Overexpressed Superoxide Dismutase and Catalase Act Synergistically to Protect the Repair of PSII during Photoinhibition in Synechococcus elongatus PCC 7942.

The repair of PSII under strong light is particularly sensitive to reactive oxygen species (ROS), such as the superoxide radical and hydrogen peroxide, and these ROS are efficiently scavenged by superoxide dismutase (SOD) and catalase. In the present study, we generated transformants of the cyanobacterium Synechococcus elongatus PCC 7942 that overexpressed an iron superoxide dismutase (Fe-SOD) from Synechocystis sp. PCC 6803; a highly active catalase (VktA) from Vibrio rumoiensis; and both enzymes together. Then we examined the sensitivity of PSII to photoinhibition in the three strains. In cells that overexpressed either Fe-SOD or VktA, PSII was more tolerant to strong light than it was in wild-type cells. Moreover, in cells that overexpressed both Fe-SOD and VktA, PSII was even more tolerant to strong light. However, the rate of photodamage to PSII, as monitored in the presence of chloramphenicol, was similar in all three transformant strains and in wild-type cells, suggesting that the overexpression of these ROS-scavenging enzymes might not protect PSII from photodamage but might protect the repair of PSII. Under strong light, intracellular levels of ROS fell significantly, and the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein, was enhanced. Our observations suggest that overexpressed Fe-SOD and VktA might act synergistically to alleviate the photoinhibition of PSII by reducing intracellular levels of ROS, with resultant protection of the repair of PSII from oxidative inhibition.

Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use.

The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

Occurrence of trans monounsaturated and polyunsaturated fatty acids in Colwellia psychrerythraea strain 34H.

Colwellia psychrerythraea strain 34H is an obligately psychrophilic bacterium that has been used as a model cold-adapted microorganism because of its psychrophilic growth profile, significant production of cold-active enzymes, and cryoprotectant extracellular polysaccharide substances. However, its fatty acid components, particularly trans unsaturated fatty acids and long-chain polyunsaturated fatty acids (LC-PUFAs), have not been fully investigated. In this study, we biochemically identified Δ9-trans hexadecenoic acid [16:1(9t)] and LC-PUFAs such as docosahexaenoic acid. These results are comparable with the fact that the strain 34H genome sequence includes pfa and cti genes that are responsible for the biosynthesis of LC-PUFAs and trans unsaturated fatty acids, respectively. Strain 34H cells grown under static conditions at 5 °C had higher levels of 16:1(9t) than those grown under shaken conditions, and this change was accompanied by an antiparallel decrease in the levels of Δ9-cis hexadecenoic acid [16:1(9c)], suggesting that the cis-to-trans isomerization reaction of 16:1(9c) is activated under static (microanaerobic) culture conditions, that is, the enzyme could be activated by the decreased dissolved oxygen concentration of cultures. On the other hand, the levels of LC-PUFAs were too low (less than 3% of the total), even for cells grown at 5 °C, to evaluate their cold-adaptive function in this bacterium.

Expression of a highly active catalase VktA in the cyanobacterium Synechococcus elongatus PCC 7942 alleviates the photoinhibition of photosystem II.

The repair of photosystem II (PSII) after photodamage is particularly sensitive to reactive oxygen species-such as H2O2, which is abundantly produced during the photoinhibition of PSII. In the present study, we generated a transformant of the cyanobacterium Synechococcus elongatus PCC 7942 that expressed a highly active catalase, VktA, which is derived from a facultatively psychrophilic bacterium Vibrio rumoiensis, and examined the effect of expression of VktA on the photoinhibition of PSII. The activity of PSII in transformed cells declined much more slowly than in wild-type cells when cells were exposed to strong light in the presence of H2O2. However, the rate of photodamage to PSII, as monitored in the presence of chloramphenicol, was the same in the two lines of cells, suggesting that the repair of PSII was protected by the expression of VktA. The de novo synthesis of the D1 protein, which is required for the repair of PSII, was activated in transformed cells under the same stress conditions. Similar protection of the repair of PSII in transformed cells was also observed under strong light at a relatively low temperature. Thus, the expression of the highly active catalase mitigates photoinhibition of PSII by protecting protein synthesis against damage by H2O2 with subsequent enhancement of the repair of PSII.

Pseudomonas toyotomiensis sp. nov., a psychrotolerant facultative alkaliphile that utilizes hydrocarbons.

A psychrotolerant, facultatively alkaliphilic strain, HT-3(T), was isolated from a sample of soil immersed in hot-spring water containing hydrocarbons in Toyotomi, Hokkaido, Japan. 16S rRNA gene sequence-based phylogeny suggested that strain HT-3(T) is a member of the genus Pseudomonas and belongs to the Pseudomonas oleovorans group. Cells of the isolate were Gram-negative, aerobic, straight rods, motile by a single polar flagellum. The strain grew at 4-42 °C, with optimum growth at 35 °C at pH 7, and at pH 6-10. It hydrolysed Tweens 20, 40, 60 and 80, but not casein, gelatin, starch or DNA. Its major isoprenoid quinone was ubiquinone-9 (Q-9) and the DNA G+C content was 65.1 mol%. The whole-cell fatty acid profile consisted mainly of C(16 : 0), C(16 : 1)ω9c and C(18 : 1)ω9c. Phylogenetic analyses based on gyrB, rpoB and rpoD sequences revealed that the isolate could be discriminated from Pseudomonas species that exhibited more than 97 % 16S rRNA gene sequence similarity and phylogenetic neighbours belonging to the P. oleovorans group including the closest relative of the isolate, Pseudomonas alcaliphila. DNA-DNA hybridization with P. alcaliphila AL15-21(T) revealed 51 ± 5 % relatedness. Owing to differences in phenotypic properties and phylogenetic analyses based on multilocus gene sequence analysis and DNA-DNA relatedness data, the isolate merits classification in a novel species, for which the name Pseudomonas toyotomiensis sp. nov. is proposed. The type strain is HT-3(T) ( = JCM 15604(T)  = NCIMB 14511(T)).

Fatty acid and hydrocarbon composition in tropical marine Shewanella amazonensis strain SB2B(T).

Shewanella amazonensis strain SB2B(T) is an isolate from shallow-water marine sediments derived from the Amazon River delta. This bacterium contained a long-chain polyunsaturated hydrocarbon, all-cis -3,6,9,12,16,19,22,25,28 hentriacontanonaene (C31:9), constituting 1-2% of the total fatty acid methyl ester and hydrocarbon fraction, which was produced dependently of decreased growth temperature. Analysis of its cellular fatty acid composition demonstrated that isopentadecanoic acid was the major fatty acid component and that all the main monounsaturated fatty acids had straight chains with a cis configuration. However, monoenoic cyclopropyl fatty acids, which were previously reported to be present in this bacterium, were not detected by mass spectrometric analysis. The growth temperature affected the content of Δ9-cis -hexadecenoic [16:1(Δ9c)], palmitic, and heptadecanoic acids. These results suggest that C31:9, as well as 16:1(Δ9c) might be involved in adaptation to low temperature in S. amazonensis strain SB2B(T) . Our result suggests that polyunsaturated fatty acid synthase protein complex may be involved in synthesis of C31:9 but not in production of eicosapentaenoic acid.

The antioxidative function of eicosapentaenoic acid in a marine bacterium, Shewanella marinintestina IK-1.

When the eicosapentaenoic acid (EPA)-deficient mutant strain IK-1Delta8 of the marine EPA-producing Shewanella marinintestina IK-1 was treated with various concentrations of hydrogen peroxide (H(2)O(2)), its colony-forming ability decreased more than that of the wild type. Protein carbonylation, induced by treating cells with 0.01 mM H(2)O(2) under bacteriostatic conditions, was enhanced only in cells lacking EPA. The amount of cells recovered from the cultures was decreased more significantly by the presence of H(2)O(2) for cells lacking EPA than for those producing EPA. Treatment of the cells with 0.1 mM H(2)O(2) resulted in much lower intracellular concentrations of H(2)O(2) being consistently detected in cells with EPA than in those without EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2) in S. marinintestina IK-1.

Glass Bead-based Genetic Transformation:An Efficient Method for Transformation of Thraustochytrid Microorganisms.

Here, we describe a new method for genetic transformation of thraustochytrids, well-known producers of polyunsaturated fatty acids (PUFAs) like docosahexaenoic acid, by combining mild glass (zirconia) bead treatment and electroporation. Because the cell wall is a barrier against transfer of exogenous DNA into cells, gentle vortexing of cells with glass beads was performed prior to electroporation for partial cell wall disruption. G418-resistant transformants of thraustochytrid cells (Aurantiochytrium limacinum strain SR21 and thraustochytrid strain 12B) were successfully obtained with good reproducibility. The method reported here is simpler than methods using enzymes to generate spheroplasts and may provide advantages for PUFA production by using genetically modified thraustochytrids.

Effects of Aerobic Growth on the Fatty Acid and Hydrocarbon Compositions of Geobacter bemidjiensis BemT.

Geobacter spp., regarded as strict anaerobes, have been reported to grow under aerobic conditions. To elucidate the role of fatty acids in aerobiosis of Geobacter spp., we studied the effect of aerobiosis on fatty acid composition and turnover in G. bemidjiensis BemT. G. bemidjiensis BemT was grown under the following different culture conditions: anaerobic culture for 4 days (type 1) and type 1 culture followed by 2-day anaerobic (type 2) or aerobic culture (anaerobic-to-aerobic shift; type 3). The mean cell weight of the type 3 culture was approximately 2.5-fold greater than that of type 1 and 2 cultures. The fatty acid methyl ester and hydrocarbon fraction contained hexadecanoic (16:0), 9-cis-hexadecenoic [16:1(9c)], tetradecanoic (14:0), tetradecenoic [14:1(7c)] acids, hentriacontanonaene, and hopanoids, but not long-chain polyunsaturated fatty acids. The type 3 culture contained higher levels of 14:0 and 14:1(7c) and lower levels of 16:0 and 16:1(9c) compared with type 1 and 2 cultures. The weight ratio of extracted lipid per dry cell was lower in the type 3 culture than in the type 1 and 2 cultures. We concluded that anaerobically-grown G. bemidjiensis BemT followed by aerobiosis were enhanced in growth, fatty acid turnover, and de novo fatty acid synthesis.

The cell membrane-shielding function of eicosapentaenoic acid for Escherichia coli against exogenously added hydrogen peroxide.

The colony-forming ability of catalase-deficient Escherichia coli mutant genetically modified to produce eicosapentaenoic acid (EPA) showed less decrease than in a control strain producing no EPA, when treated with 0.3mM hydrogen peroxide (H(2)O(2)) under non-growth conditions. H(2)O(2)-induced protein carbonylation was enhanced in cells lacking EPA. The amount of fatty acids was decreased more significantly for cells lacking EPA than for those producing EPA. Much lower intracellular concentrations of H(2)O(2) were detected for cells with EPA than those lacking EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2).

A phosphopantetheinyl transferase gene essential for biosynthesis of n-3 polyunsaturated fatty acids from Moritella marina strain MP-1.

