1887

Abstract

-Ornithine production in the alfalfa microsymbiont occurs as an intermediate step in arginine biosynthesis. Ornithine is required for effective symbiosis but its synthesis in has been little studied. Unlike most bacteria, 1021 is annotated as encoding two enzymes producing ornithine: -acetylornithine (NAO) deacetylase (ArgE) hydrolyses NAO to acetate and ornithine, and glutamate -acetyltransferase (ArgJ) transacetylates -glutamate with the acetyl group from NAO, forming ornithine and -acetylglutamate (NAG). NAG is the substrate for the second step of arginine biosynthesis catalysed by NAG kinase (ArgB). Inactivation of in strain 1021 resulted in arginine auxotrophy. The activity of purified ArgB was significantly inhibited by arginine but not by ornithine. The purified ArgJ was highly active in NAO deacetylation/glutamate transacetylation and was significantly inhibited by ornithine but not by arginine. The purified ArgE protein (with a 6His-Sumo affinity tag) was also active in deacetylating NAO. and single mutants, and an double mutant, are arginine prototrophs. Extracts of the double mutant contained aminoacylase (Ama) activity that deacetylated NAO to form ornithine. The purified products of three candidate genes ( (), () and ) all possessed NAO deacetylase activity. and , but not , expressed functionally complemented an Δ : : Km mutant. We conclude that Ama activity accounts for the arginine prototrophy of the mutant. Transcriptional assays of , and , fused to a promoterless gene, showed that their expression was not significantly affected by exogenous arginine or ornithine.

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This article contains a correction applying to the following content:
Genetic and biochemical characterization of arginine biosynthesis in 1021
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2015-08-01
2020-02-19
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