Chantal Tardif

The cellulosomes from Clostridium cellulolyticum

Cellulosomes produced by Clostridium cellulolyticum grown on cellulose were purified and separated using anion‐exchange chromatography. SDS/PAGE analysis of six fractions showed variations in their cellulosomal protein composition. Hydrolytic activity on carboxymethyl cellulose, xylan, crystalline cellulose and hatched straw differed from one fraction to another. Fraction F1 showed a high level of activity on xylan, whereas fractions F5 and F6 were most active on crystalline cellulose and carboxymethyl cellulose, respectively. Several cellulosomal components specific to fractions F1, F5 and F6 were investigated using MS analysis. Several hemicellulases were identified, including three xylanases in F1, and several cellulases belonging to glycoside hydrolase families 9 and 5 and, a cystein protease inhibitor were identified in F5 and F6. Synergies were observed when two or three fractions were combined. A mixture containing fractions F1, F3 and F6 showed the most divergent cellulosomal composition, the most synergistic effects and the highest level of activity on straw (the most heterogeneous substrate tested). These findings show that on complex substrates such as straw, synergies occur between differently composed cellulosomes and the degradation efficiency of the cellulosomes is correlated with their enzyme diversity.

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

Background: Family-9 glycoside hydrolases displaying various organizations are plethoric in cellulosome-producing bacteria. Results: The 13 family-9 enzymes synthesized by Clostridium cellulolyticum exhibit different substrate specificities, activities, and relationships with key cellulosomal cellulases in artificial cellulosomes. Conclusion: This variety is required for efficient degradation of crystalline cellulose. Significance: This first global picture provides insights on the multiplicity and diversity of family-9 enzymes in bacterial cellulosomes. The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.

A comparison of the effects of inhibitors of carbonic anhydrase on osteoclastic bone resorption and purified carbonic anhydrase isozyme II

SummaryWe have assessed the effects of five sulfonamides with widely varying inhibitory activity for carbonic anhydrase (CA) in the bone slice assay using disaggregated rat osteoclasts (OCs), and in the Maren assay where the catalytic activity of purified CA isozyme II (CA II) was measured. There was an excellent correlation between the relative potencies of the compounds in the two assays: ethoxzolamide (ETH)>acetazolamide (AZ)>M&B 21659>M&B 9811>M&B 7973. In the bone slice assay, ETH and AZ were found to be the most potent inhibitors of OC bone resorption, with IC50 values of 0.09 and 0.8 μM, respectively (from plan surface area of bone resorbed). These results support previous observations showing that OCs use CA II to generate protons during bone resorption and that CA II activity is essential for OCs to be able to resorb bone.

A Two-Component System (XydS/R) Controls the Expression of Genes Encoding CBM6-Containing Proteins in Response to Straw in Clostridium cellulolyticum

The composition of the cellulosomes (multi enzymatic complexes involved in the degradation of plant cell wall polysaccharides) produced by Clostridium cellulolyticum differs according to the growth substrate. In particular, the expression of a cluster of 14 hemicellulase-encoding genes (called xyl-doc) seems to be induced by the presence of straw and not of cellulose. Genes encoding a putative two-component regulation system (XydS/R) were found upstream of xyl-doc. First evidence for the involvement of the response regulator, XydR, part of this two-component system, in the expression of xyl-doc genes was given by the analysis of the cellulosomes produced by a regulator overproducing strain when grown on cellulose. Nano-LC MS/MS analysis allowed the detection of the products of all xyl-doc genes and of the product of the gene at locus Ccel_1656 predicted to bear a carbohydrate binding domain targeting hemicellulose. RT-PCR experiments further demonstrated that the regulation occurs at the transcriptional level and that all xyl-doc genes are transcriptionally linked. mRNA quantification in a regulator knock-out strain and in its complemented derivative confirmed the involvement of the regulator in the expression of xyl-doc genes and of the gene at locus Ccel_1656 in response to straw. Electrophoretic mobility shift assays using the purified regulator further demonstrated that the regulator binds to DNA regions located upstream of the first gene of the xyl-doc gene cluster and upstream of the gene at locus Ccel_1656.

