Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant
Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant
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DOI：
10.1111/1758-2229.12531
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发表时间：
2017-06-01
影响因子：
3.3
通讯作者：
Commichau, Fabian M.
中科院分区：
文献类型：
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作者：
Dormeyer, Miriam;Luebke, Anastasia L.;Commichau, Fabian M.
谷氨酸是合成代谢反应中氮的主要供体。革兰氏阳性土壤细菌枯草芽孢杆菌利用外源提供的谷氨酸或使用gltAB编码的谷氨酸合酶（GOGAT）合成谷氨酸。在没有谷氨酸的情况下，转录因子GltC激活GOGAT基因的表达以产生谷氨酸。因此，gltC突变株是谷氨酸营养缺陷型的。利用遗传选择和筛选系统，我们可以分离和区分gltC抑制突变体在一个步骤。所有突变体都获得了独立于GltC合成谷氨酸的能力。我们鉴定了（i）编码转录因子GltR的gltR基因中的功能获得性突变，（ii）gltAB操纵子启动子中的突变和（iii）含有gltAB操纵子的基因组位点的大规模扩增。属于前两类的突变体组成型表达gltAB基因，并产生足够的谷氨酸生长。相比之下，属于第三类的突变体出现频率最高，并通过增加低表达gltAB基因的拷贝数来解决谷氨酸限制。因此，谷氨酸营养缺陷型的一个B。枯草芽孢杆菌gltC突变体可以以多种方式缓解。此外，gltAB基因的重组依赖性扩增是指示突变层次的主要突变事件。
Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations.