scholarly journals Cloning and Inactivation of a Gene Essential to Inorganic Carbon Transport of Synechocystis PCC6803

1991 ◽  
Vol 96 (1) ◽  
pp. 280-284 ◽  
Author(s):  
Teruo Ogawa
Keyword(s):  
Inorganic Carbon ◽  
Synechocystis Pcc6803 ◽  
Inorganic Carbon Transport ◽  
Carbon Transport

Cloning and characterization of the genes required for inorganic carbon transport of Synechocystis PCC6803

1991 ◽  
Vol 69 (5) ◽  
pp. 951-956 ◽  
Author(s):  
Teruo Ogawa
Keyword(s):  
Nadh Dehydrogenase ◽  
Inorganic Carbon ◽  
Wild Type ◽  
Reading Frame ◽  
Synechocystis Pcc6803 ◽  
Dna Libraries ◽  
Insertional Inactivation ◽  
Inorganic Carbon Transport ◽  
Carbon Transport

Transformation of the high CO2-requiring mutants of Synechocystis PCC6803 defective in inorganic carbon (Ci) transport (RKa and RKb) by wild type (WT) DNA libraries restored their ability to grow under air levels of CO2. Two clones (PK-1 and HP-1), which complement RKa and RKb, respectively, were isolated from the libraries. PK-1 contained an 11.8-kilobase pair (kbp) DNA insert. The sequence of amino acid coded in the DNA in the region of the mutation showed an extensive homology to that of the ndh2 gene product of liverwort chloroplasts, which is suspected to be the subunit 2 of NADH dehydrogenase. Based on the result, we designated the gene mutated in RKa as ndh2. Inactivation of the ndh2 gene in the WT cells by inserting an aminoglycoside-3′-phosphotransferase gene generated a mutant (M57) that was unable to grow under low CO2 conditions. HP-1 contained a 5.4-kbp DNA insert. Sequencing of nucleotides in the region of the mutation revealed an open reading frame that codes a hydrophobic protein that consists of 80 amino acids. Insertional inactivation of this putative Ci transport gene, designated ictA, generated a high CO2-requiring mutant (M9). All these mutants (RKa, RKb, M9, and M57) showed very low activity of CO2 uptake into the intracellular Ci pools. The activity of HCO3− uptake was negligibly low in RKb, M9, M57 and high CO2-grown cells of RKa, and was about 10% the activity of wild type cells in low CO2-adapted cells of RKa. Key words: CO2-concentrating mechanism, inorganic carbon transport, Synechocystis PCC6803, mutant, NADH dehydrogenase, insertional inactivation.

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.

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