorescence microscope using blue exciter filters. As a control, 1% (final concentration) galactose was aAdherence-defective mutant derived from strain YS. I These strains were obtained from the culture collection of the Department added to the cell suspension together with the fluorescent of Microbiology, Tel Aviv University. lectin. Immunoblotting. The desired bacterial strain was grown on the appropriate medium under appropriate conditions. A Stripping of exocellular components from R. albus. The 1-ml sample was washed once with PBS in a tared Eppendorf stripping procedure used in this work was performed by a vial. The pellet was weighed and suspended to 40 mg (wet modification of the protocol used by Wood et al. (32) for weight) per ml. The suspension was combined with a half- releasing cell-bound . Cells were centrifuged and volume sample of 0.6% (wt/vol) sodium dodecyl sulfate (in suspended in 5 mM KH2PO4-NaOH buffer (pH 6.7). After 30 PBS), and the resultant suspension was boiled for 10 min and min at 25°C, the cells were centrifuged and suspended in the centrifuged. The supernatant fluids were collected and same buffer. The procedure was repeated once again, and cooled, and various dilutions (10-fold, 30-fold, 100-fold, etc.) the cells were processed for SEM. were applied (2 pul) to nitrocellulose sheets. Cytochemistry and SEM. A 1-ml sample of culture fluid The dot blots were dried and quenched with 0.2% (wt/vol) was centrifuged in an Eppendorf Microfuge. The cell pellet Tween 20 in PBS (PBS-Tween). The blots were incubated was suspended in 0.9% NaCl (saline). The suspension was for 2 h with cellulosome-specific (mutant AD2-adsorbed) filtered through a Nuclepore membrane filter (0.6 p.m) and antibodies (25 ,ug/ml; 2), using 5% (vol/vol) milk-PBS-Tween washed with saline. A solution (0.2 ml) of CF (1 mg/ml: as a diluent. The blots were rinsed several times with BioMakor, Rehovot, Israel) was applied to the filter for a PBS-Tween, and anti-rabbit immunoglobulin horseradish period of 10 min. The filter was washed with saline, and the peroxidase-linked Fab fragments (Amersham) were applied cells (attached to the filter) were fixed overnight with 5% (1:1,000 dilution in 5% milk-PBS-Tween). After a 2-h incu- glutaraldehyde. The material was dehydrated by a series of bation, the blots were rinsed as described above, and sub- graded solutions. The specimen was critical-point strate solution (0.5 mg of 4-chloro-1-naphthol per ml, diluted dried using liquid C02, and the preparation was coated with from a 3-mg/ml methanolic stock solution with 0.015% gold in a Polaron sputter-coater and examined with a Jeol [wt/vol] H202 in 50 mM Tris hydrochloride-saline, pH 7.4) JSM-35 scanning electron microscope. was added. Color development occurred within 5 to 10 min. Agglutination test for lectin screening. The interaction of C. Cellulose activity. The detection of cellulolytic activity in thermocellum YS with a panel of lectins was performed in washed cells was performed using the Congo red carboxy- the laboratory of R. J. Doyle (8). For this work 21 different methyl cellulose (CMC)-agar assay reported by Beguin (5). lectins (obtained from either E-Y Labs, San Mateo, Calif., or Cells were washed once and suspended in PBS to an optical Sigma Chemical Co., St. Louis, Mo.) were employed, rep- density (400 nm) of 1.0. A 10-,ul volume of each cell resenting a broad spectrum of sugar specificities. The lectins preparation was applied to wells in the agar layer which used were from the following sources: Phaseolus vulgaris contained CMC. The preparations were incubated for 20 h at (PHA-E), Griffonia simplicifolia GS-I and GS-II, G. the corresponding temperature at which the given bacterial simplicifolia isolectins GSI-A4 and GSI-B4, Ulex sp. I and II, strain was optimally