From the Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, D-37077 Göttingen, Germany.
Department of Bioanalytics, Albrecht-von-Haller Institute for Plant Sciences, Göttingen Center for Molecular Biosciences, Georg-August University Göttingen, 9747 AG Groningen, Germany.
J Biol Chem. 2014 Jul 25;289(30):21098-107. doi: 10.1074/jbc.M114.562066. Epub 2014 Jun 16.
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.
革兰氏阳性细菌枯草芽孢杆菌编码三种二腺苷环化酶，这些酶合成必需的信号核苷酸环二腺苷酸。营养期酶 DisA 和 CdaA 的活性受到与其保守伴侣蛋白的蛋白-蛋白相互作用的控制。在这里，我们分析了独特的孢子形成特异性二腺苷环化酶 CdaS 的调控。CdaS 作为唯一的二腺苷酸环化酶的低表达导致自发抑制突变的出现。该 cdaS 基因中的几个突变影响了 CdaS 的 N 端结构域。相应的 CdaS 突变蛋白表现出显著增加的酶活性。CdaS 的 N 端结构域由两个α螺旋组成，并连接到 C 端催化活性的二腺苷酸环化酶（DAC）结构域。第一个或两个螺旋的缺失也导致活性显著增加，表明 N 端结构域用于限制 DAC 结构域的酶活性。与 CdaS 高度相似的蛋白 YojJ 的结构表明，该蛋白形成六聚体，与 DAC 结构域的酶活性不兼容。相反，N 端结构域的突变和缺失导致构象变化，从而导致酶活性的显著增加。尽管全长 CdaS 蛋白被发现形成六聚体，但具有 N 端第一个螺旋缺失的截短版本形成具有高酶活性的二聚体。为了评估 CdaS 在孢子形成中的作用，我们检测了野生型和 cdaS 突变体孢子的萌发。结果表明，CdaS 形成的环二腺苷酸对于有效的萌发是必需的。
