Osmotically Controlled Synthesis of the Compatible Solute Proline Is Critical for Cellular Defense of Bacillus subtilis against High Osmolarity
Osmotically Controlled Synthesis of the Compatible Solute Proline Is Critical for Cellular Defense of Bacillus subtilis against High Osmolarity
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DOI：
10.1128/jb.05490-11
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发表时间：
2011-10-01
影响因子：
3.2
通讯作者：
Bremer, Erhard
中科院分区：
文献类型：
--
作者：
Brill, Jeanette;Hoffmann, Tamara;Bremer, Erhard
枯草芽孢杆菌在高盐环境中通过从头合成积累大量的可溶性脯氨酸作为胁迫保护剂。我们阐明了遗传决定因素所需的适应性脯氨酸生产的前体谷氨酸。该脯氨酸生物合成途径依赖于proJ编码的γ-谷氨酰激酶、proA编码的γ-谷氨酰磷酸还原酶和proH编码的Delta（1）-吡咯啉-5-羧酸还原酶。proHJ操纵子的破坏消除了自适应脯氨酸的产生，并强烈损害了B的能力。枯草杆菌以科普高渗透压的生长条件。破坏的proA基因也废除了自适应脯氨酸的生物合成，但造成的，相反，破坏proHJ，脯氨酸营养缺陷型。北方印迹分析表明proHJ操纵子的转录是可诱导的，而proBA操纵子的转录不是。报告基因融合研究表明，proHJ表达迅速诱导后，渗透上移。只要渗透刺激持续存在，表达增加就会维持，并且与生长培养基的普遍渗透压敏感相关。引物延伸分析揭示了一种类似于B的典型SigA型启动子的受遗传控制的proHJ启动子。枯草芽孢杆菌proHJ启动子区的缺失分析鉴定了一个126-bp的DNA片段，该片段携带所有顺式调节转录所需的序列。我们的数据揭示了在B中存在与ProA相关的合成代谢和自适应脯氨酸生物合成途径。枯草杆菌和证明，兼容的溶质脯氨酸的合成是一个中心方面的细胞防御高渗透压环境，这种土壤细菌。
Bacillus subtilis is known to accumulate large amounts of the compatible solute proline via de novo synthesis as a stress protectant when it faces high-salinity environments. We elucidated the genetic determinants required for the osmoadaptive proline production from the precursor glutamate. This proline biosynthesis route relies on the proJ-encoded gamma-glutamyl kinase, the proA-encoded gamma-glutamyl phosphate reductase, and the proH-encoded Delta(1)-pyrroline-5-caboxylate reductase. Disruption of the proHJ operon abolished osmoadaptive proline production and strongly impaired the ability of B. subtilis to cope with high-osmolarity growth conditions. Disruption of the proA gene also abolished osmoadaptive proline biosynthesis but caused, in contrast to the disruption of proHJ, proline auxotrophy. Northern blot analysis demonstrated that the transcription of the proHJ operon is osmotically inducible, whereas that of the proBA operon is not. Reporter gene fusion studies showed that proHJ expression is rapidly induced upon an osmotic upshift. Increased expression is maintained as long as the osmotic stimulus persists and is sensitively linked to the prevalent osmolarity of the growth medium. Primer extension analysis revealed the osmotically controlled proHJ promoter, a promoter that resembles typical SigA-type promoters of B. subtilis. Deletion analysis of the proHJ promoter region identified a 126-bp DNA segment carrying all sequences required in cis for osmoregulated transcription. Our data disclose the presence of ProA-interlinked anabolic and osmoadaptive proline biosynthetic routes in B. subtilis and demonstrate that the synthesis of the compatible solute proline is a central facet of the cellular defense to high-osmolarity surroundings for this soil bacterium.