Control of the diadenylate cyclase CdaS in Bacillus subtilis: an autoinhibitory domain limits cyclic di-AMP production

• Published in: The Journal of biological chemistry Vol. 289; no. 30; pp. 21098 - 21107
• Main Authors: Mehne, Felix M P, Schröder-Tittmann, Kathrin, Eijlander, Robyn T, Herzberg, Christina, Hewitt, Lorraine, Kaever, Volkhard, Lewis, Richard J, Kuipers, Oscar P, Tittmann, Kai, Stülke, Jörg
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25.07.2014
• Subjects: Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Dinucleoside Phosphates - chemistry; Dinucleoside Phosphates - genetics; Dinucleoside Phosphates - metabolism; Microbiology; Phosphorus-Oxygen Lyases - chemistry; Phosphorus-Oxygen Lyases - genetics; Phosphorus-Oxygen Lyases - metabolism; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Diadenylate Cyclase; Enzyme Structure; Bacterial Signal Transduction; Sporulation; Germination; Bacillus; c-di-AMP; Enzyme Mutation; DAC Domain
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.562066

The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination.