Novel Organization and Divergent Dockerin Specificities in the Cellulosome System of Ruminococcus flavefaciens

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 dockerins 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 ∼110-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.

[1]  R. Doi,et al.  The Clostridium cellulovorans cellulosome. , 1994, Critical reviews in microbiology.

[2]  R. Doi,et al.  Cohesin-Dockerin Interactions of Cellulosomal Subunits of Clostridium cellulovorans , 2001, Journal of bacteriology.

[3]  C. Tardif,et al.  Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum , 1997, Applied and environmental microbiology.

[4]  L. Ljungdahl,et al.  Two cellulases, CelA and CelC, from the polycentric anaerobic fungus Orpinomyces strain PC-2 contain N-terminal docking domains for a cellulase-hemicellulase complex , 1997, Applied and environmental microbiology.

[5]  E. Bayer,et al.  Specialized cell surface structures in cellulolytic bacteria , 1987, Journal of bacteriology.

[6]  E. Bayer,et al.  Species‐specificity of the cohesin‐dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: Prediction of specificity determinants of the dockerin domain , 1997, Proteins.

[7]  J Kirby,et al.  Dockerin-like sequences in cellulases and xylanases from the rumen cellulolytic bacterium Ruminococcus flavefaciens. , 1997, FEMS microbiology letters.

[8]  J. Aubert,et al.  Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface , 1993, Journal of bacteriology.

[9]  E. Bayer,et al.  Cohesin‐dockerin recognition in cellulosome assembly: Experiment versus hypothesis , 2000, Proteins.

[10]  C. Tardif,et al.  The cellulolytic system of Clostridium cellulolyticum. , 1997, Journal of biotechnology.

[11]  Pedro M. Coutinho,et al.  Carbohydrate-active enzymes : an integrated database approach , 1999 .

[12]  P. Gounon,et al.  OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope , 1995, Journal of bacteriology.

[13]  H. Flint,et al.  Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences. , 2000, Microbiology.

[14]  Raphael Lamed,et al.  A Scaffoldin of the Bacteroides cellulosolvens Cellulosome That Contains 11 Type II Cohesins , 2000, Journal of bacteriology.

[15]  E. Bayer,et al.  The cellulosome--a treasure-trove for biotechnology. , 1994, Trends in biotechnology.

[16]  H. Flint,et al.  Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii (formerly P. ruminicola subsp. brevis) B(1)4. , 1997, Anaerobe.

[17]  E. Bayer,et al.  Unorthodox intrasubunit interactions in the cellulosome ofClostridium thermocellum , 1992 .

[18]  P. Béguin,et al.  The cellulosome: an exocellular, multiprotein complex specialized in cellulose degradation. , 1996, Critical reviews in biochemistry and molecular biology.

[19]  P Béguin,et al.  A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA , 1996, Journal of bacteriology.

[20]  J. Aubert,et al.  Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA , 1994, Journal of bacteriology.

[21]  R. E. Hungate,et al.  Phenylpropanoic Acid: Growth Factor for Ruminococcus albus , 1982, Applied and environmental microbiology.

[22]  H. Flint,et al.  A bifunctional enzyme, with separate xylanase and beta(1,3-1,4)-glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens , 1993, Journal of bacteriology.

[23]  Raphael Lamed,et al.  Cellulosomal Scaffoldin-Like Proteins fromRuminococcus flavefaciens , 2001, Journal of bacteriology.

[24]  E. Bayer,et al.  Nonproteolytic cleavage of aspartyl proline bonds in the cellulosomal scaffoldin subunit from Clostridium thermocellum , 2001, Applied biochemistry and biotechnology.

[25]  H. Gilbert,et al.  The Conserved Noncatalytic 40-Residue Sequence in Cellulases and Hemicellulases from Anaerobic Fungi Functions as a Protein Docking Domain (*) , 1995, The Journal of Biological Chemistry.

[26]  E. Bayer,et al.  Cellulosomes-structure and ultrastructure. , 1998, Journal of structural biology.

[27]  Birte Svensson,et al.  Recent Advances in Carbohydrate Bioengineering , 1999 .

[28]  W. Schwarz The cellulosome and cellulose degradation by anaerobic bacteria , 2001, Applied Microbiology and Biotechnology.

[29]  M. P. Bryant,et al.  Commentary on the Hungate technique for culture of anaerobic bacteria. , 1972, The American journal of clinical nutrition.

[30]  L. Ljungdahl,et al.  The cellulosome: the exocellular organelle of Clostridium. , 1993, Annual review of microbiology.

[31]  D. Crothers,et al.  Improved estimation of secondary structure in ribonucleic acids. , 1973, Nature: New biology.

[32]  M. Morrison,et al.  Adherence of the Gram-Positive BacteriumRuminococcus albus to Cellulose and Identification of a Novel Form of Cellulose-Binding Protein Which Belongs to the Pil Family of Proteins , 1998, Journal of bacteriology.

[33]  O. Shoseyov,et al.  Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[34]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[35]  Karen P. Scott,et al.  EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain , 2001, Applied and Environmental Microbiology.

[36]  H. Ohara,et al.  Characterization of the Cellulolytic Complex (Cellulosome) from Ruminococcus albus , 2000, Bioscience, biotechnology, and biochemistry.

[37]  C. Tardif,et al.  Interaction between the endoglucanase CelA and the scaffolding protein CipC of the Clostridium cellulolyticum cellulosome , 1996, Journal of bacteriology.

[38]  B Henrissat,et al.  Glycoside hydrolases and glycosyltransferases: families and functional modules. , 2001, Current opinion in structural biology.

[39]  E. Bayer,et al.  Anomalous dissociative behavior of the major glycosylated component of the cellulosome of clostridium thermocellum , 1991, Applied biochemistry and biotechnology.

[40]  Raphael Lamed,et al.  A Novel Cellulosomal Scaffoldin fromAcetivibrio cellulolyticus That Contains a Family 9 Glycosyl Hydrolase , 1999, Journal of bacteriology.

[41]  A. Demain,et al.  Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL‐protein reveals an unusual degree of internal homology , 1993, Molecular microbiology.

[42]  E Setter,et al.  Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum , 1983, Journal of bacteriology.

[43]  Tetsuya Kimura,et al.  Cloning and DNA Sequencing of the Genes EncodingClostridium josui Scaffolding Protein CipA and Cellulase CelD and Identification of Their Gene Products as Major Components of the Cellulosome , 1998, Journal of bacteriology.