Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Taxonomy: Historical and Current

Short Description of the Family

Me. thy. lo. phi. la’ ce. ae. M.L. masc. n. Methylophilus type genus of the family, -aceae ending to denote family, M.L. fem. pl. n. Methylophilaceae the Methylophilus family.

Phylogenetically a member of the order Methylophilales, class Betaproteobacteia (Garrity et al. 2005). The family contains the type genus Methylophilus (Jenkins et al. 1987), Methylobacillus (Yordy and Weawer 1977: emended by Urakami and Komagata 1986), Methylovorus (Govorukhina and Trotsenko 1991: emended by Doronina et al. 2005a), Methylotenera (Kalyuzhnaya et al. 2006).

Members of the family are not halophilic obligate or restricted facultative methylotrophs, assimilate one-carbon compounds via the 2-keto-3-deoxy-6-phospogluconate (KDPG) variant of the ribulose monophosphate (RuMP) pathway. Gram-negative rods, multiply by binary fission. Motile by means of one or several polar or subpolar flagella or nonmotile. Do not form resting bodies. Do not grow in TGY, LB, and Nutrient media. Methane is not used. Aerobic, having a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor. Major cellular fatty acids are C16:1ω7c and C16:0. The major phospholipid is phosphatidylethanolamine. Ubiquinone Q-8 is the predominant isoprenoid quinone. The phylogenetic distance between the four genera is about 93–96 % 16S rRNA gene sequence similarity.

Phylogenetic Structure of the Family and Its Genera

Analysis of the nearly complete sequences of the 16S rRNA genes indicate that methylobacteria of the family Methylophilaceae are separated from sequences of species representing the genera Methylophilus, Methylobacillus, Methylovorus, and Methylotenera (Fig. 32.1 ).

Fig. 32.1
figure 1figure 1

Maximum likelihood phylogenetic tree showing the relationship of representatives of the Methylophilaceae based on 16S rRNA gene sequences. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model (Tamura and Nei 1993). The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein 1985). Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011)

Full size image

Functional gene—the mxaF gene encodes the large subunit of the classical pyrroloquinoline quinone–linked methanol dehydrogenase which is found in majority of extant Gram-negative methylobacteria (Anthony and Williams 2003). Phylogenetic tree based on mxaF amino acid sequences show high levels of similarity between the members of the family Methylophilaceae (Fig. 32.2 ).

Fig. 32.2
figure 2figure 2

Maximum likelihood tree based on mxaF amino acid sequences showing the phylogenetic position members of Methylophilaceae among methylotrophic bacteria. The numbers at the branch points are bootstrap values from 1,000 replicates. Bar, 5 % of evolutionary distance (5 amino acid substitutions per 100 amino acids). The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model (see previous Fig. 32.1 )

Full size image

Molecular Analyses

DNA-DNA Hybridization Studies

Almost all descriptions of Methylophilus, Methylobacillus, and Methylovorus species include results of DNA-DNA hybridization (DDH) studies. Levels of DNA-DNA relatedness between Methylophilus strains—M. methylotrophus NCIMB 10515T, M. leisingeri DSM 6813T, M. quaylei VKM B-2338T, M. rhizosphaerae CBMB127T, M. flavus VKM B-2547T, M. luteus VKM B-2548T, and M. glucosoxydans VKM B-1607T were 28–46 % (Madhaiyan et al. 2009; Doronina et al. 2012).

DNA-DNA relatedness between the reference strains of the genus Methylobacillus (M. glycogens ATCC 29475T, M. flagellatus DSM 6875T, M. pratensis NCIMB 13994T, M. gramineus VKM B-2591T, M. arboreus VKM B-2590T) were in the range 38–45 % (Gogleva et al. 2011).

DDH relatedness between Methylovorus glucosetrophus VKM B-1745T and Methylovorus mays VKM B-2221 was 56–58 % (Doronina et al. 2000). The levels of DNA-DNA homology between Methylovorus menthalis VKM B-2663T and M. glucosetrophus VKM B-1745T and M. mays VKM B-2221T were 40 and 58 %, respectively (Doronina et al. 2011).

