Abstract: A mesophilic marine bacterial strain (strain osh08T) that produced eicosapentaenoic acid and hentriacontanonaene, was identified by the molecular and ordinary microbiological methods. This strain had the high sequence similarity of its 16S rRNA gene to those of Shewanella basaltis JCM 14937T (EU143361; 99.8%) and Shewanella hafniensis NBRC 100975T (AB205566; 98.4%). But, strain osh08T was phenotypically different from S. basaltis J83T and S. hafniensis NBRC 100975T in that it was able to ferment glucose, produce β-glucosidase and β-galactosidase and utilize D-mannose and unable to utilize malate. However, all three strains used iron(III) and manganese(IV) as terminal electron acceptors under anaerobic conditions. The DNA-DNA relatedness between strain osh08T and S. basaltis J83T and that between strain osh08T and S. hafniensis NBRC 100975T were 4.7% and 11.8%, respectively. The G+C content of strain osh08T was 43.4 mol%. Strain osh08T had, in addition to a polar flagellum, many pilus-like structures around the cell that were not observed in the cells of S. basaltis J83T. Thus strain osh08T was identified as a new species of Shewanella. The name Shewanella oshoroensis sp. nov. was proposed and strain osh08T (=NBRC 107685T) is the type strain.
INTRODUCTION
Previously a mesophilic bacterial strain that produced eicosapentaenoic acid (EPA) was isolated from an alga sample collected on the Hokkaido coast, a cool-temperate region of Japan (Sugihara et al., 2010). This strain has been tentatively identified as Shewanella sp. strain osh08T based on the high similarity of its 16S rRNA gene sequence with those of Shewanella basaltis J83T (EU143361; 99.8%) and Shewanella hafniensis NBRC 100975T (AB205566; 98.4%). Shewanella sp. strain osh08T produced, in addition to EPA, a very long-chain polyunsaturated hydrocarbon, hentriacontanonaene (C31:9). C31:9 has been detected only from psychrophilic bacteria from polar marine (Nichols et al., 1995) and deep sea environments (Tamaoka et al., 1998). The configuration and positions of the double bonds of this compound were determined to be all cis at positions 3, 6, 9, 12, 15, 19, 22, 25 and 28 (Sugihara et al., 2010). As C31:9 have been detected only in EPA- or docosahexaenoic acid (DHA)-producing bacteria and the EPA-deficient mutant did not produce C31:9, it is speculated that the pfa genes responsible for the biosynthesis of EPA and DHA are involved in the synthesis of C31:9 (Sugihara et al., 2010). Recently it was shown that C31:9 can be generated by head-to-head condensation of two hexadecatetraenoic fatty acid molecules (Sukovich et al., 2010), although no biochemical evidence has been available. Strain osh08T is the first reported EPA and C31:9-producing bacterium isolated from non-psychrophilic environments. In this study, strain osh08T was taxonomically characterized as a new Shewanella sp. and the name Shewanella oshoroensis sp. nov. was proposed.
MATERIALS AND METHODS
Sampling, bacterial strains and culture conditions: Seaweed samples (fragments of red alga), which were used to isolate Shewanella sp. strain osh08T, were collected on the mid-latitude seashore of Oshoro, Hokkaido, Japan (43.19 °N, 141.00 °E), as described previously (Sugihara et al., 2010). Samples were subjected to isolation and purification procedures immediately after collection using ZoBell agar medium (Z medium: 0.1% peptone, 0.1% yeast extract, 0.01% Fe3(PO4)2 and 1.5% agar) in 50% (v/v) seawater (ZoBell, 1946). Filtered natural seawater was utilized to prepare media (Sugihara et al., 2010). Plates were then incubated at 20°C for several days. Bacterial colonies were directly treated with acetyl chloride in methanol for methanolysis, as described below. Shewanella sp. strain osh08T was the sole EPA-producing bacterium isolated in this study. The following type strains were used: Shewanella hafniensis NBRC100975T (Satomi et al., 2006), Shewanella basaltis J83T (Chang et al., 2008) and Shewanella oneidensis MR-1T (ATCC 700550TM) (Venkateswaran et al., 1999). Strain osh08T and these reference strains were cultivated for characterization at the indicated temperatures in Luria-Bertani (LB) medium containing 3.0% NaCl.
