Requirement of alkanes for salt tolerance of Cyanobacteria: characterization of alkane synthesis genes from salt-sensitive Synechococcus elongatus PCC7942 and salt-tolerant Aphanothece halophytica.

Cyanobacteria have been attracting great interest in the research area of biofuel production. All Cyanobacteria contain C15 -C19 hydrocarbons, but physiological roles of hydrocarbons remain to be clarified. Recently, two universal but mutually exclusive hydrocarbon production pathways in Cyanobacteria were discovered. In this study, we constructed a deletion mutant of alkane synthesis genes in fresh water cyanobacterium Synechococcus elongates PCC 7942. The mutant was incapable to produce alkanes and exhibited normal growth phenotype at low salinity. But, the mutant became salt sensitive. Overexpression of alkane synthesis genes from halotolerant Aphanothece halophytica in Synechococcus PCC7942 restored the growth defect. The alkane synthesis gene from halotolerant cyanobacterium A. halophytica was salt induced and produced a significant amount of alkanes at high salinity. These results indicate the requirement of alkanes for salt tolerance, and the alkane synthesis genes from A. halophytica could be a promising candidate for future biofuel application. SIGNIFICANCE AND IMPACT OF THE STUDY: Cyanobacteria have been attracting great interest in the research area of biofuel production. All Cyanobacteria contain C15 -C19 hydrocarbons, but physiological roles of hydrocarbons remain to be clarified. In this study, it was found that the deletion mutant of alkane synthesis genes in fresh water cyanobacterium Synechococcus elongates PCC 7942 was incapable to produce alkanes and salt sensitive. The alkane synthesis gene from halotolerant cyanobacterium Aphanothece halophytica was salt induced and produced a significant amount of alkanes at high salinity. These results demonstrate the alkane synthesis genes from A. halophytica could be a promising candidate for future biofuel application.

Thermodynamical properties of reaction intermediates during apoplastocyanin folding in time domain.

Two intermediates observed for the folding process of apoplastocyanin (apoPC) were investigated by using a photoinduced triggering system combined with the transient grating and transient lens methods. The thermodynamic quantities, enthalpy, heat capacity, partial volume, and thermal expansion volume changes during the protein folding reaction were measured in time domain for the first time. An interesting observation is the positive enthalpy changes during the folding process. This positive enthalpy change must be compensated by positive entropy changes, which could be originated from the dehydration effect of hydrophobic residues and/or the translational entropy gain of bulk water molecules. Observed negative heat capacity change was explained by the dehydration effect of hydrophilic residues and/or motional confinement of amino acid side chains and water molecules in apoPC. The signs of the volume change and thermal expansion volume were different for two processes and these changes were interpreted in terms of the different relative contributions of the hydration and the dehydration of the hydrophilic residues. These results indicated two-step hydrophobic collapses in the early stage of the apoPC folding, but the nature of the dynamics was different.

Functional analysis of salt-inducible proline transporter of barley roots.

We cloned a cDNA encoding Hordeum vulgare Proline Transporter (HvProT) from salt-stressed barley roots by differential display. HvProT was 2,161 bp long and had an open reading frame encoding 450 amino acids. The deduced amino acid sequence of HvProT was similar to those of proline transporter proteins of rice (65.7%), Arabidopsis (57.7%) and tomato (42.0%). Northern blot analysis showed that the transcript level of HvProT was induced in roots at 30 min after 200 mM NaCl treatment and its peak was observed at 3 h. However, the transcript level was very low in leaves and did not increase by salt stress. The expression level of Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), encoding a key enzyme of proline synthesis, was induced later than HvProT by salt stress. A transport assay using a yeast with mutation in proline uptake revealed that HvProT was a transporter with high affinity for L-proline (K(m) = 25 microM). HvProT was found to be a unique transporter with high affinity for L-proline. Since its transport activity was dependent on the pH gradient, HvProT was suggested to be a H(+)/amino acid symporter. In situ hybridization analysis showed that the HvProT mRNA was strongly expressed in root cap cells under salt stress. HvProT might play an important role in the transport of proline to root tip region urgently upon salt stress.

Inducers of glycinebetaine synthesis in barley.

