Methylobacillus arboreus sp. nov., and Methylobacillus gramineus sp. nov., novel non-pigmented obligately methylotrophic bacteria associated with plants

Systematic and Applied Microbiology 34 (2011) 477–481

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Methylobacillus arboreus sp. nov., and Methylobacillus gramineus sp. nov., novel non-pigmented obligately methylotrophic bacteria associated with plants Anna A. Gogleva a,b , Elena N. Kaparullina a,b , Nina V. Doronina a,b , Yuri A. Trotsenko a,b,∗ a b

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia Pushchino State University, Pushchino, Moscow Region, 142290, Russia

a r t i c l e

i n f o

Article history: Received 19 July 2010 Received in revised form 24 February 2011 Accepted 5 March 2011 Keywords: Methylotrophy Methylobacillus Rump

a b s t r a c t Two newly isolated obligate methanol-utilizing bacteria (strains IvaT and LapT ) with the ribulose monophosphate pathway of C1 assimilation are described. The isolates are strictly aerobic, Gram negative, asporogenous, motile rods multiplying by binary fission, mesophilic and neutrophilic, synthesize indole-3-acetate. The prevailing cellular fatty acids are straight-chain saturated C16:0 and unsaturated C16:1 acids. The major ubiquinone is Q-8. The predominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Ammonia is assimilated by glutamate dehydrogenase. The DNA G+C contents of strains IvaT and LapT are 54.0 and 50.5 mol% (Tm ), respectively. Based on 16S rRNA gene sequence analysis and DNA–DNA relatedness (38–45%) with type strains of the genus Methylobacillus, the novel isolates are classified as the new species of this genus and named Methylobacillus arboreus IvaT (VKM B-2590T , CCUG 59684T , DSM 23628T ) and Methylobacillus gramineus LapT (VKM B-2591T , CCUG 59687T , DSM 23629T ). The GenBank accession numbers for the 16S rRNA gene and mxaF gene sequences of the strains IvaT and LapT are GU937479, GU937478 and HM030736, HM030735, respectively. © 2011 Elsevier GmbH. All rights reserved.

To date, the group of validly published names of obligate and restricted facultative methylotrophic methanol utilizing bacteria with the ribulose monophosphate (RuMP) pathway of formaldehyde assimilation comprises four genera. Terrestrial obligate and restricted facultative methylobacteria assigned to the genera Methylobacillus [22,24], Methylophilus [11] and Methylovorus [6] are clearly distinguished from moderately halophilic the genus Methylophaga [21] by some physiological characteristics and DNA G+C content. Because of similar morphology and metabolic organization the main taxonomic criteria to classify obligate and restricted facultative methylobacteria are their genomic and phylogenetic characteristics. The genus Methylobacillus is presented by the three species M. glycogenes [22], M. flagellatus [8] and M. pratensis [5], utilizing methanol or methylamine as a sole source of carbon and energy. Here, we describe two newly isolated methylotrophic strains IvaT and LapT having the RuMP pathway for which the names

Abbreviations: KDPG, 2-keto-3-deoxy-6-phosphogluconate; MDH, methanol dehydrogenase; PQQ, pyrroloquinoline quinone; RuMP, ribulose monophosphate pathway; PMS, phenazine methosulfate. ∗ Corresponding author at: G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia. Tel.: +7 495 925 7448; fax: +7 495 956 3370. E-mail addresses: [email protected], [email protected] (Y.A. Trotsenko). 0723-2020/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2011.03.005

