Identification of a novel adenylate cyclase in the ruminal anaerobe, Prevotella ruminicola D31d

Abstract

Our previous evaluation of ruminal and other anaerobic bacteria showed only Prevotella ruminicola D31d produced detectable concentrations of cyclic AMP. In order to investigate the synthesis of this important metabolic regulator, the gene for adenylate cyclase (cya), which produces cyclic AMP, was cloned and expressed in a cyaA mutant of Escherichia coli. The cloned P. ruminicola D31d gene was able to complement the cyaA mutation and permitted fermentation of lactose on MacConkey Lactose agar plates. Analysis of the DNA sequence of the 2.5-kilobase pair insert revealed an open reading frame encoding for a 67-kDa protein. This protein was novel in that no amino acid similarity was observed with other procaryotic or eucaryotic adenylate cyclases in the GenBank database. Production of cyclic AMP in the E. coli clone was confirmed with a radioimmunoassay technique. This is the first example of an adenylate cyclase gene identified from an anaerobic bacterium.