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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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Type IV pili biogenesis cluster of R. flavefaciens 007C.The protein Pil3, encoded by the pil3 gene, shares 51% amino acid identity to the cellulose-binding protein (CbpC) of R. albus 8 and 43% amino acid identity to R. albus 20 protein GP25 (a protein that was shown to be underproduced by spontaneous cellulose adhesion-defective mutant D5). The gene downstream (pil4) encodes another protein with prepilin-type IV N-terminal domain, whereas the genes upstream encode: putative PilT−/PilB-like ATPases (pilT, pilB), prepilin peptidase (pilD), two proteins with pseudopilin PulG-like domains (pil1, pil2), type IV pilus assembly protein (pilM) and a type II secretion system protein (pilF). OrfX refers to open reading frame without recognized signal sequences.
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pone-0065333-g005: Type IV pili biogenesis cluster of R. flavefaciens 007C.The protein Pil3, encoded by the pil3 gene, shares 51% amino acid identity to the cellulose-binding protein (CbpC) of R. albus 8 and 43% amino acid identity to R. albus 20 protein GP25 (a protein that was shown to be underproduced by spontaneous cellulose adhesion-defective mutant D5). The gene downstream (pil4) encodes another protein with prepilin-type IV N-terminal domain, whereas the genes upstream encode: putative PilT−/PilB-like ATPases (pilT, pilB), prepilin peptidase (pilD), two proteins with pseudopilin PulG-like domains (pil1, pil2), type IV pilus assembly protein (pilM) and a type II secretion system protein (pilF). OrfX refers to open reading frame without recognized signal sequences.

Mentions: The R. flavefaciens pil3 gene is flanked by open reading frames that are homologous to genes known to be involved in the assembly and secretion of type IV pili and flagella (Fig. 5, Table S6). These include upstream genes encoding a putative pilus retraction (PilT-like) ATPase, followed by a PilB (Vir B11)-like ATPase, and a putative prepilin peptidase (PilD-like) gene. The first two genes in the cluster encode a putative PilM-like pilus assembly protein (involved in the export of the pilus subunit and its assembly in P. aeruginosa) and a putative F-domain of the type II secretion system (PulF homologue). One gene (pil4) with a type IV pilus signature sequence (prepilin-type N-terminal cleavage/methylation site) is present downstream of pil3, and also encodes a protein with high similarity to R. albus pilin CbpC. The nucleotide composition of the putative pilus biogenesis cluster did not differ significantly in its G+C content (47.04%) from the whole genome (45.2%). We also detected a homologous gene cluster in the genome of R. flavefaciens FD1 [5].


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)

Type IV pili biogenesis cluster of R. flavefaciens 007C.The protein Pil3, encoded by the pil3 gene, shares 51% amino acid identity to the cellulose-binding protein (CbpC) of R. albus 8 and 43% amino acid identity to R. albus 20 protein GP25 (a protein that was shown to be underproduced by spontaneous cellulose adhesion-defective mutant D5). The gene downstream (pil4) encodes another protein with prepilin-type IV N-terminal domain, whereas the genes upstream encode: putative PilT−/PilB-like ATPases (pilT, pilB), prepilin peptidase (pilD), two proteins with pseudopilin PulG-like domains (pil1, pil2), type IV pilus assembly protein (pilM) and a type II secretion system protein (pilF). OrfX refers to open reading frame without recognized signal sequences.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065333-g005: Type IV pili biogenesis cluster of R. flavefaciens 007C.The protein Pil3, encoded by the pil3 gene, shares 51% amino acid identity to the cellulose-binding protein (CbpC) of R. albus 8 and 43% amino acid identity to R. albus 20 protein GP25 (a protein that was shown to be underproduced by spontaneous cellulose adhesion-defective mutant D5). The gene downstream (pil4) encodes another protein with prepilin-type IV N-terminal domain, whereas the genes upstream encode: putative PilT−/PilB-like ATPases (pilT, pilB), prepilin peptidase (pilD), two proteins with pseudopilin PulG-like domains (pil1, pil2), type IV pilus assembly protein (pilM) and a type II secretion system protein (pilF). OrfX refers to open reading frame without recognized signal sequences.
Mentions: The R. flavefaciens pil3 gene is flanked by open reading frames that are homologous to genes known to be involved in the assembly and secretion of type IV pili and flagella (Fig. 5, Table S6). These include upstream genes encoding a putative pilus retraction (PilT-like) ATPase, followed by a PilB (Vir B11)-like ATPase, and a putative prepilin peptidase (PilD-like) gene. The first two genes in the cluster encode a putative PilM-like pilus assembly protein (involved in the export of the pilus subunit and its assembly in P. aeruginosa) and a putative F-domain of the type II secretion system (PulF homologue). One gene (pil4) with a type IV pilus signature sequence (prepilin-type N-terminal cleavage/methylation site) is present downstream of pil3, and also encodes a protein with high similarity to R. albus pilin CbpC. The nucleotide composition of the putative pilus biogenesis cluster did not differ significantly in its G+C content (47.04%) from the whole genome (45.2%). We also detected a homologous gene cluster in the genome of R. flavefaciens FD1 [5].

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