Identification of an ATP-binding cassette transporter involved in bicarbonate uptake in the cyanobacterium Synechococcus sp. strain PCC 7942

Abstract

Exposure of cells of cyanobacteria (blue-green algae) grown under high-CO(2) conditions to inorganic C-limitation induces transcription of particular genes and expression of high-affinity CO(2) and HCO(3)(-) transport systems. Among the low-CO(2)-inducible transcription units of Synechococcus sp. strain PCC 7942 is the cmpABCD operon, encoding an ATP-binding cassette transporter similar to the nitrate/nitrite transporter of the same cyanobacterium. A nitrogen-regulated promoter was used to selectively induce expression of the cmpABCD genes by growth of transgenic cells on nitrate under high CO(2) conditions. Measurements of the initial rate of HCO(3)(-) uptake after onset of light, and of the steady-state rate of HCO(3)(-) uptake in the light, showed that the controlled induction of the cmp genes resulted in selective expression of high-affinity HCO(3)(-) transport activity. The forced expression of cmpABCD did not significantly increase the CO(2) uptake capabilities of the cells. These findings demonstrated that the cmpABCD genes encode a high-affinity HCO(3)(-) transporter. A deletion mutant of cmpAB (M42) retained low CO(2)-inducible activity of HCO(3)(-) transport, indicating the occurrence of HCO(3)(-) transporter(s) distinct from the one encoded by cmpABCD. HCO(3)(-) uptake by low-CO(2)-induced M42 cells showed lower affinity for external HCO(3)(-) than for wild-type cells under the same conditions, showing that the HCO(3)(-) transporter encoded by cmpABCD has the highest affinity for HCO(3)(-) among the HCO(3)(-) transporters present in the cyanobacterium. This appears to be the first unambiguous identification and description of a primary active HCO(3)(-) transporter.

Figure 1

Comparison of the structures of the cmp-genomic region in WT and in the M42 (ΔcmpAB∷kan^r) and CMP+ (PnirA∷cmpABCD) mutants of Synechococcus sp. strain PCC7942. The bar above the map shows the probe region used for Northern hybridization analysis. The open bars represent the antibiotic-resistance gene cassettes and the hatched bars show the location and orientation of the kanamycin-resistance gene (npt) and the spe^r gene (aad). The restriction endonuclease sites are abbreviated as follows: B, BglII; N, NcoI; P, PstI; Sa, SalI; Sp, SphI; and X, XbaI.

Figure 2

(A) Northern hybridization analysis of total RNA from Synechococcus, showing the effects of CO₂ conditions on expression of the cmp operon. Synechococcus cells were grown with NO₃⁻ under high-CO₂ conditions (2% CO₂ in air) and transferred to low-CO₂ conditions (0.005% CO₂ in air). RNA samples (10 μg per lane) from WT (lanes 1 and 2) and the M42 mutant (lanes 3 and 4), extracted before (lanes 1 and 3) and 30 min after (lanes 2 and 4) the transfer, were denatured with formamide, separated on a 1.2% agarose-formaldehyde gel, transferred to a positive-charged nylon membrane (Hybond N+, Amersham), and hybridized with a ³²P-labeled cmpC-specific probe. (B) Northern hybridization analysis of total RNA, showing the nitrogen-regulated expression of the cmp gene cluster under high CO₂ in the CMP+ mutant. Synechococcus cells were grown with NH₄⁺ and transferred to NO₃⁻-containing medium under high-CO₂ conditions. RNA samples (10 μg per lane) from WT (lanes 1 and 2) and CMP+ (lanes 3 and 4), extracted before (lanes 1 and 3) and 30 min after (lanes 2 and 4) the transfer, were analyzed as in A. (C) Immunoblotting analysis of CmpA in the plasma membrane of Synechococcus grown under constant C and nitrogen conditions. Plasma membrane samples from WT cells grown under high CO₂ (lane 1) and low CO₂ (lane 2) conditions in NO₃⁻-containing medium and those from the CMP+ cells grown under high-CO₂ conditions in NH₄⁺- (lane 3) and NO₃⁻- (lane 4) containing media were compared. Membrane proteins (5 μg per sample) were solubilized with SDS, fractionated by SDS/PAGE (10% gel), and electrotransferred to poly(vinylidene difluoride) membrane for immunostaining.

Figure 3

Uptake of HCO₃⁻ by high-CO₂-grown cells (H) of WT (▾) and the CMP+ mutant (○, ●) and low-CO₂-grown cells (▴) of WT (H) in the light. Cells were grown with NH₄⁺ (○) or NO₃⁻ (●, ▾, ▴) as the nitrogen source. Uptake was initiated by illumination immediately after the addition of 100 μM H¹⁴CO₃⁻ to the cell suspensions. The amount of HCO₃⁻ taken up by the cells was determined from the total amount of ¹⁴C accumulated in the cell. Assays were done at 30°C and pH 9.0.

Figure 4

The rate of O₂ evolution (A), net HCO₃⁻ uptake (B), and gross CO₂ uptake (C) as a function of the HCO₃⁻ concentration in medium during steady-state photosynthesis in WT (▾) and the CMP+ mutant (○ and ●). Cells were grown under high-CO₂ conditions at a light intensity of 250 μmol of photons m⁻²⋅s⁻¹ with NH₄⁺ (○) or NO₃⁻ (● and ▾) as the nitrogen source. Assays were done at 30°C and pH 8.2.

Figure 5

The rate of O₂ evolution (A), net HCO₃⁻ uptake (B), and gross CO₂ uptake (C) as a function of the HCO₃⁻ concentration in medium during steady-state photosynthesis in WT (□ and ■) and the M42 mutant (▵ and ▴). Cells were grown under high-CO₂ (□ and ▵; H) and low-CO₂ (■ and ▴; L) conditions at a light intensity of 90 μmol of photons m⁻²⋅s⁻¹. Assays were done as described in Fig. 4.