grown. After Congo red staining of the Lotus tetragonolobus, Limaxflavus, Helix aspersa, Arachis residual CMC, endoglucanase activity was detected by the hypogaea, Dolichos biflorus, Ricinus communis I and II, appearance of clearing zones, the size of which was roughly Sophora japonica, Triticum vulgaris (wheat germ aggluti- proportional to the extent of CMC hydrolysis. nin), Canavalia ensiformis (concanavalin A), Pisum sativum, Solanium tuberosum, loach egg, Sarothamnus, RESULTS Maclura pomifera, and Glycine max. Surface ultrastructure of C. thermocellum strains. Several Cell suspensions were adjusted to an optical density of 1.0 strains of C. thermocellum, obtained from various sources, (1 cm; 450 nm) in (sodium) phosphate-buffered saline (PBS) were subjected to treatment with CF, and the labeled cells (pH 7.2) or 50 mM Tris hydrochloride (pH 7.3). A sample (50 were analyzed by SEM (Fig. 1). With the exception of the ,ul) was mixed with a solution (50 p.1) of the desired lectin (50 adherence-defective mutant AD2 (Fig. le), the surfaces of all p.g total) on a Boerner plate. The plate was rocked gently for cellobiose-grown strains tested were characterized by a 15 min, and the results were observed with a microscope (X4 dense distribution of protuberant structures. U- d

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3794 VOL. 169, 1987 SURFACE STRUCTURES OF CELLULOLYTIC BACTERIA 3795

FIG. 2. SEM of various CF-stained, mesophilic, anaerobic, cellulolytic bacteria. The gram-negative strains A. cellulolyticus (a) and B. cellulosolvens (b) and the gram-positive strains C. cellulovorans (c) and C. cellobioparum (d) were grown on cellobiose, stained with CF, and processed for SEM. Note the presence of large protuberant structures on all cells. Bar, 1.0 ,um.

In the case of the YS strain, the size, shape, and disposi- ferences in cellulosome and polycellulosome structure. (The tion of the structures observed by SEM in Fig. la were very terms cellulosome and polycellulosome, initially coined by similar to those of the polycellulosomal protuberances pre- us [17], are considered to be synonymous with the terms viously analyzed by TEM (3, 4). In general, the surface OBS [originally bound small cellulase complex] and OBL protuberances on cells of the NCIB strain appeared to be [originally bound large cellulase complex], respectively, as smaller in size than those of YS, but the extent of their used by others [7, 12; J. H. D. Wu and A. L. Demain, Abstr. distribution was similar (Fig. lb). Other strains of C. Annu. Meet. Am. Soc. Microbiol. 1985, 079, p. 248]). thermocellum, e.g., LQRI and Jl, also exhibited similar As stated above, these structures appeared to be entirely protuberant structures with characteristic strain-specific lacking on the cellobiose-grown, CF-stained, adherence- variations in their size and shape (Fig. lc and d). These defective mutant AD2 (Fig. le), which has been shown to differences may reflect the reported strain-specific size dif- lack surface cellulosome when grown on cellobiose (2, 4).

FIG. 1. SEM of various strains of the gram-positive, thermophilic, cellulolytic anaerobe C. thermocellum. Cells of strains YS (a), NCIB 10682 (ATCC 27405) (b), LQRI (c), Jl (d), or adherence-defective mutant AD2 (e) were grown on cellobiose, stained with CF, and processed for SEM. An unstained sample of strain YS is shown in panel f. Bar, 1.0 p.m. 3796 LAMED ET AL. J. BACTERIOL.

FIG. 3. SEM of the CF-stained, gram-positive, mesophilic, anaerobic, cellulolytic rumen bacterium R. albus. Cells were treated with CF either before (a) or after (b) implementation of the stripping procedure described in the text. Note that after stripping, there is a general reduction in the surface density of the very large protuberant structures. Bar, 1.0 ,um.

