Publications by authors named "Hidetoshi Okuyama"

48 Publications

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

J Oleo Sci 2017 Jul 13;66(7):791-795. Epub 2017 Jun 13.

Laboratory of Molecular Environmental Microbiology, Graduate School of Agriculture, Hokkaido University.

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.
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http://dx.doi.org/10.5650/jos.ess17084DOI Listing
July 2017

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

J Oleo Sci 2017 Jan 8;66(1):93-101. Epub 2016 Dec 8.

Horonobe Research Institute for the Subsurface Environment (H-RISE), NOASTEC.

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 Bem. G. bemidjiensis Bem 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 Bem followed by aerobiosis were enhanced in growth, fatty acid turnover, and de novo fatty acid synthesis.
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http://dx.doi.org/10.5650/jos.ess16122DOI Listing
January 2017

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

Plant Cell Physiol 2016 Sep 21;57(9):1899-907. Epub 2016 Jun 21.

Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama, 338-8570 Japan Institute for Environmental Science and Technology, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama, 338-8570 Japan

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.
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http://dx.doi.org/10.1093/pcp/pcw110DOI Listing
September 2016

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

Mar Drugs 2016 May 12;14(5). Epub 2016 May 12.

Laboratory of Environmental Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-8589, Japan.

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.
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http://dx.doi.org/10.3390/md14050094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882568PMC
May 2016

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

Sci Rep 2016 Jan 8;6:18990. Epub 2016 Jan 8.

Horonobe Research Institute for the Subsurface Environment, Northern Advancement Centre for Science and Technology, 5-3, Sakae-machi, Horonobe-cho, Teshio-gun, Hokkaido 098-3221, Japan.

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.
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http://dx.doi.org/10.1038/srep18990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705541PMC
January 2016

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

J Basic Microbiol 2015 Jul 24;55(7):838-45. Epub 2015 Feb 24.

Course in Molecular Biology, Division of Biosphere Science, Graduate School of Environmental Science, Hokkaido University, Kita-ku, Sapporo, Japan.

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.
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http://dx.doi.org/10.1002/jobm.201400815DOI Listing
July 2015

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

Photosynth Res 2013 Nov 2;117(1-3):509-15. Epub 2013 Mar 2.

Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Institute for Environmental Science and Technology, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama, 338-8570, Japan.

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.
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http://dx.doi.org/10.1007/s11120-013-9804-7DOI Listing
November 2013

Structural Confirmation of a Unique Carotenoid Lactoside, P457, in Symbiodinium sp. Strain nbrc 104787 Isolated from a Sea Anemone and its Distribution in Dinoflagellates and Various Marine Organisms.

J Phycol 2012 Dec 28;48(6):1392-402. Epub 2012 Sep 28.

Department of Biology, Nippon Medical School, Kawasaki, 211-0061, Japan.

The molecular structure of the carotenoid lactoside P457, (3S,5R,6R,3'S,5'R,6'S)-13'-cis-5,6-epoxy-3',5'-dihydroxy-3-(β-d-galactosyl-(1→4)-β-d-glucosyl)oxy-6',7'-didehydro-5,6,7,8,5',6'-hexahydro-β,β-caroten-20-al, was confirmed by spectroscopic methods using Symbiodinium sp. strain NBRC 104787 cells isolated from a sea anemone. Among various algae, cyanobacteria, land plants, and marine invertebrates, the distribution of this unique diglycosyl carotenoid was restricted to free-living peridinin-containing dinoflagellates and marine invertebrates that harbor peridinin-containing zooxanthellae. Neoxanthin appeared to be a common precursor for biosynthesis of peridinin and P457, although neoxanthin was not found in peridinin-containing dinoflagellates. Fucoxanthin-containing dinoflagellates did not possess peridinin or P457; green dinoflagellates, which contain chlorophyll a and b, did not contain peridinin, fucoxanthin, or P457; and no unicellular algae containing both peridinin and P457, other than peridinin-containing dinoflagellates, have been observed. Therefore, the biosynthetic pathways for peridinin and P457 may have been coestablished during the evolution of dinoflagellates after the host heterotrophic eukaryotic microorganism formed a symbiotic association with red alga that does not contain peridinin or P457.
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http://dx.doi.org/10.1111/j.1529-8817.2012.01219.xDOI Listing
December 2012

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

Acta Biochim Pol 2012 17;59(1):155-7. Epub 2012 Mar 17.

