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1 Department of Biomaterials Engineering, Chosun University, Gwangju 501-759, South Korea
2 Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia
3 Department of Environmental Engineering, Chosun University, Gwangju 501-759, South Korea
Correspondence
Si Wouk Kim
swkim{at}chosun.ac.kr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain MPT is DQ463161.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; De Zwart et al., 1996). More recently, Methylophaga alcalica and ‘Methylophaga natronica’ – moderately haloalcalophilic obligately and restricted facultatively methylotrophic bacteria were described by Doronina et al. (2003a, b). Species of the genus Methylophaga are distinguished from methylobacteria of the other RuMP pathway by their requirement for Na+, Mg2+ and vitamin B12, tolerance of NaCl and the low G+C content (38.0–49.0 mol%) of their DNA. On the other hand, two strains, Methylophaga marina KM3 and KM5, isolated recently from Red Sea algae were not found to require vitamin B12 (Li et al., 2007). Here, we report the taxonomic characterization of a neutrophilic, moderately halophilic, vitamin B12-independent, RuMP pathway restricted facultative methylotroph belonging to the genus Methylophaga. The name Methylophaga aminisulfidivorans sp. nov. is proposed for this new isolate.
The novel strain was isolated from a seawater sample collected from Mokpo, South Korea. A 1 ml sample was used to inoculate a 500 ml flask containing 50 ml mineral salts medium [MSM; 2 g KH2PO4 l–1; 2 g (NH4)2SO4 l–1; 0.2 g MgSO4 . 7H2O l–1; 30 g NaCl l–1; 0.002 g FeSO4 . 7H2O l–1; pH 7.0] supplemented with 1 % methanol (v/v) and the flask was then incubated at 150 r.p.m. for 2 days at 30 °C. An aliquot of 0.5 ml of the turbid suspension was then transferred to fresh medium with 1 % (v/v) methanol and incubated as before. After serial transfers, a small amount of the suspension was spread on an agar-containing methanol plate and incubated at 30 °C for 3 days. From the plate, fast-growing colonies were selected and transferred to fresh plates. After several purification steps, a novel fast-growing strain, MPT, was obtained. The culture purity of the isolated methylotrophic bacteria was tested by examining colonies and cell suspensions with light and electron microscopes.
The pure culture of strain MPT was stored in liquid MSM for 10 days, on agar slants at 4 °C for 2 weeks or freeze-dried with a protectant (skimmed milk) for over a year. Cell morphology was examined using batch cultures in the late-exponential growth phase. An aliquot of cell suspension was mounted on a Formvar-coated copper grid and stained with 0.2 % (w/v) phosphotungstic acid (pH 7.2). For thin sectioning, cells were collected by centrifugation and pre-fixed with 1.5 % (w/v) glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2) and washed three times with 1 % (w/v) OsO4 in the same buffer for 3 h at 20 °C. After dehydration in a series of alcohols, the cells were embedded in Spurr epoxy resin and sectioned with a microtome (LKB 2128 Ultratome; LKB). Ultrathin sections were mounted on copper grids and double-strained with uranyl acetate and lead citrate (Reynolds, 1963). Negatively stained preparations and thin sections were viewed with a transmission electron microscope (JEM-100B; JEOL) at operating voltages of 60 kV and 80 kV, respectively.
The methods, reagents and media used for phenotypic characterization were as described by Doronina et al. (1995). Utilization of a wide range of growth substrates was determined in MSM after cultivation for 2 weeks with methanol replaced by the other carbon compounds (more than 60 were tested). Organic acids, amino acids and methylated amines were added at concentrations of 0.2 % (w/v), while carbohydrates and alcohols were added at concentrations of 0.2–0.5 % (w/v or v/v). To test alternative nitrogen sources, (NH4)2SO4 was replaced by other nitrogen compounds. Methane utilization was tested in an atmosphere containing methane and air (1 : 1, v/v) in 700 ml conical flasks containing 100 ml MSM and fitted with rubber stoppers. Hydrogen utilization was tested by the same procedure but under an atmosphere consisting of H2/O2/CO2 (7 : 2 : 1, v/v).
