Isoprenoid compounds form a large ubiquitous class of natural products that fulfill a wide variety of essential cellular functions in all living organisms. In eukaryotes, isoprenoid compounds are synthesized by a mevalonate (MVA) dependent pathway. However, in many bacteria, these compounds are synthesized by an alternative, MVA-independent route whose first committed intermediate is 2-methylerythritol 4-phosphate (MEP). Both pathways converge at isopentenyl diphosphate (IPP) and subsequent steps are similar in all organisms. Since the MEP pathway is absent in mammals, it is considered an attractive target for the development of novel antibiotics. The pathogen, M. tuberculosis belongs to the MEP pathway class of organisms. The applicants will prepare a transgenic bacterial E. coli host cell whose genome contains disruptions in a first endogenous gene in the MEP pathway and a second endogenous gene which is located downstream of the first gene in the MEP pathway. A transgene from M. tuberculosis that functionally replaces the disrupted downstream gene will be cloned into the cell. The growth of the host cell in the presence of test agent is then compared to a control culture to determine the activity of the test agent. Where the test agent renders the bacterial host cell nonviable on media containing a chemical supplement to relieve the 1-deoxy-D-xylulose-5-phosphate (DXP) block and viable on the media containing MVA indicates that the test agent has the potential to be an effective antibacterial against M. tuberculosis.