A phosphopantetheinyl transferase (PPTase) gene (pfaE), cloned from the docosahexaenoic acid (DHA)-producing bacterium Moritella marina strain MP-1, has an open reading frame of 861 bp encoding a 287-amino acid protein. When the pfaE gene was expressed with pfaA-D, which are four out of five essential genes for biosynthesis of eicosapentaenoic acid (EPA) derived from Shewanella pneumatophori SCRC-2738 in Escherichia coli, the recombinant produced 12% EPA of total fatty acids. This suggests that pfaE encodes a PPTase required for producing n-3 polyunsaturated fatty acids, which is probably involved in the synthesis of DHA in M. marina strain MP-1.

Escherichia coli engineered to produce eicosapentaenoic acid becomes resistant against oxidative damages.

The colony-forming ability of Escherichia coli genetically engineered to produce eicosapentaenoic acid (EPA) grown in 3mM hydrogen peroxide (H(2)O(2)) was similar to that of untreated cells. It was rapidly lost in the absence of EPA. H(2)O(2)-induced protein carbonylation was enhanced in cells lacking EPA. The fatty acid composition of the transformants was unaffected by H(2)O(2) treatment, but the amount of fatty acids decreased in cultures of cells lacking EPA and increased in cultures of cells producing EPA, suggesting that cellular EPA is stable in the presence of H(2)O(2) in vivo and may protect cells directly against oxidative damage. We discuss the possible role of EPA in partially blocking the penetration of H(2)O(2) into cells through membranes containing EPA.

Recombinant production of docosahexaenoic acid in a polyketide biosynthesis mode in Escherichia coli.

The docosahexaenoic acid (DHA) biosynthesis gene cluster (pDHA3) from the DHA-producing Moritella marina strain MP-1 includes the genes pfaA, pfaB, pfaC, and pfaD, which are similar to the genes of polyketide biosynthesis. When this cluster was co-expressed in Escherichia coli with M. marina MP-1 pfaE, which encodes phosphopantetheinyl transferase, DHA was biosynthesized. The maximum production of DHA (5% of total fatty acids) was observed at 15 degrees C. This is the first report of the recombinant production of DHA in a polyketide biosynthesis mode.

Anaerobic decomposition of humic substances by Clostridium from the deep subsurface.

Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of (14)C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth's subsurface.

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells.

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.

Induction of high affinity phosphate transporter in the duckweed Spirodela oligorrhiza.

Duckweed plants (Spirodela oligorrhiza) grown under phosphate (Pi)-deficient conditions (- P plants) exhibited more than 50-fold higher Pi uptake activity than plants grown under Pi-sufficient conditions (+ P plants). The Pi uptake activity of - P plants measured using (32)Pi was significantly inhibited by carbonylcyanide m-chlorophenylhydrazone, indicating that Pi uptake is energized by the electrochemical proton gradient across the plasma membrane (PM). When Pi uptake was examined at various concentrations of Pi, more active uptake of Pi was observed in - P plants than in + P plants, irrespective of the Pi concentrations. An immunoblot analysis of the PM proteins using antiserum against the conserved sequence of the high-affinity Pi transporter recognized the occurrence and large accumulation of a novel protein band at 48 kDa in - P plants. The protein was almost completely extracted with chloroform-methanol (2:1, v/v), but only a trace amount of the protein was detected in + P plants. Immunohistochemical studies of plant roots using the same antiserum demonstrated a large accumulation of high-affinity Pi transporters at the outermost cortical cells of - P plants, but not of + P plants. When an immunoblot analysis of PM proteins was performed using antiserum against the PM H(+)-ATPase, a positive band of about 96 kDa was detected in both plants with a similar signal intensity. Furthermore, ATP-hydrolytic and ATP-dependent H(+)-transporting activities of PM H(+)-ATPase in - P plants were not higher than those in + P plants. However, kinetic analyses showed that the PM H(+)-ATPase in - P plants had a lower K(m) value and a higher coupling efficiency between ATP hydrolysis and H(+) pumping than the corresponding values in + P plants. These results suggest that the significant stimulation of Pi uptake in - P plants may be due mainly to the induction and accumulation of the high-affinity Pi transporter in the PM, and that the electrochemical proton gradient across the PM may be generated by the high-ATP-affinity and energy-efficient H(+) pump in - P plants. This would facilitate the acquisition of Pi in S. oligorrhiza under Pi-depleted conditions.

