Methylopila Capsulata Gen. Nov., Sp. Nov., a Novel Non-Pigmented Aerobic Facultatively Methylotrophic Bacterium

International Journal of Systematic Bacteriology (1 998), 48, 13 1 3-1 32 1 Printed in Great Britain

Methylopila capsulata gen. nov., sp. nov., a novel non-pigmented aerobic facultatively methylotrophic bacterium

Nina V. Doronina,’ Yuri A. Trotsenko,’ Valentina I. Krausova,’ Eugenia S. Boulygina’ and Tatjana P. Tourova’

Author for correspondence: Yuri A. Trotsenko. Tel: +7 95 925 74 48. Fax: +7 95 923 36 02. e-mail: trotsenkoiwibpm.serpukhov.su

~ ~ ~ Institute of Biochemistry A new genus, Methy/opi/a, and one new species are described for a group of and Physiology of seven strains of facultatively methylotrophic bacteria with the serine pathway Microorganisms, Russian Academy of Sciences, of C, assimilation. These bacteria are aerobic, Gram-negative, non-spore- Pushchino, Moscow forming, motile, colourless rods that multiply by binary fission. Their DNA base region, 142292, Russia content ranges from 66 to 70 mol% G+C. Their cellular fatty acid profile * Institute of Microbiology, consists primarily of C18:lto7cis-vaccenic and C19:ocyclopropane acids. The major Russian Academy of hydroxy acid is 3-OH C,4:o.The main ubiquinone is 9-10. The dominant cellular Sciences, Moscow, 1 17811, Russia phospholipids are phosphatidylethanolamine and phosphatidylcholine. The new isolates have a low level of DNA-DNA homology (5-10%) with the type strains of the serine pathway methylobacteria belonging to the genera Methylobacterium, Aminobacter, Hyphomicrobium and Methylorhabdus. Another approach, involving 165 rRNA gene sequence analysis of strain IMIT, has shown that the new isolates represent a separate branch within the a-2 subclass of the Proteobacteria. The type species of the new genus is Methy/opi/a capsdata sp. nov., with the type strain lMIT(= VKM B-1606T).

Keywords: serine pathway methylotrophs, Met/2j~/opi/acapsdata gen. nov., sp. nov.

INTRODUCTION colourless non-motile dichloromethane-utilizing bacteria (Doronina et al., 1995). Nevertheless, it seemed The group of aerobic Gram-negative asporogenous evident that a taxonomic structure of the serine facultatively met hylo trop hic bacteria (met hylo- pathway facultative methytobacteria should include bacteria) having the serine pathway and capable of some new taxa to cover their phenotypic and genotypic growth on a variety of reduced C, substrates, except diversity. Previously, we described three novel strains methane. is very heterogeneous. However, so far of colourless facultative methylobacteria having an only four of the previously described genera have intermediate position between the genera Methylo- been validly published : Hyphomicrohium, Methylo- bacterium and Aminohacter and proposed the generic bacterium, Aminobacter and Methylorhabdus. As name Methylomicrobium for these strains known, the representatives of the genus Hypho- (Govorukhina et al., 1989; Doronina & Trotsenko, rnicrohium form hyphae and multiply by budding 1992). Since the genus ‘ Methylomicrohiuni’ (Hirsch. 1984), the genus Methylobacterium was (Govorukhina et al., 1989) has not been validated, this emended for the pink-pigmented facultative methylo- generic name was subsequently used for a group of bacteria (PPFM)(Patt et al., 1976; Bousfield & Green, Type I methanotrophs (Bowman et al., 1995). In view 1985). The genus Aminobacter was proposed for non- of the fact that our extended study of ‘Methylo- pigmented, budding, methylamine-utilizing bacteria ~-2icrobiurn capsulatus’ (Govorukhina et al., 1989) unable to grow on methanol (Urakami et al., 1992) showed that this organism did not belong to the genus and the new genus Methylorhabdus was suggested for Methylomicrobium (Bowman et al., 1995) it would be

.....,...,...... appropriate to place this organism in a new genus, for Abbreviations: PMS, phenazine methosulfate; PPFM, pink-pigmented which we propose the name Methylopila. Our studies facultative methylobacteria. of seven non-pigmented strains of facultatively The GenBank accession number for 165 rDNA sequence for Methylopila methylotrophic bacteria which utilize the serine path- capsdata I M 1 is AF004844. way indicate that they should be placed in a single

