pH Regulation in Bacillus subtilis (AREA R15)

描述（由申请人提供）：枯草芽孢杆菌pH调节。酸碱胁迫对于细菌在pH值变化剧烈的土壤和水生环境中生存以及在pH值变化有助于毒性的人类宿主中生存是重要的。酸和碱诱导多种耐药基因。肠道中ph依赖性代谢产生酸和多胺，这些渗透碱的摄取促进肿瘤发生。在制药行业，pH值在抗生素和克隆蛋白的生长和生产中起着至关重要的作用。氢化酶的pH调节对氢燃料的生物生产具有重要意义。本项目旨在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。枯草芽孢杆菌是革兰氏阳性孢子形成菌的主要模式生物，如炭疽芽孢杆菌（炭疽的病原体）和杀虫剂苏云金芽孢杆菌。需要解决的问题包括：(1)枯草芽孢杆菌细胞内pH值如何随外部pH值变化？多药转运体和Na+/H+反转运体是否能促进细胞内pH恢复？(2)单个细胞的pH稳态是否不同？pH稳态是否依赖于细胞周期和产孢状态？(3)在酸胁迫或碱胁迫下还可能表达哪些基因？上述目的将通过使用YFP荧光法和tet -YFP结构的共聚焦显微镜测量枯草芽孢杆菌的细胞质pH来解决。大肠杆菌的初步实验表明，可以进行稳态和时程观察。对于枯草芽孢杆菌，稳态和位移恢复实验将通过将一个ter - yfp基因座转化为不同的突变菌株进行，这些突变菌株含有被认为有助于pH稳态的转运蛋白缺陷。此外，将设计微阵列研究以揭示对pH胁迫作出反应的基因。本区域研究的设计将使本科生研究人员的参与最大化。每个本科生选择一个特定的突变菌株或测试条件，然后写一份小型拨款提案。我们鼓励一年级学生和高级分子生物学专业的学生参与并考虑从事研究工作。学生们起草文章，并共同撰写每篇稿件以供发表。酸碱胁迫对细菌定殖于人类宿主是重要的，许多pH胁迫基因有助于毒力。pH调节将在枯草芽孢杆菌中观察到，枯草芽孢杆菌是一种与炭疽芽孢杆菌（炭疽的病原体）和苏云金芽孢杆菌有关的模式生物。多药转运体和钠转运体在细菌pH调节中的作用将被研究。

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.