Biology subjects

Price, G. D.

Publications and source records attributed to Price, G. D..

7 recordsLinked to original sources

bioRxiv2026

Heterologous expression in E. coli reveals the bicarbonate transporter BicA2 drives carbon uptake in marine Prochlorococcus spp.

The widespread oceanic cyanobacterial Prochlorococcus genus is a major contributor to global carbon fixation, yet mechanisms enabling this lineage to elevate intracellular inorganic carbon as a substrate for photosynthesis remain unresolved. Cyanobacterial CO2 concentrating mechanisms typically rely on membrane-bound bicarbonate (HCO -) transporters SbtA1, SbtA2, BicA and BCT1, and CO2-to-HCO3- conversion uptake systems (CO2 pumps; NDH-I3 and NDH-I4), to elevate a cellular HCO3- pool for use by Rubisco-containing carboxysomes. Evidence suggests Prochlorococcus harbours carboxysomes with a low-CO2-specificity Rubisco, implying a functional CCM dependent on active HCO3- uptake. However, canonical CO2 pumps are absent, leaving distant HCO - transporter homologues, BicA2 and SbtA2, as prime candidates for HCO3- transport in this group. Yet these have not been functionally characterised. Here we demonstrate that BicA2 from P. marinus CCMP1375 mediates Na+-dependent HCO3- uptake in E. coli, while BicA2 from P. marinus CCMP1986 is inactive in its native form but acquired transport function through a single amino acid substitution during adaptive laboratory evolution. These findings confirm BicA2 as a low-affinity, Na+-dependent bicarbonate transporter with variable flux, revealing a previously uncharacterized CCM component in Prochlorococcus. This mechanistic insight reshapes our understanding of carbon acquisition strategies in the most abundant photosynthetic organism on Earth and highlights evolutionary plasticity in transporter function with implications for global biogeochemical cycles. HighlightThis study provides the first mechanistic evidence of bicarbonate transport in Prochlorococcus, revealing functional and evolutionary flexibility in its CO2-concentrating mechanism components and reshaping our understanding of carbon acquisition in the most abundant marine photoautotroph.

microbiology↗

bioRxiv2025

Functional characterisation of bicarbonate transporters from the cyanobacterial SbtA2 family and subsequent expression in tobacco

Cyanobacteria rely on bicarbonate (HCO3-) as the primary inorganic carbon (Ci) source for photosynthesis in aquatic environments. To use of this Ci source, cyanobacteria employ CO2 concentrating mechanisms (CCMs) that elevate cytoplasmic HCO3- via plasma membrane transporters, enhancing carboxylation by carboxysomal Rubisco. The sodium-dependent SbtA1 transporter family is well-characterized in freshwater cyanobacteria, but the related SbtA2 family, prevalent in marine -cyanobacteria, remains uncharacterised. Here, we report functional characterisation of SbtA2 homologues from marine Synechococcus spp., which exhibit high Ci uptake flux with apparent chloride dependence and intermediate HCO3- affinity (Km {approx} 150 {micro}M), when assessed in E. coli. SbtA2 achieved internal Ci accumulation up to 24 mM within 30 seconds. Co-expression with the putative regulator SbtB2 reduced uptake activity, suggesting a regulatory role for this protein. These findings indicate that SbtA2 transporters contribute significantly to carbon acquisition in marine cyanobacteria. Given potential to enhance CO2 supply to Rubisco in C3 plants, we targeted SbtA2 to the tobacco chloroplast inner envelope membrane; however, this did not improve photosynthesis or growth. Our results highlight the functional diversity of cyanobacterial Ci transporters and suggest that additional components may be required for effective transfer of such systems into plant chloroplasts. HighlightSbtA2 bicarbonate transporters from some marine -cyanobacteria are high flux transporters with an unusual chloride-dependence and show intermediate uptake affinity when expressed in E. coli. Initial attempts failed to demonstrate enhanced photosynthesis when directed to tobacco chloroplasts.

plant biology↗

bioRxiv2024

Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts

The ATP-driven bicarbonate transporter 1 (BCT1), a four-component complex in the cyanobacterial CO2-concentrating mechanism, could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis. HighlightWe describe the directed evolution and rational design of a cyanobacterial four-component bicarbonate transporter and the localization of its subunits to various chloroplast sub-compartments for improving C3 plant photosynthesis.

plant biology↗

bioRxiv2023

Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally significant proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in [a]-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present the first structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate (RuBP), and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial [a]-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of -carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage. One-Sentence SummaryThe carboxysomal carbonic anhydrase, CsoSCA, is allosterically activated by the Rubisco substrate RuBP, revealing a novel mechanism controlling key enzyme activity in cyanobacterial -carboxysomes.

molecular biology↗

bioRxiv2022

A cross-scale analysis to understand and quantify effects of photosynthetic enhancement on crop growth and yield

Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying effectively how the seasonal growth and yield dynamics of target crops might be affected over a wide range of environments is limited. Using a state-of-the-art cross-scale model we predicted crop-level impacts of a broad list of promising photosynthesis manipulation strategies for C3 wheat and C4 sorghum. The manipulation targets have varying effects on the enzyme-limited (Ac) and electron transport-limited (Aj) rates of photosynthesis. In the top decile of seasonal outcomes, yield gains with the list of manipulations were predicted to be modest, ranging between 0 and 8%, depending on the crop type and manipulation. To achieve the higher yield gains, large increases in both Ac and Aj are needed. This could likely be achieved by stacking Rubisco function and electron transport chain enhancements or installing a full CO2 concentrating system. However, photosynthetic enhancement influences the timing and severity of water and nitrogen stress on the crop, confounding yield outcomes. Strategies enhancing Ac alone offers more consistent but smaller yield gains across environments, Aj enhancement alone offers higher gains but is undesirable in less favourable environments. Understanding and quantifying complex cross-scale interactions between photosynthesis and crop yield will challenge and stimulate photosynthesis and crop research. Summary StatementLeaf-canopy-crop prediction using a state-of-the-art cross-scale model improves understanding of how photosynthetic manipulation alters wheat and sorghum growth and yield dynamics. This generates novel insights for quantifying impacts of photosynthetic enhancement on crop yield across environments.

plant biology↗

bioRxiv2021

Regulatory adenylnucleotide-mediated binding of the PII-like protein SbtB to the cyanobacterial bicarbonate transporter SbtA is controlled by the cellular energy state

Cyanobacteria have evolved a remarkably powerful CO2 concentrating mechanism (CCM), enabling high photosynthetic rates in environments with limited inorganic carbon (Ci). Therefore, this CCM is a promising system for integration into higher plant chloroplasts to boost photosynthetic efficiency and yield. The CCM depends on active Ci uptake, facilitated by bicarbonate transporters and CO2 pumps, to elevate CO2 concentration around the active sites of the primary CO2 fixing enzyme, Rubisco, which is encapsulated in cytoplasmic micro-compartments (carboxysomes). The essential CCM proteins have been identified, but the molecular signals and regulators that coordinate function in response to light, Ci availability and other environmental cues are largely unknown. Here, we provide evidence, based on a novel in vitro binding system, for a role of the PII-like SbtB protein in regulating Ci uptake by the bicarbonate transporter, SbtA, in response to the cellular adenylate energy charge (AEC) through dynamic protein-protein interaction. Binding of the SbtA and SbtB proteins from two phylogenetically distant species, Cyanobium sp. PCC7001 and Synechococcus elongatus PCC7942, was inhibited by high ATP, and promoted by low [ATP]:[ADP or AMP] ratios in vitro, consistent with a sensory response to the AEC mediated through adenylnucleotide ligand-specific conformation changes in SbtB. In vivo, cell cultures of S. elongatus showed up to 70% SbtB-dependent down-regulation of SbtA bicarbonate uptake activity specifically in the light activation phase during transitions from dark to low light when low cellular AEC is expected to limit metabolic activity. This suggests SbtB may function as a curfew protein during prolonged low cellular AEC and photosynthetically unfavourable conditions to prevent energetically futile and physiologically disadvantageous activation of SbtA.

plant biology↗

bioRxiv2019

Structural basis for the allosteric regulation of the SbtA bicarbonate transporter by the PII-like protein, SbtB, from Cyanobium sp. PCC7001

Cyanobacteria have evolved a suite of enzymes and inorganic carbon (Ci) transporters that improve photosynthetic performance by increasing the localized concentration of CO2 around the primary CO2-fixating enzyme, Rubisco. This CO2-concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles and allows cyanobacteria to thrive under limiting Ci conditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate Ci transporters in response to changing conditions. Cyanobacterial sodium-dependent bicarbonate transporters (SbtAs) are inhibited by PII-like regulatory proteins (SbtBs), with the inhibitory effect being modulated by adenylnucleotides. Here, we used isothermal titration calorimetry to show that SbtB from Cyanobium sp. PCC7001 (SbtB7001) binds AMP, ADP, cAMP and ATP with micromolar-range affinities. X-ray crystal structures of apo- and nucleotide-bound SbtB7001 revealed that while AMP, ADP and cAMP have little effect on the SbtB7001 structure, binding of ATP stabilizes the otherwise flexible T-loop and that the flexible C-terminal C-loop adopts several distinct conformations. We also show that ATP binding affinity is increased ten-fold in the presence of Ca2+ and we present an X-ray crystal structure of Ca2+ATP:SbtB7001 that shows how this metal ion facilitates additional stabilizing interactions with the apex of the T-loop. We propose that the Ca2+ATP-induced conformational change observed in SbtB7001 is important for allosteric regulation of SbtA activity by SbtB and is consistent with changing adenylnucleotide levels in illumination/darkness cycles.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC=\"FIGDIR/small/762807v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (43K):\norg.highwire.dtl.DTLVardef@99bac2org.highwire.dtl.DTLVardef@b43082org.highwire.dtl.DTLVardef@1dcac4borg.highwire.dtl.DTLVardef@b38ea1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