Growth in acid and base is 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 fermentation industry, pH plays critical roles in growth and production. pH regulation of hydrogenase enzymes has implications for the biological production of hydrogen fuel.
It is hypothesized that genes whose expression responds to pH change contribute to pH homeostasis and extreme-pH survival, by several mechanisms. The project will characterize a number of putative pH stress responses arising from DNA array studies of global pH stress comparing aerobic and anaerobic cultures. The arrays revealed a set of "core pH genes" up-regulated in acid or base, of which thirty showed rapid response in an acid-shift experiment. An example is the inner membrane protein gene yagU. For yagU and other core pH genes, knockout mutants and overproducing clones will be screened for anaerobic growth in moderate acid or base (pH 5 or pH 8.5), and for survival in extreme acid or extreme base (pH 2.0 or pH 9.8). Another important class of pH-dependent genes is the hydrogenases, which are up-regulated in acid with anaerobiosis, but up in base with aeration. It is hypothesized that hydrogenases convert H2 to 2H+ at high pH, and that they may remove acidity by the reverse reaction. H2 production and consumption, at low pH vs. high pH, will be tested by microrespirometry. Hydrogenase mutants will be tested for effects on growth and survival at low pH vs. high pH. Mutants will be characterized for pH homeostasis using a new method of bacterial pH measurement devised through the current NSF-funded project, based on pH-titratable YFP and GFP reporter gene fusions.
Broader impacts. The project will contribute to the nation's human resources by continuing an innovative research program run by undergraduates. Undergraduates write their own mini-proposals, design and conduct the experiments, and participate in writing up the reports for publication. Most of the undergraduates attracted to work on the current project decide to pursue careers in science. In the wider community, the PI conducts middle-school teacher workshops on science education and addresses civic organizations on current advances in microbiology.
Our research addressed how the intestinal bacterium Escherichia coli K-12 grows at low pH (moderate acid, pH 5-7) and survives exposure to extreme low pH (extreme acid, pH 2-3). E. coli is a model for ingested bacteria that survive the extreme acid of gastric fluid in the stomach, then grow in the intestines in moderate acid. Intestinal bacteria include host flora that enhance human health, as well as strains that cause disease. E. coli is also the workhorse of biotechnology, engineered to produce many kinds of industrial products. With respect to Criterion 2: This RUI project trained 23 undergraduate researchers, including 11 under-served minorities (representative members shown in Figure 1). Five have since entered PhD or predoctoral research programs, and six others plan to do so. Seven Kenyon undergraduates plus a recent graduate from another college have co-authored four primary publications on genes that help E. coli grow and survive in acid. For more information: http://biology.kenyon.edu/slonc/slonc.htm (1) Our microarray study revealed a large number of genes whose RNA is expressed when E. coli is exposed to a permeant acid, benzoic acid, a compound used for food preservation. Benzoic acid releases hydrogen ions inside the cell, and thus acidifies the cytoplasm, causing a severe acid stress. Benzoate was shown to up-regulate (increase production of messenger RNA) genes involved in biofilm and pili formation (Kannan et al., 2008). This means that formation of biofilms and pili may involve cytoplasmic acid stress. (2) Hydrogen biofuel production is a major aim of applied bacteriology. Bacteria generate hydrogen via hydrogenase enzymes, which interconvert hydrogen ions with hydrogen gas. We showed that hydrogenase complex 3 is needed for extreme-acid survival of E. coli, but only when grown anaerobically (with very low oxygen). Our finding represents the first low-oxygen specific component of acid resistance shown in E. coli. At low external pH, the hydrogenase 3 converts 2H+ to H2 (Figure 2). Thus, hydrogenase 3 provides a way to consume excess acid. By contrast, hydrogenase 2 acts in the opposite direction, converting hydrogen gas to hydrogen ions. Thus, hydrogenase 2 is formed primarily at high external pH, whereas hydrogenase 3 is formed at low external pH, where it contributes to extreme-acid survival (Noguchi et al., 2010). A strain forming only hydrogenase 3 produces increased hydrogen gas, especially at low pH. Our findings add important insights as to the regulation of hydrogen production. (3) Cytoplasmic pH (the pH inside the bacterial cell) must be maintained within a narrow range (about pH 7.3-7.8) for the bacteria to grow. My undergraduates devised a new technique to measure cytoplasmic pH using fluorimetry of green fluorescent protein (GFP) (Wilks & Slonczewski, 2007). This technique was used to measure for the first time the periplasmic pH of E. coli. (4) We used GFP fluorimetry to show that osmolytes such as NaCl and proline provide pH homeostasis in a K+ transport-defective strain (Kitko et al., 2010). Previously, it was thought that only exogenous K+ could enable pH homeostasis in a K+ transport-defective strain.
