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Abstract
Clostridium cellulolyticum, a mesophilic anaerobic bacterium, produces highly active enzymatic complexes called cellulosomes. This strain was already shown to bind to cellulose, however the molecular mechanism(s) involved is not known. In this context we focused on the gene named hycP, encoding a 250-kDa protein of unknown function, containing a Family-3 Carbohydrate Binding Module (CBM3) along with 23 hyaline repeat modules (HYR modules). In the microbial kingdom the gene hycP is only found in C. cellulolyticum and the very close strain recently sequenced Clostridium sp BNL1100. Its presence in C. cellulolyticum guided us to analyze its function and its putative role in adhesion of the cells to cellulose. The CBM3 of HycP was shown to bind to crystalline cellulose and was assigned to the CBM3b subfamily. No hydrolytic activity on cellulose was found with a mini-protein displaying representative domains of HycP. A C. cellulolyticum inactivated hycP mutant strain was constructed, and we found that HycP is neither involved in binding of the cells to cellulose nor that the protein has an obvious role in cell growth on cellulose. We also characterized the role of the cellulosome scaffolding protein CipC in adhesion of C. cellulolyticum to cellulose, since cellulosome scaffolding protein has been proposed to mediate binding of other cellulolytic bacteria to cellulose. A second mutant was constructed, where cipC was inactivated. We unexpectedly found that CipC is only partly involved in binding of C. cellulolyticum to cellulose. Other mechanisms for cellulose adhesion may therefore exist in C. cellulolyticum. In addition, no cellulosomal protuberances were observed at the cellular surface of C. cellulolyticum, what is in contrast to reports from several other cellulosomes producing strains. These findings may suggest that C. cellulolyticum has no dedicated molecular mechanism to aggregate the cellulosomes at the cellular surface.
Highlights
Cellulose, a major polysaccharide on earth, is a linear polymer of glucose organized in a regular crystalline arrangement and forming insoluble linear microfibrils
We analyzed the properties of the new HycP CBM3 and compared them to those of the well known corresponding to bacterial microcrystalline cellulose (BMCC) Avicel Sigmacell PAS-cellulose rCBM3a (CipC) CBM3a (Table 2)
We showed that HycP contains a functional CBM3 that we classified within the CBM3b subtype according to its amino-acids sequence features
Summary
A major polysaccharide on earth, is a linear polymer of glucose organized in a regular crystalline arrangement and forming insoluble linear microfibrils. Several cellulolytic microorganisms carry out efficient deconstruction of crystalline cellulose and other polysaccharides of the plant cell wall. Clostridium cellulolyticum, a mesophilic anaerobic bacterium, produces highly active extracellular enzymatic complexes called cellulosomes together with free enzymes. In this cellulolytic strain, cellulosomes are made up of a non enzymatic scaffolding protein called CipC, composed of a CBM3, two hydrophilic modules (62) whose function remains unknown and eight type I cohesins [3]. Cellulosomal or free plant cell wall degrading enzymes display catalytic modules classified into three distinct groups in the CAZY database: the glycoside hydrolase, the pectate lyase, and the carbohydrate esterase group (http://www.cazy.org/ [5])
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