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1 Department of Microbiology, University of Washington, Seattle, WA 98195, USA
2 Department of Chemical Engineering, University of Washington, Seattle, WA 98195, USA
3 Omak High School, Omak, WA 98841, USA
Correspondence
Ludmila Chistoserdova
milachis{at}u.washington.edu
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7c, C19 : 0 cyclo and C16 : 0. The dominant cellular phospholipids are phosphatidyl acid, phosphatidylcholine and phosphatidylethanolamine. The G+C content of the DNA is 65·7±0·3 mol%. 16S rRNA gene-based phylogenetic analysis revealed that the novel isolate belongs to the 
-Proteobacteria and is closely related to the only representative of the genus Labrys, Labrys monachus (97·4 % sequence similarity). However, the level of DNA–DNA relatedness with L. monachus is less than 3 %, justifying the placement of this isolate into a novel species of the genus Labrys. The name Labrys methylaminiphilus sp. nov. is proposed (type strain JLW10T=ATCC BAA-1080T=DSM 16812T).
Published online ahead of print on 14 January 2005 as DOI 10.1099/ijs.0.63409-0.
The GenBank/EMBL/DDBJ accession number for the nearly complete sequence of the 16S rRNA gene of Labrys methylaminiphilus JLW10T is AY766152.
Present address: Department of Microbiology, University of Washington, Seattle, WA 98195, USA. 
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). Methylotrophs play an important role in biogeochemical cycling and possess a potential for use in bioremediation (Pol et al., 1994; De Marco et al., 2004). We are studying a specific habitat that is rich in methylotrophic activity: the sediment of Lake Washington at a depth of about 60 m (Kuivila et al., 1988; Costello & Lidstrom, 1999). Until recently, research at this site has focused primarily on methanotroph populations (Auman et al., 2000; Costello & Lidstrom, 1999) and no survey of broader methylotroph presence has been conducted. This study focused on enrichment, isolation and taxonomic description of methylamine-utilizing bacteria from Lake Washington sediment. One of these isolates, a novel facultative methylotroph, is described.
Sediment samples were collected on 21 July 2003, off the RV Clifford Barnes from a 63 m-deep site in Lake Washington, Seattle, WA, USA (47° 38·075' N 122° 15·993' W) using a box core that allowed collection of undisturbed sediment. Sediment cores were taken to a depth of approximately 20 cm and subsectioned into layers approximately 1·5 cm thick. Samples were transported to the laboratory on ice and used immediately. One millilitre samples of the upper 1·0 cm of the sediment cores were inoculated into 250 ml flasks containing 50 ml 0·2x salts medium (Harder et al., 1973) supplemented with 0·01 % methylamine (w/v) and were incubated for 6–8 days at room temperature with shaking (125 r.p.m.). In subsequent enrichments, 10 ml of the previous enrichment culture were diluted 1 : 4 (to a total of 50 ml) in the same medium, with the methylamine concentration increased to 0·2 %. Flasks were incubated at room temperature with shaking for 3–5 days. Strain JLW10T was obtained as a colony after the third enrichment culture was plated onto a solid medium (0·2x salts medium solidified by 1·8 % Bacto agar, supplemented with 0·2 % methylamine) and incubated at room temperature for 1 week. The isolate was further purified by triplicate streaking on solid medium. The purity of the culture was monitored by microscopy and by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, obtained by PCR amplification of DNA from either a single colony or from 5 ml of liquid culture as described below. For routine cultivation, a methylamine-supplemented minimal medium (Harder et al., 1973) was used. In addition, the following media were used: MMB medium (DSMZ, medium 628), TGY broth (Murray, 1992) and Luria–Bertani medium (Sambrook et al., 1989). For long-term storage, 200 µl DMSO was added to 1·8 ml exponentially growing liquid culture and the mixed suspensions were stored at –80 °C. Labrys monachus DSM 5896T, obtained from the DSMZ, was used as a reference strain.
For scanning electron microscopy, cells were washed with double-distilled water and fixed in modified Karnovsky's fixative (2 % paraformaldehyde, 2·5 % glutaraldehyde, 8 mM CaCl2 in 0·1 M cacodylate buffer, pH 7·4) for 2 h at 4 °C. Cells were then washed three times for 5 min with double-distilled water, spotted onto plastic coverslips coated with 1 % poly-L-lysine, dehydrated in a graded series of ethanol to 100 % and subsequently critical-point-dried. Samples were sputter-coated with gold/palladium and viewed with a JOEL, JSM 6300F scanning electron microscope at 15 kV. Cells of isolate JLW10T were Gram-negative, non-motile, capsulated, rod-shaped bacteria, 0·7–1·0 µm wide and 1·0–1·2 µm long (Fig. 1). They appeared as single cells or in pairs. Cells reproduced by budding and did not appear to form cysts or other resting bodies. Capsule formation, observed by electron microscopy, was also visualized with India ink (Pelikan) by light microscopy.
