3) To determine which glucose oxidizing pathway species of bacteria use, scienti

Question

3) To determine which glucose oxidizing pathway species of bacteria use, scientists have design respirometric experiments using glucose labeled with radioactive carbon (14C) in various position. Then, scientists collect the CO2 released soon after the bacteria were fed with the radiolabeled glucose. It is assumed that the released CO2 is the result of the decarboxylation of pyruvate (EMP and ED pathways) or 6-phosphogluconate (HMP pathway). Remember that, under aerobic conditions, the carboxyl group of pyruvate is released as CO2 when pyruvate reacts with coenzyme A to produce acetyl-coenzyme A.
a) Three separate analyses of glucose degradation by the gram-negative bacteria E. coli through radiorespirometry were performed using each time a different types of radiolabeled glucose molecules: glucose with carbon #1 labeled, or glucose with carbon #2 labeled, or glucose with carbons #3 and 4 labeled. The graph below (Figure 4) shows the results of the 3 experiments. Based on the pathways given in figures 1 to 3, rank (when possible) metabolic pathway (EMP, HMP, or ED) by order of predominance in E. coli? Briefly explain. (8 pts)

Figure 4: Graph showing the amount of 14CO2 release when glucose labeled on carbon #1(14C(#1)), #2 (14C(#2)) and #3 AND 4 (14C(#3&4) were used to feed E. coli. The Y-axis represents the amount of 14CO2 released.

b) Based on your knowledge of the fate of acetyl-coenzyme A under aerobic conditions, which metabolic pathway is likely at the origin of the 14CO2 released at the later stage of the experiment when glucose is labeled on carbon #2 or on carbon #1? Based on your answer in part b), would you modify your answer from part a)? Explain your reasoning. (6 pts)

c) The following two graphs show the same types of respirometric experiments with two strains of bacteria (A and B). Based on this graph, identify which strain is gram-negative. Briefly
explain. (6 pts)

Figure 5: Graph showing the amount of 14CO2 release at the early stage when glucose labeled on
carbon #1 (14C(1)), #3 (14C(3)) and #4 (14C(4) were used to feed two different bacterial strains.
The Y-axis represents the amount of 14CO2 released.

Explanation / Answer

Ans. A) In hexose monophosphate shunt (HMP pathway), the glucose is labeled at its 1st carbon that is released as carbon dioxide. The remaining five carbon sugars again go into into the glycolysis. In case of ED (Entner-Doudoroff) pathway, the carbon dioxide is released from the C1 and C4 of glucose, which eventually releases acetyl CoA (from 2nd and 5th carbon). On the other hand in the Embden-Meyerhof-Parnas (EMP) pathway, the carbon is released from the C3 and C4 of carbon. Therefore, as per the graph, the prevalence of pathways in E.coli is as followed

EMB pathway > HMP pathway > ED pathway

B). The acetylCoA synthesized in ED (Entner-Doudoroff) pathway, again enters into the citric acid cycle under aerobic conditions and releases two molecules of carbon dioxide. Consequently, the order of predominant pathways alters as -

EMB pathway > ED pathway > HMP pathway

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