Morwan Osman and Jamie Blaza

Morwan Osman and Jamie Blaza

Measuring metabolism in living cells with remission spectroscopy: introducing the approach and recent results on the antitubercular drug bedaquiline and germination of bacterial spores

Bioenergetic systems transduce the energy that organisms need to sustain themselves and grow. In aerobic organisms, they work by transferring electrons from donor molecules to O2, building an electrochemical gradient in the process. This gradient then drives the synthesis of ATP. Measuring this system in vivo is demanding because the high metabolic flux means any attempt to break open cells to extract and measure (‘grind and find’) will perturb the system before measurements can be made. We use remission spectroscopy to measure cytochrome/haem spectra in living cells without any disruption to the cells. As the spectrum of haems changes if they are carrying an electron or not and are integral components of electron transfer chains they provide a remarkable spectroscopic handle to measure bioenergetics in vivo. Two case studies of this approach will be presented to illustrate the approach. The first focuses on the mechanism of the antitubercular drug bedaquiline and uses the changes that occur at the electron transfer chain to infer how bedaquiline acts at ATP synthase resolving whether bedaquiline uncouples or blocks the enzyme. The second focuses on bacterial spores as they germinate and our observations on the temporal order of re-hydration and resumption of metabolic activity.