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Nitrogen sources were tested in nitrogen-free basal liquid medium (as above) in which (NH4)2SO4 was replaced with K2SO4. The following compounds were tested at a concentration of 0·1 % (w/v): KNO3, methylamine, dimethylamine, trimethylamine, urea, guanidine, glycine, serine, valine, alanine, cysteine, asparagine, arginine, aspartate, methionine, histidine, threonine, proline, glutamate, phenylalanine, tryptophan, peptone and yeast extract. For N2 fixation experiments, the same nitrogen-free basal liquid medium was used. Temperature optima were investigated in TGY medium. pH optima were investigated in the minimal salts medium supplemented with 0·1 % methylamine. Due to the poor growth exhibited by the control strain L. monachus on methylamine, growth medium for L. monachus was supplemented by 10 % TGY. NaH2PO4/Na2HPO4 at a final total concentration of 0·05 M was used as the buffering system (pH range 4·0–9·6). Culture pH was monitored at the completion of each experiment, to ensure that no shifts in pH had occurred. Growth rates were determined by monitoring cell density at 600 nm using a plate reader (Bioscreen C MBR) or in bulk cultures (250 ml flasks). Specific growth rates were calculated as the mean of triplicate determinations. The optimal concentration of methylamine for growth was tested using the plate reader at the following concentrations of methylamine (w/v): 0·0005, 0·001, 0·005, 0·01, 0·05, 0·1, 0·5, 1 and 2 %. Desiccation resistance, Voges–Proskauer reaction, oxidation/fermentation catabolism test, nitrite reduction, fluorescent pigment production, urease production, indole production and gelatin hydrolysis were tested according to Smibert & Krieg (1994). Plate assays were conducted to test ability to degrade agarose and starch. Tests for cellulose and nitrocellulose degradation were conducted in liquid media, using sterile 1 cm strips of cellulose or nitrocellulose.
Colonies of isolate JLW10T grown at 30 °C for 3 days on a basal medium supplemented with methylamine were white, 1–2 mm in diameter, circular, convex, opaque and butyrous. Specific growth rates in liquid determined using the Bioscreen plate reader were 0·067 h–1 in minimal medium supplemented with methylamine (0·2 %) and 0·24 h–1 in TGY medium. Comparable rates were obtained in flask experiments (data not shown). Isolate JLW10T grew in the temperature range 10–35 °C, with an optimum at 28–30 °C. The isolate was resistant to desiccation under the conditions tested. Isolate JLW10T grew over a pH range of 4·0–9·5, with an optimum at pH 5·0–7·0. The optimal concentration of methylamine for growth of JLW10T was 0·05 % and concentrations above 0·5 % were toxic. While isolate JLW10T did not require additional growth factors when grown on methylamine, the addition of yeast extract did stimulate growth. In addition to methylamine, the following substrates supported growth of isolate JLW10T as sole sources of carbon and energy: dimethylamine, trimethylamine, methylsuccinic acid, choline, betaine, fructose, glucose, maltose, lactose, mannose, sucrose, sarcosine, succinate, pyruvate, malate, citrate, oxalate, acetate, mannitol, ethanol, acetone, glycerol and all the tested amino acids, with the exceptions of tryptophan and methionine. No growth occurred on methane, methanol, glycolic acid, vanillin, toluene, naphthalene, formaldehyde, DMSO or trichloroethylene. In addition, methylamine, dimethylamine, trimethylamine, urea, ammonium, nitrate, and all tested amino acids served as sole nitrogen sources for growth. Isolate JLW10T did not grow without an added nitrogen source. Accordingly, no product was amplified with primers specific for the nifH gene (Zehr & McReynolds, 1989), which encodes the Fe-protein of nitrogenase, suggesting that the strain is unable to utilize N2. The isolate hydrolysed agar, agarose, humic acids and cellulose, but not starch or nitrocellulose. Indole and Voges–Proskauer tests were negative. No water-soluble fluorescent pigment was produced on King B medium. Tests for oxidase and catalase were positive. Urease activity was absent. Tests for oxidation of carbohydrates were positive. Glucose was fermented, but not sucrose or lactose. No gas production was observed.
