Carbonic anhydrases (CA) are zinc-containing metalloenzymes that catalyze the reversible hydration of CO2. The three evolutionarily unrelated families of CAs are designated α-, β-, and γ-CA. Aquatic photosynthetic organisms have evolved different forms of CO2 concentrating mechanisms (CCMs) to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CCMs is the critical roles played by various specially localized extracellular and intracellular CAs. Five CAs have previously been identified in Chlamydomonas reinhardtii, a green alga with a well-studied CCM. Here we identify a sixth gene encoding a β-type CA. This new β-CA, designated Cah6, is distinct from the two mitochondrial β-CAs in C. reinhardtii. Nucleotide sequence data show that the Cah6 cDNA contains an open reading frame encoding a polypeptide of 264 amino acids with a leader sequence likely targeting the protein to the chloroplast stroma. We have fused the Cah6 open reading frame to the coding sequence of maltose-binding protein in a pMal expression vector. The purified recombinant fusion protein is active and was used to partially characterize the Cah6 protein. The purified recombinant fusion protein was cleaved with protease Factor Xa to separate Cah6 from the maltose-binding protein and the purified Cah6 protein was used to raise an antibody. Western blots, immunolocalization studies, and northern blots collectively indicated that Cah6 is constitutively expressed in the stroma of chloroplasts. A possible role for Cah6 in the CCM of C. reinhardtii is proposed.
Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO2 and HCO3. Aquatic photosynthetic organisms have evolved different forms of CO2-concentrating mechanisms to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CO2-concentrating mechanisms is the critical roles played by various specially localized extracellular and intracellular CAs. There are three evolutionarily unrelated CA families designated α-, β-, and γ-CA. In the green alga, Chlamydomonas reinhardtii Dangeard, eight CAs have now been identified, including three α-CAs and five β-CAs. In addition, C. reinhardtii has another CA-like gene, Glp1 that is similar to known γ-CAs. To characterize these different CA isoforms, some of the CA genes have been overexpressed to determine whether the proteins have CA activity and to generate antibodies for in vivo immunolocalization. The CA proteins Cah3, Cah6, and Cah8, and the γ-CA-like protein, Glp1, have been overexpressed. Cah3, Cah6, and Cah8 have CA activity, but Glp1 does not. At least two of these proteins, Cah3 and Cah6, are localized to the chloroplast. Using immunolocalization and sequence analyses, we have determined that Cah6 is located to the chloroplast stroma and confirmed that Cah3 is localized to the chloroplast thylakoid lumen. Activity assays show that Cah3 is 100 times more sensitive to sulfonamides than Cah6. We present a model on how these two chloroplast CAs might participate in the CO2-concentrating mechanism of C. reinhardtii. Key words: carbonic anhydrase, CO2-concentrating mechanism, Chlamydomonas, immunolocalization.
The Chlamydomonas reinhardtii tla1 (truncated light-harvesting chlorophyll antenna size) mutant was generated upon DNA insertional mutagenesis and shown to specifically possess a smaller than wild type (WT) chlorophyll antenna size in both photosystems. Molecular and genetic analysis revealed that the exogenous plasmid DNA was inserted at the end of the 5' UTR and just prior to the ATG start codon of a hitherto unknown nuclear gene (termed Tla1), which encodes a protein of 213 amino acids. The Tla1 gene in the mutant is transcribed with a new 5' UTR sequence, derived from the 3' end of the transforming plasmid. This replacement of the native 5' UTR and promoter regions resulted in enhanced transcription of the tla1 gene in the mutant but inhibition in the translation of the respective tla1 mRNA. Transformation of the tla1 mutant with WT Tla1 genomic DNA successfully rescued the mutant. These results are evidence that polymorphism in the 5' UTR of the Tla1 transcripts resulted in the tla1 phenotype and that expression of the Tla1 gene is a prerequisite for the development/assembly of the Chl antenna in C. reinhardtii. A blast search with the Tla1 deduced amino acid sequence
Background: Chlamydomonas reinhardtii, a green micro-alga can be grown at the lab heterotrophically or photo-heterotrophically in Tris-Phosphate-Acetate (TAP) medium which contains acetate as the sole carbon source. When grown in TAP medium, Chlamydomonas can utilize the exogenous acetate in the medium for gluconeogenesis using the glyoxylate cycle, which is also present in many bacteria and higher plants. A novel bacterial strain, LMJ, was isolated from a contaminated TAP medium plate of Chlamydomonas. We present our work on the isolation and physiological and biochemical characterizations of LMJ. Methods: Several microbiological tests were conducted to characterize LMJ, including its sensitivity to four antibiotics. We amplified and sequenced partially the 16S rRNA gene of LMJ. We tested if LMJ can utilize cyclic alkanes, aromatic hydrocarbons, poly-hydroxyalkanoates, and fresh and combusted car motor oil as the sole carbon source on Tris-Phosphate (TP) agar medium plates for growth. Results: LMJ is a gram-negative rod, oxidase-positive, mesophilic, non-enteric, pigmented, salt-sensitive bacterium. LMJ can ferment glucose, is starch hydrolysis-negative, and is very sensitive to penicillin and chloramphenicol. Preliminary spectrophotometric analyses indicate LMJ produces pyomelanin. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of LMJ showed that it matched to that of an uncultured bacterium clone LIB091_C05_1243. The nearest genus relative of LMJ is an Acidovorax sp. strain. LMJ was able to use alkane hydrocarbons, fresh and combusted car motor oil, poly-hydroxybutyrate, phenanthrene, naphthalene, benzoic acid and phenyl acetate as the sole carbon source for growth on TP-agar medium plates. Conclusions: LMJ has 99.14% sequence identity with the Acidovorax sp. strain A16OP12 whose genome has not been sequenced yet. LMJ’s ability to use chemicals that are common environmental pollutants makes it a promising candidate for further investigation for its use in bioremediation and, provides us with an incentive to sequence its genome.
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