Fig. 5.

C. necator requires CA or Ci uptake for robust autotrophic growth in 0.5% and 1.5% CO₂. C. necator strains were grown autotrophically in minimal medium at a variety of CO₂ levels, and end point optical density was measured after 48 h (Methods). (A) Growth of the C. necator double CA knockout (ΔCA) was greatly impaired in 0.5% and 1.5% CO₂. Compared to wild-type C. necator (WT), which grew to a final OD600 of 0.73 ± 0.28 in 1.5% CO₂ (six biological replicates), growth of ΔCA was greatly impaired, reaching a final OD of 0.23 ± 0.17 (three biological replicates). Expression of either the human CA II (ΔCA+CA) or the DAB2 Ci transporter from H. neapolitanus (ΔCA+DAB) recovered robust growth which exceeded even the wild type, indicating that the wild type may not express saturating levels of CA. Panel (B) focuses on 1.5% CO₂. See SI Appendix, Fig. S9 for statistical analysis.