Exploring the onset of B₁₂-based mutualisms using a recently evolved Chlamydomonas auxotroph and B₁₂-producing bacteria

Abstract

<jats:title>Summary</jats:title><jats:p>Cobalamin (vitamin B<jats:sub>12</jats:sub>), is a cofactor for crucial metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonise the algal phycosphere, forming stable communities that gain preferential access to exudates and in return provide compounds, such as B<jats:sub>12</jats:sub>. Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B<jats:sub>12</jats:sub>-dependent strain of <jats:italic>Chlamydomonas reinhardtii</jats:italic>, metE7, forms a mutualism with certain bacteria, including the rhizobium <jats:italic>Mesorhizobium loti</jats:italic> and even a strain of the gut bacterium <jats:italic>E. coli</jats:italic> engineered to produce cobalamin. Although metE7 was supported by B<jats:sub>12</jats:sub> producers, its growth in co-culture was slower than the B<jats:sub>12</jats:sub>-independent wild-type, suggesting that high bacterial B<jats:sub>12</jats:sub> provision may be necessary to favour B<jats:sub>12</jats:sub> auxotrophs and their evolution. Moreover, we found that an <jats:italic>E. coli</jats:italic> strain that releases more B<jats:sub>12</jats:sub> makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B<jats:sub>12</jats:sub> release provided an advantage in co-cultures. We hypothesise that, given the right conditions, bacteria that release more B<jats:sub>12</jats:sub> may be selected for, particularly if they form close interactions with B<jats:sub>12</jats:sub>-dependent algae.</jats:p><jats:sec><jats:title>Originality-Significance statement</jats:title><jats:p>Microalgae are fundamental to the global carbon cycle, and yet despite being photosynthetic they often rely on other organisms for micronutrients. One of these micronutrients is vitamin B<jats:sub>12</jats:sub> (cobalamin), which they receive from bacteria. Many environmental studies support the widespread role of B<jats:sub>12</jats:sub> in algal-bacterial mutualisms, so here we wished to investigate how these mutualisms may arise evolutionarily by using an experimentally evolved B<jats:sub>12</jats:sub>-dependent alga and various B<jats:sub>12</jats:sub>-producers. A B<jats:sub>12</jats:sub>-producing rhizobium, <jats:italic>Mesorhizobium loti</jats:italic>, could stably support the B<jats:sub>12</jats:sub>-dependent <jats:italic>Chlamydomonas reinhardtii</jats:italic> metE7 strain, and vice versa, but nutrient supplementation increased growth of both species further. metE7 could also be supported by <jats:italic>E. coli</jats:italic> strains engineered to produce B<jats:sub>12</jats:sub>, and engineering a strain to release a higher proportion of B<jats:sub>12</jats:sub> led to better algal growth, which increased bacterial growth in turn. We suggest that as B<jats:sub>12</jats:sub>-based mutualisms develop, increased B<jats:sub>12</jats:sub> release may be selected for and therefore lead to more productive symbioses.</jats:p></jats:sec>