Ornithine is the central intermediate in the arginine degradative pathway and its regulation inBacillus subtilis

Abstract

The Gram-positive model bacterium Bacillus subtilis is able to utilize a variety of proteinogenic and non-proteinogenic amino acids as sources of carbon, energy and nitrogen. The utilization of the amino acids arginine, citrulline and ornithine is catalyzed by enzymes that are encoded in the rocABC and rocDEF operons and by the rocG gene. Expression of these genes is under control of the alternative sigma factor SigL. RNA polymerase associated to this sigma factor depends on an ATP-hydrolyzing transcription activator to initiate transcription. The RocR protein acts as transcription activator for the roc genes. In this work, we have studied the contributions of all enzymes of the Roc pathway to the degradation of arginine, citrulline and ornithine. This identified the previously uncharacterized RocB protein as responsible for the conversion of citrulline to ornithine. In vitro assays with the purified enzyme suggest that it acts as a manganese-dependent N-carbamoyl-L-ornithine hydrolase that cleaves citrulline to ornithine and carbamate. So far, the molecular effector that triggers transcription activation by RocR has not been unequivocally identified. Using a combination of transcription reporter assays and biochemical experiments we demonstrate that ornithine is the molecular inducer for RocR activity. Our work suggests that binding of ATP to RocR triggers its hexamerization, and binding of ornithine then allows ATP hydrolysis and activation of roc gene transcription. Thus, ornithine is the central molecule of the roc degradative pathway as it is the common intermediate of arginine and citrulline degradation and the molecular effector for the transcription regulator RocR.