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.

Characterization of genes that encode subunits of cucumber PS I complex by N-terminal sequencing.

N-terminal amino acid sequencing was carried out to characterize the genes of the cucumber PS I complex (PSI-100) that contains eight polypeptides and catalyzes the light-dependent transfer of electrons from plastocyanin to ferredoxin. The genes of all subunits except the 17.5 kDa polypeptide in PSI-100 have been identified. These are psaA/psaB (65/63 kDa), psaD (20 kDa), psaE (19.5 kDa), psaF (18.5 kDa), psaH (7.6 kDa), and psaC (5.8 kDa). The 17.5 kDa polypeptide is a new protein and is designated tentatively as the gene product of psaM. N-terminal amino-acid sequencing indicated the presence of two polypeptides in the 7.6 kDa band. One of these is the gene product of psaH and is essential for the activity of the PS I complex, and the other one is as yet unrecognized and largely depleted in the PSI-100 complex. Gene products of psaG, psaI, and psaK, which have been proposed as the components of PS I complex, are not involved in the PSI-100 complex, but are involved in the PS I complex (PSI-200), which contains 120 chlorophyll per reaction center chlorophyll (P700) and light-harvesting chlorophyll a/b protein complexes. Three polypeptides (26,23 and 22.5 kDa) are not involved in the PSI-100 and are assigned as the apo-protein of light-harvesting chlorophyll a/b protein complexes.

Reconstitution of mature plastocyanin from precursor apo-plastocyanin expressed in Escherichia coli.

The precursor plastocyanin from Silene pratensis (white campion) has been expressed in Escherichia coli. The precursor protein was accumulated in insoluble aggregates and partially purified as an apo-protein. The purified precursor apo-plastocyanin was processed to the mature apo-plastocyanin by chloroplast extracts. N-terminal amino-acid sequencing indicated that the processed protein was identical to the N-terminal amino-acid residues of mature plastocyanin that was deduced from the nucleotide sequence. The copper could be incorporated into the apo-plastocyanin of mature size in vitro, but could not into the precursor apo-plastocyanin under the same conditions. Absorption spectra and reduction potential of the reconstituted mature plastocyanin were indistinguishable from those of the purified spinach plastocyanin. The electron transfer activities of the reconstituted plastocyanin with both the Photosystem I reaction center (P700) and cytochrome f were almost the same as those of the purified spinach plastocyanin.

Synechococcus sp. PCC7942 Transformed with Escherichia coli bet Genes Produces Glycine Betaine from Choline and Acquires Resistance to Salt Stress.

Synechococcus sp. PCC7942, a fresh water cyanobacterium, was transformed by a shuttle plasmid that contains a 9-kb fragment encoding the Escherichia coli bet gene cluster, i.e. betA (choline dehydrogenase), betB (betaine aldehyde dehydrogenase), betI (a putative regulatory protein), and betT (the choline transport system). The expression of these genes was demonstrated in the cyanobacterial cells (bet-containing cells) by northern blot analysis, as well as by the detection of glycine betaine by 1H nuclear magnetic resonance in cells supplemented with choline. Endogenous choline was not detected in either control or bet-containing cells. Both control and bet-containing cyanobacterial cells were found to import choline in an energy-dependent process, although this import was restricted only to bet-containing cells in conditions of salt stress. Glycine betaine was found to accumulate to a concentration of 45 mM in bet-containing cyanobacterial cells, and this resulted in a stabilization of the photosynthetic activities of photosystems I and II, higher phycobilisome contents, and general protective effects against salt stress when compared to control cells. The growth of bet-containing cells was much faster in the presence of 0.375 M NaCl than that of control cells, indicating that the transformant acquired resistance to salt stress.

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.

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.

Sequence and expression of genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase from Chromatium vinosum.

