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

Abstract

Cobalamin (vitamin B₁₂ ) is a cofactor for essential metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonize the algal phycosphere, forming stable communities that gain preferential access to photosynthate and in return provide compounds such as B₁₂ . Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B₁₂ -dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B₁₂ producers, its growth in co-culture was slower than the B₁₂ -independent wild-type, suggesting that high bacterial B₁₂ provision may be necessary to favour B₁₂ auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B₁₂ makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B₁₂ release provided an advantage in co-cultures. We hypothesize that, given the right conditions, bacteria that release more B₁₂ may be selected for, particularly if they form close interactions with B₁₂ -dependent algae.

Fig. 1

B₁₂‐dependent strain of C. reinhardtii takes up B₁₂ produced by the heterotrophic bacterium M. loti. A. The evolved metE7 mutant of C. reinhardtii, together with its ancestral line and a revertant (Helliwell et al. 2015), were cultured with a range of B₁₂ concentrations in TP medium at 25°C with constant illumination at 100 μmol·m⁻²·s⁻¹ for 7 days, at which point the cell density in the cultures were measured. B. M. loti was cultured in TP media with 0.1% glycerol at 25°C with constant illumination at 100 μmol·m⁻²·s⁻¹ and measurements of cell density and B₁₂ concentration were made over 6 days. C. An M. loti culture, which reached stationary phase, was filtered through a 0.4 μm filter and metE7 cells starved of B₁₂ were added to the filtrate at an OD730 nm of 0.1. This culture was then further filtered at multiple time intervals over the course of 1 h to remove metE7 cells and the B₁₂ concentration measured in this filtrate. Keys to the different measurements are indicated in legends within the graphs. Error bars represent standard deviations, n > = 3.

Fig. 2

Comparison of commensal and mutualistic co‐cultures between various strains of C. reinhardtii and M. loti. M. loti was co‐cultured with the revertant or ancestral lines or metE7 in TP medium at 25°C and with illumination at 100 μmol·m⁻²·s⁻¹ over a 16:8 h light dark cycle for 20 days with periodic measurements of algal and bacterial density as well as B₁₂ concentration. A. Measurement of algal density by particle counter; the ancestral and revertant line density increases at a faster rate than metE7 density. B. M. loti cell density determined by plating serial dilutions of the cultures on TY media; M. loti density is not significantly higher in co‐culture with the ancestral or revertant lines than with metE7 on almost every day. Dotted line indicates axenic growth of M. loti in TP medium (i.e. with no C. reinhardtii strain). C. Total B₁₂ concentration measured by S. typhimurium bioassay on aliquots of the co‐cultures (media and cell fraction); B₁₂ is generally higher in co‐culture with the ancestral or revertant line. Dark grey = ancestral line, light grey = revertant line, black = metE7. Error bars = standard deviation, n = 5.

Fig. 3

metE7 and M. loti only partially support each other's nutrient requirements. M. loti and metE7 were cultured semi‐continuously (10% volume replacement per day) under the same conditions as before, but with the addition of 200 ng·L⁻¹ of B₁₂, 0.02% glycerol or nothing from day 0. Periodic measurements of algal and bacterial density as well as B₁₂ concentration in the cells and media were made. A. metE7 density increases in the B₁₂ supplemented cultures, but also in the glycerol‐supplemented cultures after a delay. B. M. loti density increases in response to glycerol but not B₁₂ supplementation. C. Total B₁₂ concentration increases more substantially in response to glycerol addition than B₁₂ addition itself. Dark grey = 200 ng·L⁻¹ of B₁₂ addition, light grey = 0.02% glycerol addition, black = control. Error bars = standard deviation, n = 4.

Fig. 4

Growth and B₁₂ production of two E. coli strains engineered to synthesize vitamin B₁₂. ED656 expresses BtuF, a protein involved in B₁₂ uptake, while ΔbtuF is a kanamycin resistant btuF knockout. E. coli cells were inoculated at 10⁷ cells·ml⁻¹ and grown for 4 days in TP media with 0.1% glycerol (v/v), at 25°C, and with illumination for a 16:8 h light: dark period at 100 μmol·m⁻²·s⁻¹. A. E. coli density in colony forming units per mL. B₁₂ measured by bioassay in the (B) whole culture, (C) supernatant after centrifugation at 10 000 g for 2 min, or (D) pellet after centrifugation. E. E. coli strains were grown as above but were initially inoculated at different starting percentages: 4, 20, 50, 80, or 96% ΔbtuF with the remainder made up with ED656. Cultures were maintained by diluting 10 000‐fold on day 3 and 6 after CFU density measurements. CFU density of ΔbtuF and both strains combined were measured over 9 days by plating a dilution series onto LB agar plates with or without kanamycin (50 μg·ml⁻¹), respectively, and counting the colonies after an overnight incubation at 37°C. The x axis indicates the percentage of E. coli that is the ΔbtuF strain on day 0 in each culture, and the y axis indicates the change in that percentage on days 3, 6 and 9 (labelled on the right of the plot) compared with day 0. For panels A–D, n = 5, for panel E, n = 10.

Fig. 5

The ΔbtuF mutant of E. coli releases more B₁₂ and is better than the isogenic parent ED656 at supporting metE7. For panels A–C, metE7 was co‐cultured with either E. coli ED656 or ΔbtuF for 3 days at 25°C with shaking at 120 rpm and constant illumination at 100 μmol·m⁻²·s⁻¹. A. metE7 colony forming unit (CFU) density on day 2 of co‐culture with either E. coli strain. B. ED656 or ΔbtuF CFU density on day three of co‐culture with metE7. C. Ratio of CFUs of E. coli:metE7 on day three of co‐culture. D. Photograph of 5 μl droplets of ED656 or ΔbtuF cultures at 10⁵ cells·ml⁻¹ spotted 10 mm from metE7 cultures at 10⁵ cells·ml⁻¹ on TP agar and incubated for 30 days at 25°C with constant illumination at 100 μmol·m⁻²·s⁻¹. For panels E–G, metE7 was co‐cultured for 9 days under the same conditions as above with a mix of ΔbtuF and ED656 strains of E. coli, where the intended starting percentage of ΔbtuF in this mix was 4, 20, 50, 80 or 96%. Cultures were maintained by diluting 10‐fold on days 3 and 6 after CFU density measurements. E. metE7 colony forming unit (CFU) density on days 0, 3, and 6 (most metE7 CFU measurements were 0 on day 9). F. Total E. coli (ED656 + ΔbtuF) CFU density on days 0, 3, 6, and 9. G. The x axis indicates the percentage of E. coli that is the ΔbtuF strain on day 0 in each culture, and the y axis indicates the change in that percentage on days 3, 6 and 9 (labelled on the right of the plot) compared with day 0. For panels A–C, n = 7–8, and for E, F, and G, n = 10.