Course in Environmental Molecular Biology and Microbial Ecology, Graduate School of Environmental Science, Hokkaido University, Sapporo, Japan.

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.
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July 2012

Hydrophilic and Hydrophobic Compounds Antithetically Affect the Growth of Eicosapentaenoic Acid-Synthesizing Escherichia coli Recombinants.

Open Microbiol J 2011 3;5:114-8. Epub 2011 Nov 3.

Course in Environmental Molecular Biology and Microbial Ecology, Division of Biosphere Science, Graduate School of Environmental Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan.

The growth of Escherichia coli DH5α recombinants producing eicosapentaenoic acid (EPA) (DH5αEPA+) and those not producing EPA (DH5αEPA-) was compared in the presence of hydrophilic or hydrophobic growth inhibitors. The minimal inhibitory concentrations of hydrophilic inhibitors such as reactive oxygen species and antibiotics were higher for DH5αEPA+ than for DH5αEPA-, and vice versa for hydrophobic inhibitors such as protonophores and radical generators. E. coli DH5α with higher levels of EPA became more resistant to ethanol. The cell surface hydrophobicity of DH5αEPA+ was higher than that of DH5αEPA-, suggesting that EPA may operate as a structural constituent in the cell membrane to affect the entry and efflux of hydrophilic and hydrophobic inhibitors.
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http://dx.doi.org/10.2174/1874285801105010114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219880PMC
August 2012

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

J Basic Microbiol 2011 Oct 24;51(5):484-9. Epub 2011 Mar 24.

Department of Biological Science, School of Science, Hokkaido University, Kita-ku, Sapporo, Japan.

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.
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http://dx.doi.org/10.1002/jobm.201000427DOI Listing
October 2011

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

Int J Syst Evol Microbiol 2011 Aug 3;61(Pt 8):1842-1848. Epub 2010 Sep 3.

Laboratory of Environmental Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan.

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)).
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http://dx.doi.org/10.1099/ijs.0.024612-0DOI Listing
August 2011

The Escherichia coli highly expressed entD gene complements the pfaE deficiency in a pfa gene clone responsible for the biosynthesis of long-chain n-3 polyunsaturated fatty acids.

FEMS Microbiol Lett 2010 Jun;307(2):207-11

Division of Biosphere Science, Graduate School of Environmental Science, Hokkaido University, Kita-ku, Sapporo, Japan.

The Escherichia coli entD gene, which encodes an Sfp-type phosphopantetheinyl transferase (PPTase) that is involved in the biosynthesis of siderophore, is available as a high-expression ASKA clone (pCA24N::entD) constructed from the E. coli K-12 strain AG1. In E. coli DH5alpha, pCA24N::entD complemented a pfaE-deficient clone that comprised pfaA, pfaB, pfaC and pfaD, which are four of the five pfa genes that are responsible for the biosynthesis of eicosapentaenoic acid derived from Shewanella pneumatophori SCRC-2738. Sfp-type PPTases are classified into the EntD and PfaE groups, based on differences between their N-terminal-domain structures. Here, we showed that all Sfp-type PPTases may have the potential to promote the biosynthesis of long-chain n-3 polyunsaturated fatty acids.
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http://dx.doi.org/10.1111/j.1574-6968.2010.01987.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901515PMC
June 2010

Enhancement of the nitrogen fixation efficiency of genetically-engineered Rhizobium with high catalase activity.

J Biosci Bioeng 2010 Oct 20;110(4):397-402. Epub 2010 May 20.

Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, 080-8555, Japan.

The vktA catalase gene, which had been cloned from Vibrio rumoiensis S-1T having extraordinarily high catalase activity, was introduced into the root nodule bacterium, Rhizobium leguminosarum bv. phaseoli USDA 2676. The catalase activity of the vktA-transformed R. leguminosarum cells (free-living) was three orders in magnitude higher than that of the parent cells and this transformant could grow in a higher concentration of exogenous hydrogen peroxide (H2O2). The vktA-transformant was inoculated to the host plant (Phaseolus vulgaris L.) and the nodulation efficiency was evaluated. The results showed that the nitrogen-fixing activity of nodules was increased 1.7 to 2.3 times as compared to the parent. The levels of H2O2 in nodules formed by the vktA-transformant were decreased by around 73%, while those of leghemoglobins (Lba and Lbb) were increased by 1.2 (Lba) and 2.1 (Lbb) times compared with the parent. These results indicated that the increase of catalase activity in rhizobia could be useful to improve the nitrogen-fixing efficiency of nodules by the reduction of H2O2 content concomitantly with the enhancement of leghemoglobins contents.
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http://dx.doi.org/10.1016/j.jbiosc.2010.04.007DOI Listing
October 2010

Membrane eicosapentaenoic acid is involved in the hydrophobicity of bacterial cells and affects the entry of hydrophilic and hydrophobic compounds.