Physiological and biochemical characterization of strain osh08T: Routine biochemical tests were performed as described in Cowan and Steel’s manual (Barrow and Feltham, 1993). Reduction of Mn(IV) under anaerobic conditions was ascertained by clear zone formation and visible growth of cells during cultivation on agar plates using the published method of Myers and Nealson (1988), with a slight modification and reduction of Fe(III) by the same method as described above but Mn(IV) was substituted with iron, using not only strain osh08T but also S. hafniensis NBRC100975T, S. basaltis J83T and S. oneidensis MR-1T (ATCC 700550TM) for comparison. Ferric citrate (100 mM) and manganese dioxide (100 mM) were used as electron acceptors. Sodium acetate (15 mM) was added to both media as electron donor. Agar plate preparation and inoculation of cells were carried out under anaerobic conditions (10% hydrogen, 10% carbon dioxide and 80% nitrogen) in an anaerobic chamber (Te-Her Anaerobox ANX-1W, Hirasawa Works, Tokyo). Plates were then incubated anaerobically at 30°C for more than two weeks.
| Table 1: | Phenotypic characteristics of strain osh08T and related type strains1 |
| 1Strains 1: osh08T (present study), 2: S. hafniensis NBRC 100975T; Satomi et al. (2006), 3: S. basaltis J83T; Chang et al. (2008), 4: S. gaetbuli LMG 19866T; Yoon et al. (2004), 5: S. denitrificans OS217T; Brettar et al. (2002), 6: S. livingstonensis LMG 19866T; Bozal et al. (2002) 7: S. oneidensis MR-1T; Venkateswaran et al. (1999), 2Result obtained in the present study, 3The same result was obtained in this study, +: Positive, -: Negative, NA: No data available, All strains grew at 4°C and were positive for cytochrome oxidase and catalase and for reduction of nitrate to nitrite | |
For the determination of other phenotypic characteristics, API 20NE tests were performed in triplicate using the methods recommended by the manufacturer (bioMérieux Japan, Tokyo, Japan). Some phenotypic characteristics unavailable for S. oneidensis MR-1T (ATCC 700550TM) were also examined in this study using API 20NE tests (Table 1).
The growth of S. hafniensis NBRC100975T and S. oneidensis MR-1T (ATCC 700550TM) in medium at pH 10 and that of S. basaltis J83T in medium containing no NaCl, which had not been previously reported, were determined as described previously (Sugihara et al., 2010). Optimum temperature for growth of S. basaltis J83T was evaluated visibly by the extent of growth on LB agar plates containing 3% NaCl incubated at 20, 25, 30 and 35°C for 24 h.
Electron microscopy: Cells of strain osh08T, which were grown on liquid Z medium as described above, were suspended in liquid Z medium. A small drop of the suspension was placed on a carbon-coated copper grid and negatively stained with 2% (w/v) uranyl acetate as described (Haschemeyer and Myers, 1972) for observation with a transmission electron microscope (JEM1200EXS, JEOL, Tokyo).
DNA procedures: Genomic DNA was extracted from strain osh08T and various type strains by the method of Marmur (1961). High-performance liquid chromatography (HPLC) was used to determine the DNA G+C content (Tamaoka and Komagata, 1984). Briefly, the DNA preparations were denatured at 100°C for 10 min, digested with nuclease P1 (Yamasa Shoyu, Tokyo) and subjected to HPLC using a Cosmosil 5C18 column (type: Waters, 4.6x150 mm, Nakalai Tesque, Kyoto, Japan). An equimolar mixture of four deoxyribonucleotides (Yamasa Shoyu) was used as the standard. The nucleotides were detected with a spectrophotometer detector set at 260 nm.
Levels of DNA relatedness were determined fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labeled DNA probes and microplates.
Amplification, cloning and sequencing of the 16S rRNA gene were performed as described previously (Sugihara et al., 2010). The sequence-comparative searches were performed using the NCBI (http://www.ncbi.nlm.nih.gov/) databases. Multiple alignments of the sequence were performed, nucleotide substitution rates (Knuc values) were calculated and a neighbour-joining phylogenetic tree was constructed using the CLUSTALW program of DDBJ (http://clustalw.ddbj.nig.ac.jp/top-j.html). The accuracy of the phylogenetic tree branches was confirmed by a bootstrap method (Felsenstein, 1985). The strain osh08T 16S rRNA gene sequence was previously deposited in DDBJ/GenBank/EMBL with the accession number AB447987 (Sugihara et al., 2010).
