Cellulosome gene cluster analysis for gauging the diversity of the ruminal cellulolytic bacterium Ruminococcus 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.