Cellulosomal Scaffoldin-Like Proteins fromRuminococcus flavefaciens

ABSTRACT Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaAwas partially sequenced. The sequenced portion of scaAcontains repeating cohesin modules and a C-terminal dockerin domain. ScaB contains seven relatively divergent cohesin modules, two extremely long T-rich linkers, and a C-terminal domain of unknown function. Collectively, the cohesins of ScaA and ScaB are phylogenetically distinct from the previously described type I and type II cohesins, and we propose that they define a new group, which we designated here type III cohesins. Selected modules from both genes were overexpressed inEscherichia coli, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate that ScaA serves as a cellulosomal scaffoldin-like protein for severalR. flavefaciens enzymes. The data are supported by the direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The evidence also demonstrates that the ScaA dockerin binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacterial cell surface. This study is the first direct demonstration in a cellulolytic rumen bacterium of a cellulosome system, mediated by distinctive cohesin-dockerin interactions.

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

[2]  H. Flint,et al.  A xylanase produced by the rumen anaerobic protozoan Polyplastron multivesiculatum shows close sequence similarity to family 11 xylanases from gram-positive bacteria. , 1999, FEMS microbiology letters.

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

[4]  B. Rost,et al.  Combining evolutionary information and neural networks to predict protein secondary structure , 1994, Proteins.

[5]  Malmqvist,et al.  Epitope Mapping by Label-Free Biomolecular Interaction Analysis , 1996, Methods.

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

[7]  E. Bayer,et al.  The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides. , 1999, Trends in microbiology.

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

[9]  H. Ohara,et al.  Sequence of egV and Properties of EgV, a Ruminococcus albus Endoglucanase Containing a Dockerin Domain , 2000, Bioscience, biotechnology, and biochemistry.

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

[11]  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.

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

[13]  B. Dalrymple,et al.  16S rDNA sequencing of Ruminococcus albus and Ruminococcus flavefaciens: design of a signature probe and its application in adult sheep. , 1999, Microbiology.

[14]  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.

[15]  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.

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

[17]  P. Cronje,et al.  Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction , 2000 .

[18]  F. Rainey,et al.  Phylogenetic analysis by 16S ribosomal DNA sequence comparison reveals two unrelated groups of species within the genus Ruminococcus. , 1995, FEMS microbiology letters.

[19]  K. Cheng,et al.  The rumen: a unique source of enzymes for enhancing livestock production. , 1996, Anaerobe.

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

[21]  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.

[22]  B. Henrissat,et al.  Cellulosome‐like sequences in Archaeoglobus fulgidus: an enigmatic vestige of cohesin and dockerin domains , 1999, FEBS letters.

[23]  W. Engelhardt,et al.  Polysaccharide degradation in the rumen: biochemistry and genetics. , 1995 .

[24]  R. E. Hungate,et al.  The Rumen and Its Microbes , 2013 .

[25]  P. Alzari,et al.  The cellulosome of Clostridium thermocellum. , 1998, Biochemical Society transactions.

[26]  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.

[27]  J. Miron,et al.  Adhesion to cellulose by Ruminococcus albus: a combination of cellulosomes and Pil-proteins? , 2000, FEMS microbiology letters.

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

[29]  P. Gounon,et al.  Characterization and Subcellular Localization of the Clostridium thermocellum Scaffoldin Dockerin Binding Protein SdbA , 1996 .

[30]  B. White,et al.  Assessment of the endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1 , 1990, Applied and environmental microbiology.

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

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

[33]  H. Flint,et al.  The rumen microbial ecosystem--some recent developments. , 1997, Trends in microbiology.

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

[35]  M. P. Bryant,et al.  The rumen bacteria , 1997 .

[36]  E. Stackebrandt,et al.  16S rDNA analysis reveals phylogenetic diversity among the polysaccharolytic clostridia. , 1993, FEMS microbiology letters.

[37]  B. Rost.  PHD: predicting one-dimensional protein structure by profile-based neural networks. , 1996, Methods in enzymology.

[38]  H. Fierobe,et al.  Sequence Analysis of Scaffolding Protein CipC and ORFXp, a New Cohesin-Containing Protein inClostridium cellulolyticum: Comparison of Various Cohesin Domains and Subcellular Localization of ORFXp , 1999, Journal of bacteriology.

[39]  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.

[40]  B. Rost,et al.  Prediction of protein secondary structure at better than 70% accuracy. , 1993, Journal of molecular biology.

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

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

[43]  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.

[44]  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.

[45]  E. Bayer,et al.  Cellulose, cellulases and cellulosomes. , 1998, Current opinion in structural biology.

[46]  P. Lawson,et al.  The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. , 1994, International journal of systematic bacteriology.

[47]  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.