Analysis of fatty acids and polyunsaturated hydrocarbons: The cellular fatty acid composition of the isolates was determined as described previously (Sugihara et al., 2010) with a minor modification. Briefly, small aliquots of wet cells were subjected to methanolysis using 1 mL of 10% (v/v) acetyl chloride in methanol at 100°C for 1 h. A 10 μg aliquot of heneicosanoic acid (21:0; Sigma-Aldrich, Tokyo) was used as an internal standard. Fatty acid methyl esters were extracted with hexane together with hydrocarbon and this was designated the fatty acid and hydrocarbon fraction. In this study, the amount of hydrocarbons was estimated using 21:0 as the internal standard, because the difference in the amount of hydrocarbons estimated using either 21:0 or hydrocarbon (n-tetracosane) as standards was negligible.
Fatty acid methyl esters and hydrocarbons were analysed by gas-liquid chromatography (GLC) as described by Sugihara et al. (2010) on a gas chromatograph (model GC-353B; GL Sciences, Tokyo) equipped with a capillary column BPX70 (25 m longx0.22 mm I.D., 0.25 mm film thickness, SGE Japan, Yokohama, Japan) and flame ionization detection with nitrogen as the carrier gas. These compounds were identified by ion-trap mode gas chromatography-mass spectrometry (GC/MS) on a Varian system (model CP-3800 gas chromatograph and a Saturn 2200 ion-trap mass spectrometer, Varian Technologies Japan, Inc., Tokyo, Japan) under the same conditions as described previously (Orikasa et al., 2006). Data were analysed using a Saturn™ Software Workstation Version 5.52. In GC/MS analysis, two modes of electron impact ionization and chemical ionization were utilized. In chemical ionization-gas chromatography-mass spectrometry, acetonitrile was used as an ionization reagent.
RESULTS AND DISCUSSION
Morphological characteristics: Strain osh08T was Gram-negative (Sugihara et al., 2010) and had a single unsheathed flagellum (Fig. 1a). Interestingly, the strain had pilus-like structures 40-500 nm in length on the cell surface (Fig. 1b). Although S. basaltis J83T is the species closest to strain osh08T in its 16S rRNA gene sequence (Fig. 2), it has no pilus-like structures (not shown). The typical form of strain osh08T cells was rods 0.3-0.6 mm wide by 0.8-1.5 mm long.
Strain osh08T formed opaque, pink-brown, glossy, convex and rounded colonies with an entire margin. The size of the colonies was less than 1 mm in diameter after incubation for 18 h at 30°C. A clear zone approximately 1 mm wide around the colony was observed only for strain osh08T. This clear zone may be generated by digestion of peptides in the medium by secreted peptidase.
Physiological and biochemical characteristics: Strain osh08T grew in a temperature range of 4 to 40°C with an optimum temperature of 30°C (Sugihara et al., 2010). It grew in media containing no NaCl and in media with 7% NaCl and growth was observed at pH 10. The physiological and biochemical characteristics of strain osh08T were compared with those of six type strains of Shewanella species (Table 1).
When strain osh08T was compared with the six reference strains, characteristics common to all strains were growth at 4°C, positive oxidase and catalase reactions and nitrate reduction. Strain osh08T, S. basaltis J83T and S. hafniensis NBRC 100975T had common features of no nitrite reduction; no indole, urease, or arginine dihydrolase production; positive utilization of glucose and maltose and no utilization of adipate. Of these four strains, fermentation of glucose, production of β-galactosidase and β-glucosidase and utilization of D-mannose were detected only for strain osh08T (Table 1).
| Fig. 1(a-b): | Electron micrographs of (a) Negatively stained cells of strain osh08T, In (b) A cell of strain osh08T with a flagellum (arrow) and pilus-like structures (arrowheads), Bar indicates 500 nm in (a) and 100 nm in (b) |
| Fig. 2: | Phylogenetic trees derived from the 16S rRNA gene sequence data of strain osh08T and related Shewanella type strains using the neighbour-joining method for calculation. Numbers indicate bootstrap values of greater than 500. Bar, 0.01 nucleotide substitution rate (Knuc) unit |
When strain osh08T was compared with the sole non-marine mesophilic bacterium S. oneidensis MR-1T (Venkateswaran et al., 1999), there were many characteristics common to these two strains, such as an optimum growth temperature of 30°C, growth at 4°C and 40°C and growth in medium with no NaCl and at pH 10. Strain osh08T was positive for reduction of iron oxide and manganese oxide under anaerobic conditions in the presence of 15 mM sodium acetate as electron donor (Table 1), as was S. oneidensis MR-1T (Venkateswaran et al., 1999). The reduction of these metal ions was also observed in this study for S. basaltis J83T and S. hafniensis NBRC 100975T (Table 1). Both strain osh08T and S. oneidensis MR-1T contained C31:9 in addition to EPA (Sugihara et al., 2010).
