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

Abstract

Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803 adapts to a wide range of adverse environments including high salinity (Joset et al., 1996). These characteristics make it a useful model for understanding stress tolerance in relation to oxygenic photosynthesis. Glucosylglycerol has been identified in Synechocystis sp. PCC 6803 as an osmoprotectant that contributes to its salt tolerance (Mikkat et al., 1996). On the other hand, the mechanisms acting against the ion toxicity of salt stress in the cyanobacterium remain to be elucidated because, not only osmotic pressure, but also ion effects account for its decrease in photosynthetic activity (Allakhverdiev et al., 1999). Na + /H+ antiporter activity appears to be involved in the intracellular Na + homeostasis of Synechococcus sp. PCC 6311 (Blumwald et al., 1984). However, salt stress depresses the activity and synthesis of Na + /H + antiporters in Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 (Allakhverdiev et al., 1999;. There is a dramatic loss of Na+ antiporter activity in Synechocystis sp. PCC 6803 in the presence of 1.0 M NaCl, although it can tolerate up to 1.2 M NaCl (Reed and Stewart, 1985;Allakhverdiev et al., 1999). Apart from Na + /H + antiporters, other membrane transporters may be involved in Na + homeostasis in this cyanobacterium. Both prokaryotic and eukaryotic cells possess primary Na + -ATPases, which extrude Na + directly (Gimmler, 2000). There are several potential Na + -ATPases in the Synechocystis sp. PCC 6803 genome, but no Na + -ATPase in any cyanobacterium has been definitively identified thus far (Ritchie, 1998). Bioenergetic analysis reveals that, under physiological ranges of pH, Na + -coupled secondary ion transport across membranes occurs in Synechococcus sp. PCC 7942 (Ritchie, 1992(Ritchie, , 1998, implying that there is an energyconsuming Na + efflux mechanism in cyanobacteria. Additionally, it has been well established that counteraction of Na + and other metal ions such as K + and Ca 2+ is also a determinant factor in Na + homeostasis. Therefore, it remains to be established whether or not the predicted energy-dependent Na + efflux protein(s) actually exist and the extent to which other cation ATPases may also affect Na + sensitivity of cyanobacteria. In addition to Na + homeostasis, Na + /H + antiporters are also involved in internal pH regulation . In cyanobacteria, light induces the transient acidification of culture medium in a process that /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 N...

mrpA, a gene with roles in resistance to Na+ and adaptation to alkaline pH in the cyanobacterium Anabaena sp. PCC7120

“…Elanskaya et al (2002) found that, with the exception of the NhaS3 mutant, which could not be segregated, none of the mutants showed a significant growth depression under high-salt and/or high-pH conditions. Wang et al (2002) also found that NhaS3 may perform essential housekeeping functions for the survival of the organism, being critical for salt tolerance. Disruption of nhaS2 and nhaS4 demonstrated that both genes could be essential for the survival of cyanobacteria in freshwater habitats characterized by large fluctuations in salt concentration and pH.…”

“…PCC6803. Recently Wang et al (2002) and Elanskaya et al (2002), independently, have mutated five genes identified as encoding putative Na + /H + antiporters in the Synechocystis genome (Kaneko et al, 1996). Elanskaya et al (2002) found that, with the exception of the NhaS3 mutant, which could not be segregated, none of the mutants showed a significant growth depression under high-salt and/or high-pH conditions.…”

Critical Roles of Bacterioferritins in Iron Storage and Proliferation of Cyanobacteria

“…2B; Supplemental Table I). Replacement of the gene with a chloramphenicol resistance cassette was achieved by a fusion PCR technique (Wang et al, 2002). The double Dsll1341Dslr1890 (DbfrADbfrB) mutant was generated by transforming the Dslr1890 strain with the Dsll1341 construct.…”

A Transcriptional Regulator Sll0794 Regulates Tolerance to Biofuel Ethanol in Photosynthetic Synechocystis sp. PCC 6803

“…Glutaredoxins are small ubiquitous glutathione-disulfide oxidoreductases that reduce disulfide bonds of target proteins and maintain the redox homoeostasis of Synechocystis cells, and disruption of a glutaredoxin coding gene ssr2061 in Synechocystis has been reported with reduced cell viability against oxidative stress (47). The sll0273 gene encoding a putative Na ϩ /H ϩ antiporter was found essential for growth at low Na ϩ /K ϩ ratios (48), and its mutant showed low tolerance to elevated pH in the BG11 medium (49). In our previous study, we found that the slr1829 gene encoding a putative poly(3-hydroxyalkanoate) synthase was inducted by butanol, and the enhanced production of carbon storage compound polyhydroxyalkanoates (PHAs) was one important mechanism for Synechocystis to combat against butanol stress (50).…”