Involvement of the cynABDS Operon and the CO₂-Concentrating Mechanism in the Light-Dependent Transport and Metabolism of Cyanate by Cyanobacteria

Typical time course (25 min) of NCO⁻ uptake and utilization by S. elongatus PCC7942 wild type (WT) and the cynA mutant (::cynA). Cells grown in standing culture with low C_i concentrations were provided with 1 mM KO¹⁴CN in the light. Uptake and accumulation of ¹⁴C was determined as described in method B. (A) Simultaneous measurements of cyanate-dependent CO₂ fixation (•) and O₂ evolution (○) for the wild type and the cynA mutant. (B) Intracellular accumulation of acid-labile NCO⁻ + C_i (▪) for the wild type and the cynA mutant (::cynA).

Effect of KOCN on HCO₃⁻ and CO₂ transport in Synechococcus sp. strain UTEX 625 cells grown in standing culture. The initial rate (20 s) of HCO₃⁻ transport was measured (method C) at substrate concentrations of 50 μM (•) and 250 μM (○) in pH 8 buffer to determine the effect of increasing [KOCN] on Na⁺-independent HCO₃⁻ transport in the presence of 100 μM NaCl (A) or Na⁺-dependent HCO₃⁻ transport in the presence 30 mM NaCl (B). (C) The initial rate (10 s) of CO₂ transport (▪) was measured at 10 μM in the presence of 100 μM NaCl (method D). Also shown is the effect of KOCN on CO₂ transport in cells grown under high-C_i conditions(⋄). The data are the average of six determinations ± standard deviations.

KOCN-dependent CO₂ efflux by cells grown under high-CO₂ conditions. Illuminated wild-type (WT) S. elongatus PCC7942 cells were provided with 1 mM KO¹³CN, and the efflux of ¹³CO₂ was followed by MS over time in the absence or presence of 25 mM NaNO₃ or NaNO₂. Bovine CA was added to the suspension to illustrate that CO₂ was the C_i species arising in the cell suspensions. Also shown are the CO₂ fluxes arising from the cynA and cynS mutants and from Synechocystis sp. strain PCC6803 grown in high-CO₂ concentrations.

Measurements of KOCN-dependent O₂ evolution (A) and Chl a fluorescence quenching (B) in S. elongatus PCC7942 wild-type (WT) cells and the cynA and cynS mutants. Cells grown under low-C_i conditions were allowed to deplete the medium of C_i in the light. Cells were then provided with 1 mM KOCN, and O₂ evolution and Chl a fluorescence quenching were measured simultaneously over time. Once a steady state was achieved, C_i-dependent O₂ evolution and Chl a fluorescence quenching were also measured following the addition of 25 μM C_i to the cell suspensions. Also shown in panel A is the response of Synechocystis sp. strain PCC6803 to the addition of 1 mM KOCN.

Effect of KOCN on CO₂ transport. S. elongatus PCC7942 cynA mutant cells grown under low-C_i conditions were placed in the reaction chamber attached to the MS and allowed to equilibrate (25 mM BTP-HCl buffer, pH 8, at 30°C) in the light (300 μmol of photons m⁻² s⁻¹) in the presence of KOCN. CO₂ transport was initiated by adding 3.5 μM CO₂ to the reaction vessel, and its concentration was followed over time. The difference between the curves obtained in the light (A, 0 mM KOCN, maximum uptake) and the dark (F, no uptake) was taken as a measure of CO₂ transport. The effect of 1, 3, 5, or 10 mM KOCN (B, C, D, and E) on CO₂ transport are also shown.

Involvement of the cyanobacterial CCM in cyanate metabolism. (A) Time course of KOCN-dependent ¹²CO₂ efflux in the light (solid lines) and dark (dashed lines). S. elongatus strain PCC7942 ΔchpX ΔchpY cells grown on 5% CO₂ were suspended in 100 mM EPPS (N-[2-hydroxyethyl]piperazine-N′-[3-propanesulfonic acid]) buffer, pH 8, and incubated in the light or dark for 5 min in the reaction cuvette, in the absence and presence of 10 mM NaH¹³CO₃ (¹³C_i). The experiment was started by the addition of 1 mM KOCN, and ¹²CO₂ efflux was measured over time by MS. (B) Dependence of ¹²C_i efflux rate on ¹³C_i concentration. The average rate of ¹²C_i efflux, at a constant cell density (7.5 μg of Chl a ml⁻¹), was calculated from ¹²CO₂ efflux measurements over an 8-min interval as a function of ¹³C_i concentration for ΔchpX ΔchpY (chpXY) cells in the light (•) and dark (○) provided with 1 mM KOCN. Also shown (dashed line and ▴) is the dependence on ¹³C_i concentration of ¹²CO₂ efflux in S. elongatus PCC7942 wild-type (WT) cells grown under low-C_i conditions.