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United States Department of Agriculture

Agricultural Research Service

Title: Complementation of An Escherichia Coli Adenylate Cyclase Mutant with a Gene from Prevotella Ruminicola D31d

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

Submitted to: Conference on Rumen Function
Publication Type: Abstract Only
Publication Acceptance Date: November 13, 1997
Publication Date: N/A

Technical Abstract: Synthesis of cyclic AMP (cAMP) by the enzyme adenylate cyclase is involved in catabolite repression in aerobic and facultative anaerobic bacteria. However, the presence of cAMP in anaerobic bacteria has been questioned. Previous work in our laboratories has demonstrated that cAMP is virtually undetectable in a variety of ruminal and other anaerobic bacteria. The only exception observed 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. Escherichia coli strain SP850 was used as the host for cloning the P. ruminicola D31d gene. This strain is deficient in adenylate cyclase, cannot ferment lactose, and produces colorless colonies on MacConkey agar. A genomic bank from 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. The clone contained a plasmid with a 2.3 kb insert. The clone is currently being analyzed for cAMP production and the DNA insert is being sequenced. Presence of cAMP in the E. coli clone and DNA sequence analyses of the insert should provide evidence for the isolation of an adenylate cyclase gene from P. ruminicola D31d, which would be the first such gene cloned from an anaerobic bacterium.

Last Modified: 4/21/2015
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