Daniel R A, Errington J
Sir William Dunn School of Pathology, University of Oxford, U.K.
J Mol Biol. 1993 Jul 20;232(2):468-83. doi: 10.1006/jmbi.1993.1403.
Dipicolinic acid (DPA) is a small polar molecule that accumulates to high concentrations in bacterial endospores, and is thought to play a role in spore heat resistance, or the maintenance of heat resistance. Previous work has shown that mutations in the spoVF locus of Bacillus subtilis prevent the formation of DPA, and give rise to heat-sensitive spores. Addition of exogenous DPA during spore development led to the restoration of heat resistance. This suggested that the spoVF locus encoded dipicolinic acid synthetase, the enzyme thought to catalyse the single reaction needed to synthesise DPA from dihydroxydipicolinic acid, an intermediate in the lysine biosynthetic pathway. We have now cloned and sequenced the spoVF locus of Bacillus subtilis and show that it comprises two coordinately regulated genes, now designated dpaA and dpaB. Expression of fragments of the dpa operon in Escherichia coli has shown that the two gene products together specify DPA synthetase activity. The promoter of the dpa operon, which lies just upstream of the first gene, has been identified by primer extension analysis. Sequences in this region show strong sequence similarity to several promoters recognized by the sigma K form of RNA polymerase. Transcription from this promoter was detected four hours after the onset of sporulation, at about the same time that sigma K activity is known to appear. Furthermore, transcription was abolished by mutations in a series of genes that are known to be required for the synthesis of active sigma K. These results are in accordance with previous work indicating that DPA synthetase activity was present only during the late stages of sporulation and specifically in the mother cell compartment. Transcription was enhanced by a gerE mutation, indicating that, like the previously described cotA gene, spoVF is negatively regulated by GerE. The mother-cell-specific synthesis of an enzyme responsible for a compound that accumulates to high concentrations in the prespore raises interesting questions about intercellular transport mechanisms.
吡啶二羧酸（DPA）是一种小的极性分子，它在细菌芽孢中积累到高浓度，并且被认为在芽孢耐热性或耐热性的维持中起作用。先前的研究表明，枯草芽孢杆菌spoVF位点的突变会阻止DPA的形成，并产生热敏性芽孢。在芽孢发育过程中添加外源DPA可恢复耐热性。这表明spoVF位点编码吡啶二羧酸合成酶，该酶被认为催化从二羟基吡啶二羧酸合成DPA所需的单一反应，二羟基吡啶二羧酸是赖氨酸生物合成途径中的一种中间体。我们现在已经克隆并测序了枯草芽孢杆菌的spoVF位点，并表明它由两个协同调控的基因组成，现在命名为dpaA和dpaB。dpa操纵子片段在大肠杆菌中的表达表明，这两个基因产物共同决定了DPA合成酶的活性。dpa操纵子的启动子位于第一个基因的上游，已通过引物延伸分析确定。该区域的序列与RNA聚合酶的σK形式识别的几个启动子具有很强的序列相似性。在芽孢形成开始后4小时检测到该启动子的转录，大约与已知σK活性出现的时间相同。此外，一系列已知是合成活性σK所必需的基因突变会消除转录。这些结果与先前的研究一致，表明DPA合成酶活性仅在芽孢形成的后期存在，并且特别存在于母细胞区室中。gerE突变增强了转录，表明与先前描述的cotA基因一样，spoVF受到GerE的负调控。负责一种在芽孢前体细胞中积累到高浓度的化合物的酶在母细胞中特异性合成，这引发了关于细胞间运输机制的有趣问题。
