|
内容記述 |
The cyanobacterium Synechocystis sp. PCC 6803 has five genes for putative Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters (designated nhaS1, nhaS2, nhaS3, nhaS4, and nhaS5). In this thesis, their products were studied by means of functional expression in Escherichia coli, targeted mutagenesis, and phylogenetic analyses.<br /> Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters are membrane proteins that exchange Na<SUP>+</SUP> for H<SUP>+</SUP>, the two most important ions in cell bioenergetics. Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters were discovered by Peter Mitchell and his colleagues in the late 60’s and, since then, it has been found that they are widely distributed throughout the biological kingdoms. Chapter 1 describes previous studies of Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters, which will provide an overview of how these universal devises are used by various organisms. It should be stressed that no Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters had been identified in cyanobacteria, despite of accumulating evidence that Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters play important roles in the maintenance of intracellular ion homeostasis during acclimation to changes in environmental conditions.<br /> Chapter 2 describes characterization of Synechocystis Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters by functional expression of the nhaS genes in Escherichia coli. The author successfully induced the expression of nhaS1, nhaS3, and nhaS4 under control of an Na<SUP>+</SUP>-dependent promoter in E. coli TO114, a strain that is deficient in Na<SUP>+</SUP>/H<SUP>+</SUP>-antiport activity. Inverted membrane vesicles prepared from nhaS1/TO114 and nhaS3/TO114 cells exhibited Na<SUP>+</SUP>(Li<SUP>+</SUP>)/H<SUP>+</SUP>-antiport activity. Kinetic analysis of the antiport activity revealed that the nhaS1 gene encodes a low-affinity Na<SUP>+</SUP>/H<SUP>+</SUP> antiporter with a K<SUB>m</SUB> value of 7.7 mM for Na<SUP>+</SUP> ions and a K<SUB>m</SUB> value of 2.5 mM for Li<SUP>+</SUP> ions, while the nhaS3 gene encodes a high-affinity Na<SUP>+</SUP>/H<SUP>+</SUP> antiporter with a K<SUB>m</SUB> value of 0.7 mM for Na<SUP>+</SUP> ions and a K<SUB>m</SUB> value of 0.01 mM for Li<SUP>+</SUP> ions. Transformation of E. coli TO114 with the nhaS1 and nhaS3 genes increased tolerance to high concentrations of Na<SUP>+</SUP> and Li<SUP>+</SUP> ions and depletion of K<SUP>+</SUP> ions. This is the first demonstration of functional characterization of Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters from cyanobacteria. Inverted membrane vesicles prepared from nhaS4/TO114 cells did not have Na<SUP>+</SUP>/H<SUP>+</SUP>-antiport activity, and the cells themselves were as sensitive as the original TO114 cells to Na<SUP>+</SUP> and Li<SUP>+</SUP> ions. However, the nhaS4/TO114 cells were tolerant to depletion of K<SUP>+</SUP> ions. This result suggested that the nhaS4 gene might encode a membrane protein that transports Na<SUP>+</SUP> and/or K<SUP>+</SUP> ions．<br /> Evaluation of effects of targeted inactivation of the nhaS genes on growth phenolypes of Synechocystis cells is described in Chapter 3. She created single and double mutants of Synechocystis in which individual nhaS genes were interrupted by insertion of an antibiotic-resistance gene cartridge. The disruption of the nhaS1, nhaS2, nhaS4, and nhaS5 genes in all copies of the chromosomal DNA was verified by PCR. She failed to disrupt the nhaS3 gene; homozygous null mutants were not recovered after the mutagenesis. The single mutants that she obtained did not show any phenotypic changes in terms of the sensitivity to growth inhibition by NaCl. nhaS1 nhaS2 cells grew slower than wild-type cells both in BG11 medium (the standard medium), that contained 18 mM Na<SUP>+</SUP>, and in a high-salt medium, prepared by adding NaCl, to 0.5 M, to the BG11 medium. The growth retardation of nhaS1 nhaS2 cells appeared to be greater in the presence of 0.5 M NaCl than in its absence. In contrast, nhaS4 nhaS5 cells grew as well as wild-type cells regardless of the presence or absence of 0.5 M NaCl. These results suggested that (i) the function of the nhaS1 and nhaS2 genes’ products might be complementary, (ii) the nhaS3 gene is essential for viability of Synechocystis cells, and (iii) products of the nhaS4 and nhaS5 genes may contribute little to high-salt stress tolerance.<br /> Chapter 4 describes phylogenetic analyses of the Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters and putative homologues of the Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters from widely divergent phyla. BLAST search results indicated that NhaS1 and NhaS2 are similar to NhaP of Pesudomonas aeruginosa and eukaryotic Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters, while NhaS3, NhaS4, and NhaS5 are similar to NapA of Enterococcus hirae. Comparison of deduced amino acid sequences of the NhaS proteins to those of the Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters revealed significant similarities within the putative fifth and sixth transmembrane segments, of the NhaS proteins, and corresponding regions of the Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters. A phylogenetic tree based on the evolutionary distances revealed that 127 Na<SUP>+</SUP>/H<SUP>+</SUP> antiporter homologues from 53 species, including eukaryotes and prokaryotes, cluster with two groups, which are named NhaP/NHE and NapA families. The ubiquitous distribution of the members of the two families throughout the biological kingdoms indicates that the two types of proteins diverged before the divergence of major lineages in prokaryotes. Na<SUP>+</SUP>/H<SUP>+</SUP> antiporter homologues from various cyanobacteria form five distinct groups, namely, NhaS1-5 subfamilies. NhaS3 orthologs exist in all cyanobacteria where the entire genome sequence is available, suggesting that NhaS3 might be of particular importance to cyanobacteria. This is in agreement with the fact that the nhaS3 gene is essential for the viability of Synechocystis (Chapter 3). The NhaS1 and NhaS2 subfamilies belong to the NhaP/NHE family, while the others belong to the NapA family. The findings that NhaS1 and NhaS3 are low-affinity and high-affinity Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters, respectively (Chapter 2), are consistent with the common kinetic properties of each type of Na<SUP>+</SUP>/H<SUP>+</SUP> antiporters. The Arabidopsis proteins cluster with six groups, and two of them (SOS1 and AtNHX1) belong to the NhaP/NHE family. SOS1 and NhaS1 form a monophyletic group with a bootstrap value less than 50% and are closely related to NhaS2. These results might indicate the possibility that the Arabidopsis genes were acquired from the ancestor of plastids, although the relationship between these proteins are not well resolved. |
要旨・審査要旨 / Abstract, Screening Result (322.3 kB)
