The extracellular xylan degradative system in Clostridium cellulolyticum cultivated on xylan: evidence for cell-free cellulosome production - PubMed (original) (raw)

The extracellular xylan degradative system in Clostridium cellulolyticum cultivated on xylan: evidence for cell-free cellulosome production

O Mohand-Oussaid et al. J Bacteriol. 1999 Jul.

Abstract

In this study, we demonstrate that the cellulosome of Clostridium cellulolyticum grown on xylan is not associated with the bacterial cell. Indeed, the large majority of the activity (about 90%) is localized in the cell-free fraction when the bacterium is grown on xylan. Furthermore, about 70% of the detected xylanase activity is associated with cell-free high-molecular-weight complexes containing avicelase activity and the cellulosomal scaffolding protein CipC. The same repartition is observed with carboxymethyl cellulase activity. The cellulose adhesion of xylan-grown cells is sharply reduced in comparison with cellulose-grown cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan- and cellulose-grown cells have different compositions. In both cases, the scaffolding protein CipC is present, but the relative proportions of the other components is dramatically changed depending on the growth substrate. We propose that, depending on the growth substrate, C. cellulolyticum is able to regulate the cell association and cellulose adhesion of cellulosomes and regulate cellulosomal composition.

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Figures

FIG. 1

Growth and production of attached (in pellet fraction after centrifugation) and cell-free xylanase activities by C. cellulolyticum cultivated on insoluble xylan. Each point is the mean ± the standard deviation (SD) (indicated by error bar) of four determinations for two different cultures. Symbols: ■, attached xylanase activities; ●, cell-free xylanase activities; ▴, cell mass.

FIG. 2

FPLC gel filtration chromatography on Superose 12 HR column of the cell-free fraction of C. cellulolyticum grown on xylan. The fractions were analyzed for protein content (■) and xylanase activity (●) under the following conditions: protein load, 0.38 mg; flow rate, 0.5 ml/min; and buffer, 100 mM Tris-HCl (pH 7.0). Arrows indicate the retention times of some standards: a, blue dextran (indicating void volume); b, thyroglobulin (669 kDa); c, apoferritin (443 kDa); d, β-amylase (200 kDa); e, albumin (66 kDa); f, carbonic anhydrase (29 kDa); g, total volume.

FIG. 3

Electrophoretic characterization of components of the cell-free fraction of C. cellulolyticum grown on xylan. The different samples were analyzed by SDS-PAGE under the conditions described in Materials and Methods. Lanes: 1, whole cell-free fraction (9 μg); 2, unattached fraction (9 μg); 3, cellulosomal fraction (fraction eluted from cellulose affinity chromatography and subjected to gel filtration chromatography) (9 μg). Molecular mass in kilodaltons is indicated on the right.

FIG. 4

Xylanase zymograms of total extracellular material (lane A) and pure cellulosome (lane B) derived from xylan-grown cultures of C. cellulolyticum. Samples were mildly denatured prior to SDS-PAGE, and proteins were analyzed for xylanase activity with a xylan-polyacrylamide gel overlay. All samples contained 9 μg of protein.

FIG. 5

Adsorption of the cellulosome to Avicel or insoluble xylan. The capacity of a constant amount (70 μg) of the purified cellulosome to bind to various amounts of crystalline cellulose or insoluble xylan (1 to 70 mg per tube) was determined as described in Materials and Methods. The results are expressed as percentages of bound xylanase activity as a function of various amounts of Avicel or xylan. Each point is the mean ± SD (indicated by error bar) of four determinations for two different assays. Symbols: ■, Avicel; ●, xylan.

FIG. 6

Relative proportions of several components of cellulosomes derived from cellulose-grown (white columns) and xylan-grown cells (black columns). The amount of the 135-kDa fraction has been arbitrarily fixed to 100%. Each set of data is the mean ± SD (indicated by error bars) of separate analyses of four separate SDS-PAGE experiments. An example gel from an SDS-PAGE experiment, performed as described in Materials and Methods, is shown. Lanes: A, cellulosome derived from cellulose-grown cells (9 μg); B, cellulosome derived from xylan-grown cells (9 μg). Molecular mass in kilodaltons is indicated.

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The extracellular xylan degradative system in Clostridium cellulolyticum cultivated on xylan: evidence for cell-free cellulosome production - PubMed (original) (raw)