Analysis of 16S rRNA gene sequences: As described previously (Sugihara et al., 2010), the 1,534 bp nucleic acid sequence of the 16S rRNA gene of strain osh08T was determined (AB447987) and showed 99.8 and 98.4% similarity to those of S. basaltis JCM 14937T (EU143361) and S. hafniensis NBRC 100975T (AB205566), respectively. Figure 2 shows the phylogenetic tree derived from the 16S rRNA gene sequences of strain osh08T and of various Shewanella type strains.
DNA base composition and DNA-DNA hybridisation: The DNA G+C content of strain Shewanella sp. strain osh08T was 43.4 mol% (Table 1). The level of DNA relatedness between strain osh08T and S. basaltis J83T and that between strain osh08T and S. hafniensis NBTC 100975T were 4.7 and 11.8%, respectively.
Fatty acids and hydrocarbons: The detailed fatty acid and hydrocarbon compositions of osh08T were described previously by Sugihara et al. (2010). The major Δ9-hexadecenoic acid, 16:1(Δ9), accounted for 28.8% of the total fatty acid and hydrocarbon fraction and this strain contained isomers of monounsaturated fatty acids with a double bond at different positions, such as Δ7-pentadecenoic acid [15:1(Δ7)], Δ9-pentadecenoic acid [15:1(Δ9)], Δ7-hexadecenoic acid [16:1(Δ7)], 16:1(Δ9), Δ9-heptadecenoic acid [17:1(Δ9)], Δ11-heptadecenoic acid [17:1(Δ11)], Δ9-octadecenoic acid [18:1(Δ9)] and Δ11-octadecenoic acid [18:1(Δ11)] (Sugihara et al., 2010). These isomers of monounsaturated fatty acids are found in most Shewanella species (Satomi et al., 2003, 2006). Normal and iso-branched odd-carbon-number fatty acids were also observed in strain osh08T. Iso-pentadecanoic (i15:0) and pentadecanoic (15:0) acids accounted for approximately 10% of the total fatty acid and hydrocarbon fraction and both EPA and C31:9 were detected at approximately 1 and 3%, respectively, in this strain (Sugihara et al., 2010), as well as in S. basaltis J83T and S. hafniensis NBTC 100975T.
Description of Shewanella oshoroensis sp. nov.: Shewanella oshoroensis (o.sho.ro.en’sis. L. adj. oshoroensis, from Oshoro, a harbour village of Hokkaido, Japan from which the sample alga was collected).
Cells are Gram-negative rods, 0.3-0.6 mm wide by 0.8-1.5 mm long, facultatively anaerobic, motile by a single unsheathed flagellum. Cells have pilus-like structures of 40-500 nm in length on the surface and are mesophilic with an optimum temperature for growth at 30°C. Growth occurs in medium with no NaCl and in that containing 7.0% NaCl and in that at pH 10. Catalase and oxidase reactions are positive, gelatinase and β-galactosidase and β-glucosidase are produced, but urease and arginine dihydrolase are not produced. Fermentation of glucose was detected. Iron oxide and manganese oxide were reduced under anaerobic conditions. The G+C content of the DNA is 43.4 mol%. Other physiological characteristics are shown in Table 1. Major fatty acids are 16:1(Δ9), 16:0, 17:1(Δ9), i15:0 and 15:0. Small amounts of EPA and C31:9 are detected. The type strain is strain osh08T (=NBRC 107685T).
CONCLUSION
In this study the mesophilic EPA and C31:9-producing bacterium was identified as a novel Shewanella species and named as Shewanella oshoroensis sp. nov. S. oshoroensis strain osh08T (NBRC 107685T) is a promising strain for the production of C31:9 at moderate temperatures, such as at 25°C (Sugihara et al., 2010).
ACKNOWLEDGEMENTS
This study was partly supported by the National Institute of Polar Research, Japan and by a Grant-in-Aid for Scientific Research ((C) no. 22570130) from the Ministry of Education, Science, Sports and Culture of Japan.