Functional characterisation of cyanobacterial bicarbonate transporters in e. Coli

Abstract

Cyanobacterial bicarbonate transporters BCT1, SbtA and BicA are important components of the cyanobacterial carbon dioxide concentrating mechanism, which cyanobacteria evolved to adapt to low carbon dioxide conditions. They also show potential in applications aimed at improving photosynthetic rates and yield when expressed in the chloroplasts of C3 crop species. However, these transporters are regulated by light availability in cyanobacteria, suggesting these transporters may require activator(s) when functionally expressed in C3 crops. Little is known about these activation/deactivation processes. This thesis investigated the feasibility of using Escherichia coli or yeast as a heterologous expression system to assess the function and regulation of a range of cyanobacterial bicarbonate transporters. Physiological measurements of radioactive bicarbonate uptake, and complementation tests with high-carbon-dioxide-requiring strains of E. coli and yeast, demonstrated that six SbtA homologs were active in E. coli, while BCT1 and BicA homologs were not; none of the tested SbtA and BicA homologs was active in yeast. The six SbtA homologs active in E. coli were then characterised in this heterologous system. The six SbtA homologs active in E. coli 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. These characteristics could potentially make it a good candidate for expression in the chloroplasts of C3 crops. Importantly, the study showed that the SbtB protein (encoded by a companion gene near sbtA) suppressed bicarbonate uptake by SbtA in E. coli, by binding to SbtA. This suggests it has a role in post-translational regulation of SbtA, possibly as an inhibitor in the dark. Due to the structural similarities of SbtB to PII protein GlnK, a model of SbtA-SbtB interaction was suggested based on the model of the regulation of ammonium transporter AmtB by GlnK. Preliminary results suggested that the loop between helix 5 and 6 of SbtA may play a role in the interaction between SbtA and SbtB, but this requires further investigation. Although BicA was not active in E. coli or yeast, its expression and quaternary structure was investigated further. BicA was detected in the plasma membrane of both E. coli and yeast, suggesting that it is not grossly misfolded, but may require an activator for function. Bacterial two-hybrid analysis suggested that BicA formed some kind of oligomer in the E. coli plasma membrane, consistent with the self-interaction seen in other members of the Sulphate Permease family to which it belongs. This study was the first to establish E. coli as a heterologous expression and analysis system for bicarbonate transporters from cyanobacteria or any other system; this study identified several SbtA transporters as useful for expression into the chloroplast envelopes of higher plants. Show more