~- ~-- ___~ 00724 0 1998 IUMS 1313 N. V. Doronina and others species, for which we propose the name Metlzylopilu medium K after 2 weeks cultivation with methanol being capsuluta. replaced by the other carbon compounds. Organic acids and amino acids were added at concentrations of 0.05-0.3 YO, while carbohydrates and alcohols were added at concen- METHODS trations of 0.2-0-5 YO.To test alternative nitrogen sources Bacterial strains. Pure cultures of seven bacterial strains (NH,),SO, was replaced by the other nitrogen compounds. (IMIT, UZ, 14, 9AA, 10AA, 26AA and 37AA) utilizing Methane utilization was tested in an atmosphere containing methanol and methylamine as well as some multicarbon CH, and air (1 : 1, v/v) in 700 ml conical flasks containing compounds as growth substrates were isolated from soil 100 ml medium K and fitted with rubber stoppers. Hydrogen samples taken from Uzbekistan (TMIT, 14, UZ) and utilization was tested by the same procedure but in an Kazakhstan (AA) as described earlier (Govorukhina et a/., atmosphere of H, + 0, + CO, (7 : 2 : 1, by vol.). Utilization of 1989; Doronina & Trotsenko, 1992). Met1iylohacteriiini mono- and dichloromethane was tested as described earlier organophilunz XX ATCC 27886' was kindly supplied by R. (Doronina et al., 1995). Liquid cultures were grown at S. Hanson (University of Minnesota, USA). K. Komagata 30 "C on a rotary shaker at 180 r.p.m. (Tokyo University, Japan) provided Protomonas strains (Urakami & Komagata, 1984) that have been reclassified Phospholipid and cellular fatty acid composition. Phospho- later into the genus Methylobacterium (Bousfield & Green, lipid composition of the cells was determined according to 1985) as Methylobacterium extorguens NCIB 9399" (= TK the methods described previously (Govorukhina & 000 1") and Methjllobacteriuni rhodesianum NCIB 1061 1 ( = Trotsenko, 1989). To determine the cellular fatty acid TK 001 6). Hyphoinicrobium zavarrinii IFAM ZV-622' was composition the cultures were grown on methanol or obtained from G. A. Zavarzin (Institute of Microbiology, methylamine with 2% agar medium K on Petri plates for Moscow, Russia). Aminobacter anzinovorans ATCC 233 14' 48 h at 29 "C. Several milligrams of cells were transferred (= JCM 7852T = NCIB 9039T), Aniinobacter anzinovorans with a loop to a screw-top microreaction jar containing ATCC 23819 (strain MA = NCIB 11590) (Urakami et a/., 150 pl of a 20 % aqueous tetramethylammonium hydroxide 1992), Methylorhabdus multivorans ATCC 5 1890' (strain solution, the mixture was heated at 100 "C for 20 min and DM 13' = VKM B-2030') and Pseudonzonas aeruginosa cooled to room temperature, and 100 pl of acetonitrile was ATCC 10145' were used as the reference strains. added. The jar was shaken, 20 pl of the top (acetonitrile) layer was withdrawn and evaporated in a stream of nitrogen The methylotrophic cultures were grown on a liquid mineral at room temperature, 20 pl 20 O/O tetramethylammonium medium K, containing 2.0 g (NH,),SO,, 2.0 g KH,PO,, 0.5 g hydroxide solution was added, the mixture was heated at NaC1,0-125g MgS0,.7H20, 0.002 g FeSO, .7H,O, and 1 1 100 "C for 1 min, and a 0.2-0-5 pl portion was introduced distilled water; the pH of this medium was adjusted to 7.2. into the chromatograph injector (heated to 385 "C). The Substrates were sterilized and added separately. Methanol analysis was carried out in a Hewlett-Packard 5830A or methylamine were added to a concentration of 0.5% chromatograph, using a 15 m x 0.2 mm glass capillary (v/v) or 0.3% (w/v), respectively. Flasks were shaken at column filled with silicone OV-101 sorbent. 