Glycinebetaine is an osmoprotectant accumulated by barley (Hordeum vulgare) plants in response to high levels of NaCl, drought, and cold stress. Using barley seedlings in hydroponic culture, we characterized additional inducers of glycinebetaine accumulation. These included other inorganic salts (KCl, MgCl(2), LiCl, and Na(2)SO(4)), oxidants (H(2)O(2) and cumene hydroperoxide), and organic compounds (abscisic acid, polymixin B, n-butanol, salicylic acid, and aspirin). Stress symptoms brought on by high NaCl and other inducers, and not necessarily correlated with glycinebetaine accumulation, include wilting, loss of chlorophyll, and increase in thiobarbituric acid reacting substances. For NaCl, Ca(2+) ions at 10 to 20 mM decrease these stress symptoms without diminishing, or even increasing, glycinebetaine induction. Abscisic acid induces glycinebetaine accumulation without causing any of the stress symptoms. NaCl, KCl, and H(2)O(2) (but not other inducers) induce glycinebetaine at concentrations below those needed for the other stress symptoms. Mg(2+) at 10 to 20 mM induces both stress symptoms and glycinebetaine, but only at low (0.2 mM) Ca(2+). Although illumination is needed for optimal induction, a significant increase in the leaf glycinebetaine level is found in complete darkness, also.

An isozyme of betaine aldehyde dehydrogenase in barley.

Betaine aldehyde dehydrogenase (BADH) is an important enzyme for Gly betaine synthesis. We isolated two types of BADH cDNAs (BBD1 and BBD2) from barley. As BBD1 contained the signal sequence (SKL) targeting to microbodies, BBD2 was more similar to previously reported genes coding for BADH in dicotyledons (chloroplast type) than those in monocotyledons (microbody type). The two barley BADH genes showed different expression patterns. The BBD1 transcript was more abundant in roots than leaves and was induced to higher levels by salt, drought and abscisic acid (ABA) treatment. BBD2 transcript was more abundant in leaves and induced by salt, drought, PEG and ABA treatment. To understand the processing of these BADH proteins, we partially purified both enzymes and determined their N-terminal sequences. Based on comparisons of the N-terminal sequences to their deduced amino acid sequence, neither BBD1 nor BBD2 is processed at the N-terminus. These results suggest that BBD2 codes for a new type of BADH, which is not localized in either chloroplasts or mitochondria.

Residue aspartate-147 from the third transmembrane region of Na(+)/H(+) antiporter NhaB of Vibrio alginolyticus plays a role in its activity.

NhaB is a bacterial Na(+)/H(+) antiporter with unique topology. The pH dependence of NhaB from Vibrio alginolyticus differs from that of the Escherichia coli NhaB homolog. Replacement of Asp-147 with Glu made high H(+) concentrations a requirement for the NhaB activity. Replacement of Asp-147 with neutral amino acids inactivated NhaB.

Halotolerant cyanobacterium Aphanothece halophytica contains an Na(+)/H(+) antiporter, homologous to eukaryotic ones, with novel ion specificity affected by C-terminal tail.

Recently, a cyanobacterium Synechocystis sp. PCC 6803 has been shown to contain an Na(+)/H(+) antiporter gene homologous to plants (SOS1 and AtNHX1 from Arabidopsis) and mammalians (NHEs from human) but not to Escherichia coli (nhaA and nhaB). Here, we examined whether a halotolerant cyanobacterium Aphanothece halophytica has homologous genes. It turned out that A. halophytica contains an Na(+)/H(+) antiporter homologous to plants, mammalians, and some bacteria (nhaP from Pseudomonas and synnhaP from Synechocystis) but with novel ion specificity. Its gene product, ApNhaP (Na(+)/H(+) antiporter from Aphanothece halophytica), exhibited the Na(+)/H(+) antiporter activity over a wide pH range between 5 and 9 and complemented the Na(+)-sensitive phenotype of the antiporter-deficient E. coli mutant. The ApNhaP had virtually no activity for the Li(+)/H(+) antiporter but showed high Ca(2+)/H(+) antiporter activity at alkaline pH. The ApNhaP complemented the Ca(2+)-sensitive phenotype of the E. coli mutant but not the Li(+)-sensitive phenotype. The replacement of a long C-terminal tail of ApNhaP with that of Synechocystis altered the ion specificity of the antiporter. These results suggest that the ion specificity of an Na(+)/H(+) antiporter is partly determined by the structural properties of the C-terminal tail, which was well exemplified in the case of A. halophytica.

