Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens

M T Rincon, S Y Ding, S I McCrae, J C Martin, V Aurilia, R Lamed, Y Shoham, E A Bayer, H J Flint

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of clockerins found in R.flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an similar to110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

Original languageEnglish
Pages (from-to)703-713
Number of pages11
JournalJournal of Bacteriology
Volume185
Issue number3
DOIs
Publication statusPublished - Feb 2003

Keywords

  • cellulolytic complex cellusome
  • scaffolding protein CIPA
  • clostridium-thermocellum
  • binding protein
  • cell-surface
  • cellulase
  • gene
  • identification
  • degradation
  • domains

Cite this

Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens. / Rincon, M T ; Ding, S Y ; McCrae, S I ; Martin, J C ; Aurilia, V ; Lamed, R ; Shoham, Y ; Bayer, E A ; Flint, H J .

In: Journal of Bacteriology, Vol. 185, No. 3, 02.2003, p. 703-713.

Research output: Contribution to journalArticle

Rincon, MT, Ding, SY, McCrae, SI, Martin, JC, Aurilia, V, Lamed, R, Shoham, Y, Bayer, EA & Flint, HJ 2003, 'Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens', Journal of Bacteriology, vol. 185, no. 3, pp. 703-713. https://doi.org/10.1128/JB.185.3.703-713.2003
Rincon, M T ; Ding, S Y ; McCrae, S I ; Martin, J C ; Aurilia, V ; Lamed, R ; Shoham, Y ; Bayer, E A ; Flint, H J . / Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens. In: Journal of Bacteriology. 2003 ; Vol. 185, No. 3. pp. 703-713.
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T1 - Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens

AU - Rincon, M T

AU - Ding, S Y

AU - McCrae, S I

AU - Martin, J C

AU - Aurilia, V

AU - Lamed, R

AU - Shoham, Y

AU - Bayer, E A

AU - Flint, H J

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N2 - The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of clockerins found in R.flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an similar to110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

AB - The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of clockerins found in R.flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an similar to110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

KW - cellulolytic complex cellusome

KW - scaffolding protein CIPA

KW - clostridium-thermocellum

KW - binding protein

KW - cell-surface

KW - cellulase

KW - gene

KW - identification

KW - degradation

KW - domains

U2 - 10.1128/JB.185.3.703-713.2003

DO - 10.1128/JB.185.3.703-713.2003

M3 - Article

VL - 185

SP - 703

EP - 713

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 3

ER -