Sensitivity to antibiotics was examined by spreading cells onto TGY agar plates and placing onto them Difco discs that contained the following antibiotics (µg ml–1): gentamicin (10), neomycin (30), streptomycin (10), ampicillin (10), chloramphenicol (30), erythromycin (15), kanamycin (30), nalidixic acid (30), penicillin (10) and tetracycline (30). The effect of antibiotics on cell growth was assessed after 2 weeks. Cells were resistant to kanamycin, ampicillin, erythromycin, nalidixic acid, tetracycline, chloramphenicol and penicillin, but were sensitive to streptomycin, neomycin and gentamicin.
For cell extract preparation, methylamine-grown cells were pelleted by centrifugation at 5000 r.p.m. at 4 °C, resuspended in 1 ml buffer (25 mM Tris/HCl, 10 mM EDTA, pH 7·2 or 25 mM KH2PO4/Na2HPO4, pH 7·2) and disrupted by passage through a French pressure cell at 1·2x108 Pa. Cell extracts were centrifuged at 20 817 g for 25 min at 4 °C to remove cell debris. Hydroxypyruvate reductase and serine glyoxylate aminotransferase were assayed as described by Goodwin (1990), hexulosephosphate synthase was assayed as described by Shishkina et al. (1976) and phosphoribulokinase and ribulose bisphosphate carboxylase/oxygenase were assayed as described by Tabita (1980). Methylenetetrahydrofolate dehydrogenase (MH4FDH) was assayed as described by Vorholt et al. (1998). Methanol dehydrogenase and methylamine dehydrogenase were assayed according to Anthony & Zatman (1965) and Eady & Large (1968), respectively. N-Methylglutamate synthase/lyase was assayed according to Kimura et al. (1995), with a modification as follows. Formaldehyde formation was followed by coupling to NADPH production in the presence of MH4FDH. The modified reaction mixture contained the following additional compounds: 0·1 mM NADP, 3 mM tetrahydrofolate and 1 U MH4FDH (all from Sigma). The activity of formate dehydrogenase was measured in the following reaction mixture: 50 mM potassium phosphate buffer, pH 7·0, 1 mM NAD and 20 mM formate. The activity of formaldehyde dehydrogenase was measured in the following reaction mixture: 50 mM potassium phosphate buffer, pH 7·0, 1 mM NAD, 10 mM formaldehyde and 2 mM glutathione. Protein concentration was determined spectrophotometrically (Dawson et al., 2002). The data on enzyme activity measurements are presented in Table 1. No activity was detected for methanol dehydrogenase. Accordingly, PCR amplification of the mxaF gene with specific primers (McDonald & Murrell, 1997) produced negative results. No methylamine dehydrogenase activity was measured; however, cell extracts possessed N-methylglutamate synthase/lyase activity, the key enzyme for an alternative pathway for methylamine oxidation, the N-methylglutamate pathway (Table 1). No activities were detected for key enzymes of either the ribulose monophosphate cycle for formaldehyde assimilation or for the Calvin–Benson–Bassham cycle, but activities of key enzymes of the serine cycle were present (Table 1). High activity of MH4FDH was present, indicating that the tetrahydrofolate-linked pathway may be involved in formaldehyde oxidation/detoxification. One of the most common formaldehyde oxidation pathways in methylotrophs is an analogous pathway linked to tetrahydromethanopterin, the so-called ‘archaeal’ pathway (Vorholt et al., 1999). We were not able to test for any enzyme activities indicative of this pathway, as tetrahydromethanopterin or its derivatives are not commercially available. However, we were able by PCR to amplify a key gene for this pathway, fae, encoding the formaldehyde activating enzyme (Vorholt et al., 2000). NAD-linked, glutathione-dependent formaldehyde dehydrogenase and formate dehydrogenase activities were also present.
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). Extractions were performed using one-phase chloroform/methanol/buffer extractant. Lipids were recovered, dissolved in chloroform and fractionated on disposable silicic acid columns into neutral, glyco- and polar lipid fractions. The polar lipid fraction was trans-esterified with mild alkali to recover the PLFA as methyl esters in hexane. The PLFAs were analysed by gas chromatography with peak confirmation performed by electron impact mass spectrometry (GC/MS). The phospholipids were separated by TLC on Kieselgel 60 F254 plates (Merck) using chloroform/methanol/7 M ammonium hydroxide/water (65 : 25 : 3 : 1) as a solvent system. Iodine stain (Dittmer & Wells, 1969), Dragendorff spray, ninhydrin reagent (Daniels et al., 1994) and phosphomolybdic acid staining were used for phospholipid identification. Fatty acids found in the polar lipid fraction of isolate JLW10T are listed in Table 2, together with fatty acids reported in the literature for two reference strains representative of budding bacteria (Labrys and Angulomicrobium) and four reference strains representative of non-pigmented facultatively methylotrophic bacteria (Methylopila, Aminobacter, Methylarcula and Methylorhabdus). The major components of the hydrolysate obtained from methylamine-grown cells of isolate JLW10T were C18 : 17c, C19 : 0 cyclo and C16 : 0. The major phospholipids were phosphatidyl acid, phosphatidylcholine and phosphatidylethanolamine.
