Provided herein are methods, and related compositions for identifying a putative essential gene which serves as an antibiotic target in a bacterium, the method, in some embodiments, comprising (a) generating an antibiotic resistant mutant of the bacterium by a method comprising the step of selecting for growth in the presence of an antibiotic to produce an antibiotic resistant mutant clone (AbR mutant)； (b) transforming the AbR mutant with one or more essential genes of the bacterium and a transposon which insertionally inactivates bacterial DNA to produce a pool of transposon mutants which are merodiploid for the one or more essential genes； (c) growing bacteria from the merodiploid pool in the presence of different amounts of the antibiotic to produce two or more test cultures； and (d) comparing the distribution of transposon insertions between the test cultures to identify a putative essential gene serving as a target of the antibiotic in the bacterium.
