Molecular Analysis of a Cellulase/xylanase gene,celD, From the Ruminal Bacterium Ruminococcus Flavefaciens Fd-1

152 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1992.A genomic library of Ruminococcus flavefaciens FD-1 DNA was constructed using the Escherichia coli bacteriophage $\lambda$ vector $\lambda$DASH. A recombinant phage exhibiting activity against both Ostazin brilliant red-hydroxyethyl cellulose and carboxymethyl cellulose (CMC) was isolated. This clone (designated FD1-1) was further analyzed by restriction endonuclease mapping and Southern blot analysis. Substrate specificity data show that the cloned gene encodes both endoglucanase and endoxylanase activities. CMC and xylan zymograms of protein produced by FD1-1 and separated by non-denaturing PAGE suggest that the endoglucanase and endoxylanase activities reside on the same polypeptide. Transposon mutagenesis using Tn5supF localized the celD gene within the insert of FD1-1 and supported the zymogram results that the cellulase and xylanase activities were located on a single polypeptide. DNA sequence analysis of celD revealed an open reading frame of 1086 nucleotides encoding a 362 amino acid polypeptide with a molecular mass of 46,329 daltons. The CelD polypeptide had common features with other cellulases e.g. a putative catalytic domain, hydroxyamino acid rich domain, a putative cellulose binding domain, and repeated amino acid sequences near the C-terminus. In addition, CelD showed amino acid sequence similarity with other Ruminococcus cellulases.Northern blot analysis of mRNA produced by R. flavefaciens FD-1 grown using either cellobiose or cellulose as the substrate indicated that celD was only expressed in cells grown with cellulose as the substrate. The cellodextrinase gene, celA, was shown to be expressed in cells grown with either cellobiose or cellulose as the substrate. The results indicate that cellulase synthesis by R. flavefaciens FD-1 is differentially regulated by the carbon source and celD is cellulose inducible.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD