Figure 1.

Isolation and characterization of LCIB-Clover expressing strain. A, Strategy to obtain the LCIB-Clover-expressing strain, MBC-3, by the genetic crossing of lcib-insertion mutant with WT strains (CC-1690 and C9) and introduction of the expression plasmid pCT1. mt^–, mating type minus; mt⁺, mating type plus. B, Accumulation of LCIB, LCIC, and LCIB-Clover fusion proteins in C9, B3, and MBC-3 cells. Cells were grown in liquid culture aerated with 5% CO₂ or 0.04% CO₂ for 12 h. Histone H3 was used as a loading control. The asterisk indicates non-specific bands from C9 and B3 cells, which appeared at the same molecular weight as LCIB-Clover. kDa, kilo Dalton. C, Spot tests for the growth of C9, B3, and MBC-3 cells. Cell suspensions were diluted to the indicated optical density at 730 nm, and 3 µL of each diluted cell suspension was spotted onto MOPS-P agar plates and incubated in HC (5% CO₂) conditions for 4 d, LC (0.04% CO₂) conditions for 5 d, or VLC (0.01% CO₂) conditions for 8 d. D, Typical responses of net O₂-evolving activities of C9 (closed circles), B3 (open circles), and MBC-3 cells (closed squares) against calculated CO₂ concentrations at pH 7.8 for the ranges of 0–400 µM CO₂ and 0–27.5 µM CO₂ (inset). Shaded areas represent different physiological CO₂-acclimation states of Chlamydomonas: VLC (<7 µM CO₂; dark gray), LC (7–70 µM; medium gray), and HC (>70 µM CO₂; light gray). CO₂ concentrations were calculated from the concentration of Ci added, assuming that CO₂ and ${\text{HCO}}_3^{–}$ are in equilibrium due to the activity of CA localized in the periplasmic space. Before measurements, cells were grown in the liquid culture aerated with 0.04% CO₂ for 12 h. E, V_max and K_0.5 (CO₂) values. The CO₂ concentration required for the half-maximal rate of V_max of C9, B3, and MBC-3 cells was calculated from the O₂-evolving activities in D. Data in all experiments are mean values ± standard deviation from three or four biological replicates. *P-value < 0.01, Student’s t test.