Dataset Information

Discovery of an algal mitochondrial carbonic anhydrase: molecular cloning and characterization of a low-CO2-induced polypeptide in Chlamydomonas reinhardtii.

ABSTRACT: In green unicellular algae, several polypeptides are induced upon exposure to limiting CO2. We report here on the localization and characterization of one of these, a 22-kDa polypeptide in Chlamydomonas reinhardtii. This nuclear-encoded polypeptide is induced in the mitochondria by a lowering of the partial pressure of CO2 in the growth medium from 5% to air CO2 levels. Sequencing of two different cDNA clones coding for the polypeptide identified it as a 20.7-kDa beta-type carbonic anhydrase (CA; carbonate dehydratase, carbonate hydro-lyase, EC 4.2.1.1). The two clones differ in their nucleotide sequences but code for identical proteins, showing that this CA is encoded by at least two genes. Northern blot hybridization reveals that mRNA transcripts are only present in cells transferred to air CO2 levels. A comparison of the deduced amino acid sequence with those of other beta-CAs shows the largest degree of similarity with CA from the cyanobacterium Synechocystis (50% identity and 66% similarity). To our knowledge, this is the first identification and characterization of a mitochondrial CA from a photosynthetic organism.

SUBMITTER: Eriksson M

PROVIDER: S-EPMC1074525 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Discovery of an algal mitochondrial carbonic anhydrase: molecular cloning and characterization of a low-CO2-induced polypeptide in Chlamydomonas reinhardtii.

Eriksson M M Karlsson J J Ramazanov Z Z Gardeström P P Samuelsson G G

Proceedings of the National Academy of Sciences of the United States of America 19961001 21

In green unicellular algae, several polypeptides are induced upon exposure to limiting CO2. We report here on the localization and characterization of one of these, a 22-kDa polypeptide in Chlamydomonas reinhardtii. This nuclear-encoded polypeptide is induced in the mitochondria by a lowering of the partial pressure of CO2 in the growth medium from 5% to air CO2 levels. Sequencing of two different cDNA clones coding for the polypeptide identified it as a 20.7-kDa beta-type carbonic anhydrase (CA ...[more]

PMID: 8876257

Similar Datasets

Project description:BackgroundCah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO(2) fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO(2) conditions.Results/conclusionsIn the present work we demonstrate that after transfer to low CO(2), Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO(2) conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO(2) conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO(2) conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO(2) the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules.SignificanceThis is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

Project description:Brucella spp. are intracellular pathogens that cause a disease known as brucellosis. Though the genus is highly monomorphic at the genetic level, species have animal host preferences and some defining physiologic characteristics. Of note is the requirement for CO2 supplementation to cultivate particular species, which confounded early efforts to isolate B. abortus from diseased cattle. Differences in the capacity of Brucella species to assimilate CO2 are determined by mutations in the carbonic anhydrase gene, bcaA Ancestral single-nucleotide insertions in bcaA have resulted in frameshifted pseudogenes in B. abortus and B. ovis lineages, which underlie their inability to grow under the low CO2 tension of a standard atmosphere. Incubation of wild-type B. ovis in air selects for mutations that "rescue" a functional bcaA reading frame, which enables growth under low CO2 and enhances the growth rate under high CO2 Accordingly, we show that heterologous expression of functional Escherichia coli carbonic anhydrases enables B. ovis growth in air. Growth of B. ovis is acutely sensitive to a reduction in CO2 tension, while frame-rescued B. ovis mutants are insensitive to CO2 shifts. B. ovis initiates a gene expression program upon CO2 downshift that resembles the stringent response and results in transcriptional activation of its type IV secretion system. Our study provides evidence that loss-of-function insertion mutations in bcaA sensitize the response of B. ovis and B. abortus to reduced CO2 tension relative to that of other Brucella lineages. CO2-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts.IMPORTANCEBrucella spp. are highly related, but they exhibit differences in animal host preference that must be determined by genome sequence differences. B. ovis and the majority of B. abortus strains require high CO2 tension to be cultivated in vitro and harbor conserved insertional mutations in the carbonic anhydrase gene, bcaA, which underlie this trait. Mutants that grow in a standard atmosphere, first reported nearly a century ago, are easily selected in the laboratory. These mutants harbor varied indel polymorphisms in bcaA that restore its consensus reading frame and rescue its function. Loss of bcaA function has evolved independently in the B. ovis and B. abortus lineages and results in a dramatically increased sensitivity to CO2 limitation.

Dataset Information

Discovery of an algal mitochondrial carbonic anhydrase: molecular cloning and characterization of a low-CO2-induced polypeptide in Chlamydomonas reinhardtii.

Publications

Discovery of an algal mitochondrial carbonic anhydrase: molecular cloning and characterization of a low-CO2-induced polypeptide in Chlamydomonas reinhardtii.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets