Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803

Primary ion pumps and antiporters exist as multigene families in the Synechocystis sp. PCC 6803 genome and show very strong homologies to those found in higher plants. The gene knock-outs of five putative Na+/H+ antiporters (slr1727, sll0273, sll0689, slr1595 and slr0415) and seven cation ATPases (sll1614, sll1920, slr0671-72, slr0822, slr1507-08-09, slr1728- 29 and slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html) were performed in this study relying on homologous recombination with mutagenenic fragments constructed using a fusion polymerase chain reaction (PCR) approach. The impacts of these gene knock-outs were evaluated in terms of Na+ and pH, and light-induced acidification and alkalization that are asso-ciated with inorganic carbon uptake. Two of the five putative antiporter mutants exhibit a characteristic interplay between the pH and Na+ dependence of growth, but only one of the antiporters appears to be necessary for high NaCl tolerance. On the other hand, the mutation of one of the two copper-trafficking ATPases produces a cell line that shows acute NaCl sensitivity. Additionally, disruptions of a putative Ca2+-ATPase and a gene cluster encoding a putative Na+-ATPase subunit also cause high NaCl sensitivity. The findings and possible mechanisms are discussed in relation to the potential roles of these transporters in Synechocystis sp. PCC 6803.