29 "C. Pseudonzonas aeruginosa was grown with 0.3 % (w/v) glucose. The bacterial strains were maintained on medium K Poly-P-hydroxybutyrate was analysed according to solidified with Difco Bacto agar or PYG agar (pH 7.0) Braunegg rt a/. (1978) by using a Pye Unicam 104 gas (Urakami et a/., 1992). chromatograph. Ubiquinones were extracted and purified according to Collins (1985). Their analysis was done by Identification methods. Cell morphology, Gram staining, using a Finnigan MX- 1310 mass spectrometer. Enzyme motility and flagellation were determined by using the assays were done as previously described (Meiberg & cultures grown on solid medium K. Production of a Harder, 1978 ; Trotsenko 1986; Doronina a/., 1995). fluorescent pigment was tested on King A and B agarized eta/., et media. Nitrate reduction was tested in liquid medium K in Electron microscopic analysis. To obtain thin sections. which (NH,),SO, was replaced with 0.5 YO(w/v) KNO, after bacterial cells were fixed for 1 h at 4 "C in 0.05 M sodium 1, 3 and 5 d incubation. The methyl red and Voges- cacodylate buffer (pH 7-2) containing 1-2O/O (v/v) glutaral- Proskauer (acetoin) reactions were tested in glucose/ dehyde, washed three times in the same buffer and refixed in phosphate/peptone broth. Indole production was deter- 1 % (w/v) OsO, in 0.05 M cacodylate buffer for 4 h at 4 "C. mined with Kovacs' reagent in 1 O/O Tryptone broth. Hy- After dehydration in a series of alcohols, the cells were drogen sulfide production was tested on triple-sugar iron embedded in Spurr epoxy resin and sectioned with an LKB agar for 2 weeks. Gelatin hydrolysis was observed in stab 2128 Ultratome. The thin sections were stained with 1 o/o cultures in yeast extract-peptone medium containing 12 O/O uranyl acetate and then with 2 YOlead citrate for 10 min and gelatin after 4 weeks incubation at 22 "C. Starch hydrolysis mounted on copper grids. To obtain negatively contrasted was determined by using an iodine solution on medium K preparations, the cells were stained in 0.3 % (w/v) phospho- agar containing 0.2% soluble starch after 1, 3 and 10 d tungstic acid (pH 7-2).Micrographs were taken with a JEM incubation. 1OOB transmission electron microscope at an operating Ammonia production was tested in peptone water by voltage of 60 kV. Nessler's reagent. Oxidase activity test was performed with a DNA isolation and characterization. DNA was isolated and 1 YO solution of tetramethyl-p-phenylenediamine dihydro- purified according to Marmur (1 961). DNA G + C contents chloride. Urease activity was observed on Christensen's were determined by the thermal denaturation method with a medium. Catalase activity was detected by pouring a 3% Beckman DU-8B spectrophotometer at a heating rate of H,O, solution into colonies on solid medium K or PYG 0.5 "C min-l and were calculated according to Owen & agar. Halotolerance was tested by inoculating the cells into Lapage (1976). The DNA from E.ycherichia coli K-12 was liquid medium K with various concentrations of NaCl used as the standard. DNA-DNA hybridization was done (1-3 %). Growth at different temperatures and pH values on nitrocellulose membrane filters (0.22 pm) at optimal were tested in liquid medium K. Utilization of a wide range conditions (incubation in 50 % formamide at 50 "C for 24 h of growth substrates (> 50) was also determined in liquid with the use of deoxy [1',2',5'-'H]CTP and the nick trans-

