Date Published: July 29, 2012
Author(s): Hironobu Morisaka, Kazuma Matsui, Yohei Tatsukami, Kouichi Kuroda, Hideo Miyake, Yutaka Tamaru, Mitsuyoshi Ueda.
We performed a focused proteome analysis of cellulosomal proteins predicted by a genome analysis of Clostridium cellulovorans [Tamaru, Y., et al.. 2010. J. Bacteriol. 192:901–902]. Our system employed a long monolithic column (300 cm), which provides better performance and higher resolution than conventional systems. Twenty-three cellulosomal proteins were, without purification, identified by direct analysis of the culture medium. Proteome analysis of the C. cellulovorans cellulosome after culture in various carbon sources demonstrated the production of carbon source-adapted cellulosome components.
The development of white biotechnology requires degradation of biomass using biocatalysts. The cellulosome, which is produced by many cellulolytic gram-positive anaerobic bacteria such as Clostridium, efficiently degrades plant cell wall polysaccharides. However, the molecular mechanism of cellulosome formation has not been characterized. We sequenced the entire genome of Clostridium cellulovorans (Tamaru et al. [2010a]) and identified all genes, including those that encode proteins of known and unknown functions, related to cellulosome composition (Tamaru et al. [2010a]). Genome analysis of C. cellulovorans indicated the presence of 57 cellulosomal enzymes-encoding genes including 4 scaffold proteins-encoding genes and 53 cellulosomal enzymes-encoding genes with dockerin domains (Doi and Tamaru ). The major scaffold protein, CbpA, is comprised of 9 cohesin domains that bind to various cellulosomal enzymes via cohesin-dockerin interactions. Most cellulosomal enzymes are glycoside hydrolases, but they possess an interesting property. The cellulosome consists not only of glycoside hydrolases but of other proteins such as proteases, protease inhibitors, and unknown proteins. This characteristic may be important in the degradation of various resources. However, we have only general subcellular proteinous information regarding the proteins that actively degrade biomass, although genome analysis has provided many interesting insights into the characteristics of C. cellulovorans. The next step was to directly analyze cellulosomal proteins in the culture media.
The quality of the mass spectrum is very important in proteome analysis. The number of identified proteins is dependent on the number of detected peptides, which is dependent on the efficiency of the separation prior to ionization. Thus, the separation performance of the liquid chromatography method directly influences the quality of the proteome analysis. De Godoy et al () reported that complete pre-separation contributed quality of proteome analysis such as the number of identified proteins. We examined the validity of a proteome analysis system using a long monolithic column (300 cm) which has higher resolution versus a conventional packed column (15 cm). The long monolithic column showed good separation and decreased ionization suppression. In the proteome analysis with the conventional system, protein purification was required because the number of detectable peptides was reduced by ionization suppression. With the new system, proteome analysis of cellulosomal proteins could be performed directly from crude extracted samples thanks to the ultra-performance separation by the long monolithic column. By this benefit, the analysis of non-cellulosomal enzyme of C. cellulovorans became possible simultaneously. The ratio of cellulosomal / non-cellulosomal genes encoding glycosyl hydrolases and polysaccharide lyases of C. cellulovorans was 0.48 (29/61) while that of C. thermocellum which is other cellulosome-producing clostridia was 3.31(53/16) (Tamaru et al. [2010b]). From proteome analysis of supernatants, total 59 proteins annotated glycosyl hydrolases and polysaccharide lyases were identified and its ratio of cellulosomal / non-cellulosomal was 0.69 (24/35). It was confirmed by proteome analysis that C. cellulovorans has much non-cellulosomal enzyme as compared with other clostridia. The degradation of various resources by C. cellulovorans is highly dependent on not only cellulosomal enzymes but also these secretory proteins.
The authors declare that they have no competing interests.