Fundamental shift in vitamin B12 eco-physiology of a model alga demonstrated by experimental evolution

Abstract

A widespread and complex distribution of vitamin requirements exists over the entire tree of life, with many species having evolved vitamin dependence, both within and between different lineages. Vitamin availability has been proposed to drive selection for vitamin dependence, in a process that links an organism's metabolism to the environment, but this has never been demonstrated directly. Moreover, understanding the physiological processes and evolutionary dynamics that influence metabolic demand for these important micronutrients has significant implications in terms of nutrient acquisition and, in microbial organisms, can affect community composition and metabolic exchange between coexisting species. Here we investigate the origins of vitamin dependence, using an experimental evolution approach with the vitamin B(12)-independent model green alga Chlamydomonas reinhardtii. In fewer than 500 generations of growth in the presence of vitamin B(12), we observe the evolution of a B(12)-dependent clone that rapidly displaces its ancestor. Genetic characterization of this line reveals a type-II Gulliver-related transposable element integrated into the B(12)-independent methionine synthase gene (METE), knocking out gene function and fundamentally altering the physiology of the alga.

Figure 1

The evolution of vitamin B₁₂ dependence in C. reinhardtii. (a) E8⁺ cells plated onto solid medium −B₁₂ give rise to two colony morphologies: healthy (H-type) colonies, and smaller (S-type) colonies (as visualized under a dissecting microscope), scale bar: 1 mm. (b) Growth of four independent H- and S-type colonies +B₁₂ (1000 ng l⁻¹) and −B₁₂ after 72 h (mean±s.e.m.) n=3. Mean optical density (OD)₇₃₀ values for H- and S-type clones at this time point were: 0.78±0.08 s.e.m. (+B₁₂) and 0.78± 0.04 (−B₁₂) and 0.64±0.009 (+B₁₂) and 0.04± 0.02 (−B₁₂), respectively. (c) OD₇₃₀ of stock-points cultures on liquid medium with (1000 ng l⁻¹; grey) and without B₁₂ after 72 h (mean±s.e.m.) n=3 and (d) maximal growth rate, μ (h⁻¹) of stock-points cultures on liquid medium with (1000 ng l⁻¹; grey) and without B₁₂ as calculated from panel (c) (mean±s.e.m.) n=3. (e) Percentage of S- vs H-type colonies within the population at independent stock-points (mean±s.e.m.) n=3. (f) Percentage of S- (red) and H-type colonies (blue) after replaying selection from T50 (where S-type cells represent <30% of the population, broken black line) for 10 transfers at different concentrations of B₁₂ (mean±s.e.m.) n=3.

Figure 2

Identification of a Gulliver-related transposable element (GR-TE) in the METE gene of E8⁺ S-type cells. (a) PCR on genomic DNA of four independent S- and H-type clones using primer pair F2b/R3b (amplifying a 1-kb region between 4.4 kb and 5.4 kb from the start codon) reveals an unexpectedly large product for S-type clones (expected product size for wild-type (WT) METE: 1003 bp). A BLAST search using the sequence from the S-type product revealed a strong (E-value: 8e⁻⁶⁷) hit for C. reinhardtii METE (Supplementary Figure S2a). Another hit (E-value: 2e⁻⁸⁷) 238 bp in size was identified as a class-II GR-TE (Kim et al., 2005, 2006; Supplementary Figure S2b). The schematic diagram shows an alignment between C. reinhardtii WT METE in this region compared with the ‘S-type' product sequence. A target-site duplication of METE (grey underline) flanks a 15-bp terminal-inverted-repeat (boxed). (b) Western blotting analysis on total protein of E8⁺ and AL cells using a polyclonal antibody against C. reinhardtii METE (∼86.5 kDa; Schneider et al., 2008; L: Ladder). To verify adequate transfer and equal loading, the membrane was stained in Ponceau stain (Ponceau S) (c) Reverse transcriptase-PCR reveals that METE is expressed and regulated by B₁₂ in E8⁺. Expected products using primers Transcript_F1/R1: AL gDNA: 902 bp (+246 bp with TE+8-bp METE repeat, that is, 1148 bp), cDNA: 371 bp (+246 bp, that is, 617 bp). (d) Schematic diagram of probe used for Southern blotting. (e) Southern blotting analysis using the METE probe (probe 1) on genomic samples for stock-points and independent S- and H-type clones.

Figure 3

Characterization of mutant phenotype revertants and isolation of a stable METE insertion mutant (a) A non-reverting colony (i) alongside three independent revertant colonies (ii–iv) visualized under a dissecting microscope, after 11 days on solid medium −B₁₂. (b) PCR screen for the presence of GR-TE insertion in METE gene of clones using primers spanning GR-TE insertion site (METE_revert F1/R1). Clone no. 7 is vitamin B₁₂ dependent yet lacks the GR-TE (expected product sizes: wild-type METE− 913 bp, and METE with GR-TE insertion 913 bp+246 bp=1159 bp). Sequencing revealed a 9-bp footprint (CACCATGCT) in this clone (c) the latter 6 bp of which (underlined grey) is a remnant of the METE repeat.

Figure 4

Characterization of growth of S-type, H-type, R-type and AL cells (a). Growth over time of S-type, H-type, R-type and AL clones in the presence of vitamin B₁₂ (1000 ng l⁻¹) (mean±s.e.m.) n=10. (b) Mean maximal growth rate, μ (h⁻¹) of S-type, H-type, R-type and AL clones as calculated from panel (a). *P⩽0.05, **P⩽0.001 compared with the S-type clones (two-tailed Student's t-test) (mean±s.e.m.) n=10.

Figure 5

Vitamin B₁₂ dependence is rescued by three B₁₂-synthesizing rhizobial species of bacteria. (a) Growth of S-type mutant (unstable) in different B₁₂ regimes, including: (i) +B₁₂ (1000 ng l⁻¹), (ii) −B₁₂, (iii) Mesorhizobium loti, (iv) Sinorhizobium meliloti and (v) Rhizobium leguminosarum. The latter three treatments were grown in the absence of B₁₂ in TAP medium (mean±s.e.m.) n=3. (b) PCR with METE primers spanning the GR-TE from DNA extracted from the different conditions at day 7. (c) Growth of stable-METE-insertion mutant clone no. 7 in B₁₂ regimes described in panel (a). This experiment was carried out in TAP medium (mean±s.e.m.) n=3.