Catalytic exchange of 18O from 13C18O-labelled CO2 by wild-type cells and ecaA, ecaB, and ccaA mutants of the cyanobacteria Synechococcus PCC7942 and Synechocystis PCC6803

1998 ◽  
Vol 76 (6) ◽  
pp. 1153-1160 ◽  
Author(s):  
Anthony KC So ◽  
Harriette GC Van Spall ◽  
John R Coleman ◽  
George S Espie
Keyword(s):  
Carbonic Anhydrase ◽  
Wild Type ◽  
Synechocystis Pcc6803 ◽  
Concentrating Mechanism ◽  
Absolute Requirement ◽  
Synechococcus Pcc7942 ◽  
Catalytic Role ◽  
State Growth ◽  
Exchange Patterns ◽  
Catalytic Exchange

Expression constructs carrying the ecaA, ecaB, and ccaA genes from the cyanobacteria Synechococcus PCC7942 and Synechocystis PCC6803 were generated and each transformed into Escherichia coli. Lysates from cells expressing recombinant protein were prepared and assayed using mass spectrometry for their ability to catalyze the exchange of 18O from 13C18O to H2O. No carbonic anhydrase (CA) activity was detected from the cell lysates containing recombinant EcaA or EcaB proteins, whereas the lysate harbouring the CcaA polypeptide clearly accelerated the rate of 18O exchange. An ecaA deletion mutant in Synechococcus and insertionally inactivated ecaB and ccaA mutants in Synechocystis were generated and similarly assessed for CA activity. All mutants displayed a transient, ethoxyzolamide-sensitive, CA-like catalysis that was also exhibited by wild-type cells. The CcaA-deficient mutant showed a reduced capacity to exchange 18O out of 13C18O-labelled CO2 in the light as well as an absolute requirement for high Ci for growth, reflecting the importance of the carboxysomal CA to the operation of the cyanobacterial CO2-concentrating mechanism. No detectable differences in 18O exchange patterns, CO2 or HCO3- transport, or steady-state growth were observed between the ecaA and ecaB mutants and wild-type cells, indicating that neither EcaA nor EcaB play an essential catalytic role in the functioning of the CO2-concentrating mechanism.Key words: carbonic anhydrase, CO2-concentrating mechanism, cyanobacteria, 18O exchange, Synechococcus PCC7942, Synechocystis PCC6803.

Studies on the carbonic anhydrase activity in Synechocystis PCC6803 wild type and an acetazolamide-resistant mutant

1991 ◽  
Vol 69 (5) ◽  
pp. 1103-1108 ◽  
Author(s):  
S. Bedu ◽  
F. Joset
Keyword(s):  
Carbonic Anhydrase ◽  
Inorganic Carbon ◽  
Physiological Role ◽  
Resistant Mutant ◽  
Carbonic Anhydrase Activity ◽  
Wild Type ◽  
Synechocystis Pcc6803 ◽  
Physiological Analysis ◽  
Carbon Concentrations

The problem of the role and the localization of carbonic anhydrase activity in cyanobacteria has been addressed by two approaches using strain Synechocystis PCC6803. Physiological analysis of the differential effects of carbonic anhydrase inhibitors on the entry and accumulation of CO2 in cells grown under low or high inorganic carbon concentrations and determination of carbonic anhydrase activities in cellular subfractions led to the hypothesis of the presence of two different enzymes in this strain. This conclusion is compatible with current models. Only the internal enzyme could be regulated by variations of the external inorganic carbon concentrations. A parallel analysis of a mutant of this strain resistant to the inhibitor acetazolamide supported the hypothesis of the presence of two enzymes. This clone would be selectively impaired in the carbonic anhydrase activity involved in the maintenance of the internal CO2 pool, while its transport capacity is unchanged. Key words: carbonic anhydrase, physiological role, localization, inhibitors, cyanobacteria, mutant.