1314 International Journal of Systematic Bacteriology 48 Methylopila, a new genus of aerobic methylobacteria _ _

lation kit N 5500 (Amersham) as described earlier (Doronina et al., 1987) 165 rDNA sequence determination and analysis. Genomic DNA was extracted and the 16s rRNA gene was selectively amplified by PCR, using the following primers: 5'-AGAG- TTTGATCCTGGCTCAG-3' as forward and 5'-TACGG- TTACCTTGTTACGACTT-3' as the reverse (Lane, 1991). The PCR reaction was carried out in a final volume of 100 pl, with 1 pg DNA template, 200 pM each primer, 200 pM each dNTP, 3 U Tet-z polymerase (BioMaster, Russia) anti reaction buffer (100 mM Tris/HCl pH 8.3, 500 mM K('1, 2 mM MgC1,). The temperature cycling was done by using 30 cycles of the following thermal profile: 1 min at 94 OC, 1 min at 42 "C, 1 min at 72 "C. The final extension mas carried out at 72 "C for 6 min. The PCR products were purified using the PCR-prep kit (Promega) according to the manufxturer's instructions. The 16s rRNA gene was sequenced in both directions with the use of forward and reverse primers listed by Lane (199 I). DNA sequencing was carried out by using Sequenase version 2 of the VSB kit (USB) with minor modifications. Phylogenetic analysis. The sequence of strain IMIT was aligned manually with sequences obtained from the database of small subunit rRNAs (Larsen et al., 1993). The sequence was compared with members of the Proteobacteria: Metliyloniic~t.obiuvlzagile ACM 3308', X72767 ; Methyl- ococcus cup sulatus Texas', X72770 ; Methylobacter luteus V KM - 5 3B". M 9 5 6 5 7 ; Methy lomonas me thanica AC M 3307T, RDP 653 ; Methylophaga marina ATCC 35842', X87338 ; Pwudomonas aeruginosa ATCC 25330, M34133 ; MethylophiIris methylotrophus NCIB 1051 Y, L I 5475 ; Metliglobacillus glycogenes ATCC 29475', M95652 ; Rhodo- bacter sphtreroides IF0 12203, D 16425; Paracoccus denitrificans LMG 42 18', X69 1 59; H.vphoniicrobium vulgare ATCC 27500, X53 182; Methylosinus trichosporiurn OB3b", M29024 ; Methylocystis parvus OBBP', M29026 ; Rhodo- pseudonionuv viridis ATCC 19567, D253 14; Blastobacter denitrrJicuns LMG 8443, X66025 ; Methylobacterium organophilum NCIMB 1 1278T,D32226; Xanthobacter agilis SA35, D 16425; Blnstohacter cupsulatus ATCC 43294, X73042; Brucella uhortus I 1 / 19, X13695 ; Acidomonas methanolica IMET 10945', D30770; Bosea thiooxidans BI-42", X8 1044; Ancylobacrrr aquaticus DSM lOl", M27803 ; Thiobacillus novellus IAM 12100, D32247; and Bacillus subtilis 168, X00007, as outgroup. Regions that were either not sequenced in one or more reference organisms or had alignment uncertainty, i.e. positions 1-45, 70-100, 183-21 8, 45 1-480,998-11048 and 1376-3' terminus (E.coli nomencla- ture) were omitted from the analyses, so in total 1078 nucleotides were used in the analysis. Pairwise evolutionary distances (expressed as estimated changes per 100 nucleotides) were computed by use of the correction of Jukes & Cantor (1963). A tentative unrooted phylogenetic tree was Fig. 1. Electron micrographs of cells of strain IMIT. (a) constructed by using a 'maximum topological similarity' Negatively stained preparation. Note the subpolar location of algorithm (Chumakov & Yushmanov, 1988) and the the single flagellum; a large amount of capsular material is program TREE of the GENEBEE package (Brodsky et al., 1991). evident. (b, c) Ultrathin sections; different stages of cell The rooted-by-outgroup phylogenetic tree was constructed division. The arrow in (b) indicates a part of the capsule by the neighbour-joining method (Saitou & Nei, 1987) with covered with spikes. (c) Constrictions in the final stage of cell division. Bars, 0.5 pm. bootstrap analysis of 100 trees and using the TREECON package programs (Van de Peer & De Wachter, 1994).

RESULTS (0.8-1.5) pm in size, with a cell shape sometimes approaching coccoid. Reproduction occurred by Morphology binary fission. The separating daughter cells were The bacterial isolates were represented by Gram- equal in size to the parent cells and had one lateral negative, asporogenous, colourless rods (0.5-0.7) x flagellum (Fig. la). After division, the cells remained

.. __ __. International Journal of Systematic Bacteriology 48 1315 N. V. Doronina and others

~- ~~~ ._

Table I. Cellular fatty acid composition of colourless and pink-pigmented serine pathway methylobacteria (% of total fatty acids)

Bacteria were grown on agarized synthetic medium with 0-5% (v/v) methanol or 0.3 % (w/v) methylamine for 24 h at 29 "C.

Strain n16:O al6: 1* n18:O c18: 1109 c18: It07 t18: lc07 cyl9:O cMel8: I Hydroxy acids 3-OHt

