ScaC, an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome

M T Rincon, Jennifer Cynthia Martin, V Aurilia, S I McCrae, Garry Jonathan Rucklidge, Martin David Reid, E A Bayer, R Lamed, Harry James Flint

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.

Original languageEnglish
Pages (from-to)2576-2585
Number of pages10
JournalJournal of Bacteriology
Volume186
Issue number9
DOIs
Publication statusPublished - May 2004

Keywords

  • layer homology domains
  • ruminococcus-flavefaciens
  • clostridium-thermocellum
  • cell-surface
  • scaffoldin
  • rumen
  • degradation
  • system
  • cellulolyticus
  • organization

Cite this

ScaC, an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome. / Rincon, M T ; Martin, Jennifer Cynthia; Aurilia, V ; McCrae, S I ; Rucklidge, Garry Jonathan; Reid, Martin David; Bayer, E A ; Lamed, R ; Flint, Harry James.

In: Journal of Bacteriology, Vol. 186, No. 9, 05.2004, p. 2576-2585.

Research output: Contribution to journalArticle

Rincon, M T ; Martin, Jennifer Cynthia ; Aurilia, V ; McCrae, S I ; Rucklidge, Garry Jonathan ; Reid, Martin David ; Bayer, E A ; Lamed, R ; Flint, Harry James. / ScaC, an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome. In: Journal of Bacteriology. 2004 ; Vol. 186, No. 9. pp. 2576-2585.
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abstract = "A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.",
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T1 - ScaC, an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome

AU - Rincon, M T

AU - Martin, Jennifer Cynthia

AU - Aurilia, V

AU - McCrae, S I

AU - Rucklidge, Garry Jonathan

AU - Reid, Martin David

AU - Bayer, E A

AU - Lamed, R

AU - Flint, Harry James

PY - 2004/5

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N2 - A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.

AB - A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.

KW - layer homology domains

KW - ruminococcus-flavefaciens

KW - clostridium-thermocellum

KW - cell-surface

KW - scaffoldin

KW - rumen

KW - degradation

KW - system

KW - cellulolyticus

KW - organization

U2 - 10.1128/JB.186.9.2576-2585.2004

DO - 10.1128/JB.186.9.2576-2585.2004

M3 - Article

VL - 186

SP - 2576

EP - 2585

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 9

ER -