Without pretreatment with CF (Fig. if), protuberances on examples of which are presented in Fig. 2 through 4. the cell surface of the wild type either were not visible or Interestingly, these structures were present on both gram- appeared only in isolated instances. negative and gram-positive bacteria. In contrast to the Surface ultrastructure of various cellulolytic bacteria. Other thermophilic C. thermocellum, the strains shown in Fig. 2 cellulolytic bacteria were grown on cellobiose and subjected through 4 are all mesophilic. As with C. thermocellum; when to CF treatment before processing and SEM analysis. These the strains were not pretreated with CF, the protuberances structures were essentially similar in appearance and dispo- were essentially lacking (data not shown). sition on the cell surface to those of the various strains of C. Various surface structures have previously been identified thermocellum, with characteristic species-specific differ- in R. albus by TEM (19, 28, 32). It has also been shown that ences in all cases. Protuberance-like structures were clearly in this organism, the cell-bound cellulase(s) could be re- demonstrated in all cellulolytic strains tested in this work, moved by a stripping process which involves repeated

FIG. 4. SEM of the CF-stained, gram-positive, cellulolytic, facultative aerobe Cellulomonas sp. Cells were grown aerobically on either cellobiose (a) or glucose (b), stained with CF, and processed for SEM. Bar, 1.0 ,um. VOL. 169, 1987 SURFACE STRUCTURES OF CELLULOLYTIC BACTERIA 3797

1c _:Cd = FIG. 5. SEM of various representative CF-stained, noncellulolytic bacteria. E. coli (a), C. thermohydrosulfuricum (b), and T. brockii (c) were grown on appropriate media as described in the text, subjected to CF treatment, and processed for SEM. The encapsulated bacterium S. marcescens, similarly treated, is shown in panel d. Note that all of these noncellulolytic strains lack the large protuberant structures which characterize all of the cellulolytic strains. Bar, 1.0 p.m. washings of the isolated cells with buffer (32). We therefore (or cellulose) were covered with a multitude of protuberant subjected R. albus to a similar procedure and compared the structures, whereas the surface of glucose-grown cells was surface architecture of the cells before and after stripping. nearly devoid of such structures (Fig. 4). The results (Fig. 3) demonstrate that the large protuberance- Surface ultrastructure of noncellulolytic bacteria. To deter- like structures which coat this bacterium can be effectively mine whether protuberant structures are uniquely character- removed by the same stripping procedure. istic of cellulolytic bacteria, we examined several noncellu- In the gram-positive aerobe Cellulomonas sp., our results lolytic bacteria. In addition to E. coli (as a representative have shown that growth on cellobiose is accompanied by the aerobic, mesophilic, gram-negative strain) we tested both T. production of cell-associated cellulases. In contrast, we brockii and C. thermohydrosulfuricum, both of which found reduced endoglucanase activity when the same strain closely resemble C. thermocellum in that they are gram- was grown on glucose (data not shown). In another work positive anaerobic thermophiles but differ from C. (31), exocellular ruthenium red-stained "sheaths" were ob- thermocellum in being noncellulolytic strains. The gram- served by TEM in cellulose-grown cells of Cellulomonas sp. negative mesophile S. marcescens was included as an exam- which were absent in glucose-grown cells of the same strain. ple of an encapsulated, noncellulolytic bacteria strain (26, We therefore grew Cellulomonas sp. on the above carbon 29). sources to examine the comparative characteristics of the CF labeling of E. coli failed to reveal protuberant surface CF-stained exocellular surfaces. Cells grown on cellobiose structures (Fig. 5a). Even though both C. thermocellum and 3798 LAMED ET AL. J. BACTERIOL.

