Acid and base affect bacterial growth and survival in soil and aquatic environments, impacting soil productivity and fisheries. In the human host, acid and base induce multidrug resistance genes. In the fermentation industry, pH (a measure of the acidity or alkalinity of a solution) plays critical roles in growth and production. For example, pH regulation of hydrogenase enzymes affects the biological production of hydrogen fuel. In industrial processes, bacteria often form biofilms in which the pH is decreased, accelerating corrosion; thus, acid stress response amplifies the role of biofilms in equipment deterioration. The proposed project addresses these questions: (1) How is pH regulated in single cells versus biofilms? (2) How do multidrug efflux pumps impact bacterial survival in extreme acid or base? (3) What novel components of acid survival and base stress are revealed at low oxygen? To address these questions, bacterial cell pH will be measured using fluorescence microscopy. The export of acidic products from drug efflux pumps will be measured. Acid and base stress will be tested during bacterial growth in low oxygen.
Broader Impacts. This Research at Undergraduate Institutions Award will contribute to the nation's human resources by continuing an innovative research program run by undergraduates. Undergraduates are recruited in their first year to start in the lab, learning basic techniques while working with more advanced peers. Students then write their own mini-proposals, design and conduct the experiments, and draft the manuscript for publication. Most undergraduates attracted to work on the current project decide to pursue careers in science. In the wider community, the PI assists middle-school teachers and conducts teacher workshops on hands-on science education.
Undergraduate students at Kenyon College investigated how bacteria survive acid-stressed environments, such as the soil acidified by rain, or the acidic interior of the stomach. Students observed the bacterium Escherichia coli K-12, a laboratory strain of the E. coli that normally inhabits our intestine and helps digest our food. Related bacteria occur in all natural environments, where they cycle nutrients and help plants grow. Students also tested the acid-stress survival of bacteria cultured in an anaerobic chamber, where oxygen is kept to extremely low levels, similar to conditions found in deep soil, or in the intestinal lumen. The students investigated whether bacterial genes required for acid survival in oxygen are also required in anoxic environments. Surprisingly, the students found that under anoxic conditions, bacteria do not require the most important acid-survival genes needed in the presence of oxygen. Thus, under anoxic conditions bacteria possess entirely different acid survival mechanisms. One important mechanism for acid survival is to maintain a constant pH inside the cell. Students used fluorescence microscopy to measure the bacterial cell’s internal pH, during culture at various levels of external pH. They watched bacteria grow in a biofilm, a community of cells attached to a slide. Surprisingly, the biofilm bacteria maintained a more steady internal pH than previous reports showed for suspended bacteria. Since many bacteria form biofilms upon soil particles, and in disease conditions such as the cystic fibrosis lung, it is important to show how their pH is maintained. pH maintenance within biofilms may be related to increased drug resistance of biofilm bacteria. As bacteria proliferate by cell fission, each new cell contains an old cell pole (from the parent cell) and generates a new cell pole (by cell division). Cell fission can thus lead to polar aging, a situation in which the older cell poles show weaker stress response than the newer cell poles. Students investigated the role of polar aging during biofilm growth under pH stress. They observed biofilm growth at moderately low pH (pH 6) and at near-neutral pH (pH 7.5). The students tracked the growth of individual cells within a biofilm, comparing the rates of cell division of cells inheriting a relatively new poles versus a pole several generations old. It was found that at pH 6, the old-pole cells grow and divide more slowly than the new-pole cells. The broader impact of this research is that understanding bacterial pH stress response may help us develop new means of biotechnology to control bacterial growth, and new insights to improve the microbial quality of soil for agriculture. In addition, the reported research trained undergraduate students who then pursued research programs at Massachusetts Institute of Technology, at University of Southern California, and at the US Food and Drug Administration.