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). The G+C content was determined by HPLC separation as described in Tamaoka & Komagata (1984) and was found to be 65·7±0·3 mol% (n=3). DNA–DNA hybridization experiments were performed using the competition procedure described by Johnson (1994). DNA (1 µg) from isolate JLW10T was labelled with [3H]dCTP (Amersham) using a nick translation kit (Roche). Competitor DNA was digested using a combination of PstI and HindIII (NEB). DNA extracted from L. monachus and JLW10T (1 mg) was immobilized onto 0·22 µm Nytran N membranes (Schleicher & Schuell) and hybridized with the labelled JLW10T DNA (specific activity 5x106 d.p.m. mg–1), as described by Sambrook et al. (1989). After hybridization, filters were counted in a liquid scintillation counter (Beckman) and the percentage of hybridization was calculated as described by Johnson (1994). Three independent tests were carried out for each pair of DNA preparations. These experiments showed that isolate JLW10T and L. monachus shared only a low level of DNA–DNA relatedness (<3 %).
The 16S rRNA gene fragment was amplified as described by Lane (1991) and cloned into the pCR 2.1 vector using the Topo-TA Cloning kit (Invitrogen). DNAs from 30 clones were digested with AluI (NEB) and subjected to RFLP analysis, resolved in 2 % agarose gels. Two different RLFP patterns were observed with a frequency of approximately 50 % each. Representatives of each pattern were sequenced. DNA sequencing was carried out using the BigDye 3.1 termination sequencing kit (Applied Biosystems). Gel analyses were performed by the Department of Biochemistry Sequencing Facility at the University of Washington, using an ABI 3700 high-throughput capillary DNA analyser. Plasmids representing the two different RFLP patterns contained inserts of identical DNA, indicating that the two RFLP patterns resulted from two different orientations of the fragment within the cloning vector. For phylogenetic analyses, this sequence was aligned with reference sequences using the CLUSTAL W program (Thompson et al., 1994) and the alignments were manually refined. The PHYLIP package (Felsenstein, 2003) was used to perform neighbour-joining and parsimony analyses, with 1000 bootstrap analyses performed for each method. The two analyses resulted in similar branching patterns. Based on these analyses, the novel isolate fell into the -subclass of Proteobacteria and was most closely related to L. monachus (97·4 % similarity; Fig. 2). Sequences from both isolate JLW10T and L. monachus exhibited 86–94 % similarity to those of other known members of the -subclass of Proteobacteria, and both were most closely related to the genera Methylosinus and Methylocystis.
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-, 
- and 
-subclasses of Proteobacteria. Within the -subclass, at least 12 genera are classified that include well-characterized methylotrophic species: Hyphomicrobium, Methylobacterium, Aminobacter, Methylorhabdus, Methylarcula, Methylopila, Xanthobacter, Paracoccus, Rhodopseudomonas, ‘Methylosulfonomonas’, Ancylobacter and Albibacter. However, the ability to grow on a variety of reduced single-carbon (C1) substrates is even more widespread, since some bacteria capable of growth on C1 compounds have never been classified as methylotrophs. For example, C1 growth has been reported for bacteria involved in polymer transformation, such as Sagittula stellata (Gonzalez et al., 1997), or oligotrophic budding bacteria such as Angulomicrobium (Fritz et al., 2004).
Isolate JLW10T, as described here, is another representative of heterotrophic bacteria with a broad range of metabolic capacities. Even though originally isolated as a dominant culture in methylamine enrichments, this bacterium is capable of growth on a variety of organic substrates, including polymers such as agarose and humic acids. Of all characterized bacteria, isolate JLW10T is most related to L. monachus (Vasil'eva & Semenov, 1984; Fritz et al., 2004). Based on genotypic analysis, both bacteria are clearly separated from all known taxa and form a distinct branch within the -subclass of Proteobacteria. However, the low DNA–DNA relatedness and the unique fatty acid profile of isolate JLW10T separate it from L. monachus. The dominance of C19 : 0 cyclo fatty acid (49·4 %) is a distinctive feature of this isolate that will potentially become a signature for this bacterium at the species level. Based on comparative analysis of JLW10T with other representative genera and species, we propose that isolate JLW10T represents a novel species within the genus Labrys. The species name proposed for isolate JLW10T, Labrys methylaminiphilus, reflects the propensity of this strain to grow on methylamine and the history of its enrichment and isolation.