Cloning of peroxisomal ascorbate peroxidase gene from barley and enhanced thermotolerance by overexpressing in Arabidopsis thaliana.

A full-length cDNA clone (HvAPX1) encoding a peroxisomal type ascorbate peroxidase was isolated from barley (Hordeum vulgare cv. Haruna-nijyo) leaves by differential display. The deduced amino acid sequence of the HvAPX1 gene had 75.3% homology to that from the Gossypium hirsutum glyoxysomal APX gene and 72.1% homology to that from the Arabidopsis thaliana peroxisomal APX gene, APX3. Southern blot analysis indicated that a single-copy gene in the barley genome encoded HvAPX1. Northern blot analysis showed that the HvAPX1 transcript increased remarkably in response to heat, salt and abscisic acid treatment. Induction was not caused by treatment with hydrogen peroxide. The HvAPX1 gene was introduced into A. thaliana under control of the 35S RNA promoter of the cauliflower mosaic virus. The transgenic plants were significantly more tolerant to heat stress as compared with the wild-type.

Molecular cloning and functional characterization of two kinds of betaine-aldehyde dehydrogenase in betaine-accumulating mangrove Avicennia marina (Forsk.) Vierh.

Glycinebetaine is an important osmoprotectant in bacteria, plants, and animals, but only little information is available on the synthesis of glycinebetaine in tree plants. Among four mangrove species, glycinebetaine could be detected only in Avicennia marina. Pinitol was the main osmoprotectant in the other three species. The level of glycinebetaine in A. marina increased under high salinity. Betaine-aldehyde dehydrogenase (BADH) was detected in all four species, but choline monooxygenase could not be detected. A cDNA library was constructed from the leaves of A. marina. Two kinds of BADH cDNAs were isolated, one homologous to the spinach chloroplast BADH, and the other with unique residues SKL at the end of C-terminus. The BADH transcription levels of the former were higher than those of the latter. The levels of the former BADH increased at high salinity whereas those of the latter were independent of salinity. BADHs were expressed in Escherichia coli and purified. Two kinds of A. marina BADHs exhibited similar kinetic and stability properties, but were significantly different from those of spinach BADH. A. marina BADHs efficiently catalyzed the oxidation of betainealdehyde, but not the oxidation of omega-aminoaldehydes and were more stable at high temperature than the spinach BADH.

Overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica enhances the high-temperatue tolerance of tobacco during germination and early growth.

DnaK1 from a halotolerant cyanobacterium Aphanothece halophytica, was overexpressed in the cytosol of tobacco. When the control and transgenic tobacco seeds were incubated at 27 degrees C, more than 95% of the control and transgenic tobacco seeds germinated. However, at a high incubation temperature, 40 degrees C, only 27% of the control seeds germinated whereas 82% of the transgenic seeds germinated. High temperature treatment during the imbibition of seeds delayed germination more in the control plants than in the transformants although the maximum percentage of germination was similar in both plants. The quantum yields of electron transport and plant elongation were higher in the transformant during high temperature treatment in young seedlings, but similar in older leaves. DnaK1 was detected in small amounts in seeds and its levels increased during germination. These data indicate that the expression of DnaK1 from a halotolerant cyanobacterium A. halophytica improved the tolerance to high temperature during germination and early growth.

Na+/H+ antiporter from Synechocystis species PCC 6803, homologous to SOS1, contains an aspartic residue and long C-terminal tail important for the carrier activity.

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 from Synechocystis sp. PCC 6803. The Synechocystis NhaP antiporter (SynNhaP) was expressed in Escherichia 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.

Photo-induction of an NADPH dehydrogenase which functions as a mediator of electron transport to the intersystem chain in the cyanobacterium Synechocystis PCC6803.

Illumination of the dark-incubated cells of Synechocystis PCC6803 caused recovery of both respiratory activity of oxygen uptake and PS I-cyclic electron flow, which was monitored by the dark reduction of P700(+) in the presence of DCMU after a 50 ms pulse light (MT) under background far-red light, but the effects were much smaller in those of the mutant M55, which has an ndh-B defective gene. Activity of an NADPH-NBT oxidoreductase with a higher molecular mass (around 380 kDa), which was only found in wild type but not in M55, became evident after the dark-incubated cells were exposed to the light. Immuno-blotting analysis indicated that the NADPH-NBT oxidoreductase contains the NdhB subunit of NDH. The expression of NdhB decreased in dark-incubated cells and increased upon transfer of the cells back to light. These results indicate that an NADPH-specific NDH participates in the light-regulated cyclic electron transport around Photosystem I as well as in respiratory electron transport to the intersystem chain in Synechocystis 6803.

Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli. Structural and functional properties of wild-type, mutants and E. coli enzymes.

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the osmoprotectant glycine betaine from choline. Although betaine aldehyde has been thought to be a specific substrate for BADH, recent studies have shown that human and sugar beet BADHs also catalyze the oxidation of omega-aminoaldehydes. To characterize the kinetic and stability properties of spinach BADH, five kinds of expression vectors encoding full length, mature, E103Q, E103K, and chimera BADHs were constructed. These enzymes together with Escherichia coli BADH were expressed in E. coli and purified. The affinities for betaine aldehyde were similar in the spinach and E. coli BADHs, whereas those for omega-aminoaldehydes were higher in spinach BADH than in E. coli BADH. A chimera BADH in which part of the Rossmann type fold in the spinach BADH was replaced with that of E. coli BADH, showed properties which resembled spinach BADH more than E. coli BADH. The spinach E103K mutant was almost inactive, whereas the E103Q mutant showed a similar activity for the oxidation of betaine aldehyde to that of wild type BADH, but a lower affinity for omega-aminoaldehydes. All spinach BADHs were dimers whereas E. coli BADH was a tetramer. E. coli BADH was more stable at high temperature than spinach BADHs. The E103Q mutant was most labile to high temperature. These properties are discussed in relation to the structure of spinach BADH.

Effects of overexpression of Escherichia coli katE and bet genes on the tolerance for salt stress in a freshwater cyanobacterium Synechococcus sp. PCC 7942.

To explore the potential role of catalase and glycine betaine in the protection of cyanobacteria from damage due to salt stress, we transformed a freshwater cyanobacterium Synechococcus sp. PCC 7942 with shuttle vectors that contained the Escherichia coli katE, bet, and katE plus bet (katE/bet) genes. The catalase activity in the cells overexpressing katE and katE/bet genes was about 1.4-1.8-fold higher than that in the control cells. The control and transformant cells had a similar growth rate in the medium with a low salinity. However, under a high-salinity condition, the cells transformed with katE grew faster than the control cells, and the cells expressing katE/bet genes grew faster than those expressing either the katE or bet gene alone. These results indicate that high-salinity caused oxidative stress and the coexpression of katE and bet genes in Synechococcus cells were superior to the expression of either katE or bet alone for the protection of the cells from the damage due to high-salinity.

Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase.

The potential role of photorespiration in the protection against salt stress was examined with transgenic rice plants. Oryza sativa L. cv. Kinuhikari was transformed with a chloroplastic glutamine synthetase (GS2) gene from rice. Each transgenic rice plant line showed a different accumulation level of GS2. A transgenic plant line, G39-2, which accumulated about 1.5-fold more GS2 than the control plant, had an increased photorespiration capacity. In another line, G241-12, GS2 was almost lost and photorespiration activity could not be detected. Fluorescence quenching analysis revealed that photorespiration could prevent the over-reduction of electron transport systems. When exposed to 150 mM NaCl for 2 weeks, the control rice plants completely lost photosystem II activity, but G39-2 plants retained more than 90% activity after the 2-week treatment, whereas G241-12 plants lost these activities within one week. In the presence of isonicotinic acid hydrazide, an inhibitor of photorespiration, G39-2 showed the same salt tolerance as the control plants. The intracellular contents of NH4+ and Na+ in the stressed plants correlated well with the levels of GS2. Thus, the enhancement of photorespiration conferred resistance to salt in rice plants. Preliminary results suggest chilling tolerance in the transformant.

Spectroscopic and electrochemical studies on structural change of plastocyanin and its tyrosine 83 mutants induced by interaction with lysine peptides.

