Characterisation of cyanobacterial bicarbonate transporters in E. coli shows that SbtA homologs are functional in this heterologous expression system

Abstract

Cyanobacterial HCO3(-) transporters BCT1, SbtA and BicA are important components of cyanobacterial CO2-concentration mechanisms. They also show potential in applications aimed at improving photosynthetic rates and yield when expressed in the chloroplasts of C3 crop species. The present study investigated the feasibility of using Escherichia coli to assess function of a range of SbtA and BicA transporters in a heterologous expression system, ultimately for selection of transporters suitable for chloroplast expression. Here, we demonstrate that six β-forms of SbtA are active in E. coli, although other tested bicarbonate transporters were inactive. The sbtA clones were derived from Synechococcus sp. WH5701, Cyanobium sp. PCC7001, Cyanobium sp. PCC6307, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp. PCC7002. The six SbtA homologs varied in bicarbonate uptake kinetics and sodium requirements in E. coli. In particular, SbtA from PCC7001 showed the lowest uptake affinity and highest flux rate and was capable of increasing the internal inorganic carbon pool by more than 8 mM relative to controls lacking transporters. Importantly, we were able to show that the SbtB protein (encoded by a companion gene near sbtA) binds to SbtA and suppresses bicarbonate uptake function of SbtA in E. coli, suggesting a role in post-translational regulation of SbtA, possibly as an inhibitor in the dark. This study established E. coli as a heterologous expression and analysis system for HCO3(-) transporters from cyanobacteria, and identified several SbtA transporters as useful for expression in the chloroplast inner envelope membranes of higher plants.

Figure 1. Construct designs involved in characterisation of SbtA transporters.

In all constructs, expression of target proteins was driven by the lac promoter on plasmid pSE2. A. Schematic of typical SbtA constructs shown in Table 4. B. Schematic of typical SbtAB constructs shown in Table 4. In order to generate 7942AnsB, the start codon of sbtB (ATG) in SbtAB7942 construct was replaced with (GGG) to form a SmaI site and a later GTG -valine 41 bp downstream of the start codon was replaced with GTC-valine. In this way, expression of sbtB is completely abolished in 7942AnsB. C. Illustrated location for the c-Myc tag in 7942AMyc. SD, Shine-Dalgarno sequence.

Figure 2. Complementation of the EDCM636 mutant by expression of various SbtA clones.

Complementation of CA-deficient strain EDCM636 expressing one of the six SbtA clones, or empty vector (pSE2) as a control. A strain with empty pSE2, EDCM636a, was selected for expressed CA. EDCM636a was used as a positive control. The top panel was for growth in LB media; bottom panel was for growth in M9 media.

Figure 3. Sodium dependency of HCO₃ ^- uptake due to expression of various SbtA clones.

Cells were spun down and washed twice with CO₂-free uptake buffer (22 mM potassium phosphate buffer, 20 mM pH 8 Bis-Tris-Propane-HCl pH 8). Additional NaCl was added to cells at various concentrations prior to uptake experiments. Net uptake rates were calculated by subtracting data of pSE2 empty (18–35 nmol mg total protein⁻¹ h⁻¹) from raw data of each transporter. Values in the figure are means ± SD (n = 6). SbtA7942, black diamond; SbtA6703, black triangle; SbtA6803, white square; SbtA5701, white circle; SbtA7001, white triangle and SbtA7002, white diamond.

Figure 4. HCO₃ ^- uptake of SbtA7942 and SbtA7001 against changes at external HCO₃ ^- levels.

Uptake was measured as described in Materials and Methods. Respiratory HCO₃ ^- levels were measured with MIMS allowing a correction for dilution of ¹⁴C-HCO₃ ^- specific activity. Values in the figures are means ± SD (n = 6). A. HCO₃ ^- uptake of SbtA7942. SbtA7942 Corrected (white diamond) uptake rates were calculated by subtraction of respiratory carbon from the SbtA7942 Raw data (black diamond). Six concentrations of Ci (20 to 500 µM) were injected to pSE2 or SbtA7942 cells. Raw uptake rates for pSE2 control were 6 to 199 nmol mg⁻¹ h⁻¹ and were corrected to 98 to 609 nmol mg⁻¹ h⁻¹. B. HCO₃ ^- uptake of SbtA7001. SbtA7001 Corrected uptake rates (white triangle) were calculated by subtraction of respiratory Ci from the SbtA7001 Raw data (black triangle). Six concentrations of Ci (20 to 1000 µM) were injected to pSE2 or SbtA7001 cells. Raw uptake rates for pSE2 control were 9 to 294 nmol mg⁻¹ h⁻¹ and were corrected to 30 to 383 nmol mg⁻¹ h⁻¹. The theoretical Michaelis-Menten curve (Broken line) was calculated from SbtA7001 Corrected data (R² = 0.9031).

Figure 5. Relative accumulation of SbtA proteins expressed in enriched E. coli membrane fractions by western blotting.

The respective sbtA genes were introduced on pSE2 plasmids under control of the IPTG inducible lac promoter. An empty vector (pSE2) served as negative control (right most lane). Gene expression was induced for 2.5 h with 1 mM IPTG. The membrane-enriched protein fractions were isolated, and 50 µg total protein per lane was separated by SDS-PAGE; bands detected by western blotting using the SbtA antibody. *  =  SbtA monomer; **  =  possible dimer of SbtA.

Figure 6. Internal Ci pool sizes of E. coli cells with the empty vector or various sbtA clones. The expression vector used was the pSE2 vector.

Internal Ci pools (mM) were calculated from maximum Ci uptake and the cell volume of each strain. Ci uptakes were measured in the presence of 500 µM injected H¹⁴CO₃ ^- (except SbtA7001 which was 1000 µM) and corrected for respiratory Ci. Data as means ± SD (n = 6). The pools size of cells with each SbtA transporter was significantly different from the pool size of cells with the empty pSE2 vector as determined with the Welch's T-test (all p<0.02).

Figure 7. HCO₃ ^- uptake capacity assessed for five separate SbtAB pairs and 7942A-nsB.

Uptake rates were calculated by subtracting data for the empty pSE2 control (∼22 nmol mg⁻¹ h⁻¹) from raw data of each strain. Data were not corrected with respiratory Ci as this is encompassed in the control value. Values in the figure are means ± SD (n = 6). The statistical significance of data was analysed with the Welch's T-test. The HCO₃ ^- uptake rates of SbtA7942, SbtA6803, SbtA7001, SbtA7002 and SbtA5701 were significantly different with or without corresponding SbtB (all p<0.01). The HCO₃ ^- uptake rates of SbtA6307 had no significant difference with or without SbtB6307 (p = 0.37). 7942A-nsB showed no significant difference in HCO₃ ^- uptake rates to SbtA7942 (p = 0.59).

Figure 8. Isolation of SbtA7942 by IMAC using SbtB-HAH6 as binding partner and detected by western blotting.

Gene expression was induced for 2.5 h with 1 mM IPTG. The membrane-enriched protein fractions of E. coli containing the empty pSE2, 7942AMyc and 7942AMBH and 7942B-HAH6 vector were used for IMAC isolation. A total of 40 µg total protein of IMAC elutes per lane was separated by SDS-PAGE and subjected to Western blotting. Proteins were detected with the Anti-c-Myc antibody.