Fatty acids were extracted from cell biomass dried by lyophilization. A 200 µl aliquot of a 5.4 M solution of anhydrous HCl in methanol was added to 30 mg of dry biomass and the mixture was heated at 70 °C for 2 h. The methyl esters of fatty acids and aldehyde derivatives obtained were extracted twice with 100 µl hexane. The extract was dried and silylated in 20 µl N,O-bis-(trimethylsilyl)trifluoroacetamide for 15 min at 65 °C. A 1 µl portion of the reaction mixture was analysed with a GC-MS system (model HP-5985B; Hewlett Packard) equipped with a capillary column (25x0.25 mm) consisting of fused quartz containing an Ultra-1 nonpolar methylsilicone phase. The temperature program was run from 150 °C (2 min isotherm) to 250 °C at 5 °C min–1 and then from 250 to 300 °C at 10 °C min–1. Data processing was carried out with a computer (HP-1000; Hewlett Packard) by using the standard programs of the GC-MS system (Hewlett Packard). Phospholipid composition of the cells was determined according to previously described methods (Govorukhina & Trotsenko, 1989). Ubiquinones were extracted and purified according to Collins (1985). Analysis of ubiquinones was carried out using a mass spectrometer (MX-1310; Finnigan). Enzyme assays were performed as described previously (Trotsenko et al., 1986; Doronina et al., 1995).
DNA was isolated and purified according to Marmur (1961). The DNA G+C content was determined by using the thermal denaturation (Tm) method with a spectrophotometer (DU-8B; Beckman) at a heating rate of 0.5 °C min–1. DNA G+C content was calculated according to Owen & Lapage (1976) using the equation: mol G+C=(Tmx2.08)–106.4. The DNA of Escherichia coli K-12 was used as the standard. DNA–DNA relatedness was defined by DNA reassociation (Johnson, 1985). M. marina ATCC 35842T, M. thalassica ATCC 33146T and M. sulfidovorans LMD 95.210T were used as the reference strains.
The 16S rRNA gene of the novel strain was amplified and sequenced (Lane, 1991). The 16S rRNA gene sequences determined were aligned against the sequences of representative methylobacteria by using the CLUSTAL program. The position of sequence uncertainties was omitted; in total, 1415 nucleotides were used in the analysis. The phylogenetic relationships were determined by the neighbour-joining method and programs from the TREECON software package (Van de Peer & DeWachter, 1994), by maximum-likelihood using the PUZZLE program (Strimmer & von Haeseler, 1996) and by maximum-parsimony with the DNAPARS program from the PHYLIP package (Felsenstein, 1989) with bootstrap analysis of 100 trees. The sequences were compared with representatives of the class Gammaproteobacteria, including recognized methylotrophic species. In preliminary trials, a total of 98 sequences were used and several phylogenetic trees were generated. Phylogenetic analyses that employed different algorithms showed similar results.
Cells of the novel isolate were Gram-negative, non-motile, non-spore-forming rods and 0.2–0.4x0.8–1.0 µm in size. Reproduction occurred by binary fission. No internal complex membrane or capsules were observed. When grown on methanol agar medium, colonies were 0.5–1.5 mm in diameter after 3 days at 30 °C and were opaque, milky and raised with a smooth surface with a round edge.
The novel isolate utilized C1-compounds: methanol, methylamine, dimethylamine, trimethylamine, dimethylsulfide, DMSO and dimethylformamide (Table 1). Fructose was utilized as a multicarbon source. Strain MPT could not utilize other sugars, organic acids, amino acids, C2–C6 alcohols, nutrient or peptone broth supplemented with 3 % (w/v) NaCl at pH 7.0. It did not grow under gas mixtures of CO2/H2/O2 or CH4/O2. Nitrate was reduced to nitrite. Hydrolysis of starch was not observed. No cell aggregation and pigmentation occurred in MSM. The novel isolate was catalase- and oxidase-positive. The isolate grew in the presence of 9 % NaCl (optimum 3 % NaCl), over the pH range of 6.0 to 8.0 and over the temperature range of 25 to 32 °C (optimum pH 7.0, optimum temperature 30 °C). No growth occurred at 45 °C. Strain MPT did not require additional growth factors when grown on methanol and the addition of vitamin B12 or yeast extract did not stimulate growth.