FEMS Microbiol Lett 2010 May 30;306(2):91-6. Epub 2010 Mar 30.

Hokkaido University, Sapporo, Japan.

Eicosapentaenoic acid (EPA)-producing Shewanella marinintestina IK-1 (IK-1) and its EPA-deficient mutant IK-1Delta8 (IK-1Delta8) were grown on microtitre plates at 20 degrees C in a nutrient medium that contained various types of growth inhibitors. The minimal inhibitory concentrations of hydrogen peroxide and tert-butyl hydroxyl peroxide were 100 microM and 1 mM, respectively, for IK-1 and 10 and 100 microM, respectively, for IK-1Delta8. IK-1 was much more resistant than IK-1Delta8 to the four water-soluble antibiotics (ampicillin sodium, kanamycin sulphate, streptomycin sulphate, and tetracycline hydrochloride) tested. In contrast, IK-1 was less resistant than IK-1Delta8 to two hydrophobic uncouplers: carbonyl cyanide m-chloro phenylhydrazone (CCCP) and N,N'-dicyclohexylcarbodiimide (DCCD). The hydrophobicity of the IK-1 and IK-1Delta8 cells grown at 20 degrees C was determined using the bacterial adhesion to hydrocarbon method. EPA-containing ( approximately 10% of total fatty acids) IK-1 cells were more hydrophobic than their counterparts with no EPA. These results suggest that the high hydrophobicity of IK-1 cells can be attributed to the presence of membrane EPA, which shields the entry of hydrophilic membrane-diffusible compounds, and that hydrophobic compounds such as CCCP and DCCD diffuse more effectively in the membranes of IK-1, where they can fulfil their inhibitory activities, than in the membranes of IK-1Delta8.
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http://dx.doi.org/10.1111/j.1574-6968.2010.01943.xDOI Listing
May 2010

Possible biosynthetic pathways for all cis-3,6,9,12,15,19,22, 25,28-hentriacontanonaene in bacteria.

Lipids 2010 Feb;45(2):167-77

Division of Biosphere Science, Graduate School of Environmental Science, Hokkaido University, Kita-ku, Sapporo, Japan.

A very long chain polyunsaturated hydrocarbon, hentriacontanonaene (C31:9), was detected in an eicosapentaenoic acid (EPA)-producing marine bacterium, which was isolated from the mid-latitude seashore of Hokkaido, Japan, and was tentatively identified as mesophilic Shewanella sp. strain osh08 from 16S rRNA gene sequencing. The geometry and position of the double bonds in this compound were determined physicochemically to be all cis at positions 3, 6, 9, 12, 15, 19, 22, 25, and 28. Although C31:9 was detected in all of the seven EPA- or/and docosahexaenoic acid-producing bacteria tested, an EPA-deficient mutant (strain IK-1Delta8) of one of these bacteria had no C31:9. Strain IK-1Delta8 had defects in the pfaD gene, one of the five pfa genes responsible for the biosynthesis of EPA. Although Escherichia coli DH5alpha does not produce EPA or DHA inherently, cells transformed with the pfa genes responsible for the biosynthesis of EPA and DHA produced EPA and DHA, respectively, but not C31:9. These results suggest that the Pfa protein complex is involved in the biosynthesis of C31:9 and that pfa genes must not be the only genes responsible for the formation of C31:9. In this report, we determined for the first time the molecular structure of the C31:9 and discuss the possible biosynthetic pathways of this compound.
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http://dx.doi.org/10.1007/s11745-009-3380-9DOI Listing
February 2010

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis.

FEMS Microbiol Lett 2009 Jun 21;295(2):170-6. Epub 2009 Apr 21.

