The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate
The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate
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DOI：
10.1099/mic.0.26256-0
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发表时间：
2003-09-01
期刊：
影响因子：
2.8
通讯作者：
Aymerich, S
中科院分区：
文献类型：
--
作者：
Doan, T;Servant, P;Aymerich, S
对在糖酵解或糖异生条件下生长的野生型枯草芽孢杆菌菌株进行转录组比较。特别是，它揭示了 ywkA 基因（假定编码苹果酸酶的四个旁系同源基因之一）在糖异生过程中被更多地转录。使用与 ywkA 启动子融合的 lacZ 报告基因，表明 ywkA 是由外部苹果酸特异性诱导的，并且不受葡萄糖分解代谢物抑制。 Northern 分析证实了这种表达模式，并证明 ywkA 与下游 ywkB 基因共转录。纯化 ywkA 基因产物，生化研究证明其苹果酸酶活性，NAD 时的苹果酸酶活性比 NADP 时高 10 倍（k(cat)/K-m 分别为 102 和 10 s(-1) mM(-1)）。然而，对受 ywkA 和/或 ywkA 旁系同源物影响的单个和多个突变株进行的生理测试表明，ywkA 不利于有效利用苹果酸进行生长。转座子诱变允许鉴定出负责控制 ywkA 表达的未表征的 YufL/YufM 双组分系统。纯化的 YufM 蛋白的遗传分析和体外研究表明，YufM 结合在 ywkA 启动子的上游，并响应细胞外介质中苹果酸的存在（由 YufL 传递）激活 ywkA 转录。因此，ywkA 和 yufL/yufM 可以分别重命名为 maeA（代表苹果酸酶）和 malK/malR（代表苹果酸激酶传感器/苹果酸反应调节器）。
A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic, enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (k(cat)/K-m 102 and 10 s(-1) mM(-1), respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.