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Title: IDENTIFICATION OF A BROAD-SPECIFICITY XYLOSIDASE/ARABINOSIDASE IMPORTANT FOR XYLOOLIGOSACCHARIDE FERMENTATION BY THE RUMINAL ANAEROBE SELENOMONAS RUMINANTIUM

Author
item Whitehead, Terence
item Cotta, Michael

Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/5/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: The breakdown and digestion of the fiber portion of plants requires several different enzymatic activities. These enzymes are produced by bacteria that live in the rumen of domestic animals such as cattle and sheep. One group of bacteria cannot break down intact fiber, but vary in their ability to grow on the smaller products of fiber breakdown from other bacteria. We ehave found that this ability is related to the production of enzymes involved in the final steps in fiber breakdown and now report on the cloning of a gene producing the enzymes. This is the first report of a gene sequence from this particular bacterium.

Technical Abstract: Strains of Selenomonas ruminantium varied considerably in their capacity to ferment xylooligosaccharides. This ability ranged from strain GA192, which completely utilized xylose through xylotetraose and was able to ferment considerable quantities of larger oligosaccharides, to strain HD4 which used only the simple sugars present in the hydrolysate. The ability of S. ruminantium GA192 to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidase activities. The production of these activities appears to be regulated in response to carbon source used for growth. Both arabinosidase and xylosidase were induced by growth on xylose or xylooligosaccharides, but no activity was detected in glucose or arabinose grown cultures. A genetic locus from S. ruminantium GA192 was cloned into Escherichia coli JM83 that produced both xylosidase and arabinosidase activities. Analyses of crude extracts from the E. coli clone and S. ruminantium GA192 using native polyacrylamide gel electrophoresis and methylumbelliferyl substrates indicated that a single protein was responsible for both activities. The enzyme expressed in E. coli was capable of degrading xylooligosaccharides derived from xylan. DNA sequencing of the locus demonstrated the presence of an open reading frame which encodes for a protein of 61,174 molecular weight. This is the first report of the cloning and expression in E. coli of a structural gene from S. ruminantium.