Fiber-degrading systems of different strains of the genus Fibrobacter

Abstract

The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the F(E) strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.

FIG. 1.

Phylogenetic tree for 16S rRNA gene sequences from Fibrobacter strains. Phylogenetic relationships were based on ClustalW alignments, optimized manually, and analyzed with the Phylip programs. A total of 1,000 bootstrap replicates were performed, and bootstrap values greater than 75 are indicated above each branch. rRNA gene sequences are shown with their species and EMBL or GenBank accession numbers.

FIG. 2.

CMCase (A) and xylanase (B) zymogram analyses of bacterial (P) and extracellular (S) proteins from several strains of the genus Fibrobacter. Lanes M, broad-range molecular mass standards (in kilodaltons) (Bio-Rad). Protein migration was performed with a 10% acrylamide gel.

FIG. 3.

CMCase (A) and xylanase (B) activity distribution between cells (□) and ECCF (▪) for several strains of Fibrobacter grown on filter paper cellulose. The CMCase activities varied between 3.6 and 10.7 nkat in cells in the initial culture and from 7.0 to 20.0 nkat in ECCF. The xylanase activities varied between 8.3 and 42.7 nkat of total culture volume in cells and from 17.3 to 175.0 nkat in ECCF.