TABLE 2. Cellulase activity, lectin binding, and antigenic lectin reacted with the majority of cellulolytic strains tested properties of bacterial surface components (only Cellulomonas sp. failed to interact). With the excep- Cellulase Lectin Immuno- tion of R. albus, the binding of this lectin was inhibited by Bacterial strain activitya bindingb blottingc the competing sugar (galactose). Neither probe reacted with the noncellulolytic strains used in this work. Neither probe C. thermocellum YS + + + + + + + the surface of the adherence- C. thermocellum AD2 + _d _d recognized cellobiose-grown, C. thermocellum NCIB 10682 + + + + + + + defective C. thermocellum mutant AD2. Transfer of mutant C. cellulovorans + + NTe NT AD2 to cellulose-containing medium led to an induction of C. cellobioparum + + + + + + the exocellular protuberances, which was accompanied by A. cellulolyticus + + + + + + + concomitant interaction with both antibody and lectin. B. cellulosolvens + + + + + + + Cellulomonas sp. + + - + + + + DISCUSSION R. albus ++ +f ++I-9 In previous publications (3, 4) we described the visualiza- E. coli - - - tion by TEM of cell surface protuberant structures in the C. thermohydrosulfuricum - - - anaerobic thermophilic cellulolytic bacterium C. thermocel- T. brockii - - - The of these which are not S. marcescens - - - lum. visualization structures, usually seen by TEM, was enabled by the general anion- a Cell-associated cellulase activity was determined by the Congo red specific histochemical stain CF. Treatment of cells before CMC-agar method. Washed cells of the given strain were applied to wells in for electron to stabilize the CMC-containing agar. After a 20-h incubation period at the desired tempera- processing microscopy appeared ture (see text), the agar was stained with Congo red. The extent of cellulolysis protuberances on the cell surface. In the present report we was determined by the size of the halo which surrounded the well. Symbols: demonstrate that the same treatment is advantageous for -, no measurable halo; +, halo of 1 to 3 mm; + +, halo greater than 3 mm. SEM studies. b Cells of the given strain were subjected to treatment with fluorescence- The evidence in this work demo strates a clear labeled isolectin 14 from G. simplicifolia (GS-I). Symbols: -, cells were not presented fluorescent; +, cells were highly fluorescent. correlation between cellulolytic activity and the appearance c Standardized quantities of cell extracts (40 mg of protein per ml) were dot of protuberance-like structures on the bacterial cell surface. blotted and labeled with anticellulosome antibody (derived from C. This correlation extends over a wide range of physiological thermocellum YS). For a given strain, the number of +'s scored designates and boundaries: the strains exam- the number of half-logarithmic dilutions (of the original extract) at which no evolutionary cellulolytic immunoreaction could be detected; -, no immunoreaction in the original ined in this work, whether gram positive or gram negative, extract. aerobic or anaerobic, mesophilic or thermophilic, all exhib- d The negative values refer to cellobiose-grown cells of the conditional ited these surface structures under conditions in which adherence-defective mutant; upon growth of cells on cellulose, the values cellulases are formed. For in the aerobic bacterium became positive. example, e NT, Not tested. Cellulomonas sp., elevated levels of cellulases are formed f The observed GS-I-induced fluorescence of R. albus could not be inhibited upon growth of the organism on cellobiose but not on by galactose. glucose. Correspondingly, in the present work, protuber- g In some experiments, no reaction could be detected with R. albus ance-like structures were prevalent on cellobiose-grown extracts. cells of this organism but not on glucose-grown cells. The correlation is further strengthened by the fact that in R. C. thermohydrosulfuricum are of a common genus and share albus, the identical stripping procedure which releases the many other common features, only the cellulolytic strain majority of cellulase activity from the cell surface (32) also bears defined protuberances on its