CO2 Concentrating Mechanism Genes Research Articles

Functional reconstitution of a bacterial CO2 concentrating mechanism in Escherichia coli.

Many photosynthetic organisms employ a CO2 concentrating mechanism (CCM) to increase the rate of CO2 fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an Escherichia coli strain engineered to depend on rubisco carboxylation for growth. Expression of 20 CCM genes enabled E. coli to grow by fixing CO2 from ambient air into biomass, with growth in ambient air depending on the components of the CCM. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO2 assimilation in diverse organisms.

Identification and Characterization of a Transient Receptor Potential Ion Channel (TRP2) Involved in Acclimation to Low CO2 Conditions in Chlamydomonas reinhardtii

The CO2 concentrating mechanism (CCM) is a key feature of green algal cells, induced in response to limiting CO2 conditions. Calcium-dependent signaling has been shown to play a role in this acclimation process to low inorganic carbon in Chlamydomonas reinhardtii, but the molecular players have not been characterized. One type of Ca2+ channel, the transient receptor potential (TRP) family of ion channels is specific to the algal members of the green lineage and not found in land plants. TRP channels generally mediate the flux of Ca2+ ions in response to environmental perturbations, and one recent study has revealed the role of Ca2+ signaling in acclimation to limiting CO2 in the green alga, C. reinhardtii. In this study, the gene Trp2, encoding a Ca2+ ion channel was identified through bioinformatics analyses as having a role in acclimation of algal cells to limiting CO2. Transcript abundance levels for this gene are significantly upregulated when cells are cultured in limiting CO2 and mutant cells missing the TRP2 protein show an impaired growth phenotype. We show that a calcium binding protein CAS, is downregulated in the trp2 mutant along with other CCM genes under the control of CAS. The results suggest that the TRP2 ion channel is involved in the acclimation of C. reinhardtii cells to limiting CO2.

Thylakoid localized bestrophin-like proteins are essential for the CO2 concentrating mechanism of Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. Current models of the CCM postulate that a series of ion transporters bring HCO3- from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO3- to CO2, raising the CO2 concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO3- transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, and BST3) belonging to the bestrophin family have been found to be up-regulated in low CO2 conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 has near-normal growth on low CO2, indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of BST1-3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO3- accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.

PII-like signaling protein SbtB links cAMP sensing with cyanobacterial inorganic carbon response

Cyanobacteria are phototrophic prokaryotes that evolved oxygenic photosynthesis ∼2.7 billion y ago and are presently responsible for ∼10% of total global photosynthetic production. To cope with the evolutionary pressure of dropping ambient CO2 concentrations, they evolved a CO2-concentrating mechanism (CCM) to augment intracellular inorganic carbon (Ci) levels for efficient CO2 fixation. However, how cyanobacteria sense the fluctuation in Ci is poorly understood. Here we present biochemical, structural, and physiological insights into SbtB, a unique PII-like signaling protein, which provides new insights into Ci sensing. SbtB is highly conserved in cyanobacteria and is coexpressed with CCM genes. The SbtB protein from the cyanobacterium Synechocystis sp. PCC 6803 bound a variety of adenosine nucleotides, including the second messenger cAMP. Cocrystal structures unraveled the individual binding modes of trimeric SbtB with AMP and cAMP. The nucleotide-binding pocket is located between the subunit clefts of SbtB, perfectly matching the structure of canonical PII proteins. This clearly indicates that proteins of the PII superfamily arose from a common ancestor, whose structurally conserved nucleotide-binding pocket has evolved to sense different adenyl nucleotides for various signaling functions. Moreover, we provide physiological and biochemical evidence for the involvement of SbtB in Ci acclimation. Collectively, our results suggest that SbtB acts as a Ci sensor protein via cAMP binding, highlighting an evolutionarily conserved role for cAMP in signaling the cellular carbon status.

The diversity of CO2-concentrating mechanisms in marine diatoms as inferred from their genetic content.

Marine diatoms are one of the most ecologically significant primary producers in the ocean. Most diatoms use a CO2-concentrating mechanism (CCM) to overcome the scarcity of CO2 in the ocean and limitations of the carbon-fixing enzyme Rubisco. However, the CCMs in model diatoms differ substantially in their genetic make-up and structural organization. To assess the extent of CCM diversity in marine diatoms more generally, we analyzed genome and transcriptome data from 31 diatom strains to identify putative CCM genes, examine the overall CCM architecture, and study CCM development in the context of the evolutionary history of these diatoms. Key CCM genes [carbonic anhydrases (CAs) and solute carrier 4 (SLC4) bicarbonate transporters] identified in the diatoms were placed into groups of likely orthologs by sequence similarity (OrthoMCL) and phylogenetic methods. These analyses indicated that diatoms seem to share similar HCO3- transporters, but possess a variety of CAs that have either undergone extensive diversification within the diatom lineage or have been acquired through horizontal gene transfer. Hierarchical clustering of the diatom species based on their CCM gene content suggests that CCM development is largely congruent with evolution of diatom species, despite some notable differences in CCM genes even among closely related species.

The influence of pCO2 and temperature on gene expression of carbon and nitrogen pathways in Trichodesmium IMS101.

Growth, protein amount, and activity levels of metabolic pathways in Trichodesmium are influenced by environmental changes such as elevated pCO2 and temperature. This study examines changes in the expression of essential metabolic genes in Trichodesmium grown under a matrix of pCO2 (400 and 900 µatm) and temperature (25 and 31°C). Using RT-qPCR, we studied 21 genes related to four metabolic functional groups: CO2 concentrating mechanism (bicA1, bicA2, ccmM, ccmK2, ccmK3, ndhF4, ndhD4, ndhL, chpX), energy metabolism (atpB, sod, prx, glcD), nitrogen metabolism (glnA, hetR, nifH), and inorganic carbon fixation and photosynthesis (rbcL, rca, psaB, psaC, psbA). nifH and most photosynthetic genes exhibited relatively high abundance and their expression was influenced by both environmental parameters. A two to three orders of magnitude increase was observed for glnA and hetR only when both pCO2 and temperature were elevated. CO2 concentrating mechanism genes were not affected by pCO2 and temperature and their expression levels were markedly lower than that of the nitrogen metabolism and photosynthetic genes. Many of the CO2 concentrating mechanism genes were co-expressed throughout the day. Our results demonstrate that in Trichodesmium, CO2 concentrating mechanism genes are constitutively expressed. Co-expression of genes from different functional groups were frequently observed during the first half of the photoperiod when oxygenic photosynthesis and N2 fixation take place, pointing at the tight and complex regulation of gene expression in Trichodesmium. Here we provide new data linking environmental changes of pCO2 and temperature to gene expression in Trichodesmium. Although gene expression indicates an active metabolic pathway, there is often an uncoupling between transcription and enzyme activity, such that transcript level cannot usually be directly extrapolated to metabolic activity.