CLONING, ANALYSIS AND INACTIVATION OF THE NDHK GENE ENCODING A SUBUNIT OF NADH QUINONE OXIDOREDUCTASE FROM ANABAENA PCC-7120

The function of the type-1 pyridine nucleotide dehydro nase (NDH-) in the cyanobacterium Anabaena PCC 7120 was investigated. Immunological a nalysis with antibodies raised against NdhK from Synechocystis PCC 680 3, a subunit of NDH-1, showed that NdhK in Anabaena PCC 7120 is only p resent on the plasma membrane, which confirms the results of previous studies [Howitt, C. A., Smith, G. D. & Day, D. A. (1993) Biochim. Biop hys. Acta 1141, 313-320]. Southern analysis with probes from the opero n encoding ndhC-K-J from Synechocystis PCC 6803 showed that this opero n is also conserved in Anabaena PCC 7120. Part of the operon was ampli fied using PCR with degenerate primers designed against two sequences encoding regions of NdhC and NdhJ that are conserved between cyanobact eria and chloroplasts. The nucleotide sequence of ndhK encodes a prote in of 245 amino acids with a predicted molecular mass of 27.5 kDa. The coding regions of ndhC and ndhK overlap by 7 bp, as found in the chlo roplasts of liverwort, maize, and rice. This is markedly different fro m the case in Synechocystis PCC 6803 where a 71-bp non-coding intergen ic spacer region lies between ndhC and ndhK. The ndhK clone was interr upted by the insertion of a kanamycin-resistance gene and used to tran sform Anabaena PCC 7120. 20 unsegregated transformants were produced, all of which died during attempts to segregate them. This indicates th at under the selection conditions used, ndhK is an essential gene in A nabaena PCC 7120.