cell surface. The surface divests the protuberance-like structures from the cell sur- of C. thermohydrosulfuricum appeared to be entirely smooth face. Interestingly, similar conditions caused the release of (Fig. 5b). The surface of the heavily flagellated T. brockii both cellulase activity as well as the protuberant structures was somewhat gnarled in appearance (Fig. 5c), but less so from the cell surface in many of the other cellulolytic strains than that of the cellulolytic bacteria. The surface topology of (including C. thermocellum) examined in this work (unpub- the encapsulated S. marcescens exhibited a furrowed ap- lished data). pearance, but essentially lacked protuberant structures (Fig. Our previous work with C. thermocellum (3, 15) has 5d). shown that the exocellular protuberances in this organism Interaction of bacterial strains with lectins and antibodies. are very complicated dynamic structures upon which the The similarity among surface structures in cellulolytic bac- multifunctional multicellulase complex, the cellulosome, is teria prompted us to examine whether a closer biochemical attached. These polycellulosomal protuberances apparently connection exists among these cellulolytic strains which play a definitive role in the interaction of the bacterium with would distinguish them from the noncellulolytic strains. In its cellulosic substrate. It is therefore tempting to speculate this context, two different approaches were used. The first that similar polycellulosomal surface organelles may be a consisted of immunoblotting of cell extracts by using the more general characteristic of cellulolytic bacteria. cellulosome-specific antibody preparation which has been The correlation is extended further in that the employment shown previously to selectively label the exocellular protu- of anticellulosome antibodies as an immunochemical probe berances on C. thermocellum. In another approach, a panel resulted in consistent cross-reactivity among cellobiose- of lectins were screened in an effort to find a complementary grown cellulolytic bacteria, independent of the evolutionary probe. Only one of these lectins (GS-I) agglutinated cells of similarity among the strains. The same antibodies failed to C. thermocellum YS, but not its adherence-defective mutant cross-react with the noncellulolytic strains used in this work; AD2 when grown on cellobiose. Further investigations re- even noncellulolytic strains (such as C. thermohydrosulfur- vealed that this lectin selectively binds the Si subunit of the icum and T. brockii) which are similar evolutionarily to the cellulosome (unpublished results). reference strain (C. thermocellum) failed to react with the The results (Table 2) showed a clear correlation between cellulosome-specific antibody preparation. cellulolytic activity and immunochemical cross-reactivity The exact origin of this cross-reactivity is not clear, but using the anticellulosome antibody. Similarly, the GS-I one may speculate that a common antigenic component VOL. 169, 1987 SURFACE STRUCTURES OF CELLULOLYTIC BACTERIA 3799

(such as the Si-associated polysaccharide or a common Bacteriol. 163:552-559. polypeptide sequence[s] either in the Si subunit or in any of 5. Beguin, P. 1983. Detection of cellulase activity in polyacryl- the cellulosomal cellulases) may be present in the cellulolytic amide gels using Congo Red-stained agar replicas. Anal. Bio- bacteria studied in this work. chem. 131:333-336. 6. Colvin, J. R., L. C. Sowden, G. B. Patel, and A. W. Khan. 1982. Not less surprising (and perhaps connected with this The ultrastructure of Acetivibrio cellulolyticus, a recently iso- phenomenon) is the fact that, of a variety of different lectins, lated cellulolytic anaerobe. Curr. Microbiol. 7:13-17. only one (GS-I) reacted with C. thermocellum. Indeed, 7. Coughlan, M. P., K. Hon-nami, H. Hon-nami, L. G. Ljungdahl, preliminary evidence (unpublished data) links the lectin- J. J. Paulin, and W. E. Rigsby. 1985. The cellulolytic enzyme binding activity in this organism with the Si subunit of the complex of Clostridium thermocellum is very large. Biochem. cellulosome, suggesting that the Si subunit is a target for the Biophys. Res. Commun. 