Mutation of ndh genes leads to inhibition of CO(2) uptake rather than HCO(3)(-) uptake in Synechocystis sp. strain PCC 6803

Abstract

Six mutants (B1 to B6) that grew poorly in air on BG11 agar plates buffered at pH 8.0 were rescued after mutations were introduced into ndhB of wild-type (WT) Synechocystis sp. strain PCC 6803. In these mutants and a mutant (M55) lacking ndhB, CO(2) uptake was much more strongly inhibited than HCO(3)(-) uptake, i.e., the activities of CO(2) and HCO(3)(-) uptake in B1 were 9 and 85% of those in the WT, respectively. Most of the mutants grew very slowly or did not grow at all at pH 6.5 or 7.0 in air, and their ability to grow under these conditions was correlated with CO(2) uptake capacity. Detailed studies of B1 and M55 indicated that the mutants grew as fast as the WT in liquid at pH 8.0 under air, although they grew poorly on agar plates. The contribution of CO(2) uptake appears to be larger on solid medium. Five mutants were constructed by inactivating each of the five ndhD genes in Synechocystis sp. strain PCC 6803. The mutant lacking ndhD3 grew much more slowly than the WT at pH 6.5 under 50 ppm CO(2), although other ndhD mutants grew like the WT under these conditions and showed low affinity for CO(2) uptake. These results indicated the presence of multiple NAD(P)H dehydrogenase type I complexes with specific roles.

FIG. 1

Time courses of uptake of ¹⁴CO₂ (left panel) and H¹⁴CO₃⁻ (right panel) into WT (circles), B1 (squares), and M55 (triangles) cells, measured by the silicone oil-filtering centrifugation method. The cells were grown under 3% CO₂ and then aerated with air for 18 h in the light at pH 8.0 (solid curves) or pH 7.0 (dashed curves). The cells were suspended in BG11 medium containing 15 mM NaCl buffered with 20 mM HEPES-KOH, pH 8.0. The concentrations of ¹⁴CO₂ and H¹⁴CO₃⁻ were 7.9 and 61 μM, respectively.

FIG. 2

Amount of C_i taken up by the mutant cells during incubation with ¹⁴CO₂ (hatched bars) for 10 s or with H¹⁴CO₃⁻ (open bars) for 20 s in the light. Each value is shown as a percentage of the value obtained for WT cells (259 nmol of CO₂/mg of Chl per 10 s and 473 nmol of HCO₃⁻/mg of Chl per 20 s), and the error bars indicate standard deviations (n = 3). Cells grown at pH 8.0 were suspended in the same medium as for Fig. 1.

FIG. 3

Effects of pH and CO₂ concentration on the growth of the WT and mutants on agar plates. The WT and ndhB mutant cells of Synechocystis were pelleted by centrifugation and resuspended in BG11 medium, pH 8.0, 7.0, or 6.5. Two microliters of the cell suspensions, OD₇₃₀ values of 1.0 (upper row of each panel), 0.1 (middle row), and 0.01 (lower row), were spotted on agar plates containing BG11 medium buffered at pH 8.0, pH 7.0, and pH 6.5. The plates were incubated under 3% (vol/vol) CO₂ in air (A to C) or under air (D to F) for 5 days at 60 μmol of photons of photosynthetically active radiation/m²s⁻¹.

FIG. 4

Effect of pH on growth rates of WT, B1, and M55 in liquid BG11 medium during aeration with 3% (vol/vol) CO₂ in air (closed symbols) or under air (open symbols). Growth rates are shown by the μ values. Doubling time (in days), 0.693/μ.

FIG. 5

Immunoblot of NdhB in the total cell membranes of Synechocystis sp. strain PCC 6803 WT, B1, and M55. Samples (10 μg of proteins) were solubilized at room temperature and boiled for several minutes and were run in a 10% gel. The antibody against GST-NdhB (partial) was used for immunoblotting.

FIG. 6

RT-PCR analysis of total RNA from Synechocystis cells showing the expression of ndhF3, ndhD3, and sll1734 as an operon. RNA was prepared from cells grown at pH 8.0 under 50 ppm CO₂. PCR amplification was performed with a set of primers specific to ndhF3 (ATTATCTGGCTAGTACC and GAATAGCTAAGAAAGGC), and the product was 1.8 kbp. The templates used for PCR were cDNAs which were synthesized by reverse transcription of mRNA by addition of RT with primers specific to ndhF3 (lane 1), ndhD3 (lane 2), sll1734 (lane 3), and sll1735 (lane 4), respectively.

FIG. 7

The K_0.5(CO₂) and V_max values for steady-state (A) and initial (B) CO₂ uptake by the WT and ndhD3 mutant (M) strains of Synechocystis sp. strain PCC 6803 grown under 2% CO₂ (shaded and hatched bars) and after 18 h of induction under 20 ppm CO₂ (open bars). Each bar shows the average value of the results of four measurements, and the error bars indicate standard deviations.