A DNA fragment bearing genes for the large (rbcL) and small (rbcS) subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was cloned from the photosynthetic purple sulfur bacterium Chromatium vinosum. Enzymatically fully active RuBisCO was synthesized in Escherichia coli cells when the cloned DNA was placed downstream of tac promoter. Nucleotide (nt) sequences of rbcL-rbcS were more homologous to cyanobacterial counterparts than to those from Alcaligenes eutrophus or higher plants. However, the amino acid (aa) sequence in a domain responsible for CO2 activation in the C. vinosum rbcL product resembled the corresponding aa sequence in higher plant RuBisCos, but not in the cyanobacterial enzymes. Chemically determined aa sequences at the N terminals of both subunits of RuBisCO purified from C. vinosum were not identical to those deduced from the nt sequences, although they were completely the same as aa sequences deduced from rbcA-rbcB, another locus encoding RuBisCO in C. vinosum. Therefore, the rbcL-rbcS locus seems to be barely expressed under a standard condition for photoautotrophic growth. The homology of the nt sequences between rbcL and rbcA was 82%, and that between rbcS and rbcB was 63%, whereas the codon usages of these genes were basically identical. The rbcL-rbcS and rbcA-rbcB loci therefore must have evolved from a common ancestral set of genes after duplication, instead of lateral gene transfer.

Expression of genes for subunits of plant-type RuBisCO from Chromatium and production of the enzymically active molecule in Escherichia coli.

A DNA fragment containing genes for both large (A) and small (B) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from a photosynthetic bacterium Chromatium vinosum was ligated with vectors for expressing unfused proteins and introduced into cells of Escherichia coli. The expressers of RuBisCO were screened on agar plates using the specific antibody raised against the native enzyme from Chromatium. The production of both subunits A and B in the expressers was demonstrated by an immunoblotting experiment. The amount of RuBisCO produced in the E. coli cells was as high as 15% of the total soluble protein after induction with isopropyl-beta-D-thiogalactoside. The specific activity of enzyme molecules produced in E. coli was nearly the same as that of the original Chromatium enzyme. On gel filtration high-performance liquid chromatography the two enzymes showed identical elution behavior, strongly indicating their similar quaternary structures.

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.

Kinetic studies on a cross-linked complex between plastocyanin cytochrome f.

A cross-linked complex between plastocyanin and cytochrome f was prepared by incubation in the presence of a water soluble carbodiimide and its kinetic properties were studied. The optical spectra, oxidation-reduction potentials and isoelectric pH of plastocyanin and cytochrome f did not change upon the formation of the cross-linked complex. Studies on the ionic strength effect on the electron transfer rate from cross-linked plastocyanin to ferricyanide indicated that the negative charge on the reaction site of plastocyanin was masked upon the cross-linking. It was also suggested that the sign of the net charge near the cytochrome f heme edge changed from positive to negative upon the cross-linking. On the other hand, electrostatic interactions between cross-linked plastocyanin and P700 seemed to be essentially the same as those in the case of native plastocyanin, although the rate of electron transfer from cross-linked plastocyanin to P700 was severely reduced. We also measured the intra-complex electron transfer from cytochrome f to plastocyanin. This suggested that the covalently cross-linked complex is a valid model of the electron transfer encounter complex. Based on these results, the reaction sites of plastocyanin with P700 and cytochrome f were discussed.

Importance of local positive charges on cytochrome f for electron transfer to plastocyanin and potassium ferricyanide.

To study the relationship between the electron transfer rate and the net or local charge of protein, chemically modified cytochrome f, in which positively charged amino groups are replaced with negatively charged carboxyl groups, has been prepared by using an arylating reagent 4-chloro-3,5-dinitrobenzoic acid. Four distinct species of chemically modified cytochrome f, having 1 to 4 mol of modified amino residues per mol of cytochrome f, were separated by preparative polyacrylamide gel electrophoresis. The rate of electron transfer from the reduced singly substituted cytochrome f to the oxidized spinach plastocyanin was only about 50% of that of the native unmodified cytochrome f. The reaction rate further decreased about 50% upon the modification of each amino residue. The biphasic oxidation of cytochrome f by plastocyanin was observed when more than 2 mol of amino residues were modified. The rate of the second phase also decreased with an increasing number of modified amino residues. On the other hand, the oxidation of chemically modified cytochrome f by potassium ferricyanide was clearly monotonic. The rate decreased about 30% upon the modification of each amino residue. The midpoint potentials of chemically modified cytochrome f were almost the same as that of the native protein. These results clearly indicate the importance of local positive charges on cytochrome f, since the overall net charge of cytochrome f is negative at neutral pH. The theory of electrostatic corrected outer-sphere electron transfer of Marcus explained the effect of charge on cytochrome f for the reaction with the small molecule of ferricyanide well, but not the reaction with the protein of plastocyanin.

