Responses of a Newly Evolved Auxotroph of Chlamydomonas to B 12 Deprivation

The corrinoid B is synthesized only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B dependence on multiple occasions, and we previously demonstrated that experimental evolution of the non-B -requiring alga in media supplemented with B generated a B -dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B auxotroph. Our analyses demonstrate that B deprivation of metE7 disrupts C1 metabolism, causes an accumulation of starch and triacylglycerides, and leads to a decrease in photosynthetic pigments, proteins, and free amino acids. B deprivation also caused a substantial increase in reactive oxygen species, which preceded rapid cell death. 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 limitation and an increase in B use efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B -producing bacterium. Therefore, although an early B -dependent alga would likely be poorly adapted to coping with B deprivation, association with B -producers can ensure long-term survival whilst also providing a suitable environment for evolving mechanisms to tolerate B limitation better.