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, the predominant cellular fatty acids of the novel isolate were straight-chain saturated C16 : 0 and unsaturated C16 : 1
7c acids. The presence of 3-hydroxy fatty acids was observed, but 2-hydroxy fatty acids were not observed. The novel isolate had a cellular fatty acid composition of type A and was similar to group 11 of the marine methylobacteria (Urakami & Komagata, 1987). Analysis of the cellular phospholipids revealed the presence of phosphatidylethanolamine and phosphatidylglycerol as the dominant phospholipids and phosphatidic acid, phosphatidylserine and cardiolipin (diphosphatidylglycerol) as minor components. The major isoprenoid ubiquinone is Q-8.
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). Methylamine dehydrogenase PQQ was present in methylamine-grown cells. Formaldehyde and formate dehydrogenases were active with phenazine methosulfate (PMS) while NAD+-dependent activities were absent. Formaldehyde assimilation occurred via the RuMP cycle (Entner–Doudoroff variant), as confirmed by the presence of 3-hexulosephosphate synthase and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenases were active with both NAD+ and NADP+. Rather high levels of these enzymes indicated the preferential oxidation of formaldehyde to CO2 via the dissimilatory hexulosephosphate cycle which provided the methylotroph with the reduced equivalents and energy for biosynthesis. However, we could not rule out the same potential role of the tetrahydromethanopterin (H4MPT)-dependent oxidation pathway in formaldehyde dissimilation in the novel isolate because high activities of methenyl H4MPT cyclohydrolase and NAD(P)-dependent methylene H4MPT dehydrogenases are found in Methylobacillus flagellatus KTT (Vorholt et al., 1999).
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The tricarboxylic acid cycle was deficient in 
-ketoglutarate dehydrogenase activity. The absence of isocitrate lyase and malate synthase activities indicated a non-functional glyoxylate shunt in the novel isolate. Primary ammonia assimilation occurred by reductive amination of -ketoglutarate to glutamate and via the glutamate cycle (GS/GOGAT system).
The DNA G+C content of strain MPT was estimated via Tm as 44.9 mol% (Table 1
). The level of DNA–DNA relatedness between the novel isolate and reference strains of the genus Methylophaga (M. marina ATCC 35842T, M. thalassica ATCC 33146T and M. sulfidovorans LMD 95.210T) did not exceed 25–42 %, consistent with the assignment of the novel strain to a separate species of the genus Methylophaga. According to 16S rRNA gene sequence analysis, strain MPT had gene sequence similarity of 96–98 % with M. marina, M. thalassica and M. sulfidovorans.
In the phylogenetic tree derived from 16S rRNA gene sequences (Fig. 1), strain MPT consistently branched together with the cluster of species within the genus Methylophaga within the class Gammaproteobacteria. The relatively high level of 16S rRNA gene sequence identity (94–98 %) between strain MPT and members of the genus Methylophaga indicated their close relationship. Thus, phenotypic and genotypic data allow the separation of strain MPT as a distinct genospecies of the genus Methylophaga. Consequently, strain MPT is classified as the type strain of a novel species of the genus Methylophaga, Methylophaga aminisulfidivorans sp. nov.
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Cells are Gram-negative rods, 0.8–1.2 µm in length and 0.2–0.4 µm in diameter. Colonies on mineral salts/methanol agar are white to slightly cream and 1–2 mm in diameter. Non-motile, strictly aerobic and does not require vitamin B12. Moderately halophilic. Able to grow at 20–37 °C, at pH 6.0–8.0 on MSM with 1.5–9 % (w/v) NaCl. Optimal growth takes place at 30 °C, pH 6.8–7.0 and 3 % NaCl. Catalase- and oxidase-positive. Reduces nitrate to nitrite. Restricted facultative methylotroph that utilizes methanol, monomethylamine, dimethylamine, trimethylamine, dimethylsulfide, DMSO, dimethylformamide and fructose. Utilizes C1-compounds via the RuMP pathway. Does not grow on peptone-yeast extract medium with or without NaCl. The prevailing cellular fatty acids are C16 : 0 and C16 : 1. The major ubiquinone is Q-8. DNA G+C content is 44.9 mol%.
