Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37 degrees C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30 degrees C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37 degrees C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37 degrees C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37 degrees C than at 30 degrees C. These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen.