Investigation of the role of putative inorganic carbon transporters in the carbon dioxide concentrating mechanisms of Chlamydomonas reinhardtii

Photosynthetic microalgae optimize the utilization of inorganic carbon by active uptake and concentration of inorganic carbon (Ci) around the carbon-fixing enzyme Rubisco. This process, mostly induced under limiting carbon conditions is called the Carbon Concentrating Mechanism or CCM. The photosynthetic green alga Chlamydomonas reinhardtii serves as an excellent model organism for the study of eukaryotic CCMs. However, unlike the prokaryotic cyanobacterial CCM, which has a well-characterized Ci uptake system, the Ci uptake and transport system of the C. reinhardtii CCM is not well understood. Since CO2 is a small neutral molecule, it is believed to be able to passively diffuse into the cell with or without any assistance from membrane bound transport proteins. However, the charged HCO3- ion needs transporters to facilitate its uptake across each membrane barrier. The hydrophobic barriers posed by the plasma membrane, chloroplast envelope and thylakoid membranes in the path of the charged HCO3- ion’s ultimate destination to the thylakoid lumen has led to the proposed existence of one or more transport proteins at each of these membrane locations. The roles of the C. reinhardtii NAR gene family, showing sequence homology to the Formate/Nitrite transporter family, were investigated for any changes with respect to the induction of the CCM. NAR1.2 was found to be the most interesting NAR protein with respect to the CCM. NAR1.2 was localized to the chloroplast envelope and is believed to be part of the chloroplastic Ci uptake system. The previously identified putative Ci transporter, LCI1 was localized to the plasma membrane. The expression of this protein in a LCI1 deficient background showed a significant increase in Ci uptake and Ci affinity of cells even in the absence of a functional CCM. However, the RNAi mediated knockdown of the protein failed to show any growth deficiencies or changes in photosynthetic rates at different pH levels. LCI1 is a transporter that is part of the plasma membrane Ci uptake system. In the quest for mutations in either previously identified or novel Ci transporters, an insertional mutagenesis project was also undertaken. The sequenced wild-type strain, CC-503, was found to be a natural mutant for the periplasmic carbonic anhydrase, CAH1

Identification and characterization of a solute carrier, CIA8, involved in inorganic carbon acclimation in Chlamydomonas reinhardtii

© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. The supply of inorganic carbon (Ci) at the site of fixation by Rubisco is a key parameter for efficient CO2 fixation in aquatic organisms including the green alga, Chlamydomonas reinhardtii. Chlamydomonas reinhardtii cells, when grown on limiting CO2, have a CO2-concentrating mechanism (CCM) that functions to concentrate CO2 at the site of Rubisco. Proteins thought to be involved in inorganic carbon uptake have been identified and localized to the plasma membrane or chloroplast envelope. However, current CCM models suggest that additional molecular components are involved in Ci uptake. In this study, the gene Cia8 was identified in an insertional mutagenesis screen and characterized. The protein encoded by Cia8 belongs to the sodium bile acid symporter subfamily. Transcript levels for this gene were significantly up-regulated when the cells were grown on low CO2. The cia8 mutant exhibited reduced growth and reduced affinity for Ci when grown in limiting CO2 conditions. Prediction programs localize this protein to the chloroplast. Ci uptake and the photosynthetic rate, particularly at high external pH, were reduced in the mutant. The results are consistent with the model that CIA8 is involved in Ci uptake in C. reinhardtii

The carbon concentrating mechanism in Chlamydomonas reinhardtii: Finding the missing pieces

The photosynthetic, unicellular green alga, Chlamydomonas reinhardtii, lives in environments that often contain low concentrations of CO2 and HCO3-, the utilizable forms of inorganic carbon (Ci). C. reinhardtii possesses a carbon concentrating mechanism (CCM) which can provide suitable amounts of Ci for growth and development. This CCM is induced when the CO2 concentration is at air levels or lower and is comprised of a set of proteins that allow the efficient uptake of Ci into the cell as well as its directed transport to the site where Rubisco fixes CO2 into biomolecules. While several components of the CCM have been identified in recent years, the picture is still far from complete. To further improve our knowledge of the CCM, we undertook a mutagenesis project where an antibiotic resistance cassette was randomly inserted into the C. reinhardtii genome resulting in the generation of 22,000 mutants. The mutant collection was screened using both a published PCR-based approach (Gonzalez-Ballester et al. 2011) and a phenotypic growth screen. The PCR-based screen did not rely on a colony having an altered growth phenotype and was used to identify colonies with disruptions in genes previously identified as being associated with the CCM-related gene. Eleven independent insertional mutations were identified in eight different genes showing the usefulness of this approach in generating mutations in CCM-related genes of interest as well as identifying new CCM components. Further improvements of this method are also discussed. © 2014 Springer Science+Business Media Dordrecht

Identification and characterization of a solute carrier, CIA8,involved in inorganic carbon acclimation in Chlamydomonas reinhardtii

The supply of inorganic carbon (Ci) at the site of fixation by Rubisco is a key parameter for efficient CO2 fixation in aquatic organisms including the green alga, Chlamydomonas reinhardtii. Chlamydomonas reinhardtii cells, when grown on limiting CO2, have a CO2-concentrating mechanism (CCM) that functions to concentrate CO2 at the site of Rubisco. Proteins thought to be involved in inorganic carbon uptake have been identified and localized to the plasma membrane or chloroplast envelope. However, current CCM models suggest that additional molecular components are involved in Ci uptake. In this study, the gene Cia8 was identified in an insertional mutagenesis screen and characterized. The protein encoded by Cia8 belongs to the sodium bile acid symporter subfamily. Transcript levels for this gene were significantly up-regulated when the cells were grown on low CO2. The cia8 mutant exhibited reduced growth and reduced affinity for Ci when grown in limiting CO2 conditions. Prediction programs localize this protein to the chloroplast. Ci uptake and the photosynthetic rate, particularly at high external pH, were reduced in the mutant. The results are consistent with the model that CIA8 is involved in Ci uptake in C. reinhardtii

A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii

Abstract Background Random insertional mutagenesis of Chlamydomonas reinhardtii using drug resistance cassettes has contributed to the generation of tens of thousands of transformants in dozens of labs around the world. In many instances these insertional mutants have helped elucidate the genetic basis of various physiological processes in this model organism. Unfortunately, the insertion sites of many interesting mutants are never defined due to experimental difficulties in establishing the location of the inserted cassette in the Chlamydomonas genome. It is fairly common that several months, or even years of work are conducted with no result. Here we describe a robust method to identify the location of the inserted DNA cassette in the Chlamydomonas genome. Results Insertional mutants were generated using a DNA cassette that confers paromomycin resistance. This protocol identified the cassette insertion site for greater than 80% of the transformants. In the majority of cases the insertion event was found to be simple, without large deletions of flanking genomic DNA. Multiple insertions were observed in less than 10% of recovered transformants. Conclusion The method is quick, relatively inexpensive and does not require any special equipment beyond an electroporator. The protocol was tailored to ensure that the sequence of the Chlamydomonas genomic DNA flanking the random insertion is consistently obtained in a high proportion of transformants. A detailed protocol is presented to aid in the experimental design and implementation of mutant screens in Chlamydomonas

Expression of a Low CO2–Inducible Protein, LCI1, Increases Inorganic Carbon Uptake in the Green Alga Chlamydomonas reinhardtii[W][OA]

Microalgae induce a carbon-concentrating mechanism to acclimate to CO2-limiting stress. This work provides evidence that a low CO2–inducible plasma membrane protein, LCI1, contributes to the carbon-concentrating mechanism as a component of the inorganic carbon transport machinery

MOESM1 of A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii

Additional file 1. A simple Protocol for obtaining Chlamydomonas genomic DNA