The type strain, MPT (=KCTC 12909T=VKM B-2441T=JCM 14647T), was isolated from a seawater sample collected from Mokpo, South Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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De Zwart, J. M. M., Nelisse, P. N. & Kuenen, J. G. (1996). Isolation and characterization of Methylophaga sulfidovorans sp. nov.: an obligately methylotrophic, aerobic, dimethylsulfide oxidizing bacterium from a microbial mat. FEMS Microbiol Ecol 20, 261–270.
Doronina, N. V., Braus-Stromeyer, S. A., Leisinger, T. & Trotsenko, Y. A. (1995). Isolation and characterization of a new facultatively methylotrophic bacterium: description of Methylorhabdus multivorans, gen. nov., sp. nov. Syst Appl Microbiol 18, 92–98.
Doronina, N. V., Darmaeva, Ts. D. & Trotsenko, Y. A. (2003a). Methylophaga alcalica sp. nov., a new alkaliphilic and moderately halophilic, obligately methylotrophic bacterium from the East Mongolian saline soda lake. Int J Syst Evol Microbiol 53, 223–229.
Doronina, N. V., Darmaeva, T. D. & Trotsenko, Y. A. (2003b). Methylophaga natronica sp. nov., a new alkaliphilic and moderately halophilic, restricted-facultatively methylotrophic bacterium from soda lake of the Southern Transbaikal region. Syst Appl Microbiol 26, 382–389.[CrossRef][Medline]
Felsenstein, J. (1989). PHYLIP – phylogeny inference package (version 3.2). Cladistics 5, 164–166.
Govorukhina, N. I. & Trotsenko, Y. A. (1989). Phospholipid composition of methylotrophic bacteria. Mikrobiologiya 58, 318–323 (English translation).
Janvier, M., Frehel, C., Grimont, F. & Gasser, F. (1985). Methylophaga marina gen. nov., sp. nov. and Methylophaga thalassica sp. nov., marine methylotrophs. Int J Syst Bacteriol 35, 131–139.
Johnson, J. L. (1985). DNA reassociation and RNA hybridization of bacterial nucleic acids. Methods Microbiol 28, 33–74.
Lane, D. S. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: John Wiley & Sons.
Li, T. D., Doronina, N. V., Ivanova, E. G. & Trotsenko, Y. A. (2007). B12-independent strains of Methylophaga marina from Red Sea algae. Mikrobiologiya 76, 18–23 (English translation).
Marmur, J. A. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Owen, R. J. & Lapage, S. P. (1976). The thermal denaturation of partly purified bacterial deoxyribonucleic acid and its taxonomic applications. J Appl Bacteriol 41, 335–340.[Medline]
Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque strain in electron microscopy. J Cell Biol 17, 208–219.
Strimmer, K. & von Haeseler, A. (1996). Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies. Mol Biol Evol 13, 964–969.
Trotsenko, Y. A., Doronina, N. V. & Govorukhina, N. I. (1986). Metabolism of non-motile obligately methylotrophic bacteria. FEMS Microbiol Lett 3, 293–297.
Urakami, T. & Komagata, K. (1987). Characterization of species of marine methylotrophs of the genus Methylophaga. Int J Syst Bacteriol 37, 402–406.
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
Vorholt, J. A., Chistoserdova, L., Stolyar, S. M., Thauer, R. K. & Lidstrom, M. E. (1999). Distribution of tetrahydromethanopterin-dependent enzymes in methylotrophic bacteria and phylogeny of methenyltetrahydromethanopterine cyclohydrolases. J Bacteriol 181, 5750–5757.
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