Interactions of wild-type and Tyr83 mutant (Y83F, Y83S, Y83L, and Y83H) plastocyanins (PCs) with lysine peptides as models for the PC interacting site of cytochrome f have been studied by absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies and electrochemical measurements. The spectral and electrochemical properties of PCs corresponded well with each other; species having a longer wavelength maximum for the S(Cys) pi --> Cu 3d(x)()()2(-)(y)()()2 charge transfer (CT) band observed around 600 nm and a stronger intensity for the 460-nm absorption band exhibited stronger intensities for the positive Met --> Cu 3d(x)()()2(-)(y)()()2 and negative His pi(1) --> Cu 3d(x)()()2(-)(y)()()2 circular dichroism (CD) bands at about 420 and 470 nm, respectively, a lower average nu(Cu)(-)(S) frequency, a smaller |A( parallel)| EPR parameter, and a higher redox potential, properties all related to a weaker Cu-S(Cys) bond and a more tetrahedral planar geometry for the Cu site. Similarly, on oligolysine binding to wild-type and several Tyr83 mutant PCs, a longer absorption maximum for the 600-nm CT band, a stronger intensity for the 460-nm absorption band, stronger 420-nm positive and 470-nm negative CD bands, and a lower average nu(Cu)(-)(S) frequency were observed, suggesting that PC assumes a slight more tetrahedral geometry on binding of oligolysine. Since changes were observed for both wild-type and Tyr83 mutant PCs, the structural change due to binding of oligolysine to PC may not be transmitted through the path of Tyr83-Cys84-copper by a cation-pi interaction which is proposed for electron transfer.

Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, and copper binding under various salinity conditions.

Previously, it was found that the dnaK1 gene of the halotolerant cyanobacterium Aphanothece halophytica encodes a polypeptide of 721 amino acids which has a long C-terminal region rich in acidic amino acid residues. To understand whether the A. halophytica DnaK1 possesses chaperone activity at high salinity and to clarify the role of the extra C-terminal amino acids, a comparative study examined three kinds of DnaK molecules for ATPase activity as well as the refolding activity of other urea-denatured proteins under various salinity conditions. DnaK1s from A. halophytica and Synechococcus sp. PCC 7942 and the C-terminal deleted A. halophytica DnaK1 were expressed in Escherichia coli and purified. The ATPase activity of A. halophytica DnaK1 was very high even at high salinity ( 1.0 M NaCl or KCl), whereas this activity in Synechococcus PCC 7942 DnaK1 decreased with increasing concentrations of NaCl or KCl. The salt dependence on the refolding activity of urea-denatured lactate dehydrogenase by DnaK1s was similar to that of ATPase activity of the respective DnaK1s. The deletion of the C-terminal amino acids of A. halophytica DnaK had no effect on the ATPase activity, but caused a significant decrease in the refolding activity of other denatured proteins. These facts indicate that the extra C-terminal region of A. halophytica DnaK1 plays an important role in the refolding of other urea-denatured proteins at high salinity. Furthermore, it was shown that DnaK1 could assist the copper binding of precursor apo-plastocyanin as well as that of mature apo-plastocyanin during the folding of these copper proteins.

Enhanced expression of a nuclease gene in leaves of barley plants under salt stress.

We isolated a cDNA clone, Bnuc1, encoding a nuclease I from leaves of salt-stressed barley (Hordeum vulgare L. cv. Haruna-nijyo) by the differential display method. Northern blot analysis revealed that the transcript of Bnuc1 gene was increased dramatically in barley leaves under salt stress. The expression of Bnuc1 gene was also increased by exogenously applied abscisic acid (ABA) in leaves, but not by gibberellic acid (GA) during seed germination. Furthermore, Bnuc1 gene was expressed more in old leaves than in young leaves during both salt stress and natural senescence. Salt-inducible nuclease activity possibly corresponding to the Bnuc1 gene was detected, and was much higher in old leaves than in young leaves under salt stress.

Crystal structures of wild-type and mutant plastocyanins from a higher plant, Silene.

Plastocyanin functions as an electron carrier between the cytochrome b6f complex and photosystem I. The crystal structures of the wild-type and E43K/D44K double mutant from the higher plant, Silene, have been determined at 2.0 and 1.75 A resolution, respectively. The wild-type plastocyanin comprises two monomers per asymmetric unit, one of which shows the unusually great distance between the copper ion and the Ndelta1 atom of H87 because of the hydrogen bond network formation between H87 and symmetry-related G10. The root mean square deviation for Ca atoms between the wild-type and mutant plastocyanins is 0.44 A, however, the electrostatic potential maps of their molecular surfaces are remarkably different. The low electron-transfer rate in the E43K/D44K mutant results from the hindrance of electrostatic interactions, not from the structural change due to the mutation.