Laboratory of Environmental Molecular Biology, Faculty of Environmental Earth Science, Hokkaido University, Sapporo, Japan.

When pDHA4, a vector carrying all five pfaA-pfaE genes responsible for docosahexaenoic acid (DHA; 22:6) biosynthesis in Moritella marina MP-1, was coexpressed in Escherichia coli with the individual pfaA-pfaD genes for eicosapentaenoic acid (EPA; 20:5) biosynthesis from Shewanella pneumatophori SCRC-2738, both polyunsaturated fatty acids were synthesized only in the recombinant carrying pfaB for EPA synthesis. Escherichia coli coexpressing a deleted construct comprising pfaA, pfaC, pfaD and pfaE for EPA and pfaB for DHA produced EPA and DHA. Both EPA and DHA were detected in bacteria that inherently contained pfa genes for DHA. These results suggest that PfaB is the key enzyme determining the final product in EPA or DHA biosynthesis.
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http://dx.doi.org/10.1111/j.1574-6968.2009.01582.xDOI Listing
June 2009

H2O2 tolerance of Vibrio rumoiensis S-1(T) is attributable to the cellular catalase activity.

J Biosci Bioeng 2008 Jul;106(1):39-45

Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.

The extraordinarily high level of H2O2 tolerance of Vibrio rumoiensis strain S-1(T) when compared with the tolerance levels of strain S-4, a probable catalase-deficient derivative of strain S-1(T), was demonstrated by the introduction of 0-100 mM H2O2 during the mid-exponential growth phase. The contribution of catalase to the H2O2 tolerance was also demonstrated by comparing the catalase-deficient mutant Escherichia coli strain UM2 with a UM2 strain, harboring the plasmid pBSsa1, which carried the strain S-1(T) catalase gene vktA. The decomposition rates of 23-25 mM H2O2 that was introduced in the culture fluids of strain S-1(T) and E. coli UM2 harboring pBSsa1 corresponded to the calatase activities of the cells by spectrophotometric measurements. The presence of cell surface catalase was observed by immunoelectron microscopy, using an antibody for intracellular catalase in strain S-1(T). The high level of H2O2 tolerance of strain S-1(T) was attributable to the catalase activity of the cells. Cell surface catalase is considered to contribute to the catalase activity of strain S-1(T) cells.
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http://dx.doi.org/10.1263/jbb.106.39DOI Listing
July 2008

Significance of antioxidative functions of eicosapentaenoic and docosahexaenoic acids in marine microorganisms.

Appl Environ Microbiol 2008 Feb 7;74(3):570-4. Epub 2007 Dec 7.

Faculty of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan.

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http://dx.doi.org/10.1128/AEM.02256-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2227742PMC
February 2008

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation.

World J Microbiol Biotechnol 2007 Dec 15;23(12):1739-45. Epub 2007 May 15.

Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo, 060-0810, Japan.

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria-Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.
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http://dx.doi.org/10.1007/s11274-007-9423-6DOI Listing
December 2007

An EntD-like phosphopantetheinyl transferase gene from Photobacterium profundum SS9 complements pfa genes of Moritella marina strain MP-1 involved in biosynthesis of docosahexaenoic acid.

Biotechnol Lett 2008 Mar 1;30(3):411-4. Epub 2007 Nov 1.

Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo, Japan.

The EntD-like phosphopantetheinyl transferase (PPTase) gene, cloned from the eicosapentaenoic acid-producing bacterium Photobacterium profundum strain SS9, has an ORF of 690 bp encoding a 230-amino acid protein. When this PPTase gene was expressed in Escherichia coli with pfaA, pfaB, pfaC and pfaD derived from Moritella marina MP-1, which were four of five essential genes for biosynthesis of docosahexaenoic acid (DHA), the DHA production of the recombinant was 2% (w/w) of total fatty acids. This is the first report showing that the EntD-like PPTase is involved in producing n-3 polyunsaturated fatty acids.
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http://dx.doi.org/10.1007/s10529-007-9579-zDOI Listing
March 2008

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

FEBS Lett 2007 Sep 7;581(22):4212-6. Epub 2007 Aug 7.

Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan.

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.
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http://dx.doi.org/10.1016/j.febslet.2007.07.065DOI Listing
September 2007

In vivo conversion of triacylglycerol to docosahexaenoic acid-containing phospholipids in a thraustochytrid-like microorganism, strain 12B.

