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Specialized Cell Surface Structures in Cellulolytic Bacteria RAPHAEL LAMED,'* JENNY NAIMARK,' ELY MORGENSTERN,' and EDWARD A

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Specialized Cell Surface Structures in Cellulolytic Bacteria RAPHAEL LAMED,'* JENNY NAIMARK,' ELY MORGENSTERN,' and EDWARD A 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 Bacteria 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 cellulase-containing polycellulosomes of Clostridium 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 cellulose. 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 cellulases. 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
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