1 Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA
2 Research Institute of Innovative Technology for the Earth, Kyoto 619-0292, Japan
Correspondence
Roy H. Doi
rhdoi{at}ucdavis.edu
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ABSTRACT |
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INTRODUCTION |
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Naturally occurring plant cell wall compounds are structurally heterogeneous polymers and are composed primarily of cellulose, hemicellulose, pectin and lignin (Taiz & Zeiger, 1991). The existence of various carbon-containing polymers in plant cell walls may require the C. cellulovorans cellulosomes to have different subunit compositions and subpopulations to degrade plant cell walls efficiently. Recent transcriptional and proteomic studies show that different carbon sources in the C. cellulovorans culture medium have a significant effect on cellulosomal enzymic composition and activity (Han et al., 2003a
, b
, 2004b
). Previously, Pohlschroder et al. (1994
, 1995)
found that the extracellular cellulase system of Clostridium papyrosolvens consisted of at least seven distinct high-molecular-mass protein complexes (500660 kDa). All the complexes had endoglucanase-active protein subunits, but only two had xylanase-active subunits.
In this study, we fractionated partially purified cellulosomes from C. cellulovorans into subpopulations and determined their subunit compositions and enzymic activities. The data showed the presence of heterogeneous populations of cellulosomes isolated from cells grown on different carbon sources. What are the prerequisites for the formation of functional subpopulations of cellulosomes? A partial answer may involve enzyme induction and heterogeneous populations of cohesins and dockerins.
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METHODS |
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Preparation of cellulosomal (sub)populations.
Cellulosomes were purified from culture supernatants of C. cellulovorans, as described previously (Shoseyov & Doi, 1990). The culture supernatants were obtained during the stationary phase (47 days) by centrifugation, precipitated by 80 % (w/v) ammonium sulfate saturation and dialysed. The extracellular material was then mixed with Avicel, which resulted in binding of the cellulosome complex to Avicel. After incubation for 1 h at 4 °C, the suspension was poured into a column which was washed with three volumes 50 mM Tris/HCl buffer (pH 7·5) to elute the unattached fractions. The bound fraction was eluted from the cellulose column with deionized water and concentrated with Ultrafree Biomax (10 kDa cut-off, Millipore) before being subjected to gel filtration on a HiLoad 26/60 Superdex 200 prep grade column (320 ml, Amersham Biosciences) equilibrated with 50 mM Tris/HCl buffer (pH 7·5). The high-molecular-mass fractions were collected as the cellulosomal fraction by using FPLC (Amersham Biosciences), and concentrated with Ultrafree Biomax (10 kDa cut-off, Millipore). The concentrated solution was applied to a RESOURCE Q (1 ml, Amersham Biosciences) equilibrated with 50 mM Tris/HCl buffer (pH 7·5). After the column was washed with 10 ml of the same buffer, it was eluted with a continuous gradient of NaCl (01 M) by using FPLC. The eluates were concentrated, and their protein content measured by the method of Bradford (1976)
with a protein assay kit (Bio-Rad), using BSA as the standard.
One/two-dimensional (2-D) SDS-PAGE and Western blot analysis.
IEF gels were cast using ReadyPrep Rehydration/Sample Buffer (Bio-Rad) (Anderson & Anderson, 1978; Gorg et al., 2000
). Aliquots containing 50 µg protein were loaded onto each gel. Each sample was subjected to 2-D gel electrophoresis in duplicate to control for gel-to-gel variations. Following IEF, the gels were equilibrated in an equilibration buffer [6 M urea, 2 % (w/v) SDS, 0·375 M Tris/HCl (pH 8·8), 20 % (w/v) glycerol] with 2 % (w/v) dithiothreitol, followed by equilibration buffer with 2·5 % (w/v) iodoacetamide. The second-dimension gels were cast using a linear gradient of 415 % polyacrylamide. The equilibrated tube gels were secured to the 2-D gels using agarose, and SDS-PAGE was carried out as described previously (Gorg et al., 2000
; Laemmli, 1970
). Proteins were fixed in the gels by soaking in a solution containing 40 % (v/v) methanol and 10 % (v/v) acetic acid for approximately 1 h and subsequently visualized by Coomassie blue staining (Genomic Solutions). For Western blot analysis, proteins (1 µg) were separated by SDS-PAGE and blotted onto a PVDF membrane (Immobilon-P, Millipore). The membrane was treated with antibody (diluted 1 : 5000) and stained as described previously (Tamaru & Doi, 1999
).