Biotechnol Lett 2007 Dec 28;29(12):1977-81. Epub 2007 Jul 28.

Faculty of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo, 060-0810, Japan.

The thraustochytrid-like microorganism, strain 12B, cultivated in peptone, yeast extract, and 8% (w/v) glucose in 50% (v/v) seawater, accumulated docosahexaenoic acid (DHA)-rich triacylglycerol (TAG) at 67% of total lipid. When these TAG-accumulated cells were cultivated in glucose-deficient medium, dry cell weight (3 mg per ml culture) increased approximately 3-fold relative to baseline but the TAG/total lipid decreased to 5%. At the same time, the amount of phospholipid (5 mg) per whole culture also increased 3-fold. Hence, phospholipid/total lipid increased from 13% to 67%. High levels of DHA (more than 50% of total) were maintained in phosphatidylcholine.
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http://dx.doi.org/10.1007/s10529-007-9492-5DOI Listing
December 2007

Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA fragments including a high-performance catalase gene, vktA.

Biotechnol Lett 2007 May 13;29(5):803-9. Epub 2007 Feb 13.

Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo, 060-0810, Japan.

Cellular eicosapentaenoic acid (EPA) makes up approximately 3% of total fatty acids in Escherichia coli DH5alpha, a strain that carries EPA biosynthesis genes (pEPADelta1). EPA was increased to 12% of total fatty acids when the host cell co-expressed the vector pGBM3::sa1(vktA), which carried the high-performance catalase gene, vktA. Where this vector was co-expressed, the transformant accumulated a large amount of VktA protein. However, the EPA production of cells carrying the vector, that included the insert lacking almost the entire vktA gene, was approximately 6%. This suggests that the retention of a large DNA insert in the vector and the accumulation of the resulting protein, but not the catalytic activity of VktA catalase, would potentially be able to increase the content of EPA.
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http://dx.doi.org/10.1007/s10529-007-9310-0DOI Listing
May 2007

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

FEBS Lett 2006 Dec 20;580(28-29):6690-4. Epub 2006 Nov 20.

Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan.

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).
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http://dx.doi.org/10.1016/j.febslet.2006.11.030DOI Listing
December 2006

Bacterial genes responsible for the biosynthesis of eicosapentaenoic and docosahexaenoic acids and their heterologous expression.

Appl Environ Microbiol 2007 Feb 22;73(3):665-70. Epub 2006 Nov 22.

Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan.

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http://dx.doi.org/10.1128/AEM.02270-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1800774PMC
February 2007

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

Biotechnol Lett 2006 Nov 19;28(22):1841-7. Epub 2006 Sep 19.

Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, 060-0810, Japan.

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.
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http://dx.doi.org/10.1007/s10529-006-9168-6DOI Listing
November 2006

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

FEBS Lett 2006 Aug 13;580(18):4423-9. Epub 2006 Jul 13.

Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan.

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.
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http://dx.doi.org/10.1016/j.febslet.2006.07.008DOI Listing
August 2006

Bacterial community changes in diesel-oil-contaminated soil microcosms biostimulated with Luria-Bertani medium or bioaugmented with a petroleum-degrading bacterium, Pseudomonas aeruginosa strain WatG.

J Basic Microbiol 2006 ;46(4):310-7

Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University, Kita 10 Nishi 6 Kita-ku, Sapporo 060-0810, Japan.

The microbial community structure of diesel-oil-contaminated soil microcosms biostimulated with Luria-Bertani medium (LB-BS) or bioaugmented with a petroleum-degrading bacterium, Pseudomonas aeruginosa strain WatG (WatG-BA), was investigated by denaturing gradient gel electrophoresis (DGGE) and by monitoring diesel oil degradation. The degradation in WatG-BA (64.0% +/- 4.2%) was higher than that in LB-BS (49.5% +/- 12.0%) during the first two weeks. The microbial community in WatG-BA, which was markedly dominated by strain WatG, was much simpler than that in LB-BS, where hydrocarbon degraders occurred after a lag of 3-7 days after the addition of diesel oil. The clustering profiles of the DGGE banding patterns of the two soil microcosms were only 12% similar. This difference is probably due to antibacterial substances, such as rhamnolipids, secreted by strain WatG.
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http://dx.doi.org/10.1002/jobm.200510116DOI Listing
September 2006