The functioning of the CO2 concentrating mechanism in several cyanobacterial strains: a review of general physiological characteristics, genes, proteins, and recent advances

1998 ◽  
Vol 76 (6) ◽  
pp. 973-1002 ◽  
Author(s):  
G Dean Price ◽  
Dieter Sültemeyer ◽  
Barbara Klughammer ◽  
Martha Ludwig ◽  
Murray R Badger
Keyword(s):  
Distinct Type ◽  
Ribulose Bisphosphate Carboxylase ◽  
Genome Database ◽  
Bisphosphate Carboxylase ◽  
Blue Green Algae ◽  
Synechocystis Pcc6803 ◽  
Concentrating Mechanism ◽  
Recent Advances ◽  
Ribulose Bisphosphate Carboxylase Oxygenase ◽  
Synechococcus Pcc7942

Cyanobacteria (blue-green algae) possess an environmental adaptation for survival at low CO2 concentrations. The adaptation is known as a CO2 concentrating mechanism (CCM), and it functions to actively transport and accumulate inorganic carbon ( and CO2; Ci) within the cell and then uses this Ci pool to provide elevated CO2 concentrations around the primary CO2-fixing enzyme, ribulose bisphosphate carboxylase-oxygenase (Rubisco). It appears that the site of CO2 elevation is within a unique microcompartment known as the carboxysome, which is a proteinaceous polyhedral body that contains most, if not all, of the Rubisco within the cell. This review covers comparative aspects of physiology, genetics, and proteins involved in the cyanobacterial CCM with particular focus on recent advances. This review highlights information on three strains of unicellular cyanobacteria, namely Synechocystis PCC6803 (freshwater strain; for which a full genome database is now available), Synechococcus PCC7002 (coastal marine strain) and Synechococcus PCC7942 (freshwater strain). Genes that may be involved in the CCM, directly or indirectly, are summarized in tabular form. For Synechocystis PCC6803, the number of genes related to CCM activity is now in excess of 50; however, 19 of these components have the potential to code for several distinct type-1, NADH dehydrogenase complexes.Key words: cyanobacteria, CO2 concentrating mechanism, carboxysomes, genes, photosynthesis, transporters.

The use of mutants in the analysis of the CO2-concentrating mechanism in cyanobacteria

1998 ◽  
Vol 76 (6) ◽  
pp. 1035-1042 ◽  
Author(s):  
Hiroshi Ohkawa ◽  
Masatoshi Sonoda ◽  
Hirokazu Katoh ◽  
Teruo Ogawa
Keyword(s):  
Inorganic Carbon ◽  
Cytoplasmic Membrane ◽  
Wild Type ◽  
Proton Extrusion ◽  
Synechocystis Pcc6803 ◽  
Concentrating Mechanism ◽  
In The Wild ◽  
New Type ◽  
Inorganic Carbon Transport ◽  
Carbon Transport

Mutants of cyanobacteria defective in parts of the CO2-concentrating mechanism are classified into three types. (i) Mutants defective in inorganic carbon transporters. One of these mutants was constructed by inactivating cmpA encoding 42 kDa protein in the cytoplasmic membrane. (ii) Mutants defective in NAD(P)H dehydrogenase(s). There are five ndhD genes in Synechocystis PCC6803, two of them expressed constitutively and three inducible by low CO2. Two kinds of NAD(P)H dehydrogenase appear to be involved in energizing and inducing the high affinity inorganic carbon transport system. (iii) Mutants defective in carboxysome with impaired ccm or icfA genes. New type of mutants with impaired cotA (renamed as pxcA) have also been isolated. These mutants did not show light-induced proton extrusion and were unable to grow at acidic pHs. A mutant constructed by inactivating cotA (pxcA) in the wild-type Synechocystis was unable to transport CO2 at pH 6.5. We concluded that cotA (pxcA) has a role in light-induced proton extrusion that is essential at acidic pHs to extrude protons produced during CO2 transport.Key words: CO2-concentrating mechanism (CCM), CO2 transport, NAD(P)H dehydrogenase, proton extrusion, carboxysome, mutant.

Evidence for the role of carboxysomes in the cyanobacterial CO2-concentrating mechanism

1991 ◽  
Vol 69 (5) ◽  
pp. 963-973 ◽  
Author(s):  
G. Dean Price ◽  
Murray R. Badger
Keyword(s):  
Carbonic Anhydrase ◽  
Open Reading Frame ◽  
Carbonic Anhydrase Ii ◽  
Reading Frame ◽  
Electron Microscopy Data ◽  
Concentrating Mechanism ◽  
Synechococcus Pcc7942 ◽  
Microscopy Data

