Isolation of ccmKLMN genes from the marine cyanobacterium, Synechococcus sp. PCC7002 (Cyanophyceae), and evidence that CcmM is essential for carboxysome assembly

A high CO2 requiring mutant of the marine cyanobacterium Synechococcus PCC7002 was generated using a random gene-tagging procedure. This mutant demonstrated a reduced photosynthetic affinity for inorganic carbon (Ci) and accumulated high internal levels of Ci that could not be used for photosynthesis. Analysis of the mutant genomic DNA showed that the mutagenesis had disrupted a cluster of genes involved in the cyanobacterial CO2 concentrating mechanism (CCM), the so-called ccm genes. These characteristics are consistent with a cyanobacterial mutant with defects in carboxysome assembly and/or functioning. Further genomic analyses indicated that the genes of the Synechococcus PCC7002 operon, ccmKLMN, are structurally similar to those of two closely related cyanobacteria, Synechococcus PCC7942 and Synechocystis PCC6803. The Synechococcus PCC7002 ccmM gene, which encodes a polypeptide with a predicted size of 70 kDa, was the direct target of the mutagenesis event. The CcmM protein has two distinct regions: an N-terminal region that shows similarity to an archaeon gamma carbonic anhydrase and a C-terminal region that contains repeated domains demonstrating sequence similarity to the small subunit of Rubisco. Physiological analysis of a ccmM-defined mutant showed that these cells were essentially identical to the original mutant; they required high CO2 concentrations for growth, they had a low photosynthetic affinity for Ci, and they internalized Ci to high levels. Moreover, ultrastructural examination showed that both the original and the defined mutants lack carboxysomes. Thus, our results demonstrate that the ccmM gene of Synechococcus PCC7002 encodes a polypeptide that is essential for carboxysome assembly and therefore for proper functioning of the cyanobacterial CCM

The involvement of NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in Synechococcus sp PCC7002 gives evidence for multiple NDH-1 complexes with specific roles in cyanobacteria

Random gene tagging was used to obtain new mutants of the marine cyanobacterium, Synechococcus sp. PCC7002, with defects in the CO2-concentrating mechanism (CCM). Two of these mutants, K22 and A41, showed poor growth at limiting CO2. Isolation and sequencing of a 6.6 kb genomic region revealed the existence of five potential protein-coding regions, all arranged in the same transcriptional direction. These regions code for an RbcR homologue, NdhF3 (subunit 5 of type 1 NAD(P)H dehydrogenase; NDH-1 complex), NdhD3 (subunit 4 of NDH-1), ORF427 and ORF133 (hypothetical proteins). Insertional mutants in ndhD3, ndhF3 and ORF427, like A41 and K22, were all incapable of inducing high-affinity CO2 uptake and were not fully capable of inducing high-affinity HCO3- transport. ndhD3 and ndhF3 mutants displayed P700 re-reduction rates identical to wild-type cells, suggesting that NdhD3 is part of a specific NDH-1 complex that is not involved in photosynthetic cyclic electron transport. Thus, it is feasible that NdhD3, NdhF3 and ORF427 might form part of a novel NDH-1 complex located on the cytoplasmic membrane and involved in tightly coupled energization of high-affinity CO2 transport. The possibility of multiple, functionally distinct NDH-1 complexes in cyanobacteria is discussed