Alkaline phosphatases (APases) are important enzymes in organophosphate utilization. Three prokaryotic APase gene families, PhoA, PhoX, and PhoD, are known; however, their functional characterization in cyanobacteria largely remains to be clarified. In this study, we cloned the phoD gene from a halotolerant cyanobacterium, Aphanothece halophytica ( phoD _Ap ). The deduced protein, PhoD _Ap , contains Tat consensus motifs and a peptidase cleavage site at the N terminus. The PhoD _Ap enzyme was activated by Ca ²⁺ and exhibited APase and phosphodiesterase (APDase) activities. Subcellular localization experiments revealed the secretion and processing of PhoD _Ap in a transformed cyanobacterium. Expression of the phoD _Ap gene in A. halophytica cells was upregulated not only by phosphorus (P) starvation but also under salt stress conditions. Our results suggest that A. halophytica cells possess a PhoD that participates in the assimilation of P under salinity stress.

Mycosporine-like amino acids (MAAs) are valuable molecules that are the basis for important photoprotective constituents. Here we report molecular analysis of mycosporine-like amino acid biosynthetic genes from the halotolerant cyanobacterium Aphanothece halophytica , which can survive at high salinity and alkaline pH. This extremophile was found to have a unique MAA core (4-deoxygadusol)-synthesizing gene separated from three other genes. In vivo analysis showed accumulation of the mycosporine-2-glycine but not shinorine or mycosporine-glycine. Mycosporine-2-glycine accumulation was stimulated more under the stress condition of high salinity than UV-B radiation. The Aphanothece MAA biosynthetic genes also manifested a strong transcript level response to salt stress. Furthermore, the transformed Escherichia coli and Synechococcus strains expressing four putative Aphanothece MAA genes under the control of a native promoter were found to be capable of synthesizing mycosporine-2-glycine. The accumulation level of mycosporine-2-glycine was again higher under the high-salinity condition. In the transformed E. coli cells, its level was approximately 85.2 ± 0.7 μmol/g (dry weight). Successful production of a large amount of mycosporine in these cells provides a new opportunity in the search for an alternative natural sunscreen compound source.

A putative Na+/H+ antiporter gene whose deduced amino acid sequence was highly homologous to the NhaP antiporter from Pseudomonas aeruginosa and SOS1 antiporter from Arabidopsis was isolated fromSynechocystis sp. PCC 6803. TheSynechocystis NhaP antiporter (SynNhaP) was expressed inEscherichia coli mutant cells, which were deficient in Na+/H+ antiporters. It was found that the SynNhaP complemented the salt-sensitive phenotype of the E. coli mutant. Membrane vesicles prepared from the E. coli mutant transformed with the SynNhaP exhibited the Na+/H+ and Li+/H+antiporter activities, and their activities were insensitive to amiloride. Moreover, its activity was very high between pH 5 and 9. The replacement of aspartate-138 in SynNhaP with glutamate or tyrosine inactivated the SynNhaP antiporter activity. The deletion of a part of the long C-terminal hydrophilic tail significantly inhibited the antiporter activity. A topological model suggests that aspartate-138 in SynNhaP is conserved in NhaP, SOS1, and AtNHX1 and is involved in the exchange activity. Thus, it appeared that the SynNhaP would provide a model system for the study of structural and functional properties of eucaryotic Na+/H+antiporters.

Hypersaline environments are extreme habitats on the planet and have a diverse microbial population formed by halophilic microorganisms. They are considered to be actual or potential sources for discovery bioactive compounds, compatible solutes including novel and/or extraordinarily enzymes. To date, a number of bioactive compounds for the use in various fields of biotechnology which show assorted biological activities ranging from antioxidant, sunscreen and antibiotic actions have been reported. In addition, some halophilic microorganisms are capable of producing massive amounts of compatible solutes that are useful as stabilizers for biomolecules or stress-protective agents. The present review will impart knowledge and discuss on (i) potential biotechnological applications of bioactive compounds, compatible solutes and some novel hydrolytic enzymes; (ii) recent efforts on discovery and utilization of halophiles for biotechnological interest; (iii) future perspective of aforementioned points.

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium that can grow in media of up to 3.0 m NaCl and pH 11. Here, we show that in addition to a typical H⁺-ATP synthase, Aphanothece halophytica contains a putative F₁F₀-type Na⁺-ATP synthase (ApNa⁺-ATPase) operon (ApNa⁺-atp). The operon consists of nine genes organized in the order of putative subunits β, ϵ, I, hypothetical protein, a, c, b, α, and γ. Homologous operons could also be found in some cyanobacteria such as Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017. The ApNa⁺-atp operon was isolated from the A. halophytica genome and transferred into an Escherichia coli mutant DK8 (Δatp) deficient in ATP synthase. The inverted membrane vesicles of E. coli DK8 expressing ApNa⁺-ATPase exhibited Na⁺-dependent ATP hydrolysis activity, which was inhibited by monensin and tributyltin chloride, but not by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The Na⁺ ion protected the inhibition of ApNa⁺-ATPase by N,N′-dicyclohexylcarbodiimide. The ATP synthesis activity was also observed using the Na⁺-loaded inverted membrane vesicles. Expression of the ApNa⁺-atp operon in the heterologous cyanobacterium Synechococcus sp. PCC 7942 showed its localization in the cytoplasmic membrane fractions and increased tolerance to salt stress. These results indicate that A. halophytica has additional Na⁺-dependent F₁F₀-ATPase in the cytoplasmic membrane playing a potential role in salt-stress tolerance.