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United States Department of Agriculture

Agricultural Research Service

Title: Cloning, Expression and Sequencing of An Adenylate Cyclase Gene from the Ruminal Anaerobic Bacterium, Prevotella Ruminicola

item Whitehead, Terence
item Cotta, Michael
item Wheeler, Matthew - UNIV OF IL

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: May 21, 1998
Publication Date: N/A

Technical Abstract: Cyclic AMP (cAMP) is involved in the process of catabolite repression in a variety of aerobic and facultative anaerobic bacteria. Previous work in our laboratories has demonstrated that cAMP is virtually undetectable in a variety of ruminal and other anaerobic bacteria, even under growth conditions where catabolite repression-like regulatory phenomena were demonstrated. The only exception noted was with the ruminal bacterium Prevotella ruminicola D31d, which produced increased levels of cAMP during early growth and early stationary phases. In order to further study the presence of cAMP in this strain, attempts were made to clone the gene for adenylate cyclase. Complementation of the Escherichia coli mutant strain SP850 was used for the cloning experiments. This strain is deficient in adenylate cyclase and cAMP and cannot ferment lactose. The strain thereby produces colorless colonies on MacConkey agar. A genomic bank from P. ruminicola D31d was prepared with pUC18 and introduced into SP850 by electroporation. One clone was isolated that produced dark red colonies when plated onto MacConkey agar, indicative of lactose fermentation and production of cAMP. The clone contained a plasmid with a 2.8-kb insert. cAMP was detected in the clone, indicative of the presence of adenylate cyclase activity. The DNA insert is currently being sequenced. The data indicate that the adenylate cyclase gene has been cloned from P. ruminicola D31d, which would be the first such gene cloned from an anaerobic bacterium.

Last Modified: 4/22/2015
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