Expression and characterization of Met92Gln mutant plastocyanin from Silene pratensis.

To investigate the role of the copper-ligand Met92 in the structural and functional properties of silene plastocyanin (PC), Met92 was replaced with Gln, which is the purposed fourth copper-ligand in another blue copper protein, stellacyanin. By use of the recently developed expression system [Hibino et al. (1994) J. Biochem. 116, 826-832], the Met92Gln mutant of intermediate precursor plastocyanin was successfully expressed in Escherichia coli and accumulated in the periplasmic space as a mature protein. In contrast to the wild type, most of the Met92Gln mutant PC accumulated as an apoprotein. After purification, mutant apoprotein could incorporate copper ions, although less efficiently than the wild-type apoprotein. The absorption peak of Met92Gln mutant PC was blue-shifted from 597 nm in the wild type to 591 nm. The rhombic type EPR spectrum was obtained for the mutant in place of the axial spectrum in the wild type. Compared with that of the wild-type PC, the oxidation-reduction potentials of the Met92Gln mutant PC were lower by about 35 mV over the whole pH range examined. These results indicate that the Met92Gln mutant exhibited "stellacyanin-like" spectroscopic properties. Interestingly, the electron-transfer activities of the mutant PC with the physiological electron donor (cytochrome f) and acceptor (Photosystem I) were similar to that of the wild-type PC. Since Met92 is conserved in all the plastocyanins whose primary structures are known, we propose that the primarily function of Met as a copper-ligand is in the uptake of copper ions during folding rather than in electron-transfer activities.

Autoreduction of spinach plastocyanin at alkaline pH.

The autoreduction of spinach plastocyanin has been studied by fluorescence, absorption, and paramagnetic resonance spectroscopy. Decolorization of oxidized plastocyanin, due to the autoreduction of plastocyanin, occurred rapidly at alkaline pH (half-life 20 min at pH 10.2). Its rate also increased with increasing ionic strength. In the presence of a hydrophobic fluorescent probe such as 2-p-toluidino-naphthalene-6-sulfonate (TNS), the fluorescence intensity of TNS due to binding to oxidized plastocyanin gradually increased following the autoreduction of plastocyanin, which suggests that copper is autoreduced in parallel with the exposure of a hydrophobic site (probably near Cys 84) to the solvent. The EPR spectra also supported the change of coordination geometry of copper. The reduction rate of plastocyanin by ferrocyanide at alkaline pH was almost the same as that at neutral pH, and TNS did not alter the exogenous reduction rate even at alkaline pH. These results suggest that exogenous and endogenous reduction take place independently at different reduction sites in spinach plastocyanin.

Site-directed mutagenetic study on the role of negative patches on silene plastocyanin in the interactions with cytochrome f and photosystem I.

To investigate the role of two highly conserved negative patches, residues #42-45 and #59-61, on the surface of plant plastocyanin, six mutants were constructed by site-directed mutagenesis of the intermediate precursor gene from Silene pratensis. The mutants were designed systematically to incorporate positive charges into the negative patches, and the net charge on negative patches was modified from -4 to +1. Upon expression in Escherichia coli, the mutant proteins were correctly processed to the mature size and accumulated as holo-proteins. Absorption spectra, EPR, and redox potentials of the purified mutant proteins were almost indistinguishable from those of the wild-type. It was found that the electron transfer rate from cytochrome f to plastocyanin decreased exponentially as the net charge on the negative patch (#42-45) was increased, whereas the modification of the other negative patch (#59-61) had no effect. Ionic strength dependence studies indicated that the rate constants at infinite ionic strength did not change significantly among the wild-type and the six mutants, and the electrostatic attraction energies between plastocyanin and cytochrome f decreased when residues #42-45 were modified, whereas the modification of residues #59-61 had no effect. These results clearly indicated that only one (#42-45) of the two negative patches is involved in the transient complex formation with cytochrome f. Essentially similar results were observed for the electron transfer from plastocyanin to the photosystem I reaction center (P700), although in this case, slight participation of the negative patch (#59-61) is suggested.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of transit peptide sequence of plastocyanin for its expression, processing, and copper-binding activity in Escherichia coli.