In this paper we provide a brief summary of recent work that supports the notion that the carboxysome, a polyhedral body containing Rubisco, plays a pivotal role in the cyanobacterial CO2-concentrating mechanism. This work includes (i) experiments in which active human carbonic anhydrase II was expressed in the cytosol of the cyanobacterium Synechococcus PCC7942 resulting in a high CO2-requiring phenotype, and (iii) characterization of two types of high CO2 requiring mutants of Synechococcus that appear to be incapable of generating CO2 within the carboxysomes. Carboxysomes appear to serve as a microcompartment where CO2 can be generated and elevated at the site of carboxylation. We also report on the identification by complementation and sequence analysis of a 300 base pair open reading frame that is located upstream of rbcL and that is involved in the correct functioning of the carboxysome. Preliminary electron microscopy data is also considered on the biogenesis of carboxysomes in Anabaena variabilis M3. Key words: carboxysome, cyanobacteria, carbonic anhydrase, CO2-concentrating mechanism, genetic analysis, mutants.

scholarly journals N-terminal fatty acylation of the α-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation

1994 ◽  
Vol 303 (3) ◽  
pp. 697-700 ◽  
Author(s):  
F Galbiati ◽  
F Guzzi ◽  
A I Magee ◽  
G Milligan ◽  
M Parenti
Keyword(s):  
Palmitic Acid ◽  
G Protein ◽  
Fatty Acyl ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Α Subunit ◽  
Metabolic Labelling ◽  
Fatty Acylation ◽  
Absolute Requirement ◽  
Cdna Construct

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

scholarly journals The Dependence of the Oxygen-Concentrating Mechanism of the Teleost Eye (Salmo gairdneri) on the Enzyme Carbonic Anhydrase

1969 ◽  
Vol 54 (2) ◽  
pp. 203-211 ◽  
Author(s):  
Michael B. Fairbanks ◽  
J. Russell Hoffert ◽  
Paul O. Fromm
Keyword(s):  
Carbonic Anhydrase ◽  
Environmental Water ◽  
Salmo Gairdneri ◽  
Complete Suppression ◽  
Oxygen Inhibition ◽  
Rete Mirabile ◽  
Arterial Blood ◽  
Concentrating Mechanism ◽  
The One ◽  
Erythrocyte Carbonic Anhydrase

Microoxygen polarographic electrodes were constructed and used to measure oxygen tension (POO2) in the eyes of rainbow trout (Salmo gairdneri). The values obtained are compared with arterial blood and environmental water POO2 and indicate that there is an oxygen-concentrating mechanism in the eye supplying oxygen to the avascular retina. Anatomically similar retes suggest that the mechanism is similar to the one which exists in the swim bladder. Elimination of the arterial blood supply to the choroidal gland rete mirabile of the eye (through pseudobranchectomy) and the consequent lowering of ocular oxygen tensions implicate the choroidal gland as one of the major components of the oxygen-concentrating mechanism. After pseudobranchectomy the presence of ocular POO2 above that of arterial blood is indicative of a secondary structure in the eye capable of concentrating oxygen. Inhibition of carbonic anhydrase, using acetazolamide, is shown to result in complete suppression of the oxygen-concentrating mechanism. A hypothesis is advanced for the participation of retinal-choroidal and erythrocyte carbonic anhydrase in the oxygen-concentrating mechanism.

Evolution of ethylene by Saccharomyces cerevisiae as influenced by the carbon source for growth and the presence of air

1978 ◽  
Vol 24 (6) ◽  
pp. 637-642 ◽  
Author(s):  
K. C. Thomas ◽  
Mary Spencer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Ethylene Production ◽  
Atp Synthesis ◽  
Yeast Cells ◽  
Yeast Culture ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Absolute Requirement ◽  
Wild Type Yeast

Effects of the carbon source and oxygen on ethylene production by the yeast Saccharomyces cerevisiae have been studied. The amounts of ethylene evolved by the yeast culture were less than those detected in the blank (an equal volume of uninoculated medium), suggesting a net absorption of ethylene by the yeast cells. Addition of glucose to the lactate-grown yeast culture induced ethylene production. This glucose-induced stimulation of ethylene production was inhibited to a great extent by cycloheximide. Results suggested that the yeast cells in the presence of glucose synthesized an ethylene precursor and passed it into the medium. The conversion of this precursor to ethylene might be stimulated by oxygen. The fact that ethylene was produced by the yeast growing anaerobically and also by respiration-deficient mutants isolated from the wild-type yeast suggested that mitochondrial ATP synthesis was not an absolute requirement for ethylene biogenesis.