Moderate DDH relatedness of 12–18 % between type strains of the genera Methylophilus, Methylobacillus, and Methylovorus is a proof for intrageneric membership (Govorukhina and Trotsenko 1991; Doronina and Trotsenko 1994).

Genome Comparison

The complete genome sequence of five species branching within the 16S rRNA gene tree of Methylophilaceae have been released: the type strain of Methylobacillus flagellatus KTT (Chistoserdova et al. 2007), Methylotenera mobilis JLW8T, Methylotenera versatilis 301T, and Methylovorus glucosetrophus SIP3-4 (Lapidus et al. 2011) and unclassified Methylophilales strain HTCC 2181T (Giovannoni et al. 2008) (Tables 32.1 – 32.3 ). The genome of Methylobacillus flagellatus KTT represented by a single circular chromosome of approximately 3 Mbp, potentially enoding a total of 2,766 proteins.

Table 32.1 Genome statistics and general features of Methylophilaceae species
Full size table
Table 32.2 Relationships between the five Methylophilaceae strains compared on the basis of 16S rRNA gene identity and on percentage of common proteins (Lapidus et al. 2011)
Full size table
Table 32.3 Major pathways and enzymes for carbon and nitrogen metabolism predicted from genomes (Lapidus et al. 2011)
Full size table

Based on genome analysis as well as the results of mutational analyses, their methylotrophy is enabled by methanol and methylamine dehydrogenases and their specific electron transport chain components, the tetrahydromethanopterin (H4MPT)-linked formaldehyde oxidation pathway and the assimilatory and dissimilatory RuMP cycles, and by a formate dehydrogenase. Some of the methylotrophy genes are present in more than one (identical or nonidentical copy). The obligate dependence on single-carbon compounds appears to be due to the incomplete tricarboxylic acid cycle (TCA), as no genes potentially encoding alpha-ketoglutarate, malate or succinate dehydrogenases are identifiable. The genome of M. flagellatus was compared in terms of methylotrophy functions to the previously sequenced genomes of three methylotrophs, Methylobacterium extorquens (an alphaproteobacterium, 7 Mbp) (Chistoserdova et al. 2003), Methylibium petroleiphilum (a betaproteobacterium, 4 Mbp) (Kane et al. 2007), and Methylococcus capsulatus Bath (a gammaproteobacterium, 3.3 Mbp) (Ward et al. 2004). Strikingly, metabolically and/or phylogenetically, the methylotrophy functions in M. flagellatus were more similar to those in M. capsulatus and M. extorquens than to the ones in the more closely related Methylibium petroleiphilum species, providing the first genomic evidence for the polyphyletic origin of methylotrophy in Betaproteobacteria.

Comprehensive proteomics to assess the expressed portion of the genome of Methylobacillus flagellatus was implemented (Chistoserdova et al. 2009; Chistoserdova 2011; Hendrickson et al. 2010). A total of 1,671 proteins (64 % of the inferred proteome) were detected, including all the predicted essential proteins. Nonrandom patterns observed with the nondetectable proteins appeared to the corresponding silent genomic islands, as inferred through the functional profiling and genome localization. The protein contents in methylamine- and methanol-grown cells showed a significant overlap, confirming the commonality of methylotrophic metabolism downstream of the primary oxidation reactions. The new insights into methylotrophy include detection of proteins for the N-methylglutamate pathway of methylamine oxidation that appears to be auxiliary. Two alternative enzymes for the 6-phosphogluconate dehydrogenase reaction (GndA and GndB) and the formate dehydrogenase reaction (FDH1 and FDH4) were detected. Mutant analysis revealed that GndA and FDH4 are crucial for the organism’s fitness, while GndB and FDH1 are auxiliary.

