Cellulosome gene cluster analysis for gauging the diversity of the ruminal cellulolytic bacteriumRuminococcus flavefaciens

Abstract

Ruminococcus flavefaciens is a vital cellulosome-producing fibrolytic rumen bacterium. The arrangement of the cellulosomal scaffoldin gene cluster (scaC-scaA-scaB-cttA-scaE) is conserved in two R. flavefaciens strains (17 and FD-1). Sequence analysis revealed a high mosaic conservation of the intergenic regions in the two strains that contrasted sharply with the divergence of the structural sca gene sequences. Based on the conserved intergenic regions, we designed PCR primers in order to examine the sca gene cluster in additional R. flavefaciens strains (C94, B34b, C1a and JM1). Using these conserved and/or degenerate primers, the scaC, scaA and scaB genes were amplified in all six strains, while the entire sca gene cluster and the proximal genes cttA and scaE were successfully amplified in four of the strains (17, FD-1, C94 and JM1). The sequencing of scaA and scaC genes in all the strains yielded additional insight into the variability of the structural genes with regard to the number and type of cohesin modules contained in a conserved molecular skeleton. Moreover, the scaC gene, being short and variable, appears to be a promising functional phylotyping target for metagenomic population studies of R. flavefaciens in the rumen as a function of the individual host animal.

Atypical Cohesin-Dockerin Complex Responsible for Cell Surface Attachment of Cellulosomal Components

“…It thus seems that their sequence features that would account for the observed specific coupling and promiscuous behavior may be a function of simple evolutionary events. Nevertheless, there is evidence that the various subtypes may represent distinct lineages whose descendants are maintained in the rumen of subsequent generations (20). Indeed, such distinct lineages related to R. flavefaciens strains 17 and FD-1 have been identified in the rumens of contemporary animals, and the possibility would exist for similar cross-strain interactions with some strains exhibiting a propensity for sharing their components.…”

“…Both strains share marked similarities in their properties. They produce elaborate cellulosomes of similar overall architecture and exhibit high sequence homology within the sca gene cluster (19,20). Surprisingly, affinity-based assays showed that CohE from R. flavefaciens strain 17 recognizes dockerins of its own strain exclusively (supplemental Fig.…”

“…This rumen bacterium was discovered to bear one of the most elaborate complexes in the cellulosomal world; it is much more elaborate than those reported previously for cellulolytic Clostridium species (17,18). Gene clustering analysis of several R. flavefaciens strains revealed that the cellulosome system is generally composed of four major scaffoldin subunits, ScaA, ScaB, ScaC, and ScaE, and of a cellulose-binding protein, CttA (19,20). The cohesin, dockerin, and X-modules of these cellulosomal components were found to be divergent in sequence from previously known type I and type II cellulosomal modules, and the Coh-Doc interactions were therefore collectively designated type III on the respective phylogenetic trees (21)(22)(23)(24).…”

Synergy, Structure and Conformational Flexibility of Hybrid Cellulosomes Displaying Various Inter-cohesins Linkers

“…[1][2][3] Nevertheless, the sequencing of the genome of cellulosome-producing bacteria revealed that their organisation varies significantly; for instance, some cellulosomes contain several interacting scaffoldins and their assembly requires several specific cohesin/dockerin docking systems. [4][5][6] At the opposite end of the scale are the simplest cellulosomes, such as those produced by some mesophilic Clostridia that are characterized by a unique scaffoldin and only one type of cohesin/dockerin docking system. 7 A typical example of "simple" cellulosome is constituted by the cellulolytic complexes secreted by C. cellulolyticum.…”

Structural characterization of a novel autonomous cohesin fromRuminococcus flavefaciens

“…The presence of this gene cluster has been documented in at least five different strains of R. flavefaciens (Jindou et al, 2008). Additionally, one peculiar and very interesting ORF containing only a signal peptide and an autonomous cohesin module (i.e.…”