* Total hexadecenoic acids shown, including palmitoleic, palmitelaidinic and 16: 1 with position of double bond not established. -i- Analysed as methoxy derivative form. connected by a constriction apparently formed betaine or under the gas mixture of C0,+H,+02. by the outer membrane (Fig. lb, c). The bacteria Strain 14 also failed to grow on butanol, sorbitol and studied had a distinct capsule covered with spikes fructose, but could utilize N,N-dimethylglycine. All (Fig. la). strains utilized methylamine, some amino acids, urea, peptone, ammonium salts and nitrates as nitrogen Ph y siolog ica I characteristics sources. The optimum growth temperature was 28-30 OC, while pH optimum was 7.0-7.2. Growth did Colonies on glucose-potato agar (2 d) were punctate, not occur in the presence of 3 Yo NaCl. The generation round, convex, lustrous, white, translucent, uniform in time was 7-12 h on medium K with 0.5 YOCH,OH. consistency and mucoid. Streak cultures on meat- peptone agar were distinct and white, with even edges, Chemotaxonomic characteristics a smooth surface and a mucoid consistency. Growth was moderate and odourless. Growth on agarized Table 1 shows cellular fatty acid profiles for the new medium with methanol was profuse, diffuse and isolates and members of the genera Methylobacterium, mucoid, and the colonies had even edges and a smooth Aminohacter, Methylorhabdus and Hyphomicrobiiini. surfkce. The strains hydrolysed starch and gelatin The methylation of cis-vaccenic acid (c18:1c,J7) to lacto- (weakly), but not cellulose and casein milk, although bacillic acid (cy19:J must be considered as a remark- they caused the latter to become alkaline (pH 6.5-8.8). able difference between colourless methylobacteria Acidification was observed on glucose-containing me- and the PPFMs. The virtual absence of lactobacillic dium without gas formation. All strains were strict acid in the fatty acid spectra of the PPFMs has also aerobes, although they reduced nitrates to nitrites been reported by other authors (Urakami & slowly (2 weeks). They did not form indole or Komagata, 1984). Another feature of the fatty acid ammonia, but evolved hydrogen sulfide on meat- spectra of all the strains is the occurrence of cis- infusion broth. Methyl red and Voges-Proskauer tests methyloctadec- 1 1-enoic acid, an isomer of lactobacillic were negative. No water-soluble fluorescent pigment acid which is also formed by methylation of cis- was produced on King A and B media. Urease and vaccenic acid. This fatty acid is synthesized by some oxidase were present, catalase activity was very low. Gram-negative stalked bacteria (Prosthecomicrobiu~11, The strains grew well on mineral salt liquid medium Caulohacter, Asticaccaulis), but it is completely absent with methanol (optimal concentration 0.5 YO)or meth- in members of the genera Methylobacterium, Amirio- ylamine (0.3%) as the carbon and energy sources. bacter and Hyphomicrobium (Andreev et al., 1986). Additional growth factors (yeast autolysate, biotin, The PPFMs, Methylorhabdus multivorans and the new thiamin, nicotinic acid and riboflavin) were not isolates contained primarily 3-OH C,,: acid, whereas required. Bacterial growth was also supported by some Hyphomicrobium spp. contained 3-OH C,, : ,) and alcohols (ethanol, butanol, glycerol and sorbitol), Aminobacter strains contained large amounts of 3-OH organic acids (pyruvic, malic, succinic and fumaric C,,: acid (Urakami et al., 1992). Remarkably, Hypho- acids), carbohydrates (maltose, glucose, fructose, su- microbium zavarzinii had ubiquinone Q-9, while all the crose and arabinose), as well as some amino acids other serine pathway methylobacteria possessed (glutamate and sarcosine). Strains IM IT, UZ, 9AA, ubiquinone Q- 10. IOAA, 26AA and 37AA did not grow on methane, As seen from Table 2, the major phospholipids in the mono- and dichloromethane, citrate, methionine, as- serine pathway facultative methylobacteria are paragine, lysine, arginine, histidine, glycine, valine, phosphatidylethanolamine ( 12-50 "/o) and phospha-

~ 1316 International Journal of Systematic Bacteriology 48 Methylopila, a new genus of aerobic methylobacteria ____~

Table 2. Phospholipid composition of colourless and pink-pigmented serine pathway methylobacteria ...... ) Abbreviations : PEA, phosphatidylethanolamine ; PG, phosphatidylglycerol ; DPG, diphosphatidylglycerol ; PA, phosphatidic acid; PS, phosphatidylserine ; PC, phosphatidylcholine; LL, lysolycetine ; 8-1 2, unidentified phospholipids.

Strain Phospholipid (YO)

PEA PG DPG PA PS PC LL 8 9 10 11 12

IMIT 40 12 8 3 1 18 7 500 50 uz 42 11 8 3 0 17 6 400 60 14 23 20 5 3 0 36 422212 Aininohucter aminovorans 37 16 10 2 0 25 112053 ATCC 23314T Methj-lorlrabdus multivorans 24 13 4 1 0 43 2 221 33 ATCC 5 I 890T Met hylo hm t er uim o rganoplz ilum 12 21 5 2 1 47 6201 11 ATCC 27886T Methj*lohtrcteriumextorquens NCIB 9399T 26 14 4 2 3 35 2 204 44

Table 3. Enzyme activities in cell extracts of the new isolates of methylobacteria grown on different substrates ...... % PMS, phenazine methosulfate ; PEP, phosphoenolpyruvate. There was no activity with any of the following enzymes and cofactors : formaldehyde dehydrogenase (NAD'), formate dehydrogenase (NAD'), isocitrate dehydrogenase (NAD+), isocitrate lyase and pl'ruvate carboxylase.

