Pan-Cellulosomics of Mesophilic Clostridia: Variations on a Theme

Abstract

The bacterial cellulosome is an extracellular, multi-enzyme machinery, which efficiently depolymerizes plant biomass by degrading plant cell wall polysaccharides. Several cellulolytic bacteria have evolved various elaborate modular architectures of active cellulosomes. We present here a genome-wide analysis of a dozen mesophilic clostridia species, including both well-studied and yet-undescribed cellulosome-producing bacteria. We first report here, the presence of cellulosomal elements, thus expanding our knowledge regarding the prevalence of the cellulosomal paradigm in nature. We explored the genomic organization of key cellulosome components by comparing the cellulosomal gene clusters in each bacterial species, and the conserved sequence features of the specific cellulosomal modules (cohesins and dockerins), on the background of their phylogenetic relationship. Additionally, we performed comparative analyses of the species-specific repertoire of carbohydrate-degrading enzymes for each of the clostridial species, and classified each cellulosomal enzyme into a specific CAZy family, thus indicating their putative enzymatic activity (e.g., cellulases, hemicellulases, and pectinases). Our work provides, for this large group of bacteria, a broad overview of the blueprints of their multi-component cellulosomal complexes. The high similarity of their scaffoldin clusters and dockerin-based recognition residues suggests a common ancestor, and/or extensive horizontal gene transfer, and potential cross-species recognition. In addition, the sporadic spatial organization of the numerous dockerin-containing genes in several of the genomes, suggests the importance of the cellulosome paradigm in the given bacterial species. The information gained in this work may be utilized directly or developed further by genetically engineering and optimizing designer cellulosome systems for enhanced biotechnological biomass deconstruction and biofuel production.

Catalytic subunit exchanges in the cellulosomes produced by Ruminiclostridium cellulolyticum suggest unexpected dynamics and adaptability of their enzymatic composition

“…In contrast to the species specificity residues at positions 11–12 and 47–48, which vary among the cellulosome‐producing bacteria to fit with the corresponding cohesin interface specificity , Leu at positions 22 and 58 is highly conserved among the dockerin‐bearing proteins of other bacterial species since the consensus sequences of the dockerins from R. papyrosolvens, R. josui, C. sufflavum , R. sp. BNL1100 , C. cellobioparum , R. termitidis , C. cellulovorans , and H. thermocellum harbor the same conserved Leu residue in the two segments . It is interesting to notice that Met residue is mainly the second residue at the critical position of the dockerin consensus in all these species, suggesting our data on R. cellulolyticum may apply to other bacterial species, and possibly be the basis of a general mechanism governing cellulosome dynamics.…”

“…Most of the CAZymes secreted by anaerobic Gram‐positive bacteria such as Ruminiclostridium cellulolyticum (formerly known as Clostridium cellulolyticum ), Hungateiclostridium thermocellum and Hungateiclostridium clariflavum (formerly known as Clostridium thermocellum and Clostridium clariflavum) , Clostridium cellulovorans , Ruminococcus albus , or Ruminococcus flavefaciens are gathered into large multienzyme complexes termed ‘cellulosomes’ . Most simple cellulosomes share similar architectures based on a central noncatalytic scaffolding subunit, called the scaffoldin, harboring a family 3a CBM, at least one ‘X’ module of unknown function, and 3–15 repeated type I cohesin modules . Cohesins allow the anchoring of cellulosomal CAZymes through their specific dockerin module.…”

Minimalistic Cellulosome of the Butanologenic Bacterium Clostridium saccharoperbutylacetonicum

“…The C. clariflavum cellulosomal system has 13 scaffoldins and 75 dockerin-containing proteins (mainly enzymes) (3), whereas B. cellulosolvens has 212 dockerin-bearing proteins and 32 scaffoldins (4). On the other hand, much simpler and smaller cellulosome systems can be found in the mesophilic bacteria Clostridium bornimense and Clostridium acetobutylicum, which have 5 dockerins and 2 scaffoldins in the former and 10 dockerins and 2 scaffoldins in the latter (5). In addition to the major scaffoldin, which bears multiple cohesins and one or two CBMs, the latter mesophilic cellulosome-producing bacteria each contains a gene termed orfX encoding a single cohesin.…”

“…This progress contributes to the discovery of new cellulosomal species, thus expanding our knowledge of the cellulosomal paradigm in nature. In a recent review, a genome-wide analysis of a dozen mesophilic clostridial species revealed the presence of heretofore undescribed cellulosome systems in three mesophilic clostridia (5). In particular, the cellulosomal system of Clostridium saccharoperbutylacetonicum was of special interest to us.…”

“…Like C. beijerinckii, C. saccharoperbutylacetonicum possesses an aldehyde dehydrogenase (ald) gene, whereas C. acetobutylicum has an alcohol/aldehyde dehydrogenase-encoding gene (adhE) together with acetoacetate decarboxylase (adc), which are located on separate operons (12). The recently reported cellulosomal elements of C. saccharoperbutylacetonicum (5) are located in the cellulosome gene cluster of the chromosome. Theoretically, its cellulosome is among the smallest known in nature, comprising a "minimalistic" cellulosome with only eight dockerin-bearing enzymes (five of which are located in the major cellulosome gene cluster (Fig.…”