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 FindingsThe 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/SignificanceThe 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.
Highlights
Ruminococci are cellulolytic Gram-positive cocci in the order ‘Clostridiales’, which inhabit the rumen community
In combination with suppressive subtractive hybridization (SSH) sequences obtained from our previous comparative studies of R. flavefaciens FD-1 and JM1 [30], 430,226 sequence reads from GS FLX pyrosequencing and 28,681 ESTs from Sanger sequencing were assembled using the PHRED/PHRAP system [31,32,33], producing 119 primary contiguous sequences (Table 1)
The results of this study showed a large variety and abundance of glycoside hydrolases (GHs) families, most of which can be found within the genomes of R. flavefaciens FD-1 and C. thermocellum (Table 2a)
Summary
Ruminococci are cellulolytic Gram-positive cocci in the order ‘Clostridiales’, which inhabit the rumen community. They are responsible for degrading cellulosic plant cell wall material, and for solubilizing components that can be utilized by other rumen bacteria [1]. The R. flavefaciens type strain is C94, its cellulolytic activity is much lower than that of FD-1, on more crystalline forms of cellulose [5]. 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
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