Comparative analysis revealed that the core genome of Methylophilaceae may be as small as approximately 600 genes, while the pangenome may be as large as approximately 6,000 genes. Significant divergence between the genomes in terms both gene content and gene and protein conservation was uncovered, including the varied presence of certain genes involved in methylotrophy. Overall, data demonstrate that metabolic potentials can vary significantly between different species of Methylophilaceae, including organisms inhabiting the very same environment. These data suggest that genetic divergence among the members of the family may be responsible for their specialized and nonredundant functions in C1 cycling, which in turn suggests means for their successful coexistence in the specific ecological niches.

Phenotypic Analyses

The main features of members of Methylophilaceae are listed in Tables 32.4 – 32.7

Table 32.4 Comparison of selected characteristics of members of the genus Methylophilus
Full size table
Table 32.5 Major characteristics that allow differentiation among members of the genus Methylobacillus (Gogleva et al. 2011)
Full size table
Table 32.6 Characteristics of the type strains of the genus Methylovorus (Doronina et al. 2011)
Full size table

.

Table 32.7 Characteristics of the type strains of the genus Methylotenera (Kalyuzhnaya et al. 2012)
Full size table

Methylophilus Jenkins et al. 1987, 447VP; Doronina et al. 2010, 2012

Me.thy.lo.phi’lus. Fr.n. methyle the methyl radical; Gr.adj. philos loving; M.L. masc.n. Methylophilus methyl radical loving.

Gram-negative, asporogenous, motile by polar flagella or nonmotile rods that are 0.2–0.6 × 1.0–2.5 μm in size. Some strains produce exopolysaccharide. Colonies on methanol—minimal salt agar plates incubated for 2 days at 29 °C—are pigmented or nonpigmented, 1–4 mm in diameter, with entire edge, convex, translucent to opaque, smooth. No or extremely poor growth on nutrient or blood agar; no hemolysis. Optimal temperature 24–37 °C; no growth occurs at 4 °C and 45 °C. Optimal pH 6.5–7.8. Aerobic. Metabolism respiratory; very little or no acid is produced from glucose. Methanol is oxidized as the sole carbon and energy source by all the strains. In addition, a very limited range of other carbon compounds such as methylated amines, formate, glucose, and fructose may be utilized as sole carbon and energy sources. Methane is not used. No vitamins or other growth factors are required. Catalase- and oxidase positive. Nitrate and ammonium salts are used as nitrogen sources. Produce indole from tryptophan on medium with nitrate as nitrogen source. The predominant cellular fatty acids are C16:0 and C16:1. The major isoprenoid quinone component is ubiquinone with eight isoprene units (Q-8). The predominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol; diphosphatidylglycerol (cardiolipin) is absent.

Formaldehyde is assimilated via the RuMP pathway. Assimilate NH4+ via the glutamate cycle. The tricarboxylic acid cycle is incomplete due to the absence of α-ketoglutarate dehydrogenase. Glyoxylate shunt enzymes are absent. The mol% G + C of DNA is 48–54.

The type species is Methylophilus methylotrophus Jenkins, Byron, and Jones 1987. The type strain is AS1 (ATCC 53528T = DSM 46235T = LMG 6787T = NCIMB 10515T = VKM B-1623T).

Comparison of selected characteristics of members of the genus Methylophilus are listed in Table. 32.4 .

Methylobacillus Yordy and Weawer, 1977, 254VP, Emend. Urakami and Komagata 1986, 509

Meth.yl.o.ba.cil’lus. Fr. methyle the methyl radical; L.dim.n. bacillus a small rod; M.L. masc.n Methylobacillus methyl rodlet.

