Over-expression of the β-carboxysomal CcmM protein in Synechococcus PCC7942 reveals a tight co-regulation of carboxysomal carbonic anhydrase (CcaA) and M58 content

Pathological cardiac hypertrophy, the maladaptive remodelling of the myocardium, often progresses to heart failure. The sodium–proton exchanger (NHE1) and chloride–bicarbonate exchanger (AE3) have been implicated as important in the hypertrophic cascade. Carbonic anhydrase II (CAII) provides substrates for these transporters (protons and bicarbonate, respectively). CAII physically interacts with NHE1 and AE3, enhancing their respective ion transport activities by increasing the concentration of substrate at their transport sites. Earlier studies found that a broad-spectrum carbonic anhydrase inhibitor prevented cardiomyocyte hypertrophy (CH), suggesting that carbonic anhydrase is important in the development of hypertrophy. Here we investigated whether cytosolic CAII was the CA isoform involved in hypertrophy. Neonatal rat ventricular myocytes (NRVMs) were transduced with recombinant adenoviral constructs to over-express wild-type or catalytically inactive CAII (CAII-V143Y). Over-expression of wild-type CAII in NRVMs did not affect CH development. In contrast, CAII-V143Y over-expression suppressed the response to hypertrophic stimuli, suggesting that CAII-V143Y behaves in a dominant negative fashion over endogenous CAII to suppress hypertrophy. We also examined CAII-deficient (Car2) mice, whose hearts exhibit physiological hypertrophy without any decrease in cardiac function. Moreover, cardiomyocytes from Car2 mice do not respond to prohypertrophic stimulation. Together, these findings support a role of CAII in promoting CH.

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Association of Carbonic Anhydrase Activity with Carboxysomes Isolated from the Cyanobacterium Synechococcus PCC7942

PLANT PHYSIOLOGY ◽

10.1104/pp.100.2.784 ◽

1992 ◽

Vol 100 (2) ◽

pp. 784-793 ◽

Cited By ~ 128

Author(s):

G. Dean Price ◽

John R. Coleman ◽

Murray R. Badger

Keyword(s):

Carbonic Anhydrase ◽

Carbonic Anhydrase Activity ◽

Synechococcus Pcc7942

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Cloning, Over-Expression, and Purification of Carbonic Anhydrase from an Extremophilic Bacterium: An Introduction to Advanced Molecular Biology

The American Biology Teacher ◽

10.1525/abt.2018.80.1.29 ◽

2018 ◽

Vol 80 (1) ◽

pp. 29-34 ◽

Cited By ~ 1

Author(s):

Vijayakumar Somalinga ◽

Hannah Klemmer ◽

Ashikha Arun ◽

Stephanie Mathews ◽

Hannah Wapshott ◽

...

Keyword(s):

High School ◽

High School Students ◽

Molecular Biology ◽

Carbonic Anhydrase ◽

Research Program ◽

Boot Camp ◽

Biofuel Production ◽

Carbonic Anhydrases ◽

School Students ◽

Over Expression

The photosynthetic bioreactor research program is a training platform appropriate for introducing advanced molecular biology techniques to undergraduate students and advanced high school biology students. For this advanced molecular biology training exercise, the enzyme carbonic anhydrase was cloned, over-expressed, purified, and functionally characterized. Carbonic anhydrases are industrially important enzymes with potential use in carbon sequestration and biofuel production. Alpha and beta carbonic anhydrases from Photobacterium profundum, a psychrophilic, halotolerant bacterium, were characterized in this study. Carbonic anhydrases that can withstand high salinity and are active at low temperatures can be transformed into oleaginous marine microalgae to enhance biofuel production. Our research program started with a three-day boot camp with lectures in relevant topics of molecular biology, microbiology, and research methods. After the boot camp, the lab phase of the project involved training students to perform polymerase chain reaction, DNA gel electrophoresis, DNA ligation, and bacterial transformation. In the final phase of the project, students were trained in recombinant protein over-expression and protein purification techniques. Here we report successful cloning and over-expression by high school students of two novel carbonic anhydrases from a psychrohalophile with application in biofuel production.

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A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.10.4437 ◽

1992 ◽

Vol 89 (10) ◽

pp. 4437-4441 ◽

Cited By ~ 129

Author(s):

H. Fukuzawa ◽

E. Suzuki ◽

Y. Komukai ◽

S. Miyachi

Keyword(s):

Carbon Dioxide ◽

Carbonic Anhydrase ◽

Carbon Dioxide Fixation ◽

Photosynthetic Carbon ◽

Synechococcus Pcc7942

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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

Canadian Journal of Botany ◽

10.1139/b98-063 ◽

1998 ◽

Vol 76 (6) ◽

pp. 1153-1160 ◽

Cited By ~ 5

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.

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