A Novel Cell Surface-Anchored Cellulose-Binding Protein Encoded by the sca Gene Cluster of Ruminococcus flavefaciens

ABSTRACT Ruminococcus flavefaciens produces a cellulosomal enzyme complex, based on the structural proteins ScaA, -B, and -C, that was recently shown to attach to the bacterial cell surface via the wall-anchored protein ScaE. ScaA, -B, -C, and -E are all cohesin-bearing proteins encoded by linked genes in the sca cluster. The product of an unknown open reading frame within the sca cluster, herein designated CttA, is similar in sequence at its C terminus to the corresponding region of ScaB, which contains an X module together with a dockerin sequence. The ScaB-XDoc dyad was shown previously to interact tenaciously with the cohesin of ScaE. Likewise, avid binding was confirmed between purified recombinant fragments of the CttA-XDoc dyad and the ScaE cohesin. In addition, the N-terminal regions of CttA were shown to bind to cellulose, thus suggesting that CttA is a cell wall-anchored, cellulose-binding protein. Proteomic analysis showed that the native CttA protein (∼130 kDa) corresponds to one of the three most abundant polypeptides binding tightly to insoluble cellulose in cellulose-grown R. flavefaciens 17 cultures. Interestingly, this protein was also detected among cellulose-bound proteins in the related strain R. flavefaciens 007C but not in a mutant derivative, 007S, that was previously shown to have lost the ability to grow on dewaxed cotton fibers. In R. flavefaciens, the presence of CttA on the cell surface is likely to provide an important mechanism for substrate binding, perhaps compensating for the absence of an identified cellulose-binding module in the major cellulosomal scaffolding proteins of this species.

[1]  K. Nelson,et al.  Phylogenetic analysis of the microbial populations in the wild herbivore gastrointestinal tract: insights into an unexplored niche. , 2003, Environmental microbiology.

[2]  Z. Jia,et al.  Mechanism of bacterial cell-surface attachment revealed by the structure of cellulosomal type II cohesin-dockerin complex. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[3]  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.

[4]  V. Varel,et al.  Microbial perspective on fiber utilization by swine. , 1997, Journal of animal science.

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

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

[7]  O. Shoseyov,et al.  Carbohydrate Binding Modules: Biochemical Properties and Novel Applications , 2006, Microbiology and Molecular Biology Reviews.

[8]  H. Flint,et al.  Molecular cloning of genes from Ruminococcus flavefaciens encoding xylanase and beta(1-3,1-4)glucanase activities , 1989, Applied and environmental microbiology.

[9]  H. Harmsen,et al.  Effects of Alternative Dietary Substrates on Competition between Human Colonic Bacteria in an Anaerobic Fermentor System , 2003, Applied and Environmental Microbiology.

[10]  N. Gilkes,et al.  Cellulose hydrolysis by bacteria and fungi. , 1995, Advances in microbial physiology.

[11]  Zhongtang Yu,et al.  Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract, as revealed by ribosomal intergenic spacer analysis and rrs gene sequencing. , 2005, Environmental microbiology.

[12]  Raphael Lamed,et al.  Conservation and Divergence in Cellulosome Architecture between Two Strains of Ruminococcus flavefaciens , 2006, Journal of bacteriology.

[13]  J. Aubert,et al.  The biological degradation of cellulose. , 1994, FEMS microbiology reviews.

[14]  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.

[15]  Raphael Lamed,et al.  Novel Organization and Divergent Dockerin Specificities in the Cellulosome System of Ruminococcus flavefaciens , 2003, Journal of bacteriology.

[16]  G. Fonty,et al.  Identification of Ruminococcus flavefaciens as the Predominant Cellulolytic Bacterial Species of the Equine Cecum , 1999, Applied and Environmental Microbiology.

[17]  D. Bolam,et al.  Carbohydrate-binding modules: fine-tuning polysaccharide recognition. , 2004, The Biochemical journal.

[18]  E. Bayer,et al.  Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin–dockerin interaction , 2005, Journal of molecular recognition : JMR.

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

[20]  K. Wedekind,et al.  Enumeration and isolation of cellulolytic and hemicellulolytic bacteria from human feces , 1988, Applied and environmental microbiology.

[21]  Raphael Lamed,et al.  ScaC, an Adaptor Protein Carrying a Novel Cohesin That Expands the Dockerin-Binding Repertoire of the Ruminococcus flavefaciens 17 Cellulosome , 2004, Journal of bacteriology.

[22]  Raphael Lamed,et al.  Ruminococcus albus 8 Mutants Defective in Cellulose Degradation Are Deficient in Two Processive Endocellulases, Cel48A and Cel9B, Both of Which Possess a Novel Modular Architecture , 2004, Journal of bacteriology.

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

[24]  E. Bayer,et al.  Unconventional Mode of Attachment of the Ruminococcus flavefaciens Cellulosome to the Cell Surface , 2005, Journal of bacteriology.

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

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

[27]  R. Haser,et al.  Cel9M, a New Family 9 Cellulase of the Clostridium cellulolyticum Cellulosome , 2002, Journal of bacteriology.

[28]  D. Kilburn,et al.  C1-Cx revisited: intramolecular synergism in a cellulase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[29]  J. Costerton,et al.  Electron microscopic study of the methylcellulose-mediated detachment of cellulolytic rumen bacteria from cellulose fibers. , 1987, Canadian journal of microbiology.

[30]  A. Richardson,et al.  The implications of the loss and regain of cotton‐degrading activity for the degradation of straw by Ruminococcus flavefaciens strain 007 , 1990 .

[31]  E. Bayer,et al.  The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. , 2004, Annual review of microbiology.

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

[33]  B. Michalet-Doreau,et al.  A comparison of enzymatic and molecular approaches to characterize the cellulolytic microbial ecosystems of the rumen and the cecum. , 2002, Journal of animal science.

[34]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

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