Cells are Gram-negative, asporogenous rods 0.3–0.6 × 0.8–2.0 μm motile or nonmotile, multiplying by binary fission, mesophilic and neutrophilic. Most strains are obligate methylotrophs; however, some strains can also use fructose. Colonies are shiny, smooth, raised, entire, white to light yellow, 1–4 mm in diameter on methanol-containing agar. Methanol is oxidized as the sole carbon and energy source by all strains. Aerobic, having a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor. No vitamins or other growth factors are required. Nitrate and ammonia are used as the nitrogen sources. 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 (diphosphatidylglycerol). Ammonia is assimilated by glutamate dehydrogenase. The tricarboxylic acid cycle is incomplete due to the absence of α-ketoglutarate dehydrogenase. Glyoxylate shunt enzymes are absent. The mol. G + C of DNA is 50–61. The type species is Methylobacillus glycogenes Yordy and Weawer 1977. The type strain TK 0113 = Yordy and Weaver T-11 (= ATCC 29475T = DSM 5685T = JCM 2850T = LMG 6082T = NCIMB 11375T = VKM B-2060T).

Table. 32.5 lists the differentiating properties of Methylobacillus species.

Methylovorus Govorukhina and Trotsenko 1991, 161VP, Emend Doronina, Ivanova, and Trotsenko, 2005, 903

Me.thy.lo.vo’rus. N.L. methyl the methyl radical; N.L. masc.n adj. vorus consuming; N.L. masc.n. Methylovorus the methyl consumer.

Gram-negative rods, 0.4–0.6 × 1.0–1.4 μm. Motile by a single polar flagellum. Do not form endospores or complex intracellular membranes, either sheath or prosthecae. Some strains produce slime. Multiply by binary fission. No aggeregation or pigmentation in liquid medium. Colonies on methanol mineral salt agar incubated for 2 days at 30 °C are circular, 0.5–2.0 mm in diameter, with entire edges, convex and translucent to opaque, creamy or milky in color. No growth under an atmosphere of CH4 + O2 or H2 + CO2 + O2. No growth in the presence of 3 % NaCl. Strictly aerobic with respiratory metabolism. Obligate or restricted facultative methylotrophs. Utilize methanol as the carbon and energy source. Some strains can grow poorly on glucose. Nitrates, ammonium salts, and glutamate serve as the nitrogen sources. Acetoine, H2S, and NH3 are not produced in test medium. Urease-, catalase-, and oxidase-positive. Peroxidase is variable. Do not degrade cellulose, gelatin, Tween 80. Indole is formed from l-tryptophan in mineral medium with methanol as the sole carbon and energy source with KNO3 as a nitrogen source. Ammonium ions inhibit tryptophan deamination. Assimilate C1 compounds through the RuMP pathway (Entner-Doudoroff variant) and ammonia via the glutamate cycle (glutamate synthase and glutamine synthetase). Neither α-ketoglutarate dehydrogenase nor the glyoxylate shunt enzymes are present. 6-phosphogluconate dehydrogenase is specific for NAD+ (not NADP+). The prevailing cellular fatty acids are C16:0 C16:1ω7. The major ubiquinone is Q-8. The predominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. Cardiolipin is present.

The G + C content of DNA is 54–58 mol%. The type species is Methylovorus glucosotrophus Govorukhina and Trotsenko 1991. The type strain is 6B1 (VKM B-1745T = ATCC 49758T = DSM 6874T = NCIMB 13222T = UCM B-1745T). Table. 32.5 lists characteristics of the type strains of the genus Methylovorus.

Methylotenera Kalyuzhnaya, Bowerman, Lara, Lidstrom, and Chistoserdova 2006, 289VP, Emend. Kalyuzhnaya, Beck, Vorob’ev, Smalley, Kunkell, Lidstrom, Chistoserdova, 2012

Me.thy.lo.te’ner.a. N.L. methylum (from French me´thyle, back-formation from French méthylène, coined from Gr. n. methu wine and Gr. n. hulê wood) the methyl group radical; N.L. pref. methylo- pertaining to the methyl radical; L. fem. adj. tenera delicate; N.L. fem. n. Methylotenera a methyl group-oxidizing delicate bacterium.

Gram-negative rods. Some strains are motile. Do not form resting bodies and multiply by binary fission. Utilize methylamine. In addition, may utilize methanol, betaine, fructose, ethanol, and pyruvate as the sole sources of carbon and energy. Some strains are positive for urease and nitrate reduction. Oxidize methylamine by methylamine dehydrogenase or via N-methylglutamate pathway and assimilate C1 compounds via the RuMP pathway. The major cellular fatty acids are C16:1ω7c and C16:0. The DNA G + C content is in the range 42.6–45.5 mol%.

