title: Control of the diadenylate cyclase CdaS in Bacillus subtilis: an autoinhibitory domain limits cyclic di-AMP production.
abstract: 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.
authors.affiliation: From the Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, D-37077 Göttingen, Germany.
authors.forename: Felix M P
authors.initials: FM
authors.lastname: Mehne
authors.affiliation: Department of Bioanalytics, Albrecht-von-Haller Institute for Plant Sciences, Göttingen Center for Molecular Biosciences, Georg-August University Göttingen, 9747 AG Groningen, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Schröder-Tittmann
authors.affiliation: Department for Molecular Genetics, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, 9747 AG Groningen, The Netherlands, Top Institute Food and Nutrition (TIFN), Nieuwe Kanaal 9A, 6709 PA Wageningen, The Netherlands.
authors.forename: Robyn T
authors.initials: RT
authors.lastname: Eijlander
authors.affiliation: From the Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, D-37077 Göttingen, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Herzberg
authors.affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom, and.
authors.forename: Lorraine
authors.initials: L
authors.lastname: Hewitt
authors.affiliation: Research Core Unit for Mass Spectrometry, Metabolomics and Institute of Pharmacology, Hannover Medical School, D-30625 Hannover, Germany, and.
authors.forename: Volkhard
authors.initials: V
authors.lastname: Kaever
authors.affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom, and.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lewis
authors.affiliation: Department for Molecular Genetics, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, 9747 AG Groningen, The Netherlands, Top Institute Food and Nutrition (TIFN), Nieuwe Kanaal 9A, 6709 PA Wageningen, The Netherlands.
authors.forename: Oscar P
authors.initials: OP
authors.lastname: Kuipers
authors.affiliation: Department of Bioanalytics, Albrecht-von-Haller Institute for Plant Sciences, Göttingen Center for Molecular Biosciences, Georg-August University Göttingen, 9747 AG Groningen, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Tittmann
authors.affiliation: From the Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, D-37077 Göttingen, Germany, jstuelk@gwdg.de.
authors.forename: Jörg
authors.initials: J
authors.lastname: Stülke
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D015226:Dinucleoside Phosphates; C528998:cyclic diadenosine phosphate; D019761:Phosphorus-Oxygen Lyases; C528997:diadenylate cyclase, Bacillus subtilis
keywords: Bacillus; Bacterial Signal Transduction; DAC Domain; Diadenylate Cyclase; Enzyme Mutation; Enzyme Structure; Germination; Sporulation; c-di-AMP