Methylobacillus arboreus and Methylobacillus gramineus spp. nov. are proposed. Strains IvaT and LapT were isolated from willow buds (Salix fragilis L.) and phyllosphere of silverweed (Potentilla anserina L.), respectively, sampled from the city park in Pushchino (Moscow Region, Russia) and grown on liquid mineral medium “K” containing g l−1 : KH2 PO4 – 2.0; (NH4 )2 SO4 – 2.0; MgSO4 ·7H2 O – 0.025; NaCl – 0.5; FeSO4 ·7H2 O – 0.002, pH 7.2, and 0.5% (v/v) CH3 OH. Solidified medium “K” was prepared by adding 2.0% (w/v) agar Difco (USA). Representative species of the genus Methylobacillus (M. glycogenes ATCC 29475T , M. flagellatus DSM 6875T , M. pratensis NCIMB 13994T ) were used as the reference strains. Cell morphology, Gram staining, motility and flagellation were studied for the isolates IvaT and LapT grown on “K” agar medium with methanol (0.5%, v/v). Negatively stained preparations and thin sections were imaged in JEOL JEM-100B transmission electron microscope (Japan) at operating voltages of 60 and 80 kV, respectively [10]. Biochemical tests were carried out by using API 20NE and API 20E strips (Biomerieux, France) following the manufacturer’s instructions. Indole production from 1 mM l-tryptophan was determined with the Salkowski reagent (0.05 M FeCl3 in 35% HClO4 ) on the medium “K”, in which (NH4 )2 SO4 was replaced with 0.5% (w/v) KNO3 [7]. The concentration of auxins was determined from the calibration curve constructed using standard indole-3-acetic acid solutions. The indole compounds from culture medium of the new isolates were identified by TLC and HPLC primarily as indole-3-acetic acid

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[3]. Additionally, the auxin structure was confirmed by the MS spectra (Finnigan MAT 8430, Germany). Halotolerance was tested by inoculating the cells into liquid K medium with various concentrations of NaCl (0–3%). Growth at different temperatures (4–43 ◦ C), pH values and various concentrations of methanol (0.5–5%, v/v) were tested in a liquid medium “K”. Utilization of a wide range of growth substrates was determined after cultivation on “K” medium for 2 weeks with methanol replaced by other carbon compounds (more than 65 were tested). Carbohydrates, organic acids, aminoacids and methylated amines were added at concentration of 0.05–0.3% (w/v), while alcohols were added at concentration of 0.2–0.5% (v/v). To test alternative nitrogen sources, (NH4 )2 SO4 was replaced by other nitrogen compounds (1%, w/v). Methane utilization was tested under atmosphere containing methane and air (1:1, v/v) in flasks (700 ml) fitted with rubber stoppers containing 100 ml of mineral K medium. Utilization of dichlormethane was tested as described earlier [2]. Enzyme assays of primary and intermediary pathways of C1 metabolism were done as described Trotsenko et al. [19]. Sensitivity to antibiotics was examined by placing Difco discs on cells spread on agar plates. The discs contained the following antibiotics (␮g): gentamicin (10), neomycin (30), streptomycin (10), ampicillin (10), nalidixic acid (30), lincomycin (2), oxacillin (10), neomycin (30), novobiocin (5). The effect of antibiotics on cell growth was assessed after 2 days. For the fatty acids determination the cells were grown on solidified medium “K” with methanol (0.5%, v/v) at 29 ◦ C for 30 h. Fatty acid extraction from 40 to 60 mg cells was carried out by the use of the whole biomass acid methanolysis in 1.2 N of 0.4 ml HCl in methanol by heating to 80 ◦ C for 1 h. Resulting fatty acid methyl esters were extracted twice with 0.2 ml hexane and processed on Agillent Technologies AT-5850/5973 GC–MS system with a cross-linked methyl silicone capillary column HP-5ms. The oven temperature was programmed from 140 to 320 ◦ C at 7 grad/min. 1–2 mkl of derivatised sample were injected in gas chromatograph at 280 ◦ C. Fatty acids and other lipid components were ionised by electron impact at 70 eV after separation in GC column and analysed in the scan mode. The quadrupole mass spectrometer has a resolution of 0.5 mass units over the whole mass range of 2–950 amu. The sensitivity of GC–MS system is 0.01 ng of methyl stearate. Each substance was confirmed by its mass-spectrum and NIST mass spectral database library search. Polar lipids and ubiquinones were determined as described previously [4]. DNA was isolated and purified according to Marmur [14]. The DNA G+C content was determined by using the thermal denaturating (Tm ) method with a Beckman DU-8B spectrophotometer (USA) at a heating rate of 0.5 ◦ C min−1 and calculation was according to Owen and Lapage [16] using the equation: mol G+C = (Tm × 2.08) − 106.4. The DNA of Escherichia coli K-12 was used as the standard. DNA–DNA hybridization experiments were performed using the procedure described by Johnson [12]. PCR amplification and sequencing of 16S rRNA gene was carried out as previously described [13]. The preliminary screening for similarity was done with BLAST (http://www.ncbi.nlm.nih.gov/blast). Then the determined 16S rRNA gene sequence was aligned against those of closely related strains obtained from the recent GenBank releases using the CLUSTALW software [18]. Positions of the sequence uncertainties were omitted and a total of 1397 and 1406 nucleotides (strains IvaT and LapT , respectively) were used in the analysis. Phylogenetic relationships were determined by the neighbour joining method and the programs of the TREECON package (version 1.3b) [23], with bootstrap analysis of 100 trees. Phylogenetic trees were also constructed by maximum parsimony, minimum evolution and UPGMA methods using the software packages MEGA (Molecular Evolutionary Genetics Analyses) version 4 [17]. Sequence similarity matrix was determined using BioEdit 7.0.9.0 [9]. The 16S rRNA gene

Table 1 Cellular fatty acid composition of strains IvaT and LapT and members of the genus Methylobacillus. Fatty acid Straight-chain acids C14:0 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 Cyclopropane acid C17:0 Hydroxy acid 3-OH C10:0

1

2

3

4

5

0.6 0.4 39.2 49.7 0.6 0.4 7.7

0.5 0.4 40.6 53.2 0.4 1.0 2.9

1.1 0.9 41.6 46.6 0.7 1.2 1.3

1.1 0.5 43.4 42.5 0.5 0.8 4.6

1.0 0.6 41.4 44.6 0.5 0.7 4.0

0.6

0.4

1.4

1.3

2.1

0.8

0.6

5.2

5.3

5.1

Strain: 1, Methylobacillus arboreus IvaT ; 2, Methylobacillus gramineus LapT ; 3, Methylobacillus pratensis F31T ; 4, Methylobacillus glycogenes ATCC 29475T ; 5, Methylobacillus flagellatus DSM 6875T . Results are given as a percentage of total fatty acids. All data were obtained from this study.

sequences of strains IvaT and LapT were submitted to the GenBank under accession numbers GU937479 and GU937478, correspondingly. The mxaF gene encodes the large subunit of the classical pyrroloquinoline quinone (PQQ)-linked methanol dehydrogenase (MDH), which catalyzes the oxidation of methanol to formaldehyde in majority of extant Gram-negative methylotrophic bacteria [1]. Using primers f1003 and r1561 and the protocol developed by McDonald and Murrell [15] we were able to amplify an approximately 550-bp mxaF gene fragment from DNA of strains IvaT and LapT . The mxaF gene amplicons were purified using Promega columns (USA) and they were sequenced on an automatic sequencer CEQ2000 XL (Beckman Coulter, USA) using the enzyme CEQ Dye Terminator Cycle Sequencing kit (Beckman Coulter, USA). Processing and translation of nucleotide sequences to the amino acid sequences were performed using Gene Runner (version 3.05) (Hastings Software, Inc.). Then the determined MxaF sequence was aligned using the CLUSTALW software. Phylogenetic analyses were carried out using the TREECON programs of the package with bootstrap analysis of 100 trees [23]. Tree based on mxaF amino acid sequences showing the phylogenetic position of the new strains among methylotrophic bacteria were constructed by using the neighbour joining method implemented by the TREECON software package (version 1.3b). The mxaF sequences of strains IvaT and LapT were submitted to the GenBank under accession numbers HM030736 and HM030735, respectively. Cells of strains IvaT and LapT are Gram-negative (Figs. S1 and S2), asporogenous, motile rods (0.30–0.35 × 1.50–1.52 ␮m; 0.50–0.55 × 1.10–1.20 ␮m, correspondingly) that occur either singly or in pairs or in small groups. Reproduction occurs by binary fission. Strictly aerobic, catalase and oxidase positive, methylotrophic bacteria, utilizing only methanol as a growth substrate, but not methylamine, dimethylamine, trimethylamine, formate, dichloromethane, methane, organic acids, sugars, aminoacids or C2 –C6 alcohols. No growth occurred on TGY, LB or nutrient media. Test (API strips) for nitrate reduction, urease activity, H2 S production, gelatin hydrolysis were negative, but it is for ␤-galactosidase activity and acetoin production were positive. Ammonia and nitrate were used as nitrogen sources. As shown in Table 1 (our results), the predominant cellular fatty acids of strains IvaT and LapT are unsaturated C16:1 ␻7 acid (49.66% and 53.21%) and straight-chain saturated C16:0 acid (39.16% and 40.58%). The presence of 3-hydroxy fatty acids was observed, but not 2-hydroxy fatty acids were found. The data indicate a considerable similarity in the fatty acids composition of strains IvaT and LapT and the type cultures of the genus Methylobacillus, i.e.