Zymography.
Zymography for xylanase and carboxymethylcellulase (CMCase) was performed by using 0·1 % (w/v) of each substrate incorporated into the polyacrylamide. After proteins (1 µg) were separated by SDS-PAGE, the gels were renatured and incubated in a renaturation buffer (100 mM succinic acid, 10 mM CaCl2, 1 mM dithiothreitol, pH 6·3) for 1 h at 37 °C with gentle shaking. The clearing zones corresponding to enzyme activities were visualized with 0·3 % (w/v) Congo red (stained for 10 min and destained with 1 M NaCl solution) (Béguin, 1983).
Enzyme assays.
The activities on Avicel (for cellulase), carboxymethylcellulose (CMC) (for endoglucanase), pectin (for pectate lyase) and xylan (for xylanase) were assayed at pH 6·0 and 37 °C by measuring the liberated reducing sugars as D-glucose equivalents by the SomogyiNelson method (Somogyi, 1952; Wood & Bhat, 1988
). Each reaction mixture consisted of 250 µl 1 % substrate solution, 100 µl 250 mM sodium acetate buffer (pH 6·0), and 150 µl enzyme solution. The incubation times were 30 min for endoglucanase, pectate lyase and xylanase activities and 18 h for cellulase activity. One unit of each enzyme activity was defined as the amount of enzyme which released 1 µmol reducing sugar min1 (h1 for Avicelase assays) under the condition indicated.
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RESULTS |
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To determine the effects of carbon sources in the medium on the subpopulations of cellulosomes, C. cellulovorans was cultured with four different carbon substrates. Each sample was subjected to anion-exchange chromatography in duplicate to control for variations of elution profiles (data not shown). The elution profiles of the cellulosomal subpopulations after anion-exchange chromatography were dramatically different depending on the culture conditions used (Fig. 1).
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Subunit composition of cellulosomal subpopulations from cells grown on different carbon sources
To compare the subunit composition of cellulosomal subpopulations, SDS-PAGE and Western blot patterns were determined for cellulosomes obtained from cells grown under different culture conditions (Fig. 2). The SDS-PAGE analysis demonstrated that the cellulosomal subpopulations fractionated by anion-exchange column chromatography significantly differed quantitatively and qualitatively in enzyme subunit composition (Figs 2A
, F, K and P). For example, the cellulosome subpopulation from Avicel-grown cells contained two to five major polypeptides of molecular mass 64200 kDa (Fig. 2A
). The relative amounts of individual polypeptides differed significantly, especially subunit ExgS/EngH/EngY (around 80 kDa) or XynB (around 66 kDa) (Doi et al., 2003
; Han et al., 2004a
). The cellulosome fraction 1 (Fig. 1
, F1) which had the highest Avicelase and CMCase activity contained relatively high amounts of subunits around 80 kDa or 66 kDa (Fig. 2A
). Moreover, cellulosome fraction 4 (Fig. 1
, F4) contained an additional five polypeptides of 3064 kDa which were virtually absent from cellulosome fractions 13 (Fig. 2A
). Interestingly, cellulosome fraction F4 (Fig. 1
, F4) had the highest xylanase activity. The subunit composition of cellulosomal subpopulations from xylan, pectin or mixed-carbon cultures also showed dramatically different enzyme subunit compositions (Figs 2F
, K and P).
