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Cyanobacteria possess a CO2-concentating mechanism that involves active CO2 uptake and HCO 3 ؊ transport. For CO2 uptake, we have identified two systems in the cyanobacterium Synechocystis sp. strain PCC 6803, one induced at low CO 2 and one constitutive. The low CO2-induced system showed higher maximal activity and higher affinity for CO2 than the constitutive system. On the basis of speculation that separate NAD(P)H dehydrogenase complexes were essential for each of these systems, we reasoned that inactivation of one system would allow selection of mutants defective in the other. Thus, mutants unable to grow at pH 7.0 in air were recovered after transformation of a ⌬ndhD3 mutant with a transposon-bearing library. Four of them had tags within slr1302 (designated cupB), a homologue of sll1734 (cupA), which is cotranscribed with ndhF3 and ndhD3. The ⌬cupB, ⌬ndhD4, and ⌬ndhF4 mutants showed CO2-uptake characteristics of the low CO2-induced system observed in wild type. In contrast, mutants ⌬cupA, ⌬ndhD3, and ⌬ndhF3 showed characteristics of the constitutive CO2-uptake system. Double mutants impaired in one component of each of the systems were unable to take up CO 2 and required high CO2 for growth. Phylogenetic analysis indicated that the ndhD3͞ ndhD4-, ndhF3͞ndhF4-, and cupA͞cupB-type genes are present only in cyanobacteria. Most of the cyanobacterial strains studied possess the ndhD3͞ndhD4-, ndhF3͞ndhF4-, and cupA͞cupB-type genes in pairs. Thus, the two types of NAD(P)H dehydrogenase complexes essential for low CO2-induced and constitutive CO2-uptake systems associated with the NdhD3͞NdhF3͞CupA-homologues and NdhD4͞NdhF4͞CupB-homologues, respectively, appear to be present in these cyanobacterial strains but not in other organisms.NAD(P)H dehydrogenase ͉ constitutive CO2 uptake ͉ affinity to CO2 ͉ CO2-concentrating mechanism I n cyanobacteria, NAD(P)H dehydrogenase (NDH-1) is essential for both CO 2 uptake (1-3) and photosystem-1 (PSI) cyclic electron transport (4). It has been postulated that uptake of CO 2 is energized by NDH-1-dependent PSI-cyclic electron transport (1). However, observations that mutants defective in ndhD3 display normal cyclic electron transport but are unable to induce high-affinity CO 2 uptake suggest the presence of multiple NDH-1 complexes (5-7). Two types of functionally distinct NDH-1 complexes were recently recognized in Synechocystis sp. strain PCC 6803 with the aid of mutants impaired in one or more subunits of NDH-1 (7). One complex, containing NdhD1 or NdhD2, plays a major role in PSI-cyclic electron f low but is not involved in CO 2 uptake (7). When the second type of NDH-1 complex is inactivated (in the double mutant ⌬ndhD3͞⌬ndhD4), nearly normal PSI-cyclic electron f low is observed, but the mutant does not take up CO 2 and is unable to grow under an air level of CO 2 (7). The single mutants ⌬ndhD3 and ⌬ndhD4, on the other hand, possess CO 2 -uptake activity and can grow under low CO 2 conditions (7). These results raised the possibility of multiple systems for CO 2 uptake. In...