Physiological and molecular responses of a newly evolved auxotroph of Chlamydomonas to B12 deprivation

The corrinoid B 12 is synthesised only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B 12 dependence on multiple occasions and we previously demonstrated that experimental evolution of the non-requiring alga Chlamydomonas reinhardtii in media supplemented with B 12 generated a B 12 -dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B 12 auxotroph. Our analyses demonstrate that B 12 deprivation of metE7 disrupted C1 metabolism, caused an accumulation of starch and triacylglycerides and a decrease in photosynthetic pigments, proteins and free amino acids. B 12 deprivation also caused a substantial increase in reactive oxygen species (ROS), which preceded rapid cell death. Surprisingly, survival could be improved without compromising growth by simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found further improvements in survival under B 12 limitation and an increase in B 12 use-efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B 12 -producing bacterium. Therefore, although an early B 12 -dependent alga would likely be poorly adapted to B 12 deprivation, association with B 12 -producers can ensure long-term survival whilst also providing the environment to evolve mechanisms to better tolerate B 12 limitation.