Researchers examined the ability of cultures of the bacterium Pseudomonas aeruginosa to adapt to the antibiotics ceftazidime and avibactam when they are administered together. The researchers grew four replicate cultures in growth medium with no antibiotics added (control) and four additional replicate cultures in the same growth medium with added ceftazidime and avibactam. At the conclusion of the experiment, samples of the replicates exposed to the antibiotics were removed and their genomes were sequenced. All of the antibiotic-resistant mutants were missing three genes (hmgA, galu, mexXY). It is known that one of these three genes makes wild-type P. aeruginosa susceptible to the two antibiotics. However, the researchers do not know which gene it is. Which of the following experiments would specifically determine which gene is responsible for ceftazidime- avibactam sensitivity in P. aeruginosa ?
A. Clone the three deleted genes from a wild-type P. aeruginosa strain, and insert these sequences into the mutant bacterial strains to restore sensitivity to ceftazidime-avibactam.
B. Delete genes that are homologous to the three deleted genes in other bacterial species, and determine if doing so also confers ceftazidime-avibactam resistance in those bacteria.
C. Perform qene knockout (targeted mutation) of either the hmgA, galu, or mexXY gene on separate cultures of the wild-type P. aeruginosa. For each individual mutant, determine whether or not ceftazidime-avibactam resistance has occurred.
D. Perform gene knockout (targeted mutation) of both the hmgA andgalu genes of the wild-type P. aeruginosa in a single culture, and then determine if ceftazidime-avibactam resistance has occurred in the mutant strain.