Combining free and aggregated cellulolytic systems in the cellulosome-producing bacterium Ruminiclostridium cellulolyticum

BackgroundRuminiclostridium cellulolyticum and Lachnoclostridium phytofermentans (formerly known as Clostridium cellulolyticum and Clostridium phytofermentans, respectively) are anaerobic bacteria that developed different strategies to depolymerize the cellulose and the related plant cell wall polysaccharides. Thus, R. cellulolyticum produces large extracellular multi-enzyme complexes termed cellulosomes, while L. phytofermentans secretes in the environment some cellulose-degrading enzymes as free enzymes. In the present study, the major cellulase from L. phytofermentans was introduced as a free enzyme or as a cellulosomal component in R. cellulolyticum to improve its cellulolytic capacities.ResultsThe gene at locus Cphy_3367 encoding the major cellulase Cel9A from L. phytofermentans and an engineered gene coding for a modified enzyme harboring a R. cellulolyticum C-terminal dockerin were cloned in an expression vector. After electrotransformation of R. cellulolyticum, both forms of Cel9A were found to be secreted by the corresponding recombinant strains. On minimal medium containing microcrystalline cellulose as the sole source of carbon, the strain secreting the free Cel9A started to grow sooner and consumed cellulose faster than the strain producing the cellulosomal form of Cel9A, or the control strain carrying an empty expression vector. All strains reached the same final cell density but the strain producing the cellulosomal form of Cel9A was unable to completely consume the available cellulose even after an extended cultivation time, conversely to the two other strains. Analyses of their cellulosomes showed that the engineered form of Cel9A bearing a dockerin was successfully incorporated in the complexes, but its integration induced an important release of regular cellulosomal components such as the major cellulase Cel48F, which severely impaired the activity of the complexes on cellulose. In contrast, the cellulosomes synthesized by the control and the free Cel9A-secreting strains displayed similar composition and activity. Finally, the most cellulolytic strain secreting free Cel9A, was also characterized by an early production of lactate, acetate and ethanol as compared to the control strain.ConclusionsOur study shows that the cellulolytic capacity of R. cellulolyticum can be augmented by supplementing the cellulosomes with a free cellulase originating from L. phytofermentans, whereas integration of the heterologous enzyme in the cellulosomes is rather unfavorable.

Are Cellulosome Scaffolding Protein CipC and CBM3-Containing Protein HycP, Involved in Adherence of Clostridium cellulolyticum to Cellulose?

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.

Regulation of cel Genes of C. cellulolyticum: Identification of GlyR2, a Transcriptional Regulator Regulating cel5D Gene Expression

Transcription and expression regulation of some individual cel genes (cel5A, cel5I, cel5D and cel44O) of Clostridium cellulolyticum were investigated. Unlike the cip-cel operon, these genes are transcribed as monocistronic units of transcription, except cel5D. The location of the transcription initiation sites was determined using RT-PCR and the mRNA 5′-end extremities were detected using primer extension experiments. Similarly to the cip-cel operon, cel5A and cel5I expressions are regulated by a carbon catabolite repression mechanism, whereas cel44O and cel5D expressions do not seem to be submitted to this regulation. The role of the putative transcriptional regulator GlyR2 in the regulation of cel5D expression was investigated. The recombinant protein GlyR2 was produced and was shown to bind in vitro to the cel5D and glyR2 promoter regions, suggesting that besides regulating its own expression, GlyR2 may regulate cel5D expression. To test this hypothesis in vivo, an insertional glyR2 mutant was generated and the effect of this disruption on cel5D expression was evaluated. Levels of cel5D mRNAs in the mutant were 16 fold lower than that of the wild-type strain suggesting that GlyR2 acts as an activator of cel5D expression.

Restoration of cellulase activity in the inactive cellulosomal protein Cel9V from Ruminiclostridium cellulolyticum

Ruminiclostridium cellulolyticum produces extracellular cellulosomes which contain interalia numerous family‐9 glycoside hydrolases, including the inactive Cel9V. The latter shares the same organization and 79% sequence identity with the active cellulase Cel9E. Nevertheless, two aromatic residues and a four‐residue stretch putatively critical for the activity are missing in Cel9V. Introduction of one Trytophan and the four‐residue stretch restored some weak activity in Cel9V, whereas the replacement of its catalytic domain by that of Cel9E generated a fully active cellulase. Altogether our data indicate that a series of mutations in the catalytic domain of Cel9V lead to an essentially inactive cellulase.