The type species is Methylotenera mobilis Kalyuzhnaya et al. 2006. The type strain is JLW8 (= ATCC BAA-1282T = DSM 17540T).

Comparison of selected characteristics of the type strains of the genus Methylotenera are listed in Tables 32.7 and 32.8 .

Table 32.8 Differential characteristics of methylobacteria of the family Methylophilaceae
Full size table

Colonies of Methylotenera mobilis JLW8T (= ATCC BAA-1282T = DSM 17540 T) were cream to light brown and 1–2 mm in diameter when grown at 30 °C for 4–7 days. No pigmentation was observed when cells were grown in liquid culture (Kalyuzhnaya et al. 2006).

Colonies of Methylotenera versatilis 301T were white (slightly yellowish in old cultures), mucoid, undulate, circular, convex, viscous, and up to 5 mm in diameter. The isolate grew well on solid media but not in a liquid culture. Only cultures incubated without shaking showed some growth (Kalyuzhnaya et al. 2012).

Isolation, Enrichment, and Maintenance Procedures

Isolation, Enrichment

Strains of Methylophilus methylotrophus have been isolated from activated sludge, mud, river, and pond water (MacLenman et al., British patent 1370892). The type strain AS1T was isolated from activated sludge.

Methylophilus leisingeri DM11T was isolated in Switzerland from groundwater contaminated with dichloromethane (Gaelli and Leisinger 1985; Doronina and Trotsenko 1994). 20 ml groundwater was passed through a 0.2 μm pore diameter membrane filter, and the filter was incubated in 50 ml mineral medium containing 10 mM dichloromethane. The medium contained (g/l): K2HPO4 · 3H2O, 4.1; КH2PO4, 1.4; (NH4)2SO4, 0.2; MgSO4 · 7H2O, 0.2. Prior autoclaving, the pH of the medium was adjusted to 7.2. After sterilization dichloromethane was added, and 1 l of the medium was supplemented with 1 ml trace element solution (g/l): FeSO4 · 7H2O, 1.0; MnSO4 · H2O, 1.0; (NH4)6Mo7O24 4H2O, 0.25; H3BO3, 1.0; CuCl2 · 2H2O, 0.25; ZnCl2, 0.25; NH4VO3, 0.1; Co(NO3)2 · 6H2O, 0.25; and Ca(NO3)2, 0.25. The bacteria were grown at 29 °C in 750-ml Erlenmeyer flasks containing 100 ml of the medium on a rotary shaker at 180 rpm. The flasks were tightly closed with rubber stoppers. Since the pH of the medium decreased when dichloromethane was used, dichloromethane was added in three portions to a final concentration of 10 mM (0.85 g/l) each after the pH of the medium had been adjusted to 7.2 by NaOH. Pure culture was isolated by the method of exhausting plating of enrichment onto the same medium, containing 2.0 % purified agar Difco and 0.1 g/l of Bromothymol Blue. Petri plates were incubated in a 2.5 l desiccator, 0.2 ml portions of dichloromethane being added after 24 h. Colonies of bacteria that decompose dichloromethane are surrounded by yellow zones due to formation of HCl.

Methylophilus quaylei MTT was isolated as an airborne contaminant during cultivation of methanotroph Methylocystis methanolicus on methanol (Doronina et al. 2005b). The sample of mixed liquid culture grown on medium “K” containing gl−1: КH2PO4—2.0; (NH4)2SO4—2.0; MgSO4 · 7H2O—0.025; NaCl—0.5; FeSO4 · 7H2O—0.002, pH 7.2 in Petri dish (h = 5 mm) was UV incubated for 10 min. After UV treatment, the culture was serially diluted and grown on “K” agar (Difco 2.0 % w/v) with 2 % (v/v) CH3OH. A successive isolation of a single colonies resulted in the isolation of a pure culture of obligately methylotrophic strain MTT, which could not grow on Difco nutrient agar or in atmosphere of methane: air (1:1).