Plastocyanin is a copper protein that functions as an electron carrier in the thylakoid lumen of the chloroplast. To characterize the transit peptide of plastocyanin and develop expression systems for it in Escherichia coli, three kinds of expression vectors which encode different size precursor plastocyanin molecules were constructed. Their expression, processing, and copper-binding activity have been examined. When the full-length cDNA encoding the precursor plastocyanin from Silene pratensis was expressed in E. coli, a large amount of precursor plastocyanin accumulated in insoluble aggregates. Its accumulation level was increased by the addition of copper ions. About six percent of precursor plastocyanin molecules were transported into the periplasmic space and processed to the mature protein. On the other hand, expression of the intermediate size cDNA, which contains the hydrophobic domain and basic amino acid of C-terminal transit peptide, caused exclusive translocation to the periplasmic space and correct processing to the mature size. The addition of copper ions increased the holo-protein content, but did not change the polypeptide content of mature plastocyanin, indicating that translocation and processing are independent of the incorporation of copper ions. The mature plastocyanin content corresponds to 8% (w/w) of the total E. coli protein content (123 mg per liter of culture). The purified mature holo-protein showed almost the same spectroscopic and kinetic properties as those of purified spinach plastocyanin. Expression of the cDNA encoding the mature polypeptide and two preceeding amino acid residues caused the accumulation of only a small amount of plastocyanin.(ABSTRACT TRUNCATED AT 250 WORDS)

Subunit composition of Photosystem I complex that catalyzes light-dependent transfer of electrons from plastocyanin to ferredoxin.

The PSI core complex prepared from cucumber cotyledons, which contains 80 chlorophylls per reaction center (P700) and eight polypeptides with apparent molecular masses of 65/63, 20, 19.5, 18.5, 17.5, 7.6, and 5.8 kDa, has been shown to catalyze the light-dependent transfer of electrons from plastocyanin to ferredoxin. The "native" PSI complex, which contains more than fifteen polypeptides and 120 chlorophylls per P700, did not show higher activity. Any attempt to deplete subunit(s) of the core complex decreased its activity. These results suggest that in addition to light-harvesting chlorophyll a/b protein complexes, several genes of psaA-psaK, which have been proposed as components of PSI complex, are not involved in the activity of PSI complex. It was also found that the amount of 18.5-kDa polypeptide in the PSI complex affects the activity: when this polypeptide was largely depleted, the complex was almost inactive. The inactivation was due to inhibition of electron transfer from plastocyanin to photooxidized P700. Chemical cross-linking and N-terminal amino acid sequencing experiments indicated that the 18.5-kDa polypeptide is the plastocyanin-docking protein and the psaF gene product. The function of the psaF gene product was discussed.

Charges on proteins and distances of electron transfer in metalloprotein redox reactions.

A modified form of the Debye-Marcus equation relating electron transfer rate constants to charges on proteins and distances of electron transfer has been applied to the reaction of chemically modified cytochrome f, in which positively charged amino groups are replaced with negatively charged carboxyl groups. The rate of electron transfer from reduced cytochrome f to ferricyanide decreased with increasing ionic strength when the native and singly substituted cytochrome f were used, although a sharp decrease was observed in the former case. When doubly or more than triply substituted cytochrome f was used, the rate of electron transfer was almost constant or increased with increasing ionic strength, respectively. The kinetic-ionic strength effects on this reaction can be well explained by the Debye-Marcus equation in which the charge and radius of the protein are treated as variable parameters. The results show the importance of local positive charges of about 2.0 on native cytochrome f and effective radius of about 11 A of cytochrome f for the electron transfer to ferricyanide. Since the net charge on the native cytochrome f is negative and the calculated radius of the protein is 22.8 A, the above results indicate that positive charges on the electron transfer site control the electrostatic interactions in this reaction. Previously reported data which had been analyzed by using the total net charge and full radius of the protein, were also well explained by the local charge and effective radius of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody.