Cloning and characterization of cDNA of the GPI-anchored purple acid phosphatase and its root tissue distribution in Spirodela oligorrhiza.

A cDNA clone of the glycosylphosphatidylinositol (GPI)-anchored purple acid phosphatase (PAP) has been obtained by a combination of cDNA library screening and 5' rapid amplification of cDNA ends from Spirodela oligorrhiza plants grown under phosphate-deficient (-P) conditions. The open reading frame of the S. oligorrhiza PAP cDNA consists of 1 365 bp encoding a 455 amino acid protein. Its deduced amino acid sequence shows 82 and 80% similarity to Arabidopsis thaliana and Phaseolus vulgaris PAP, respectively. The amino acid residue, Ala439, followed by two more small amino acid residues, Asp and Ser, is predicted to be the GPI-anchoring (omega) site. The absence of a dibasic motif upstream of the putative omega site suggests that the PAP is a cell wall protein. This presumption is supported by the finding that PAP was released by digestion of the cell wall fraction with cellulase. The GPI anchor is speculated to be a signal for transporting PAP to the cell wall. Immunohistochemical results using -P plant roots demonstrate that PAP is preferentially distributed in the outermost cortical cells of roots but not in the epidermis, suggesting its role in acquiring inorganic phosphate under phosphate-deficient conditions. Northern blot analysis using the S. oligorrhiza PAP cDNA as a probe demonstrates that expression of the PAP gene increased during growth of -P plants and this time-dependent occurrence in mRNA levels of the PAP in -P plants was also observed in their protein and activity levels.

Simple synthesis of diastereomerically pure phosphatidylglycerols by phospholipase D-catalyzed transphosphatidylation.

A simple method for synthesizing diastereomerically pure phosphatidylglycerols (PtdGro), namely, 1,2-diacyl-sn-glycero-3-phospho-3'-sn-glycerol (R,R configuration) and 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol (R,S configuration), was established. For this purpose, diastereomeric 1,2-O-isopropylidene PtdGro were prepared from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho) and enantiomeric 1,2-O-isopropylideneglycerols by transphosphatidylation with phospholipase D (PLD) from Actinomadura sp. This species was selected because of its higher transphosphatidylation activity and lower phosphatidic acid (PtdOH) formation than PLD from some Streptomyces species tested. The reaction proceeded well, giving almost no hydrolysis of PtdCho to PtdOH in a biphasic system consisting of diethyl ether and acetate buffer at 30 degrees C. The isopropylidene protective group was removed by heating the diastereomeric isopropylidene PtdGro at 100 degrees C in trimethyl borate in the presence of boric acid to obtain the desired PtdGro diastereomers. The purities of the products, which were determined by chiral-phase HPLC, were exclusively dependent on the optical purities of the original isopropylidene-glycerols used. The present method is simple and can be utilized for the synthesis of pure PtdGro diastereomers having saturated and unsaturated acyl chains.

Unique carotenoid lactoside, P457, in Symbiodinium sp. of dinoflagellate.

The dinoflagellates are a large group of unicellular alge in marine and fresh water. Some are an endosymbiont of marine animals. Photosynthetic dinoflagellates have peridinin, a light-harvesting carotenoid. In addition, a unique carotenoid, P457, was found from Amphinidium. The presence of P457 in Symbiodinium derived from marine animals has not been reported. We reconfirmed the molecular structure of P457, a neoxanthin-like carotenoid with an aldehyde group and a lactoside, from Symbiodinium sp. NBRC 104787 isolated from a sea anemone. In addition, we investigated the distribution of P457 and peridinin in various Symbiodinium and scleractinian coral species, and possible biosynthetic pathways of these carotenoids are proposed.