pH Regulation in Bacillus subtilis (AREA R15)

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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.