Betaine-aldehyde dehydrogenase was purified from spinach leaves and characterized. The molecular weight of the enzyme was estimated to be 120 kDa by a gel filtration chromatography. The enzyme was judged to consist of two identical pieces of the monomeric subunit with molecular weight of 60 kDa. A specific polyclonal antibody was raised against the enzyme subunit.

Electron transfer between plastocyanin and P700 in highly-purified photosystem I reaction center complex. Effects of pH, cations, and subunit peptide composition.

Treatment of isolated spinach thylakoid fragments with Triton X-100 followed by repeated sucrose density gradient centrifugations and Sephacryl S-300 and DEAE-Sephacel chromatographies yielded a highly purified P700-chlorophyll a protein complex complex which consists of five polypeptides. The protein complex is virtually free of chlorophyll b (Ch1 alpha/Ch1 b greater than 10) with approximately 30 chlorophylls per P700, and contains iron-sulfur centers A, B, and X. At pH values higher than 6, divalent cations, but not monovalent or trivalent cations, efficiently accelerated the electron transfer from reduced spinach plastocyanin to the photooxidized P700 in the P700-chlorophyll alpha protein complex. At pH values lower than 6, the reaction rate drastically increased with decreasing pH with a maximum at about pH 4.3 without cations. Divalent salts as well as monovalent or trivalent salts decreased the P700 reduction rate at low pH, indicating the involvement of electrostatic interaction in those pH regions. The rate of electron transfer from plastocyanin to the photooxidized P700 in the reaction center protein, which consists of only the largest peptide subunit and no iron-sulfur centers, was reduced only 50% at pH 7.0 in the presence of MgCl2 as compared to the case of P700-chlorophyll alpha protein complex. Essentially similar effects of pH and metal ions on this electron transfer reaction were observed as in the case of P700-chlorophyll alpha protein complex. These results strongly suggest that plastocyanin donates electrons directly to the largest peptide of P700-chlorophyll alpha protein complex and the observed effects of pH and cations are mainly due to the interaction between the largest peptide of P700-chlorophyll alpha protein complex and plastocyanin. The four small subunits in the protein complex seemed to have only a minor role in the reaction with plastocyanin.

Electron transfer reactions of chemically modified plastocyanin with P700 and cytochrome f. Importance of local charges.

Chemically modified spinach plastocyanin, in which negatively charged carboxyl residues are replaced with positively charged amino residues, has been prepared. Four distinct species of chemically modified plastocyanin, having 1 to 4 mol of modified carboxyl residue per mol of plastocyanin, could be separated by ion-exchange chromatography on DEAE-Sephacel. The rate of electron transfer from reduced cytochrome f to oxidized singly substituted plastocyanin was 30% of that of the native unmodified plastocyanin, and the reaction rate decreased further with increasing number of modified carboxyl residues. These results indicate the importance of electrostatic interactions between the negative charges on plastocyanin and the positive charges on cytochrome f in this reaction. Since the overall net charge of cytochrome f is negative at neutral pH, the positive charges on cytochrome f involved in the reaction should be localized ones. On the other hand, the rates of electron transfer from reduced singly and doubly substituted plastocyanin to photooxidized P700 in the P700-chlorophyll alpha protein complex were similar to that of native plastocyanin, which suggests that these carboxyl residues have only a minor role in the electron transfer to P700. Although divalent cation is essential for the electron transfer from native plastocyanin to P700 at neutral pH, the triply substituted plastocyanin could donate electrons to P700 even without MgCl2, and the rate of this reaction reached the maximum at a low concentration of MgCl2 (less than 2.5 mM). The modification of four carboxyl residues per plastocyanin molecule activated this reaction to the maximum level without MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)