Enzyme (cofactor) Enzyme activity [nmol min-' (mg protein)-']

Strain lMIT Strain UZ Strain 14

Methanol Succinate Methanol Succinate Methanol Succinate

Methanol dehydrogenase (PMS) 362 118 250 71 310 102 N-Methylglutamate dehydrogenase (PMS) 0" 0 0" 0 0" 0 Met h y lami ne de hydrogenase (PMS) 226" 21 180" 14 210" 22 Formaldehyde dehydrogenase (PMS) 77 9 68 7 82 10 Formate dehydrogenase (PMS) 129 22 98 11 135 15 Hydroxypyruvate reductase (NADH) 1930 497 1910 329 2100 310 Hydroxypvruvate reductase (NADPH) 759 387 720 250 815 200 Serine-glyoxylate aminotransferase (NADH) 237 90 240 52 330 100 Malate lyase (ATP, CoA) 52 19 49 15 67 11 Glycerate kinase (ATP) 28 12 34 10 35 12 Pyruvate dehydrogenase (NAD+) 47 63 52 74 49 76 Citrate syn thase 10 35 15 55 11 65 lsocitrate dehydrogenase (NADP') 41 72 49 84 56 90 Malate dehydrogenase (NADH) 51 215 68 290 70 325 Malate dehydrogenase (N ADPH) 55 94 72 110 69 120 a-Ketoglut arate dehydrogenase (NAD +) 8 14 8 17 10 21 Succinate dehydrogenase (PMS) 27 88 30 85 25 76 PEP carbo vylase + Acetyl-CoA 88 31 85 28 90 27 - Acetyi-CoA 39 19 35 15 42 15 Methylamine-grown cells. tidylcholine (17-47 Yo). Strains IMIT, UZ, 14, 9AA, hydroxybutyrate (20-40 YO,dry cell weight) under 26AA and 34AA have been shown (Trotsenko et al., nitrogen limitation and excess of carbon source (meth- 1993) to accumulate a large amount of poly-/3- anol). _ __ International Journal of Systematic Bacteriology 48 1317 N. V. Doronina and others

Table 4. DNA-DNA relatedness between colourless and pink-pigmented serine pathway methylobacteria

Strain Hybridization (%) with labelled DNA from strains :

IMIT UZ ATCC 23314 ATCC 51890 ATCC 27886 NCIB 9399

IMIT 100 78 5 5 4 4 uz 83 100 5 5 3 3 14 80 88 6 4 3 4 9AA 78 76 6 5 3 3 1OAA 86 81 5 5 3 3 26AA 80 78 4 5 3 3 37AA 80 82 5 5 3 3 Aininobacter aminovorans ATCC 233 14' 44 100 4 3 3 Methylorhabdus multivorans ATCC 5 1890T 5 5 5 100 4 3 Methylobacterium organophilum ATCC 27886' 54 5 4 100 16 Methylobacterium extorquens NCIB 9399T 5 5 4 3 15 100 Hyphomicrobiunz zavarzinii IFAM ZV-622T 44 4 4 3 3

Metabolic characteristics degree of genomic similarity between the strains and members of the other taxa was assessed by To establish the routes for primary and intermediary DNA-DNA hybridization (Table 4). The DNA metabolism in our isolates, the enzyme activities were hybridization values among the new strains were very determined in extracts of cells grown on methanol, high (76-90 YO),making it possible to assign all of them methylamine or succinate. Since similar results were to the same taxonomic cluster (genus and species). On obtained for all the isolates tested the enzyme activities the other hand, they had a low degree of DNA are presented for only three strains. As shown in Table hybridization with the representatives of the genera they oxidize methanol and methylamine to CO, 3 Meth y lobac t er ium, Amin o bac t er , Hyph omicro b iuni , through formaldehyde and formate by the appropriate Methylorhabdus and Pseudomonas. dehydrogenases active with phenazine methosulfate (PMS). The enzymes of the N-methylglutamate pathway were not detected. The serine pathway specific Comparative sequence analysis enzymes (hydroxypyruvate reductase and serine- glyoxylate aminotransferase) were found. Due to the The 1331 nucleotides of the strain IMIT 16s rRNA absence of the isocitrate lyase (ICL) activity the strains gene were sequenced, corresponding to positions 8- consequently employed the ICL-negative variant of 1406 of the E. coli sequence. The phylogenetic analysis the serine pathway. Alternatively, the key enzymes employing different algorithms showed similar results. of the ribulose monophosphate and ribulose The branching pattern of the methylotrophic strains bisphosphate pathways, i.e. 3-hexulose phosphate correlated perfectly with previous results of Bratina et synthase, ribulose bisphosphate carboxylase and al. (1992) and Janvier & Grimont (1995). According to phosphoribulokinase were not present. The new iso- our data strain IMIT appears to be related to the lates had low or zero activities for the pentose members of the a-subclass of Pvoteobacteria. The phosphate pathway enzymes (glucose-6-phosphate affiliation of this strain to the a-subclass is supported and 6-phosphogluconate dehydrogenases as well as by signature analysis. Some features characteristic for the 16s rDNA molecule of the a-subclass, such as a 2-keto-3-deoxy-6-phosphogluconatealdolase). How- ever, they contained a complete set of enzymes of shortened helix in nucleotide positions 184193 (E.coli the tricarboxylic acid cycle playing primarily a bio- numbering) and all specific signature positions (Woese, synthetic role during methylotrophic growth. CO, was 1987) were found in the sequences analysed. fixed by phosphoenolpyruvate carboxylase stimulated The matrix of full sequence similarity values for strain by acetyl-CoA. Pyruvate carboxylase was absent. IM1' and representative members of the Proteo- bacteria indicate that for strain IM 1 the highest Genomic characteristics degree of relationship is found with certain members of the a-2 subclass, e.g. Xanthobacter agilis (92.5% The G + C contents of the DNA of strains IM lT,UZ, similarity) and Brucella abortus (92.7 % similarity). 14, 9AA, lOAA, 26AA and 37AA were estimated via These values, however, are not necessarily an indi- melting point ( TnJ as 66.5-69.2 mol %. Because DNA cation of specific relationship with these organisms, hybridization remains the optimal method for because they belong to different clusters of the a-2 measuring the degree of relatedness between highly subclass of Proteobacteria. In the unrooted tree related organisms (Stackebrandt & Goebel, 1994), the constructed (data not shown), strain IMIT forms a