Methylophilus rhizosphaerae CBMB127T was isolated from rhizosphere soils of rice cultivars (Oryza sativa L. cv O-dae and Nam-pyeoung, respectively) on selective ammonium mineral salt (AMS) medium (Whittenbury et al. 1970) with 0.5 % methanol. Cells were maintained on nutrient agar (NA, Difco) with 1 % (v/v) methanol or on AMS medium (Madhaiyan et al. 2009).

Strains Methylophilus flavus ShipT and Methylophilus luteus MimT were isolated from the phyllosphere of dog rose (Rosa cinnamomea L.) and coltsfoot (Tussilago farfara L.), respectively, sampled from the city park in Pushchino (Moscow region, Russia) (Gogleva et al. 2010). The strains were grown on medium “K.” Solidified medium “K” was prepared by adding 2.0 % (w/v) Difco agar.

Methylophilus glucosoxydans BT was isolated from rhizosphere rice (Oryza sativa L.) sampled from Vietnam on “K” agar with 2.0 % methanol (Doronina et al. 2012).

An enrichment culture of Methylobacillus glycogenes T-11T was prepared by adding a small amount of partially decayed tomato to a liquid mineral salt medium containing 2 % methanol (v/v) (Yordy and Weawer 1977). The bacterium was isolated from the enrichment culture by streaking for isolated colonies on a mineral salt agar medium containing 2 % methanol. A colony was picked and cultured at 30 °C with shaking (200 rpm) in a liquid mineral salt medium (pH 7.0), containing 2 % methanol. The procedure was repeated until a pure culture was obtained.

The strain Methylobacillus flagellatus KTT was isolated by the same procedure from sewage (Govorukhina et al. 1987). Methylobacillus pratensis F31T was isolated on mineral salt medium, containing 0.5 % (v/v) methanol from meadow grass (Poa trivialis L.) sampled from the city park in Helsinki (Finland) (Doronina et al. 2004).

Stains Methylobacillus arboreus IvaT and Methylobacillus gramineus 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) (Gogleva et al. 2011).

Strain of the genus Methylovorus were isolated from activated sludge, mud, soil, pond water, and plants (Govorukhina and Trotsenko 1991; Seo and Kim 1993; Doronina et al. 2000, 2005a, 2011).

Methylovorus glucosotrophus 6B1T was isolated from pond water (Govorukhina and Trotsenko 1991). Methylovorus mays CT was isolated from maize phyllosphere (Zea mays L.) (Doronina et al. 2000). Methylovorus menthalis MMT was isolated from the root of corn mint (Mentha arvensis L.) (Doronina et al. 2011).

The root was washed three times with sterile distilled water and placed into an Erlenmeyer flask (750 ml) with 200 ml of K medium and 0.5 % (v/v) methanol. After three transfers for 2 days on a rotary shaker (180 rpm) at 29 °С, the suspension of the methylobacterial enrichment culture was plated to obtaining single colonies (exhaustive inoculation) onto agarized “K” medium with methanol. Isolated methylobacterial colonies were reinoculated on agar slants, transferred into liquid medium, and then again on solid medium for exhaustive inoculation. Reisolated methylobacterial colonies were reinoculated on slant agar. The purity of the isolated culture was controlled by light and electron microscopy, as well as by the uniformity of colonies on the agarized medium with methanol.

An obligate methylamine utilizer, Methylotenera mobilis JLW8T, was isolated from Lake Washington sediment (Washington State, USA) after enrichment in a basal salt medium (Harder et al. 1973) diluted fivefold and supplemented with 0.1 % methylamine (Kalyuzhnaya et al. 2006). Also the restricted facultative methylotroph Methylotenera versatilis 301T was isolated from this ecosystem by a dilution-plating approach (Kalyuzhnaya et al. 2012). To isolate strain 301T, 1 ml aliquot of sediment samples was mixed with 9 ml filtered lake water, and serial dilutions were plated onto plates containing lake water-based medium solidified with agar (2 %; Difco) supplemented with 5 mM methylamine. After 2 weeks incubation at room temperature, individual colonies were restreaked onto fresh agar plates.

