A METE mutant of Chlamydomonas reinhardtii provides new perspectives on the evolution of vitamin B12 auxotrophy

Vitamin B12 is synthesised only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Roughly half of all algae require vitamin B12, and the phylogenetic distribution of this trait suggests that it has evolved on multiple occasions. Previous work using artificial evolution generated a metE mutant of Chlamydomonas reinhardtii (hereafter metE7) that requires B12 for growth. Here, I use metE7 to investigate how a newly-evolved B12 auxotroph might cope with B12 limitation and interact with B12-producing bacteria. Compared to the closely related natural B12 auxotroph Lobomonas rostrata, metE7 has a higher requirement and lower binding affinity for B12. B12 deprivation of metE7 caused an increase in cell diameter, indicative of a decreased rate of cell division relative to growth. Other responses included an accumulation of starch and triacylglycerides at the expense of polar lipids and free fatty acids, and a decrease in photosynthetic pigments, proteins and free amino acids. This is reminiscent of nitrogen deprivation, but closer investigation revealed that B12 deprivation caused a substantial increase in reactive oxygen species, which preceded a rapid decline in cell viability. This might be explained by the observation that there was no induction of non-photochemical quenching, unlike under nitrogen deprivation. The metabolite S-adenosyl homocysteine, a potent inhibitor of methylation, increased substantially during B12 deprivation, as did the transcripts for several enzymes of one-carbon metabolism. The rhizobial bacterium Mesorhizobium loti formed a commensal relationship with wild-type C. reinhardtii, benefiting from the alga’s photosynthate. When co-cultured with metE7 this interaction should be considered a mutualism as the alga was dependent on bacterial B12. Adding B12 or glycerol to the coculture increased the cell density of metE7 and M. loti respectively, revealing that the rate of nutrient transfer between species was the factor limiting growth. It was unsurprising therefore that when grown in triculture, the wild type soon outcompeted metE7, even when M. loti growth and B12 production were fuelled by added glycerol. B12 release was also shown to be critical for mutualism: a mutant of E. coli that released more B12 into the media was, in contrast to its parental strain, able to support metE7. The mutual support of metE7 and M. loti over 6 months during an artificial evolution experiment illustrates that newly-evolved B12 auxotrophs could survive in the presence of B12producers, but it was also demonstrated that M. loti could not produce sufficient B12 to favour the evolution of B12 dependence in C. reinhardtii. In summary, this work has provided an insight into the challenges of evolving B12 auxotrophy making it all the more remarkable that it is such a common trait.Biotechnology and Biological Sciences Research Council (BBSRC