Molecular genetic analysis of the zwf region of cyanobacterial genomes
The oxidative pentose phosphate (OPP) cycle is the main route of carbohydrate dissimilation in cyanobacteria in the dark. In heterocysts, the OPP is a supplier of reductant to nitrogenase, thus playing an important role in nitrogen fixation. Molecular genetic analysis of glucose-6-phosphate dehydrogenase (G6PDH) and the other components of the opp cycle have been focused on recently in an effort to understand the function and regulation of the cycle in vegetative cells and in heterocysts of cyanobacteria. This study presents data on nucleotide and amino acid sequences of three enzymes of the OPP cycle (glucose-6-phosphate dehydrogenase, transaldolase and fructose-l ,6-bisphosphatase) of the filamentous, heterocystous strain Anabaena sp. PCC7120. Mutagenesis studies on the transaldolase gene (tal) of Anabaena sp. PCC7120 and the opcA gene of the unicellular strain Synechococcus sp. PCC7942 are also reported. A 4,169 bp region around the zwf gene of Anabaena sp. PCC7120 was sequenced. Three genes are located in the region: fop, tal and zwf, which encode fructose-l,6-bisphosphatase (FBPase), transaldolase and glucose-6-phosphate dehydrogenase (G6PDH), respectively. The fop gene encodes a polypeptide of 349 amino acids. The product of the tal gene consists of 381 amino acids. The zwJ gene encodes a protein of 509 amino acids. Four cysteine residues are present in the enzyme. Two of these cysteines (Cys-187 and Cys-445) are absolutely conserved in cyanobacterial G6PDH. This result reinforces the likelihood of their role in the regulation of enzyme activity. Subclones carrying a range of zwf fragments of Anabaena sp. PCC7120 did not complement the zwJmutant Escherichia coli strain DF214. Western blot analysis showed that Anabaena sp. PCC7120 G6PDH did not express in E. coli DF214. Attempts aimed at production of a deletion/insertion zwf mutant of Anabaena sp. PCC7120 were not successful. The tal gene of Anabaena sp. PCC7120 was mutated to investigate growth and survival of the mutant cells in the presence and absence of combined nitrogen. Transaldolase activity in the tal mutant cells was similar to that in the wild-type cells. The growth rates of the tal mutant cells were not significantly different from that of the wild-type cells. These results imply that more than one copy of the tal gene is present in the Anabaena sp. PCC7120 genome. A gene, designated opcA, downstream from the zwf gene of Synechococcus sp. PCC7942 was mutated to investigate the possible effect of the gene on G6PDH activity. G6PDH activity in the mutant cells was reduced by 98.6%. Western blot analysis showed that the zwf was expressed in the opcA mutant cells, but the active form of the enzyme was a considerable reduced. All the results suggest that the opcA gene affects the assembly of G6PDH or enzyme activity, but possibly not expression of the zwf gene.