How Hemorrhagic E. coll pH Sensitivity of the Glu-GABA Antiporter Resists the Acid Environment of the Stomach il.. 300 GadC AdiC 250 Recent years have been marked by a series of food poisoning outbreaks involving hemorrhagic (producing internal bleeding) strains of the bacterium Escherichia coli (E. coli). Bacteria are often a source of food poisoning. typically milder infections caused by food-borne strepto- coccal bacteria. Less able to bear the extremely acidic conditions encountered by food in the human stomach 200 150 t00- (pH 2), E. coli has not been as common a problem. The 50 hemorrhagic strains of E. coli responsible for recent out- breaks seem to have evolved more elaborate acid-resistance systems. 5 pH How do hemorrhagic E. coli bacteria survive in the acid environment of the stomach? The problem they face, in essence, is that they are submerged in a sea of hydrogen ions, many of which diffuse into their cells. To rid them- selves of these excess hydrogen ions, the E. coli cells use a clever system to pump hydrogen ions back out of their cells. Analysis 1. Applying Concepts a. Variable. In the graph, what is the dependent variable? First, the hemorrhagic E. coli cells take up cellular hydrogen ions by using the enzyme glutamic acid decarbox- ylase (GAD) to convert the amino acid glutamate to y-aminobutyric acid (GABA), a decarboxylation reaction that consumes a hydrogen ion. Second, the hemorrhagic E. coli export this GABA from their cell cytoplasm using a Glu-GABA antiporter called GadC (this transmembrane protein channel is called an antiporter because it transports two molecules across the membrane in opposite directions). However, to survive elsewhere in the human body, it is important that the Glu-GABA antiporter of hemorrhagic E. coli not function, lest it short-circuit metabolism. To see if investigation, what are the substrates that are accumulating? pH. What is the difference in hydrogen ion concentration between pH 5 and pH 7? How many times more (or less) is that? Explain. b. Substrate. What is a substrate? In this C. a. Does the amount of amino acid transported in the 10-minute experimental interval (expressed as substrate accumulation) vary with pH for the arginine-transporting AdiC antiporter? For the glutamate-transporting GadC antiporter? b. Compare the amount of substrate accumulated by AdiC in 10 minutes at pH 9.0 with that accumulated at pH 5.0. What fraction of the low pH activity is observed at the higher pH? c. In a similar fashion, compare the amount of substrate accumulated 2. Interpreting Data the GadC antiporter indeed functions only in acid environ- ments, investigators compared its activity at a variety of pHs with that of a different amino acid antiporter called AdiC, which transports arginine out of cells under a broad range of conditions. The results of monitoring transport for 10 minutes are presented in the graph. GadC at pH9.0 by with that accumulated at pH 5.0. What fraction of the low pH activity is observed at the higher pH? Making Inferences Would you antiporter exhibits the same pH dependence as the AdiC antiporter? If not, which antiporter is less active at nonacid pHs? 4. Drawing Conclusions Is the glutamate-GABA antiporter GadC active at nonacid pHs? 5. Further Analysis The GadC antiporter also transports the amino acid glutamine (Gln). Do you think this activity has any role to play in combating low pH environments? How would you test this hypothesis? Inside Outside say that the GadC cell 3. cell GABA Glutamate Chapter 5 Membranes Substrate accumulation (nmol per mg protein) Inquiry& Analysis