Identification and Characterization of the ictA/ndhL Gene

The ictA gene, renamed ndhL in this paper, essential to inorganic carbon transport of Synechocystis PCC6803, was expressed in Eschericia coli as a fusion protein with glutathione S-transferase. An antibody was raised against this fusion protein. Western analysis of the thylakoid membrane of wild-type (WT) Synechocystis revealed that a protein with an apparent molecular mass of 6.7 kilodaltons cross-reacted with this antibody. No immunoreactive protein was present in the thylakoid membranes of the Synechocystis mutants, RKb and M9, which have defects in the ictA/ndhL gene, or in the cytoplasmic membranes of the WT and mutant cells. Thus, the protein reacted with the antibody is the ictA gene product (IctA) and is localized in the thylakoid membrane of WT cells. IctA was absent in the thylakoid membranes of the M55 mutant, in which the ndhB gene is inactivated, and was poorly immunostained in the membranes of the mutants (M-ndhC and M-ndhK) constructed by inactivating the ndhC and ndhK genes of WT Synechocystis, respectively. The carbon dioxide uptake activity was nearly zero in M-ndhK and was about 40% of the activity of WT cells in M-ndhC. The RKb, M-ndhC, and M-ndhK mutants were unable to grow or grew very slowly under photoheterotrophic conditions. These results indicated that NADH dehydrogenase is essential to inorganic carbon transport and photoheterotrophic growth of Synechocystis and that IctA is one of the subunits of this enzyme. encodes subunit 2 of NADH dehydrogenase. The ictA gene encodes a hydrophobic protein consisting of 80 amino acids (11). No homologous gene has been found in the database. In addition to the ndhB gene, several ndh genes have recently been cloned from Synechocystis PCC6803 (1, 21). The previous study on the ndhB gene (10) suggested that inactivation of other ndh genes might also have significant effect on the Ci-transporting activity of the cells. The present study was undertaken in an attempt to identify and characterize the ictA gene product. For this purpose, an antibody was raised against the product of the ictA gene expressed in Escherichia coli. In addition, Synechocystis mutants (M-ndhC and M-ndhK) were constructed by inactivating the ndhC and ndhK genes of WT cells, respectively. Western analyses of the membranes fromWT and mutant cells obtained in the present and previous studies enabled me to identify the ictA gene product and to characterize it as a component of the cyanobacterial NADH dehydrogenase. Based on the evidence presented here, I propose to rename the ictA gene ndhL. A possible role of NADH dehydrogenase in C, transport is discussed. MATERIALS AND METHODS