Maintenance Procedures

Generally, strains of the family Methylophilaceae are maintained on basal salts media as agar slants at 4 °C for 1 month. Some members of the genus Methylophilus must be subculturing every 10 days. For long-term storage at −20 °C or −80 °C, cells are suspended in the basal salts media and supplemented with 10 % DMSO or 20 % glycerol. Long-term preservation methods include freeze-drying in skim milk and maintenance in liquid nitrogen at −196 °C.

Ecology

Strains of the family Methylophilaceae are obligate or restricted facultative methylotrophs (methylobacteria) capable of growth on single-carbon compounds (methanol, methylamines, dichloromethane) and play an important role for the aerobic conversion of C1 compounds in different ecological niches. They have been isolated from activated sludge, mud, river, lake and pond waters, plant rhizosphere and phyllosphere. Association of aerobic methylobacteria with plants are permanent and results from the fact that methylobacteria consume methanol released by plant into the environment through leaf stomata (Nemecek-Marshall et al. 1995; Fedorov et al. 2011). Methanol is formed during demethylation of cell wall pectin under active growth of plant cells. Plants are therefore the main source of methanol in the biosphere (Galbally and Kirstine 2002). Association between plants and methylotrophs is mutually advantageous, because methylobacteria stimulate plant growth and development due to production of bioactive substances: phytohormones (auxins, cytokinins) and vitamins (Fedorov et al. 2011).

Methylophilus rhizosphaerae (Madhaiyan et al. 2009), Methylophilus flavus and Methylophilus luteus (Gogleva et al. 2010), Methylophilus glucosoxydans (Doronina et al. 2012), Methylobacillus pratensis (Doronina et al. 2004), Methylobacillus arboreus and Methylobacillus gramineus (Gogleva et al. 2011), Methylovorus mays (Doronina et al. 2000), and Methylovorus menthalis (Doronina et al. 2011) were isolated from plants.

Pathogenicity

No reports of the family Methylophilaceae causing overt or opportunistic infections in humans, animals, or insects have been published. The members of this family are obligate or restricted facultative methylotrophs and unable to grow on blood agar or other complicated media.

The type strain Methylophilus methylotrophus AS1 is resistant to penicillin, oleandomycin, and susceptible to nalidixic acid and streptomycin (Jenkins et al. 1987). Methylophilus leisingeri DM11T (VKM B-2013T = DSM 6813T) is resistant to erythromycin and sensitive to nalidixic acid, novobiocin, and kanamycin (Doronina and Trotsenko 1994). Methylophilus flavus ShipT (VKM B-2547T = DSM 23073T = CCUG 58411T) is resistant to streptomycin and oxacillin and sensitive to ampicillin, novobiocin, nalidixic acid, gentamicin, neomycin, and lincomycin (Gogleva et al. 2010). Methylobacillus flagellatus KTT (ATCC 51484 = DSM 6875 = VKM B-1610) is resistant to streptomycin, erythromycin, ampicillin, neomycin, and lincomycin and sensitive to novobiocin, nalidixic acid, kanamycin, and gentamicin (Govorukhina et al. 1987). Methylobacillus arboreus IvaT (VKM B-2590T = CCUG 59684T = DSM 23628T) is resistant to ampicillin, oxacillin, novobiocin, streptomycin, and neomycin and sensitive to nalidixic acid, gentamicin, kanamycin, and lincomycin (Gogleva et al. 2011). Methylobacillus gramineus LapT (=VKM B-2590T= =CCUG 59683T = DSM 23629T) is resistant to novobiocin, nalidixic acid, and neomycin and sensitive to ampicillin, oxacillin, gentamicin, streptomycin, kanamycin, and lincomycin (Gogleva et al. 2011).