Description of Labrys methylaminiphilus sp. nov.
Labrys methylaminiphilus (me.thy'la.mi.ni.phi'lus. N.L. n. methyl the methyl radical; N.L. n. amini the amine group; Gr. adj. philos loving; N.L. masc. adj. methylaminiphilus methylamine-loving, referring to the methylamine-utilizing activity of the bacterium).
Cells are Gram-negative, non-motile, non-spore-forming, capsulated, multiply by budding and are oxidase- and catalase-positive. Growth is aerobic but may be microaerophilic. Grows on methylamine, dimethylamine and trimethylamine as sole sources of energy and carbon. Methylamine is oxidized to formaldehyde via the N-MG pathway. Carbon is assimilated via the serine cycle. Other substrates supporting growth include a wide range of sugars, organic acids, amino acids, aromatic compounds and alcohols. Nitrogen sources include methylated amines, most amino acids, urea, peptone, ammonium salts and nitrates. No additional growth factors are required, but yeast extract has a stimulatory effect. The major components of the fatty acid profile are C18 : 17c, C19 : 0 cyclo and C16 : 0. The major phospholipids are phosphatidyl acid, phosphatidylcholine and phosphatidylethanolamine. The G+C content of the DNA is 65·7±0·3 mol%. Cells are 0·7–1·0 µm wide and 1·0–1·2 µm long. The temperature range for growth is 10–35 °C, with optimal growth at 28–30 °C. The pH range is 4·0–9·5, with optimal pH at 5·0–7·0. Does not produce indole and the Voges–Proskauer test is negative. No water-soluble fluorescent pigments are produced on King B medium. Oxidase and catalase activities are present, but not urease activity.
The type strain, JLW10T (=ATCC BAA-1080T=DSM 16812T), was isolated from the sediment of Lake Washington, Seattle, WA, USA.
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ACKNOWLEDGEMENTS |
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M. S. Islam, H. Kawasaki, Y. Nakagawa, T. Hattori, and T. Seki Labrys okinawensis sp. nov. and Labrys miyagiensis sp. nov., budding bacteria isolated from rhizosphere habitats in Japan, and emended descriptions of the genus Labrys and Labrys monachus Int J Syst Evol Microbiol, March 1, 2007; 57(3): 552 - 557. [Abstract] [Full Text] [PDF]
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Y.-J. Chou, G. N. Elliott, E. K. James, K.-Y. Lin, J.-H. Chou, S.-Y. Sheu, D.-S. Sheu, J. I. Sprent, and W.-M. Chen Labrys neptuniae sp. nov., isolated from root nodules of the aquatic legume Neptunia oleracea Int J Syst Evol Microbiol, March 1, 2007; 57(3): 577 - 581. [Abstract] [Full Text] [PDF]
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M. G. Kalyuzhnaya, S. Bowerman, J. C. Lara, M. E. Lidstrom, and L. Chistoserdova Methylotenera mobilis gen. nov., sp. nov., an obligately methylamine-utilizing bacterium within the family Methylophilaceae Int J Syst Evol Microbiol, December 1, 2006; 56(12): 2819 - 2823. [Abstract] [Full Text] [PDF]
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M. G. Kalyuzhnaya, P. De Marco, S. Bowerman, C. C. Pacheco, J. C. Lara, M. E. Lidstrom, and L. Chistoserdova Methyloversatilis universalis gen. nov., sp. nov., a novel taxon within the Betaproteobacteria represented by three methylotrophic isolates. Int J Syst Evol Microbiol, November 1, 2006; 56(Pt 11): 2517 - 2522. [Abstract] [Full Text] [PDF]
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P. Kampfer, C.-C. Young, A. B. Arun, F.-T. Shen, U. Jackel, R. Rossello-Mora, W.-A. Lai, and P. D. Rekha Pseudolabrys taiwanensis gen. nov., sp. nov., an alphaproteobacterium isolated from soil. Int J Syst Evol Microbiol, October 1, 2006; 56(Pt 10): 2469 - 2472. [Abstract] [Full Text] [PDF]
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