Effect of PCMBS on CO2permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant

1998 ◽  
Vol 275 (6) ◽  
pp. C1481-C1486 ◽  
Author(s):  
Gordon J. Cooper ◽  
Walter F. Boron
Keyword(s):  
Carbonic Anhydrase ◽  
Intracellular Ph ◽  
Xenopus Oocytes ◽  
Membrane Lipid ◽  
Wild Type ◽  
Aquaporin 1 ◽  
Gas Channel ◽  
A Cell ◽  
Pass Through ◽  
Rule Out

A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 ( Cell Physiol. 43): C543–548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by ∼40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane’s lipid, or 4) increasing the expression of a native “gas channel.” The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2permeability is unaffected by treatment with PCMBS. These data rule out options 2–4 listed above. Thus our results suggest that CO2passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.

scholarly journals Alpha Carbonic Anhydrase 5 Mediates Stimulation of ATP Synthesis by Bicarbonate in Isolated Arabidopsis Thylakoids

2021 ◽  
Vol 12 ◽  
Author(s):  
Tatiana P. Fedorchuk ◽  
Inga A. Kireeva ◽  
Vera K. Opanasenko ◽  
Vasily V. Terentyev ◽  
Natalia N. Rudenko ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Arabidopsis Thaliana ◽  
Carbonic Anhydrase ◽  
Atp Synthesis ◽  
Thylakoid Membranes ◽  
Wild Type ◽  
Gene Encoding ◽  
Mutant Lines ◽  
Stimulation Of ◽  
Soluble Carbonic Anhydrase

We studied bicarbonate-induced stimulation of photophosphorylation in thylakoids isolated from leaves of Arabidopsis thaliana plants. This stimulation was not observed in thylakoids of wild-type in the presence of mafenide, a soluble carbonic anhydrase inhibitor, and was absent in thylakoids of two mutant lines lacking the gene encoding alpha carbonic anhydrase 5 (αCA5). Using mass spectrometry, we revealed the presence of αCA5 in stromal thylakoid membranes of wild-type plants. A possible mechanism of the photophosphorylation stimulation by bicarbonate that involves αCA5 is proposed.

scholarly journals The switch of intestinal Slc26 exchangers from anion absorptive to HCO3−secretory mode is dependent on CFTR anion channel function

2010 ◽  
Vol 298 (5) ◽  
pp. C1057-C1065 ◽  
Author(s):  
Anurag Kumar Singh ◽  
Brigitte Riederer ◽  
Mingmin Chen ◽  
Fang Xiao ◽  
Anja Krabbenhöft ◽  
...  
Keyword(s):  
Anion Channel ◽  
Transport Activity ◽  
Wild Type ◽  
Regulatory Relationship ◽  
Genetic Ablation ◽  
Anion Transporters ◽  
Absolute Requirement ◽  
Heterologous Expression Systems ◽  
Deficient Mice ◽  
Nhe3 Inhibitor

CFTR has been recognized to function as both an anion channel and a key regulator of Slc26 anion transporters in heterologous expression systems. Whether this regulatory relationship between CFTR and Slc26 transporters is seen in native intestine, and whether this effect is coupled to CFTR transport function or other features of this protein, has not been studied. The duodena of anesthetized CFTR-, NHE3-, Slc26a6-, and Scl26a3-deficient mice and wild-type (WT) littermates were perfused, and duodenal bicarbonate (HCO3−) secretion (DBS) and fluid absorptive or secretory rates were measured. The selective NHE3 inhibitor S1611 or genetic ablation of NHE3 significantly reduced fluid absorptive rates and increased DBS. Slc26a6 (PAT1) or Slc26a3 (DRA) ablation reduced the S1611-induced DBS increase and reduced fluid absorptive rates, suggesting that the effect of S1611 or NHE3 ablation on HCO3−secretion may be an unmasking of Slc26a6- and Slc26a3-mediated Cl−/HCO3−exchange activity. In the absence of CFTR expression or after application of the CFTR(inh)-172, fluid absorptive rates were similar to those of WT, but S1611 induced virtually no increase in DBS, demonstrating that CFTR transport activity, and not just its presence, is required for Slc26-mediated duodenal HCO3−secretion. A functionally active CFTR is an absolute requirement for Slc26-mediated duodenal HCO3−secretion, but not for Slc26-mediated fluid absorption, in which these transporters operate in conjunction with the Na+/H+exchanger NHE3. This suggests that Slc26a6 and Slc26a3 need proton recycling via NHE3 to operate in the Cl−absorptive mode and Cl−exit via CFTR to operate in the HCO3−secretory mode.

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