Mehylovorus glucosetrophus 6B1T (=ATCC 49758T = VKM B-1745T = NCIMB 13222T) is resistant to neomycin, lincomycin, erythromycin, and ampicillin, but sensitive to novobiocin, nalidixic acid, kanamycin, and streptomycin (Govorukhina and Trotsenko 1991).

Methylovorus mays CT (=VKM B-2221T = NCIMB 13922T) is resistant to ampicillin and lincomycin and sensitive to gentamicin, kanamycin, nalidixic acid, neomycin, novobiocin, streptomycin, and erythromycin (Doronina et al. 2000).

Methylotenera mobilis JLW8T (=ATCC BAA-1285T = DSM17540T) is sensitive to gramicidin, kanamycin, and tetracycline (Kalyuzhnaya et al. 2006). Methylotenera versatilis 301T (=JCM 17579T = VKM B-2679T) is resistant to kanamycin, ampicillin, streptomycin, neomycin, and erythromycin (Kalyuzhnaya et al. 2012).

Application

Forage protein methylobacteria of the genera Methylophilus and Methylobacillus are characterized by higher values of specific growth rate, economic coefficient, protein and lysine content, and favorable composition of elements and fatty acids (MacLennan et al. 1974; Doronina and Trotsenko 1986; Large and Bamforth 1988; Trotsenko et al. 2005). For all the reasons above, they are preferable for large-scale cultivation and forage protein production.

Exopolysaccharides (EPSs)

Methylobacteria of the genera Methylobacillus, Methylophilus, and Methylovorus synthesize EPSs from methanol; of note, EPSs yield may be regulated by varying the composition of the medium and growth conditions (Doronina et al. 2005b; Gogleva et al. 2010).

Phytosymbiosis

Aerobic methylobacteria are ubiquitous in the phyllosphere and rhizosphere of plants and often colonize their seeds (Trotsenko et al. 2001; Fedorov et al. 2011). Biological testing (using Amarantus caudatus L. seedlings), TLC, HPLC, and solid-phase enzyme immunoassays established the presence of zeatin and zeatin riboside in the culture liquid of Methylovorus mays (Ivanova et al. 2000). It was also demonstrated that representatives of the genera Methylophilus, Methylobacillus, and Methylovorus synthesize indole compounds, particularly indoleacetic acid (IAA) when grown in media containing methanol or methylamine in the presence of 5 mM l-tryptophan (Doronina et al. 2002; Gogleva et al. 2010, 2011).

The synthesis of auxins in methylobacteria depended on the composition of the medium: when (NH4)2SO4 was replaced by KNO3, the amount of IAA synthesized increased three- to fivefold.

Methylovorus mays exerted a beneficial effect on the growth and morphogenesis of tobacco, potato, and fiber flax grown in vitro (Kalyaeva et al. 2001). This obligate methylobaterium stimulated morphogenesis and antifungal resistance of Chinese cabbage Brassica chinensis L. (Doronina et al. 2009). The ability of methylobacteria to stimulate the growth and morphogenesis of plants indicates their promise in experimental plant physiology and agrobiotechnology.

Enzymes

Enzymological studies demonstrated that a number of enzymes of methylobacteria exhibit very high activity. These results provided a basis for isolating pure preparations of various dehydrogenases and other enzymes, which have a potential as research reagent and analytical tools: glucose-6-phosphate dehydrogenase (EC 1.1.1.49; 250U/mg protein) (Sokolov et al. 1980); NADP+ glutamate:dehydrogenase (EC 1.4.1.4; 180 U/mg protein) (Sokolov and Trotsenko 1987).

Biodegradation of Toxic Compounds

Methylobacteria of the family Methylophilaceae degrade a broad spectrum of highly toxic compounds: methanol, formaldehyde, methylated amines, and dichloromethane. The culture of Methylobacillus sp. is appropriate for elimination of methanol from industrial sewage (cellulose sulfate manufacturing facilities) (Trotsenko et al. 2005).