2. You isolate a Gram positive alkalinophile that grows at a pH = 10. At this external pH, the intracellular pH is maintained at 7.3. Using the equation proton motive force (pmf), determine the contributions of the electrical and chemical energy components to the pmf in this bacterium. 

3. You are studying the type VI secretion system in Pseudomonas aeruginosa. You analyze the genome and find an operon comprised of 5 genes that you believe is involved building the secretion apparatus. You name the genes tssA, tssB, tssC, tssD, and tssE based simply on their order. You believe that tssA(the first gene in the operon) encodes the crucial component of the secretion apparatus. Design an experiment to make a knockout of tssA. How will you determine if the mutation in tssA is a polar mutation? 

4. You obtain a clinical isolate of the Gram positive pathogen Clostridium difficile that is highly resistant to vancomycin. This is a significant health concern as vancomycin is a ‘last line of defense’ for treatment of C. difficile infections. You notice that all of the clinical isolates were taken from patients suffering from a multispecies infection, including a vancomycin resistant strain of E. faecalis. What couldexplain this? State your hypothesis and develop experiments to test it. 

5. You treat 107Pseudomonas aeruginosa growing planktonically and as a biofilm with high levels of the antibiotic tobramycin (10 micrograms/ml) for 2 hours. No surviving planktonic bacteria are recovered, although 104 biofilm bacteria survive this treatment. Design an experiment to identify genes important for biofilm resistance in P. aeruginosa.