Biochemical and mutational analysis of a gingipain-like peptidase activity from Prevotella ruminicola B(1)4 and its role in ammonia production by ruminal bacteria.

Abstract

A chemical mutagenesis protocol was used with the ruminal bacterium Prevotella ruminicola strain B(1)4 to generate mutant strains defective in peptidase activity. Compared with the wild-type parent strain, the isolated mutants possessed 1/10 of the enzyme activity responsible for cleavage of glycine-arginine-4-methoxy-beta-naphthylamide (Gly-Arg-MNA). A concomitant loss in activity against arginine-arginine-4-methoxy-beta-naphthylamide (Arg-Arg-MNA) was also observed. Both activities were similarly affected by various proteinase inhibitors, suggesting that the same enzyme is responsible for the Arg-Arg-MNA peptidase and Gly-Arg-MNA peptidase activities. Growth rates of wild-type and mutant strains grown in batch culture with various nitrogen sources did not differ. However, a role for the Gly-Arg-MNA peptidase activity was demonstrated in coculture experiments with gram-positive, ammonia-producing ruminal bacteria. The rate and extent of ammonia production were reduced by approximately 25% in cocultures containing the mutants when compared with that of wild-type-containing cultures. These reductions could not be accounted for simply by the decrease in ammonia production by the mutant strain alone. To our knowledge, this paper reports the first successful use of chemical mutagenesis with ruminal microorganisms.