Whole Proteome Analyses on Ruminiclostridium cellulolyticum Show a Modulation of the Cellulolysis Machinery in Response to Cellulosic Materials with Subtle Differences in Chemical and Structural Properties

Abstract

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the “xyl-doc” cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics.

Comparison of enzymatic activities and proteomic profiles of Butyrivibrio fibrisolvens grown on different carbon sources

“…The proteins involved in glycolysis, protein synthesis and carbohydrate metabolism were identified as the predominant pathways also by Hart et al [58], who moreover also described the protein family containing the elongation factor Tu as being the most highly abundant in the bovine rumen ecosystem. Upregulated proteins of central carbon catabolic pathways have also been described by Ruminiclostridium cellulolyticum cultivated on different sources of fibre [59]. We find very interesting that proteomic studies performed on rumen fluid of different animals [21, 58] brought the same results as our work studying one pure bacterial strain.…”

“…Such a result was unexpected but not exceptional. Higher levels of the enzymes from the central carbon catabolic pathways were obtained in a bacterial monoculture of Ruminiclostridium cellulolyticum [59], as well as in the whole rumen microbial community [21, 58]. This is the first proteomic study of B. fibrisolvens 3071 indicating that this strain can play the important role for the central rumen catabolic metabolism.…”

Unraveling essential cellulosomal components of the (Pseudo)Bacteroides cellulosolvens reveals an extensive reservoir of novel catalytic enzymes

“…Intriguingly, in the lower-molecular-weight fractions of both substrates, the molar amounts of the enzymes were about twice those of the higher molecular-weight fractions, meaning that there were twice the available enzymes than vacant cohesins, indicating large numbers of excess enzymes that will not associate with cohesins at a specific point. In other cellulosomal models, the enzymes are usually found in significant excess, depending on the substrate used [30, 38, 41, 42].…”

The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action

“…62 annotated CAZymes contain a type I-dockerin module and are integrated into the cellulosomes through interactions with the scaffolding protein CipC. Many cellulosomal enzymes display activity against cellulose, hemicelluloses (arabinoxylans and xyloglucans), and pectins, and it was shown, by transcriptomic and proteomic analyses, that the composition of R. cellulolyticum cellulosomes is modulated according to the growth substrates [13, 19, 38, 39]. Genes encoding cellulases, especially those gathered in the cip - cel cluster, are regulated by carbon catabolite repression, while the expression of most of the genes encoding other CAZymes and accessory enzymes is activated via two-component regulation systems as it was shown for the xyl - doc gene cluster [17–19, 39].…”