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Identification and enzyme activities of subunits of cellulosomal subpopulations
The 2-D SDS-PAGE profiles indicated that the cellulosomal fractions (F1 to F4) had different components (Fig. 3). Based on protein size and pI determined previously by MS (Han et al., 2004b
), 2-D spots were determined to be CbpA, EngE, EngK, ExgS, EngH, EngL and EngB. Fraction 1 contained larger amounts of EngL than the other fractions, while fractions 2 and 3 contained larger amounts of EngE (Fig. 3
, F1F3). The subunits EngK and ExgS were clearly found in fraction 4 (Fig. 3
, F4).
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DISCUSSION |
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The subunit compositions of cellulosomal subpopulations were also different when cells were grown with different carbon substrates. However, the composition of the major subunits CbpA (Shoseyov et al., 1992), EngE (Tamaru & Doi, 1999
) and ExgS (Liu & Doi, 1998
) was relatively constant for all the subpopulations. The cellulosomal fractions which showed higher cellulase activity contained CbpA, EngE/EngK, ExgS/EngH and EngL (Liu & Doi, 1998
; Shoseyov & Doi, 1990
; Shoseyov et al., 1992
; Tamaru & Doi, 1999
, 2000
; Tamaru et al., 2000
). All these enzymes are encoded in the large cbpA gene cluster cbpA-exgS-engH-engK-hbpA-engL (Tamaru et al., 2000
), except for EngE. cbpA is the first gene in the large gene cluster, encoding several enzymic subunits of the cellulosomes, including the endoglucanases (EngH, EngK and EngL) and cellobiohydrolase (ExgS). These enzymes are known to synergistically degrade crystalline cellulose (Murashima et al., 2002b
). A previous genetic study in Clostridium cellulolyticum found that some of the enzymic subunits encoded by genes located in the large cluster are essential for the degradation of crystalline cellulose (Maamar et al., 2004
). Thus, enzymes encoded by the large cbpA gene cluster are expected to play an important role in degrading cellulose in plant cell walls.
In order to determine whether the composition of complex plant cell walls influenced cellulosomal subpopulations, artificially mixed substrates containing cellulose, xylan and pectin were also investigated in this study. The highest xylanase activity was detected with the specific cellulosomal fractions which had XynB (Han et al., 2004a), XynA (Kosugi et al., 2002
) and four unknown xylanase proteins (3545 kDa) (Fig. 4C
, F3 and F4). This subpopulation of cellulosomes may play a major role in degrading the hemicellulose network and allowing access of other subpopulations to cellulose microfibrils in plant cell walls.
These results suggest that a regulatory system in C. cellulovorans controls the ratio of cellulosomal subpopulations that make up the cellulosomal population. How are specific subpopulations of cellulosomes assembled? Several factors may play a role. One factor is the induction of certain enzymes by substrates that determine the relative amounts of the various cellulosomal enzymes present for cellulosome assembly (Han et al., 2003b, 2004b
; Kosugi et al., 2001
; Murashima et al., 2002a
). Another factor may involve the specificity of interaction between dockerins and their cognate cohesins in CbpA (Doi et al., 2003
; Park et al., 2001
). Some cohesins may contain a specific region that is bound by the dockerins of certain enzymes which is absent in other cohesins (Bayer et al., 2004
; Doi et al., 2003
; Park et al., 2001
). Thus the composition of subpopulations may depend on the amount of various enzymes present and the interaction of different categories of cohesins and dockerins. Preliminary data show that different cellulosomal enzymes do interact differently with various cellulosomal cohesins (H. Y. Cho & R. H. Doi, unpublished data). The results presented indicate that cellulosome assembly occurs in a non-random fashion. The characterization of the cellulosomal subpopulations provides new insights into the construction of complex cellulasexylanase cellulosomes that are capable of degrading plant cell walls more effectively.
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ACKNOWLEDGEMENTS |
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Received 4 September 2004;
revised 15 December 2004;
accepted 2 February 2005.
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