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Methylobacillus glycogens ATCC 29475T , M. flagellatus DSM 6875T , M. pratensis NCIMB 13994T . The major ubiquinone was Q8. Analysis of the cellular phospholipids revealed the presence of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and minor amounts of phosphatidic acid. Phosphatidylcholine was not found. The metabolic linkage of the new methanol-utilizing strains was studied with plants because they were isolated from plants. Quantitative analysis of the spend medium of strains IvaT and LapT grown on K medium with l-tryptophan showed that the amount of synthesized IAA varied from 4.4 to 15 ␮g ml−1 . The methanol is a common volatile metabolite of plants, due to which methylotrophs are ubiquitous in plant phyllosphere and rhizosphere. Methylotrophic bacteria not merely colonize plants but are symbiotically related to them [20]. Our recent studies showed that many aerobic methylotrophic bacteria belonging to genus Methylobacillus are able to synthesize auxins – the plant hormones [3]. One of the most important auxins is indole-3-acetic acid, which is synthesized from tryptophan. In plant tissue cultures, auxins promote cell differentiation and induce the formation of roots. The enzymic profiles of methanol-grown cells indicate that strains IvaT and LapT oxidize methanol to formaldehyde by means of pyrroloquinoline quinone (PQQ)-linked methanol dehydrogenase (Table 2). Two formaldehyde oxidizing enzymes were present, i.e. NAD+ -dependent formaldehyde dehydrogenase and a PMS-linked one. Also, PMS- and NAD+ -linked formate dehydrogenases were detected in both strains. Thus, the strains IvaT and LapT appear to possess the complete set of the enzymes involved in methanol oxidation to CO2 through formaldehyde and formate that provides metabolic energy for methylotrophic growth. Both strains assimilate formaldehyde via the RuMP cycle (Entner-Doudoroff variant), as confirmed by the presence of 3-hexulose phosphate synthase and 2-keto-3-deoxy-6phosphogluconate aldolase. Glucose-6-phosphate dehydrogenase was active with both NAD+ and NADP+ . Rather high levels of these enzymes indicate the preferential oxidation of formaldehyde to CO2 via the dissimilatory hexulose phosphate cycle, which provides the methylobacteria studied with the reduced equivalents and energy for biosynthesis. The absence of the serine pathway-specific enzymes (hydroxypyruvate reductase and serine-glyoxylate transaminase) means that its operation can be excluded in the case of the methylotrophs studied. The tricarboxylic acid cycle is deficient in ␣-ketoglutarate

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Table 2 Enzyme activities (nmol min−1 mg−1 protein) in cell extracts of strains IvaT and LapT grown on methanol. Enzyme

