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. 1999 Nov 9;96(23):13571-6.
doi: 10.1073/pnas.96.23.13571.

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

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

T Omata et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Figure 1
Figure 1
Comparison of the structures of the cmp-genomic region in WT and in the M42 (ΔcmpABkanr) and CMP+ (PnirAcmpABCD) 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 sper 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
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
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
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
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.

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