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The Gram-positive bacterium Bacillus subtilis can utilize several proteinogenic and non-proteinogenic amino acids as sources of carbon, nitrogen, and energy. The utilization of the amino acids arginine, citrulline, and ornithine is catalyzed by enzymes encoded in the rocABC and rocDEF operons and by the rocG gene. The expression of these genes is controlled by the alternative sigma factor SigL. RNA polymerase associated with this sigma factor depends on ATP-hydrolyzing transcription activators to initiate transcription. The RocR protein acts as a transcription activator for the roc genes. However, the details of amino acid metabolism via this pathway are unknown. Here, we investigated the contributions of all enzymes of the Roc pathway to the degradation of arginine, citrulline, and ornithine. We identified the previously uncharacterized RocB protein as responsible for the conversion of citrulline to ornithine. In vitro assays with the purified enzyme suggest that RocB acts as a manganese-dependent N-carbamoyl-L-ornithine hydrolase that cleaves citrulline to form ornithine and carbamate. Moreover, 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 of RocR activity. Taken together, 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 of RocR., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)