pH Regulation in Bacillus subtilis (AREA R15)

描述(申请人提供)：枯草芽孢杆菌的pH调节。酸碱应激对细菌在土壤和水环境中生存非常重要，在土壤和水环境中，pH值变化很大，在人类宿主中，pH值变化会导致毒力。酸、碱可诱导多种多药耐药基因。肠道中依赖于pH的新陈代谢会产生酸和多胺，也就是说碱，它们的吸收促进了肿瘤的发生。在制药工业中，pH对抗生素和克隆蛋白的生长和生产起着至关重要的作用。氢酶的pH调节对氢燃料的生物生产具有重要意义。本项目的目的是研究革兰氏阳性模式生物枯草芽孢杆菌的pH调节及其对pH胁迫的响应。枯草杆菌是革兰氏阳性芽孢杆菌的主要模式生物，如炭疽杆菌、炭疽杆菌和杀虫剂苏云金杆菌。要解决的问题包括：(1)枯草杆菌胞内pH值如何随外界pH值变化？多药转运体和Na+/H+逆向转运体是否促进了细胞内的pH恢复？(2)不同细胞间的pH动态平衡是否存在差异？PH动态平衡取决于细胞周期和产孢量状态吗？(3)在酸胁迫或碱胁迫期间还可能表达哪些其他基因？上述目标将通过使用YFP荧光法和TetR-YFP构建的共聚焦显微镜测量枯草芽孢杆菌细胞质pH来实现。对大肠杆菌的初步实验表明，既可以进行稳态观察，也可以进行时间过程观察。对于枯草杆菌，将使用一个TetR-YFP基因座转化到不同的突变株中进行稳态和位移恢复实验，该突变株含有可能有助于pH动态平衡的转运蛋白缺陷。此外，还将设计一项微阵列研究，以揭示对pH压力做出反应的基因。这一领域的研究将被设计为最大限度地增加本科生研究人员的参与。每个本科生挑选一种特定的突变株或测试条件，然后写一份迷你助学金提案。鼓励一年级学生和高级分子生物学专业的学生参加，并考虑从事研究事业。学生起草稿件，并共同撰写每份手稿以供出版。酸和碱应激对细菌在宿主中的定植起重要作用，许多pH应激基因与毒力有关。将在枯草芽孢杆菌中观察到pH调节，这是一种与炭疽杆菌、炭疽杆菌和杀虫剂苏云金杆菌有关的模式生物。多药转运体和钠转运体在细菌pH调节中的作用将被调查。

DESCRIPTION (provided by applicant): pH Regulation in Bacillus subtilis. Acid and base stress are important for bacteria to survive in soil and aquatic environments, where pH varies drastically, as well as in the human host where pH change contributes to virulence. Acid and base induce numerous multidrug resistance genes. pH-dependent metabolism in the gut generates acids as well as polyamines, permeant bases whose uptake promotes tumorigenesis. In the pharmaceutical industry, pH plays critical roles in growth and production of antibiotics and cloned proteins. pH regulation of hydrogenase enzymes has implications for the biological production of hydrogen fuel. The aim of this project is to study pH regulation and response to pH stress in the gram-positive model organism, Bacillus subtilis. B. subtilis is a major model organism for gram-positive sporeformers such as B. anthracis, the causative agent of anthrax, and the insecticide B. thuringiensis. Questions to address include: (1) How does B. subtilis intracellular pH vary with external pH? Is intracellular pH recovery enhanced by multidrug transporters and Na+/H+ antiporters? (2) Does pH homeostasis vary among individual cells? Does pH homeostasis depend on the cell cycle and sporulation state? (3) What other genes may be expressed during acid stress or during base stress? The above aims will be addressed by measuring Bacillus subtilis cytoplasmic pH using YFP fluorimetry and confocal microscopy of a TetR-YFP construct. Preliminary experiments with E. coli indicate that both steady-state and time-course observation can be performed. For B. subtilis, the steady-state and shift-recovery experiments will be performed using a TetR-YFP locus transformed into different mutant strains containing defects in transporters proposed to contribute to pH homeostasis. In addition, a microarray study will be designed to reveal genes responding to pH stress. This AREA study will be designed so as to maximize participation of undergraduate researchers. Each undergraduate picks a particular mutant strain or test condition, and then writes a mini-grant proposal. First-year students as well as advanced molecular biology majors are encouraged to participate and consider pursuing research careers. Students draft the writeup and coauthor each manuscript for publication. Acid and base stress are important for bacteria colonizing the human host, and many pH stress genes contribute to virulence. pH regulation will be observed in Bacillus subtilis, a model organism related to B. anthracis, the causative agent of anthrax, and to the insecticide B. thuringiensis. The role of multidrug transporters and sodium transporters in bacterial pH regulation will be investigated.