Cofactor

Strain IvaT

Strain LapT

Methanol dehydrogenase Formaldehyde dehydrogenase

PMS PMS NAD+ PMS NAD+

153 155 158 62 61 340 0 0 768 536 225 140 0 77 2 12 0 194 91 0 0 452 7 0 0

276 57 4 19 9 278 0 0 941 1600 81 83 0 57 12 3 0 22 32 0 0 376 11 0 0

Formate dehydrogenase 3-Hexulosephosphate synthase Hydroxypyruvate reductase Serine-glyoxylate aminotransferase Glucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Fructose-1.6-bisphosphate aldolase KDPG aldolase Pyruvate dehydrogenase Citrate synthase ␣-Ketoglutarate dehydrogenase Isocitrate dehydrogenase Isocitrate lyase Malate synthase Glutamate dehydrogenase Glutamate synthase Glutamine synthetase

NAD(P)H NAP(P)H NAD+ NADP+ NAD+ NADP+

NAD+ NAD+ NAD+ NADP+

NADPH NADH NADH ATP, Mn2+

dehydrogenase. The absence of isocitrate lyase and malate synthase indicates the non-functional glyoxylate shunt in the strains IvaT and LapT . Primary ammonia assimilation occurs by reductive amination of 2-oxoglutarate to glutamate, since high activities of glutamate dehydrogenase are present in both strains. The glutamine synthetase/glutamate synthase pathway is not operative in the strains studied. In the phylogenetic tree derived from the 16S rRNA gene sequences (Fig. 1, Figs. S3–S5), strains IvaT and LapT consistently branched with the Betaproteobacteria. The relatively high level of 16S rRNA gene sequence similarity (97.8–98.5%) between the strains and the members of the genus Methylobacillus showed a close relationship. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that the two isolates formed a branch with the type strains of genus Methylobacillus. This branching pattern was also supported by the tree constructed with the maximum-parsimony method. Comparative analysis of amino acid sequences of mxaF protein confirmed these data showing high

0.1 Methylophilus luteus MimT VKM B-2547T (FJ872109) 61 Methylophilus flavus ShipT VKM B-2548T (FJ872108) 60 Methylophilus methylotrophus ATCC 53528 T (L15475) Methylophilus leisingeri DM11 T (AF250333) 100 “Methylophilus quaylei” MTT (AY772089) 96 T 94 92 Methylophilus rhizosphaerae CBMB 127 (EU 194887) ‘Methylophilus freyburgensis’ I 42T (AJ517772) 76 Methylotenera mobilis JLW8 T (DQ287786) Methylovorus mays CT (AY486132) 100 100 Methylovorus glucosotrophus 6B1T (AY486133) Methylobacillus pratensis F31T (AY29890) 96 86 Methylobacillus arboreus IvaT (GU937479) Methylobacillus flagellatus KTT(M95651) 75 T 86 Methylobacillus gramineus Lap (GU937478) Methylobacillus glycogenes ATCC 29475 T (M95652) Escherichia coli 0157: H7 (AY513502) Fig. 1. Neighbour-joining tree based on 16S rRNA gene showing the phylogenetic position of Methylobacillus arboreus IvaT and Methylobacillus gramineus LapT among obligate and restricted facultative methylotrophs of Betaproteobacteria. The numbers at the branch points are bootstrap values from 100 replicates. Bar, 10% of evolutionary distance (10 nucleotide substitutions per 100 nucleotides).

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0.05 58 Methylophilus rhizospherae CBMB127 T(EU194904) “Methylophilus quaylei” MTT (FJ 904268) Methylophilus methylotrophus NCIMB 10515T (FJ 904267) 100 Methylophilus luteus MimT (FJ872111) 100 Methylophilus flavus ShipT (FJ872110) Methylobacillus arboreus IvaT (HM030736) 100 Met hylobacillus gramineus LapT (HM030735) 99 53 Met hylobacillus glycogenes ATCC 29475 T (AJ878073) 94 Methylobacillus flagellatus KTT (CP000284) Methylopila capsulata ATCC 700716 (CAI47581) 100 Methylobacterium nodulans ORS 2060T (AF220764) 98 Methylobacterium extorquens DSM 6343 (AJ878068) 99 Methylobacterium organophilum ATCC 27886 T (M22629) Ancylobacter dichloromethanicus DM16 (EU589387) Angulomicrobium tetraedrale DSM 5895T (DQ652142) Fig. 2. Neighbour-joining tree based on mxaF amino acid sequences showing the phylogenetic position of Methylobacillus arboreus IvaT and Methylobacillus gramineus LapT among methylotrophic bacteria. The numbers at the branch points are bootstrap values from 100 replicates. Bar, 5% of evolutionary distance (0.05 substitutions per amino acid position).

Table 3 DNA–DNA reassociation between the new strains and the type cultures of the genus Methylobacillus. DNA–DNA relatedness with strain StrainT

Lap

Iva

M. glycogenes ATCC 29475T

M. flagellatus DSM 6875T

M. pratensis NCIMB 13994T

Lap Iva

100 44

44 100

38 44

45 43

39 41

levels of similarity between strains IvaT , LapT and M. glycogenes ATCC 29475T (89.7–98.7%), as well as M. flagellatus DSM 685T (90.4–99.3%) (Fig. 2). The level of DNA relatedness between the strain IvaT and LapT was 44%. The level of DNA relatedness between the novel and the reference strains of the genus Methylobacillus (M. glycogenes ATCC 29475T , M. flagellatus DSM 6875T , M. pratensis NCIMB 13994T ) were in range 38–45% (Table 3). Strains IvaT and LapT were motile rods that occurred singly, in pairs or in small groups but were slightly differed in size. The cell sizes were for LapT (0.50–0.55 × 1.10–1.20 ␮m) and IvaT (0.30–0.35 × 1.20–1.52 ␮m). However, such dimensions are in good agreement with the other members of the genus Methylobacillus. In addition both the strains had different pH, NaCl optima and resistant to antibiotics. Strain LapT was resistant to novobiocin, nalidixic acid, neomycin whereas strain IvaT was resistant to ampicillin, oxacillin, novobiocin, streptomycin and neomycin. Characteristics of the type cultures of the genus Methylobacillus are summarized in Table 4. Formal taxonomic description for the new species is given below.

Description of M. gramineus sp. nov. M. gramineus (gra.mi’ne.us L. masc. adj. gramineus, grassy). Obligate methanol-utilizing Gram-negative, asporogenous motile rods (0.50–0.55 × 1.10–1.20 ␮m) that occur singly, in pairs or in small groups. Capsules are not produced. Do not accumulate poly-␤-hydroxybutyrate. Reproduce by binary fission. Colonies on mineral salts/methanol agar are white, circular 4–5 mm in diameter with smooth edge, convex, semitransparent, shining. Strictly aerobic, catalase- and oxidase positive; urease negative. Growth occurs at pH 6.0–9.0 and temperature range of 10–39 ◦ C. Optimal growth at 19–24 ◦ C and pH 7.2–7.8 with 0.05% (w/v) NaCl. No growth occurred in the presence of 3% (w/v) NaCl. Produce indole 3-acetic acid from tryptophan on the medium with nitrate as a nitrogen source. Acetoin is produced, gelatin is not liquefied, H2 S is not produced. No vitamins or other growth factors are required. Oxidize methanol by PQQ-MDH to formaldehyde and assimilate it via the RuMP pathway. The tricarboxylic cycle is incomplete due to the absence of ␣-ketoglutarate dehydrogenase activity. The glyoxylate shunt enzymes and ribulosebisphosphate carboxylase are absent. Ammonia is assimilated by glutamate dehydrogenase. Nitrate

Table 4 Major characteristics that allow differentiation among members of the genus Methylobacillus (data from our results). Characteristic

