Biochemical Pathways
Below are representative transmission electron micrographs (TEM) of the WT and mutant P. gingivalis strains as well as quantitative data that measures the quantity of OMVs produced by these bacterial strains. To generate the graph, please note that the Alaei lab used a fluorescent probe to quantitate the relative levels of OMVs produced by the WT or mutant P. gingivalis strains. Use this data to answer the following questions.
(2 points) Briefly summarize the data shown in the figure. Be sure to compare the phenotypes from the bacterial strains where the GOI was deleted vs. the bacterial strains where individual amino acid substitutions were introduced into the GOI.
(2 points) Provide a hypothesis that could have been tested using this experiment. Your hypothesis should include a potential function for the protein encoded by the GOI. In other words, how might this gene be involved in OMV production?
Let’s consider the possibility that GOI encodes a phosphatase. Recall that phosphatases are enzymes that dephosphorylate proteins or other biomolecules. For the sake of simplicity, assume that this phosphatase catalyzed half of the reaction shown below (i.e. the enzyme removed one of the phosphate groups on the molecule shown on the left): 2H2O ++ 2HPO42-
(3 points) Given that the GOI product is a phosphatase, would you assume that the reaction shown above is exergonic or endergonic? Explain your reasoning.
(3 points) Porphyromonas gingivalis lives exclusively in mammalian hosts. Would you expect the phosphatase encoded by GOI to be active at -20oC? How about 150oC? Explain your reasoning.
(3 points) Given their phenotypes, did the single amino acid substitutions (R88A, H116A and H157A) alter the catalytic activity of this phosphatase? What level(s) of protein structure (primary, secondary, tertiary, quaternary) would you expect to be altered by mutations that impact phosphatase activity? Briefly explain your answer.
(3 points) Based on what you know about enzymes (and the phenotypes associated with the mutant gingivalis strains), where in the protein might the amino acid substitutions be located? Why would altering protein structure at this site result in the phenotypes that you see illustrated in the figure?
Hint: what is a common structural feature of enzymes that facilitates their role in catalyzing chemical reactions?
(4 points) Do the single amino acid substitution (R88A, H116A and H157A) mutants display the same phenotype as the null mutant strain (i.e. the mutant strain where the GOI was deleted)? What about genotypes (are they the same or different)? Briefly explain your answer.
(4 points) If Dr. Alaei’s research group had already discovered that deleting the GOI caused the phenotype shown above, why would they want to study the single amino acid substitution mutants? What would this add to our understanding of the mechanism driving OMV formation.
(4 points) Consider the specific amino acids that were mutated in the gingivalis mutants (R8A, H116A and H157A). Describe the nature of the mutations (how do the R-groups of substituted amino acids differ from the amino acids that they replaced) and predict whether these specific mutations are likely to have impacted the primary, secondary, tertiary and/or quaternary structure of the phosphatase encoded by the GOI. Explain your reasoning.
(2 points) What type of point mutations do you think underlie the amino acid substitutions in the GOI (conservative/nonconservative missense, nonsense, silent, frameshift)? Explain your reasoning.
BONUS (1 point): The substrate of the phosphatase encoded by the GOI is not a protein (see Q3). What larger macromolecule or cell structure might that molecule be a part of and how could modulating its phosphorylation level modulate budding of vesicles off the bacterial cell surface?