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

Berg Miller ME, Antonopoulos DA, Rincon MT, Band M, Bari A, Akraiko T, Hernandez A, Thimmapuram J, Henrissat B, Coutinho PM, Borovok I, Jindou S, Lamed R, Flint HJ, Bayer EA, White BA - PLoS ONE (2009)

Bottom Line: Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

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.

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Related in: MedlinePlus

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.
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pone-0006650-g003: 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.

Mentions: ORFs that include GH10 or GH11 xylanase modules commonly showed multiple catalytic modules. In one case, GH modules representing family 10 and 43 are detected in the same ORF (ORF03865; Table S2). One larger ORF (ORF03896; 4.5 kb) appears to encode a tetrafunctional endo-1,4-β-xylanase/acetyl xylan esterase, with a predicted molecular weight of 167,983 Da. The ORF contains several modules separated by glutamine-asparagine-rich linkers – two glycoside hydrolase 11 modules, a GH family 10 module, a CBM family 22 module, and a carbohydrate deacetylase at the C-terminal end. Additionally, a dockerin module is present indicating that it is cellulosome associated. This ORF was previously identified in the suppressive subtractive hybridization comparisons with R. flavefaciens JM1; [30]. Southern blots had indicated that both the GH 10 and 11 modules appeared in at least two separate EcoRI restriction fragments, and support the modular arrangement described in Table S2. A comparison of the modular organization inferred for xylanolytic enzymes from R. flavefaciens strains FD-1 and 17 is shown in Figure 3, which shows that while similar features are present, no two modular arrangements are identical between the two strains. The non-cellulosomal (ie. non dockerin-containing) enzyme XynA from R. flavefaciens 17 was previously reported to include a large NQ-rich linker, interconnecting GH11 and GH10 modules [21]. Although T-rich linkers are predominant in glycoside hydrolases from FD-1, three gene products were detected that carry NQ-rich linkers, or in one case a mixture of T-rich and NQ-rich linkers (Figure 4). The average amino acid composition of the five linkers within FD-1-ORF03896 (33% N, 35% Q, 10% W) was quite similar to that of the single large linker in R. flavefaciens 17 XynA (45% N, 26% Q, 16% W) [21]. The presence of the aromatic residue tryptophan in such linker regions is particularly unusual.


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

Berg Miller ME, Antonopoulos DA, Rincon MT, Band M, Bari A, Akraiko T, Hernandez A, Thimmapuram J, Henrissat B, Coutinho PM, Borovok I, Jindou S, Lamed R, Flint HJ, Bayer EA, White BA - PLoS ONE (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2721979&req=5

pone-0006650-g003: 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.
Mentions: ORFs that include GH10 or GH11 xylanase modules commonly showed multiple catalytic modules. In one case, GH modules representing family 10 and 43 are detected in the same ORF (ORF03865; Table S2). One larger ORF (ORF03896; 4.5 kb) appears to encode a tetrafunctional endo-1,4-β-xylanase/acetyl xylan esterase, with a predicted molecular weight of 167,983 Da. The ORF contains several modules separated by glutamine-asparagine-rich linkers – two glycoside hydrolase 11 modules, a GH family 10 module, a CBM family 22 module, and a carbohydrate deacetylase at the C-terminal end. Additionally, a dockerin module is present indicating that it is cellulosome associated. This ORF was previously identified in the suppressive subtractive hybridization comparisons with R. flavefaciens JM1; [30]. Southern blots had indicated that both the GH 10 and 11 modules appeared in at least two separate EcoRI restriction fragments, and support the modular arrangement described in Table S2. A comparison of the modular organization inferred for xylanolytic enzymes from R. flavefaciens strains FD-1 and 17 is shown in Figure 3, which shows that while similar features are present, no two modular arrangements are identical between the two strains. The non-cellulosomal (ie. non dockerin-containing) enzyme XynA from R. flavefaciens 17 was previously reported to include a large NQ-rich linker, interconnecting GH11 and GH10 modules [21]. Although T-rich linkers are predominant in glycoside hydrolases from FD-1, three gene products were detected that carry NQ-rich linkers, or in one case a mixture of T-rich and NQ-rich linkers (Figure 4). The average amino acid composition of the five linkers within FD-1-ORF03896 (33% N, 35% Q, 10% W) was quite similar to that of the single large linker in R. flavefaciens 17 XynA (45% N, 26% Q, 16% W) [21]. The presence of the aromatic residue tryptophan in such linker regions is particularly unusual.

Bottom Line: Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

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.

Show MeSH
Related in: MedlinePlus