M. arboreus IvaT (VKM B-2590)

M. gramineus LapT (VKM B-2591)

M. flagellatus DSM 6875T

M. glycogenes ATCC 29475T

M. pratensis F31T (VKM B-2224)

Flagellation Utilization of methylamine Isocitrate dehydrogenase NADP+ Urease Acetoin production Starch hydrolysis Nitrate reduction Growth at: 37 ◦ C 42 ◦ C 3% (w/v) NaCl pH optimum DNA G+C content (mol%)

1 − + − + + −

1–4 − + − + + −

1–4 + − + − + +

− + + + − − +

1 + + + − + +

+ − + 7.9–8.5 54.0

+ − − 7.2–7.8 50.5

+ − − 7.2–7.3 55.5

+ − − 6.0–8.0 53.2

+ − − 6.5–7.5 61.5

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and ammonia are used as the nitrogen sources. The prevailing cellular fatty acids are C16:0 and C16:1 . The major ubiquinone is Q8 . The predominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and cardiolipin (diphospatidylglycerol). Resistant to novobiocin, nalidixic acid and neomycin, sensitive to ampicillin, oxacillin, gentamicin, streptomycin, kanamicin and lincomycin. Unable to grow on peptone-yeast extract medium. DNA G+C content is 50.5 mol% (Tm ). The GenBank accession numbers for the 16S rRNA and mxaF genes sequences of strain LapT are GU937478 and HM030735, respectively. The type strain M. gramineus LapT (=VKM-B-2590T = CCUG 59683T = DSM 23629T ) was isolated from phyllosphere of silverweed (P. anserina L.) sampled from the city park in Pushchino (Russia, Moscow Region). Description of M. arboreus sp. nov. M. arboreus (ar.bo’re.us L. masc. adj. arboreus, pertaining to a tree). Obligate methanol-utilizing Gram-negative, asporogenous, motile rods (0.30–0.35 × 1.20–1.52 ␮m) that occur singly, in pairs or in small groups. Capsules are not produced. Do not accumulate poly-␤-hydroxybutyrate. Reproduce by binary fission. Colonies on mineral salts/methanol agar are white, circular 2 mm in diameter with smooth edge, convex, semitransparent, shining, rough. Strictly aerobic, catalase- and oxidase positive; urease negative. Growth occurs at pH 6.0–10.5, temperature range of 10–39 ◦ C and with 0.05–3% (w/v) NaCl. Optimal growth at 19–24 ◦ C and pH 7.8–9.5 with 0.5–2% (w/v) NaCl. Produce indole 3-acetic acid from tryptophan on the medium with nitrate as a nitrogen source. Acetoin is produced, gelatin is not liquefied, H2 S is not produced. No vitamins or other growth factors are required. Oxidize methanol by PQQ-MDH to formaldehyde and assimilate it via the RuMP pathway. The tricarboxylic cycle is incomplete due to the absence of ␣-ketoglutarate dehydrogenase activity. The glyoxylate shunt enzymes and ribulosebisphosphate carboxylase are absent. Nitrate and ammonia are used as the nitrogen sources. Ammonia is assimilated by glutamate dehydrogenase. The prevailing cellular fatty acids are C16:0 and C16:1 . The major ubiquinone is Q8 . The predominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and cardiolipin (diphospatidylglycerol). Resistant to ampicillin, oxacillin, novobiocin, streptomycin and neomycin, but sensitive to nalidixic acid, gentamicin, kanamicin and lincomycin. Unable to grow on peptone-yeast extract medium. DNA G+C content is 54.0 mol% (Tm ). The GenBank accession number for the 16S rRNA and mxaF genes sequences of strain IvaT are GU937479 and HM030736, respectively. The type strain M. arboreus IvaT (=VKM B-2590T = CCUG 59684T = DSM 23628T ) was isolated from willow buds (S. fragilis L.) sampled from the city park in Pushchino (Russia, Moscow Region). Acknowledgement This work was supported by the Russian Federation Basic Research Grant 10-04-00808-a.

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