1318 International Journal of Systematic Bacteriology 48 Methylopila, a new genus of aerobic methylobacteria ~- -~

Bosea thiooxidans - Methylobacterium organophilum Blastobacter denitrificans 100 Methylocystis parvus Methylosin us trichosporium a-2

. Brucella abortus 100 Blastobacter capsulatus Hyphomicrobium vulgare /

Fig. 2. Rooted-by-outgroup phylogenetic tree showing relationships among Methylopila capsulata strain lMIT and other members of the Proteobacteria including meth y Iot ro ph ic genera. Bacillus sub tilis is the outgroup. Numbers refer to bootstrap values (more than 50). Bar indicates a distance of 0.05.

separate branch within the a-2 subclass. In the rooted- serine pathway methylobacteria are summarized in by-outgroup tree (Fig. 2), the bootstrap value is too Table 5. low (Woese, 1987) for the branching point of strain IMIT. Hence, strain IMIT is a new branch of the The results of our 16s rDNA sequence analysis showed that the genus forms a distinct branch a-2 subclass of Proteobacteria. Methylopila within the a-subclass of the Proteobacteria, separated from known genera of aerobic methylotrophic bac- DISCUSSION teria. Phylogenetic relationships that were derived from the 16s rDNA sequence analysis correlate well The new isolates were distinguished clearly from with the results of 5s rDNA sequencing (Boulygina et members of the genus Pseudomonas sensu stricto, on al., 1993). Genotypic data also demonstrated that a the basis of the following characteristics : utilization of new genus was clearly separated from all other C,-compounds (methanol and methylated amines), described taxa of the serine pathway methylobacteria. quinone system, cellular fatty acid composition On the basis of the data presented here and that (Urakami et al., 1992), and low level of DNA-DNA published previously by Govorukhina et al. (1989) we hybridization. As mentioned above, only four generic propose that the seven isolates studied be placed in a names have been validly published for Gram-negative new genus, for which we propose the name Methylopila serine pathway methylobacteria : Methylobacterium gen. nov. The genus contains a single species, which is (Patt et d.,1976), Aminobacter (Urakami et al., 1992), automatically the type species of the genus. The strains, Hyphomic *rohium(Hirsch, 1984), and Methylorhabdus currently placed within the genus Methylopila were (Doronina et al., 1995). However, our isolates differed originally described under the name ' Methylo- from the PPFMs belonging to the genus Methylo- microhium capsulatum ' (Govorukhina et al., 1989), bacteriun~by some morphological features, colour of and we therefore propose that the new species be colonies, hydrolysis of gelatin and starch, cellular fatty named Methylopila capsulata sp. nov. acid composition and low DNA similarity. The new strains were distinguished from non-pigmented members of the genus Aminobacter by methanol Description of the genus Methylopila gen. nov. utilization, primary C -met a boli sm, cellular fatty acid Methylopila (Me.thyl.o.pi.la. M.L.n. methyl the composition, DNA G+C content and low DNA methyl group; Gr. adj. pila ball or sphere; M.L. similarit) levels. They also differed from members of Methylopila methyl-using sphere). the genus Hyphomicrobium by their inability to form hyphae, quinone system, primary methylamine metab- When grown on methanol mineral salt medium, the olism, cellular fatty acid composition and low DNA cells are motile rods, usually 0.5-0.7 ym in diameter similari ti and from Methylorhabdus multivorans by and 1-0-1.3 ym in length, which occur singly or in morphological features, the presence of oxidase and pairs. Gram-negative. Non-pigmented. Endospores low DNA similarity. Based on these results, our are absent. Multiply by fission. No prosthecae isolates are close neighbours and might be placed in a detected. Colonies are white. Pyocyanin and separate taxon, for which we propose the generic name fluorescein are not produced. Growth is supported by Methy lop ill, with the type species Meth y lop ila nutrient agar and PYG agar. The methyl red and capsulata. The major characteristics differentiating the Voges-Proskauer tests are negative. Oxidase- and genus Mcthylopila from the other related genera of urease-positive. Acids are produced from sugars

