Cellulosomics of the cellulolytic thermophile Clostridium clariflavum

Abstract

BackgroundClostridium clariflavum is an anaerobic, thermophilic, Gram-positive bacterium, capable of growth on crystalline cellulose as a single carbon source. The genome of C. clariflavum has been sequenced to completion, and numerous cellulosomal genes were identified, including putative scaffoldin and enzyme subunits.ResultsBioinformatic analysis of the C. clariflavum genome revealed 49 cohesin modules distributed on 13 different scaffoldins and 79 dockerin-containing proteins, suggesting an abundance of putative cellulosome assemblies. The 13-scaffoldin system of C. clariflavum is highly reminiscent of the proposed cellulosome system of Acetivibrio cellulolyticus. Analysis of the C. clariflavum type I dockerin sequences indicated a very high level of conservation, wherein the putative recognition residues are remarkably similar to those of A. cellulolyticus. The numerous interactions among the cellulosomal components were elucidated using a standardized affinity ELISA-based fusion-protein system. The results revealed a rather simplistic recognition pattern of cohesin-dockerin interaction, whereby the type I and type II cohesins generally recognized the dockerins of the same type. The anticipated exception to this rule was the type I dockerin of the ScaB adaptor scaffoldin which bound selectively to the type I cohesins of ScaC and ScaJ.ConclusionsThe findings reveal an intricate picture of predicted cellulosome assemblies in C. clariflavum. The network of cohesin-dockerin pairs provides a thermophilic alternative to those of C. thermocellum and a basis for subsequent utilization of the C. clariflavum cellulosomal system for biotechnological application.

The cohesin module is a major determinant of cellulosome mechanical stability

“…In fact, even those microorganisms that produce cell surface-anchored cellulosomes were found to produce free cellulosomes (14,(28)(29)(30).…”

Nanoscale Engineering of Designer Cellulosomes

“…[88,109] CipA binds directly to an anchoring scaffoldin (SdbA), but ScaA from A. cellulolyticus binds to an intermediate adaptor scaffoldin (ScaB) made of four type-II cohesins which binds via a type-I dockerin to three specialized cohesins of the anchoring scaffoldin (ScaC), [107,108] Intriguingly, the cellulosome system of C. clariflavum closely emulates the A. cellulolyticus system, wherein ScaA harbours eight type-I cohesins, ScaB bears five type-II cohesins and ScaC presents four specialized cohesins. [86] Some primary scaffoldins (C. cellulovorans, C. cellulolyticum, C. josui and C. acetobutylum) may not be attached to the bacterial cell wall and are free in the extra-cellular environment. [20,80] The primary scaffoldin of A. cellulolyticus also differs from the CipA paradigm by an extra enzyme.…”

“…[71,72] It is a multi-enzyme complex which was later found in other cellulolytic bacteria, [18,78] including C. cellulovorans, [79,80] C. cellulolyticus, [81] C. josui, [82] C. acetobutylicum, [83] C. papyrosolvens, [84] C. clariflavum, [85,86] Acetivibrio cellulolyticus, [87] Bacteroides cellulosolvens, [88] Ruminococcus albus, [89] and R. flavefaciens. [90,91] Cellulosomes are multi-enzyme complexes whose building block is the intermodular cohesin-dockerin pair ( Figure 4).…”

Understanding the structure and function of bacterial expansins: a prerequisite towards practical applications for the bioenergy and agricultural industries

“…Nevertheless, the biological function for these multi‐modular proteins is still unknown, given that the cellulase activity of C. michiganensis protein is unaffected by the removal of the expansin domain, supporting the notion that expansins and cellulases are non‐synergistic. Recently, Chen et al ., ; analysed two expansins from the anaerobe C. clariflavum naturally encoded as fusion proteins with N‐terminal dockerin domains for possible interactions into the cellulosome (Artzi et al ., , ; Chen et al ., ). However, for a reason not specified by the authors the native dockerin domain of both expansins (Clocl_1298 and Cocl_1862) was replaced with a dockerin domain from Bacteroides cellulosolvens allowing assemble into designer cellulosome, showing an increase of release of reducing sugars detected at low cellulase (cellulosome) concentration and short reaction time (Chen et al ., ).…”