Reading_Transport - Transport of Nutrients by Microbial...

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Unformatted text preview: Transport of Nutrients by Microbial Cells In the lecture we will talk about some of the experimental evidence for transporters and what are the advantages of having so many different types of transporters. Textbook Reading Page 133-138, “Uptake of Nutrients into Cells” What you should get out of this text: 1. Understand why energy-dependent transport of nutrients into cells is necessary to support growth of most bacteria. (Why would a transporter be needed even for a nutrient that could passively diffuse across the membrane?) 2. Be familiar with facilitated diffusion. 3. What is the function of energy in transport systems? 4. Know that ABC transporters are comprised of a solute-binding protein, a transmembrane protein, and an ATPase. 5. Know the location of the solute-binding protein in Gram-negative versus Gram-positive bacteria. 6. Be able to explain what can be the energy sources for the secondary transporters (uniporter, symporter, and antiporter). 7. What is the energy source for the PTS transporter? 8. What happens to a sugar as it is transported by the PTS? 9. Know the role of a siderophore in transporting iron and how iron is released from the siderophore. Measuring the Rate of Transport The rate at which a transporter moves a substrate across a membrane can be measured using whole cells. Use of a radioactive substrate allows for easy monitoring for the location of the substrate. Cells and a particular concentration (also called an initial concentration) of the radioactive substrate are incubated together for a set period of time. The free, extracellular substrate is then separated from the cells, which contain the substrate that was transported into the cells. To separate the free substrate from the cells, the reaction is filtered over a 0.2 µm filter. This filter will retain cells but not the free substrate. The amount of radioactivity associated with the cells on the filter is then measured using a scintillation counter. The change in the amount of radioactivity associated with the cells divided by the amount of time the cells were incubated with the substrate is equal to the “rate of transport”. The “rate of transport” at different initial concentrations of substrate is then measured and plotted. If a substrate enters the cell using a transporter, then the rate of reaction with initially increase with increasing substrate concentration but will plateau at a maximal rate. For a substrate that can passively diffuse into the cell, the rate increase with increasing concentration of the substrate, but the rate does not plateau. Functional Differences Between Classes of Transporters We will compare binding-protein dependent (BPD) transporters versus electrochemical gradient (ECG) transporters. BPD transporters have a higher affinity for their substrate. BDP can achieve a concentration gradient of substrate of 105 inside/1 outside, whereas ECG can achieve a gradient of 102-103 inside/1 outside. However, ECG transporters have a faster rate of transport than the BPD transporters. ...
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