Responses of a Newly Evolved Auxotroph of Chlamydomonas to B <sub>12</sub> Deprivation
Freddy Bunbury, Katherine E. Helliwell, Payam Mehrshahi, Matthew P. Davey, Deborah L. Salmon, Andre Holzer, Nicholas Smirnoff, Alison G. Smith
Abstract
The corrinoid B 12 is synthesized 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-B 12requiring 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 disrupts C1 metabolism, causes an accumulation of starch and triacylglycerides, and leads to a decrease in photosynthetic pigments, proteins, and free amino acids. B 12 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 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 coping with B 12 deprivation, association with B 12 -producers can ensure long-term survival whilst also providing a suitable environment for evolving mechanisms to tolerate B 12 limitation better.