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Expression of cellulosome components and type IV pili within the extracellular proteome of Ruminococcus flavefaciens 007.

Vodovnik M, Duncan SH, Reid MD, Cantlay L, Turner K, Parkhill J, Lamed R, Yeoman CJ, Miller ME, White BA, Bayer EA, Marinšek-Logar R, Flint HJ - PLoS ONE (2013)

Bottom Line: The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources.Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose.One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.

View Article: PubMed Central - PubMed

Affiliation: Chair for Microbiology and Microbial Biotechnology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.

ABSTRACT

Background: Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose.

Methodology/principal findings: The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel.

Conclusions/significance: This study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.

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Cellulose-specific changes in expression of culture supernatant proteins in R. flavefaciens strains grown on Avicel (7.5 days), xylan (48 hours) or cellobiose (24 hours).(A) supernatant (CCSUP) fraction of strain 007C, (B) supernatant fraction of strain 007S. 1-fold change indicates no change, >1-fold change indicates upregulation in comparison to cellobiose-grown cultures, <1-fold change indicates downregulation in comparison to cellobiose-grown cultures. Results are based on duplicated biological experiments, and three technical replicates for each gel separation.
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pone-0065333-g004: Cellulose-specific changes in expression of culture supernatant proteins in R. flavefaciens strains grown on Avicel (7.5 days), xylan (48 hours) or cellobiose (24 hours).(A) supernatant (CCSUP) fraction of strain 007C, (B) supernatant fraction of strain 007S. 1-fold change indicates no change, >1-fold change indicates upregulation in comparison to cellobiose-grown cultures, <1-fold change indicates downregulation in comparison to cellobiose-grown cultures. Results are based on duplicated biological experiments, and three technical replicates for each gel separation.

Mentions: We detected strong up-regulation of three proteins in culture supernatants of strain 007C (>10 fold) that was specific to Avicel-grown relative to cellobiose-grown cultures, with little or no increase in xylan-grown cultures (Fig. 3, 4A). These proteins were a GH48 cellulase, a type IV pilin homologue and a rubrerythrin-like protein; in contrast, the cellulosomal proteins ScaC (Fig. 3) and GH9 and GH26 enzymes (Fig. 3, 4A) were less affected by the growth substrate. There was some evidence for substrate-dependent expression of CttA and ScaA, especially among cell wall associated proteins (Fig. S2), but these appeared to increase in both xylan and Avicel-grown cultures relative to cellobiose.


Expression of cellulosome components and type IV pili within the extracellular proteome of Ruminococcus flavefaciens 007.

Vodovnik M, Duncan SH, Reid MD, Cantlay L, Turner K, Parkhill J, Lamed R, Yeoman CJ, Miller ME, White BA, Bayer EA, Marinšek-Logar R, Flint HJ - PLoS ONE (2013)

Cellulose-specific changes in expression of culture supernatant proteins in R. flavefaciens strains grown on Avicel (7.5 days), xylan (48 hours) or cellobiose (24 hours).(A) supernatant (CCSUP) fraction of strain 007C, (B) supernatant fraction of strain 007S. 1-fold change indicates no change, >1-fold change indicates upregulation in comparison to cellobiose-grown cultures, <1-fold change indicates downregulation in comparison to cellobiose-grown cultures. Results are based on duplicated biological experiments, and three technical replicates for each gel separation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672088&req=5

pone-0065333-g004: Cellulose-specific changes in expression of culture supernatant proteins in R. flavefaciens strains grown on Avicel (7.5 days), xylan (48 hours) or cellobiose (24 hours).(A) supernatant (CCSUP) fraction of strain 007C, (B) supernatant fraction of strain 007S. 1-fold change indicates no change, >1-fold change indicates upregulation in comparison to cellobiose-grown cultures, <1-fold change indicates downregulation in comparison to cellobiose-grown cultures. Results are based on duplicated biological experiments, and three technical replicates for each gel separation.
Mentions: We detected strong up-regulation of three proteins in culture supernatants of strain 007C (>10 fold) that was specific to Avicel-grown relative to cellobiose-grown cultures, with little or no increase in xylan-grown cultures (Fig. 3, 4A). These proteins were a GH48 cellulase, a type IV pilin homologue and a rubrerythrin-like protein; in contrast, the cellulosomal proteins ScaC (Fig. 3) and GH9 and GH26 enzymes (Fig. 3, 4A) were less affected by the growth substrate. There was some evidence for substrate-dependent expression of CttA and ScaA, especially among cell wall associated proteins (Fig. S2), but these appeared to increase in both xylan and Avicel-grown cultures relative to cellobiose.

Bottom Line: The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources.Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose.One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.

View Article: PubMed Central - PubMed

Affiliation: Chair for Microbiology and Microbial Biotechnology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.

ABSTRACT

Background: Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose.

Methodology/principal findings: The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel.

Conclusions/significance: This study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.

Show MeSH
Related in: MedlinePlus