~~ ~ International Journal of Systematic Bacteriology 48 1319 N. V. Doronina and others

Table 5. Major differentiating characteristics of the serine pathway facultative methylobacteria belonging to various genera

Character Methylobacterium Aminobactev Hyphomicrobium Methylorhabdus Methylopila

Morphology (flagella) + + + - + Reproduction by : Budding - + + - - Division + - - + + Hyphae formation - - + - - Oxidase + + - - + Catalase + + + + - Carotenoids + - - - - Methylamine metabolism : Amine dehydrogenase + - - + + N-Methylglutamate + + + - - Formaldehyde dehydrogenase - - - + - (NAD+/GSH) Isocitrate lyase - + + - - Cyclopropane acid cy,,: ,, Trace + + + + Major ubiquinone Q-10 Q-10 Q-9 Q-10 Q-10 Utilization of methanol + - + + + DNA G + C content (mol %) 60-70 62-64 61-65 66-67 66-70 oxidatively, but not fermentatively. Nitrates are pairs and having a capsule covered with spikes. reduced to nitrites slowly. Chemo-organotrophic and Colonies grown on nutrient agar or glucose-potato facultatively methylotrophic assimilating C, com- agar are round ;viscous, semitransparent, convex, even pounds by the isocitrate lyase- serine pathway. edged, 2 mm in diameter. Cells accumulate poly-p- Vitamins are not required for growth. Ammonium hydroxybutyrate. Does not form indole or NH,. salts, nitrate, urea, peptone, some amino acids and Catalase activity is very low. Produce H,S during methylated amines are utilized as nitrogen sources. growth in nutrient broth. Utilizable carbon sources are Strictly aerobic, with respiratory metabolism. Good methanol, me th ylated amines, but anol, ethanol, growth occurs between pH 6.5 and 7.5, but not above glycerol, maltose, sucrose, L-arabinose, D-fructose, D- pH 9.0 and below pH 5.0. Grows well at 28 and 35 "C glucose, succinate, fumarate, pyruvate. Gelatin and but not at 42 "C. No growth in the presence of 3% starch are hydrolysed weakly. Methanol and methyl- NaCl. The cell fatty acid profile is characterized by the amine are oxidized by the appropriate PMS-linked presence of 60-70 YO cis-vaccenic acid (c18:lw,). The dehydrogenases to formaldehyde which is assimilated major hydroxy acid is 3-OH C,,: o. The quinone system by the isocitrate lyase- serine pathway. The mol% is ubiquinone Q- 10. Dominant phospholipids are G + C of the DNA is 67-2 (T,). The type strain IMIT phosphatidylethanolamine and phosphatidylcholine. was isolated from soil of Tashkent city (Uzbekistan) The DNA G + C content ranges from 66 to 70 mol %. and is deposited in the Russian Collection of Micro- The levels of DNA-DNA hybridization with members organisms as Methylopila capsulata VKM B- 1606T. of the genera Methylorhabdus, Methylobacterium, The species also includes strains UZ, 14, 9AA, IOAA, Aminobac t er, Hyph om icro b ium and Pseudom onas are 26AA and 37AA. less than 10%. The genus Methylop~labelongs to the a-2 subclass of the Proteobacteria, as revealed by ACKNOWLEDGEMENTS comparative sequence analysis of the 16s rDNA, and is phylogenetically distinct from the other methylo- The authors are indebted to the referees for valuable trophic genera. The type species is Methylopila comments on the manuscript. This work was financially capsula ta. supported by the RFBR grants 96-04-50820 and 98-04- 48144. Description of Methylopila capsulata sp. nov. REFERENCES Methylopila capsulata (cap.su.la'ta. L.n. capsule a small chest, capsule; M.L. adj. capsulata having a Andreev, L. B., Akimov, V. N. & Nikitin, D. 1. (1986). Peculiarities of fatty acid composition of the genus Caulobacter. Folia capsule). Microbiot 31, 144-153. In addition to the characteristics that define the genus, Boulygina, E. S., Chumakov, K. M. & Netrusov, A. 1. (1993). it has the characteristics described below. Cells are Systematics of gram-negative methylotrophic bacteria based on motile pleomorphic rods or cocci occurring singly or in 5s rRNA sequences. In Microbial Growth on C, Compounds, pp.

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