International Journal of Systematic Bacteriology (1 998), 48, 13 13-1 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 bac- teria (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- 13 10 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.