130:904-909. lectin and comprises an a-D-galactose-rich glycoconjugate. 8. Doyle, R., and K. Keller. 1984. Lectins in diagnostic microbiol- The surfaces of several other cellulolytic bacteria were also ogy. Eur. J. Clin. Microbiol. 3:4-9. labeled extensively with the fluorescein-derivatized lectin, 9. Giuliano, C., and A. W. Khan. 1985. Conversion of cellulose to sugars by resting cells of a mesophilic anaerobe, Bacteroides but none of the noncellulolytic strains (including the encap- cellulosolvens. Biotechnol. Bioeng. 27:980-983. sulated bacterium) interacted with this lectin. 10. Groleau, D., and C. W. Forsberg. 1981. Cellulolytic activity of The possibility of functional conservation correlated with the rumen bacterium Bacteroides succinogenes. Can. J. Micro- structural homologies at the molecular level has previously biol. 27:517-530. been considered for cellulolytic and cellulose-recognizing 11. Han, Y. W., and V. R. Srinivasan. 1968. Isolation and charac- bacteria (23). Our results may indicate an expression of such terization of a cellulose-utilizing bacterium. Appl. Microbiol. a conservation among the cellulolytic bacterial strains exam- 16:1140-1145. ined in this study. This does not necessarily imply that all 12. Hon-nami, K., and M. P. Coughlan, H. Hon-nami, L. H. cellulolytic bacteria require such a common molecular appa- Carreira, and L. G. Ljungdahl. 1985. Properties of the cellulolytic enzyme system of Clostridium thermocellum. Bio- ratus; the expression of the system using cellobiose as a technol. Bioeng. Symp. 15:191-205. substrate may not always be the rule. Indeed, C. thermo- 13. Hungate, R. E. 1944. Studies on cellulose . I. The cellum AD2 does not express the exocellular cellulosome in culture and physiology of an anaerobic cellulose-digesting bac- the absence of cellulose. Moreover, these results (particu- terium. J. Bacteriol. 48:499-513. larly regarding the lectin) may not reflect a rigid correlation, 14. Klaushofer, H., and E. Parkinen. 1965. Zur frage de Bedeutung i.e., that these probes are definitive markers of cellulolytic or aerober und anaerober thermophiler Sporenbildner als Infekti- cellulose-binding activity; as more cellulolytic and onsursache in Rubenzukerfabriken. I. Clostridium thermohydro- noncellulolytic strains are examined in the future, many sulfuricum eine neue Art eines saccharoseabbauenden, exceptions may accumulate. thermophilen, schwefelwasserstoffbidenden Clostridiums. Z. In general terms, this work supports the hypothesis made Zuckerind. Boehm. 15:445-451. 15. Lamed, R., and E. A. Bayer. 1986. Contact and cellulolysis in earlier for the systems of C. thermocellum (2, 17) and B. Clostridium thermocellum via extensile surface organelles. cellulosolvens (9) that the more relevant form of the cellulase Experientia 42:72-73. is not the cell-free enzyme system, but rather the surface- 16. Lamed, R., E. Setter, and E. A. Bayer. 1983. Characterization of associated cellulase-containing complexes which mediate a cellulose-binding, cellulase-containing complex in Clostridium contact with the insoluble polymeric substrate. The possible thermocellum. J. Bacteriol. 156:828-836. appearance of cell-free cellulases, either in solution or at- 17. Lamed, R., E. Setter, R. Kenig, and E. A. Bayer. 1983. The tached to the cellulosic substrate, may reflect the partial cellulosome-a discrete cell surface organelle of Clostridium disassembly of the exocellular structures into soluble or thermocellum which exhibits separate antigenic, cellulose- cell-free particulate fractions which retain some but not all of binding and various cellulolytic activities. Biotechnol. Bioeng. the cellulolytic properties. The molecular nature Symp. 13:163-181. of these 18. Lamed, R., and J. G. Zeikus. 1980. Glucose fermentation associations is as yet unclear and will be the subject of pathway of Thermoanaerobium brockii. J. Bacteriol. 141: further studies. 1251-1257. 19. Leatherwood, J. M. 1973. Cellulose degradation by Ruminococ- cus. Fed. Proc. 32:1814-1818. ACKNOWLEDGMENTS 20. Murray, W. D., and L. C. Sowden, and J. R. Colvin. 1984. We thank R. J. 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