Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1

Abstract

Background: Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels.

Methodology/principal findings: The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation.

Conclusions/significance: The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.

Figure 1. Abundance of glycoside hydrolase modules and carbohydrate-binding modules detected in R. flavefaciens FD-1.

A. The 101 GH family modules predicted in R. flavefaciens FD-1. B. The 68 detected CBMs, according to family type.

Figure 2. Comparison of chromosomal regions encoding xylose isomerase and associated genes involved in utilization of xylo-oligosaccharides between R. flavefaciens strains FD-1 and 17.

Figure 3. Modular structures of multi-modular enzymes involved in xylan breakdown from R. flavefaciens FD-1 and 17.

Catalytic modules are indicated by glycoside hydrolase enzyme family (GH10, GH11, CE3 etc). Families of carbohydrate binding modules (CBM22 etc) and dockerin modules (Doc) are also indicated. All complete ORFs carry a predicted signal peptide at the N terminus (not shown). Incomplete ORFs are indicated by an asterisk.

Figure 4. Instances of unusual NQ-rich linker regions in enzymes from R. flavefaciens FD-1 and 17.

The linker sequences are shown in full, while the catalytic modules and binding modules that they connect are indicated by appropriate abbreviations (GH10 etc).

Figure 5. Proportions of cellulases, enzymes cleaving non-cellulosic plant cell wall polysaccharides (including carbohydrate esterases) and other predicted ORFs among the total cellulosome-associated genes and the up-regulated cellulosome-associated ORFs.

Up-regulated genes are those dockerin-containing ORFs that have fold changes of 2-fold or greater when grown on cellulose. For the purposes of this work, the putative cellulases include any ORF containing glycoside hydrolase (GH) families 5, 8, 9, and 48. The enzymes cleaving non-cellulosic plant cell wall polysaccharides (mainly hemicellulases) include ORFs containing GH families 10, 11, 16, 26, 43, 44, 53, 74 105, some subfamilies of GH5, all families of polysaccharide lysases (PL) and carbohydrate esterases (CE). ORFs that did not have any significant hits in the database are grouped as “unknown,” and ORFs that do not fall into any of the previous categories are grouped as “other.” Putative β-glucosidases and β-xylosidases were ORFs containing sequences consistent with GH family 3.

Figure 6. Comparison of microarray data to qRT-PCR data in terms of relative expression (fold change) of five selected ORFs.

Each number on the x-axis corresponds to the ORF designation assigned by TIGR's annotation engine.