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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Research » Publications at this Location » Publication #80971

Title: A PREVOTELLA RUMINICOLA B14 OPERON ENCODING EXTRACELLULAR POLYSACCHARIDE HYDROLASES

Author
item GARDNER, RICHARD
item WELLS, JAMES
item FIELDS, MATTHEW
item WILSON, DAVID
item Russell, James

Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/2/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Cattle lose their capacity to digest cellulose which is an important part of the diet when large amounts of starch are fed. Starch causes a decrease in forestomach (rumen) pH, and the cellulose-digesting bacteria cannot tolerate even modest decline in pH. We have undertaken a project to produce an acid-resistant bacterium that can digest cellulose at low pH. We noted that an acid-resistant starch-digesting ruminal bacterium had an enzyme similar to those digesting cellulose, but this enzyme could not bind cellulose. We used gene reconstruction to add a cellulose binding site to the enzyme. By sequencing the DNA we identified an enzyme that degraded another plant polymer (mannan). When the rumen bacterium reads its DNA, both enzymes are always produced, and we were able to identify the start site of DNA reading, which we can use with our reconstructed gene. If this project is successful, we will be able to re-inoculate the rumen with a genetically reconstructed, pH-resistant, cellulolytic bacterium and increase the rate of cellulose digestion in cattle that have low rumen pH.

Technical Abstract: When Escherichia coli XL1-Blue MRA (P2) was infected with lambda DNA containing Prevotella ruminicola B14 chromosomal DNA, only a few plaques produced B-1,4-endoglucanase activity and all of these had mananas activity. Positive phage contained a 17 kb Sac I DNA fragment that gave six bands after EcoRI digestion. The EcoRI fragments were ligated into pBluescript and sequenced. The order of the fragments was verified by PCR and by restriction mapping. The DNA sequence contained 6 open reading frames (ORFs). The 4th and 5th ORFs encoded two related B-1,4- endoglucanases. E. coli clones carrying ORF5 and ORF6 had B-1,4-endogluca- nase and mannanase activities, while a clone carrying only ORF6 hydrolyzed mannan but no carboxymethylcellulose. The 6th ORF had three regions of homology to mannanase A from Pseudomonas fluorescens. Based on these results, ORF6 encoded the mannanase gene. The 3rd ORF had 10 regions of homology with cellulose binding protein A from Clostridium cellulovorans. The 1st and 2nd ORF had no significant homology to genes or amino acid sequences in GeneBank or SwissPort. All of the ORFs except 1 encoded a potential signal peptide sequence. The upstream region of ORF1 contained four direct repeats, and four inverted repeat elements, but no apparant o70 sequence like promoter was present. The segment of DNA containing the 6 ORFs was preceded and followed by potential transcription termination signals suggesting a single transcriptional unit.