Abstract
The rumen bacterium Cellulosilyticum ruminicola H1 efficiently hydrolyzes cellulose. To gain insights into the regulatory mechanisms of cellulase synthesis, comparative transcriptome analysis was conducted for cultures grown on 2% filter paper, 0.5 and 0.05% cellobiose, and 0.5% birchwood xylan. It was found that cellulose induced a majority of (hemi)cellulases, including 33 cellulases and a cellulosomal scaffoldin (1.3- to 22.7-fold); seven endoxylanases, two mannanases, and two pectatelyases (2- to 16-fold); and pyruvate formate-lyase (PFL, 1.5- to 7-fold). Noticeably, 3- and 2.5-fold increased transcription of a cellobiohydrolase and the cellulosomal scaffoldin precursor were detected in 0.05% than in 0.5% cellobiose. Consistently, 9- and 4-fold higher specific cellobiohydrolase activities were detected in the filter paper and 0.05% cellobiose culture. SDS- and native-PAGE zymograms of cellulose-enriched proteins from the filter paper culture displayed cellulase activities, and cellulolytic “complexes” were enriched from the filter paper- and 0.05% cellobiose-cultures, but not from the 0.5% cellobiose culture. LC-MS/MS identified the cellulosomal scaffoldin precursor in the “complexes” in addition to cellulase, hemicellulase, and PFL proteins. The addition of 0.5% cellobiose, but not 0.05% cellobiose remarkably inhibited strain H1 to degrade filter paper. Therefore, this work reveals a cellobiose-dose related regulatory mechanism of cellulase synthesis by lower for induction and higher for repression, which has extended our understanding of the regulation of microbial cellulase synthesis.
Highlights
Numerous studies on microbial cellulase components and hydrolysis mechanisms have revealed 14 glycoside hydrolase (GH) clans of related 135 GH families on the basis of protein sequence similarity and folding homologs
To obtain an overview of the gene categories induced by cellulose and its hydrolytic product, cellobiose in the rumen cellulolytic bacterium H1, comparative transcriptome analysis was conducted for the cultures grown on 2% filter paper, 0.05 and 0.5% cellobiose, and 0.5% xylan
Through a combination of differential transcriptome and zymogram assays, this study determined that the rumen cellulolytic bacterium C. ruminicola H1 employs a mechanism of substrate cellulose, trace contents of its hydrolytic product, cellobiose, induced the synthesis of cellulases, in particular cellobiohydrolase, and a presumable cellulosome, an efficient fibrolytic protein complex
Summary
Numerous studies on microbial cellulase components and hydrolysis mechanisms have revealed 14 glycoside hydrolase (GH) clans of related 135 GH families on the basis of protein sequence similarity and folding homologs. Unlike the free system cellulases, the cellulosome comprises a characteristic scaffoldin protein, which contains multiple cohesins and a variety of cellulases and hemicellulases that are frequently embedded in a C-terminal dockerin domain to anchor to the cognate cohesins. Some cellulosome producers, such as C. thermocellum, produce free cellulases (Schwarz, 2001; Schwarz et al, 2004)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have