Development of a RT-qPCR method for the quantification of Fibrobacter succinogenes S85 glycoside hydrolase transcripts in the rumen content of gnotobiotic and conventional sheep.

An improved RNA isolation method based on the acid guanidinium-phenol-chloroform (AGPC) procedure using saline precipitation but no column purification was evaluated for quantifying microbial gene expression using reverse transcription quantitative PCR (RT-qPCR) in rumen contents. The method provided good RNA integrity and quantity extracts. The transcript levels of eight glycoside hydrolase (GH) genes of the major rumen fibrolytic bacterium Fibrobacter succinogenes were quantified in the complex microbiota of a conventional sheep and in a gnotobiotic lamb harboring a microflora containing F. succinogenes S85 as the sole cellulolytic microorganism. This study validated the improved RNA isolation method, RT-qPCR conditions to quantify GH transcripts using either the F. succinogenes S85 tuf gene or the 16S rRNA-encoding gene (rrs) as the reference gene, and demonstrated the need to work with good quality RNAs. Transcripts from all the selected genes cel3, endA(FS), celF and endB endoglucanase genes, cedA cellodextrinase gene, mlg lichenase gene, and xynC and xynD xylanase genes of F. succinogenes S85 were detected and quantified at varying levels in the rumen content of the two animal models. This study opens new perspectives in studying microbial gene expression in the rumen of both conventional and gnotobiotic sheep.

Fiber-degrading systems of different strains of the genus Fibrobacter.

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.