Hyphomicrobium Chloromethanicum Sp. Nov. and Methylobacterium

International Journal of Systematic and Evolutionary Microbiology (2001), 51, 119–122 Printed in Great Britain

Hyphomicrobium chloromethanicum sp. nov. NOTE and Methylobacterium chloromethanicum sp. nov., chloromethane-utilizing bacteria isolated from a polluted environment

Ian R. McDonald,1 Nina V. Doronina,2 Yuri A. Trotsenko,2 Craig McAnulla1 and J. Colin Murrell1

Author for correspondence: Ian R. McDonald. Tel: j44 24 765 28362. Fax: j44 24 765 23568. e-mail: ip!dna.bio.warwick.ac.uk

1 Department of Biological Two chloromethane-utilizing facultatively methylotrophic bacteria, strains Sciences, University of CM2T and CM4T, were isolated from soil at a petrochemical factory. On the Warwick, Coventry CV4 7AL, UK basis of their morphological, physiological and genotypical properties, strain CM2T (l VKM B-2176T l NCIMB 13687T) is proposed as a new species of the 2 Institute of Biochemistry T and Physiology of genus Hyphomicrobium, Hyphomicrobium chloromethanicum, and strain CM4 Microorganisms, Russian (l VKM B-2223T l NCIMB 13688T) as a new species of the genus Academy of Sciences, Methylobacterium, Methylobacterium chloromethanicum. 142292 Puschino, Moscow region, Russia

Keywords: chloromethane, Hyphomicrobium, Methylobacterium

Chloromethane (methyl chloride; CH$Cl) is the most chloromethane. Cells were rod-shaped, 0n8–1n0i1n0– abundant halocarbon in the atmosphere and is re- 8n0 µm, occurring singly or occasionally in rosettes. sponsible for 15–20% of chlorine-catalysed ozone Motility occurs by a single flagellum, cells stain Gram- destruction in the stratosphere. It is released at an negative and colonies are pigmented pale pink on ' estimated global rate of 3n5–5i10 tons per year, methanol salts agar plates. They are strictly aerobic, primarily from natural sources, with less than 1% of catalase- and oxidase-positive, and the optimum tem- the flux from industrial sources (Khalil et al., 1999). perature for growth is 25–30 mC (Green & Bousfield, Eight strains of chloromethane-utilizing bacteria 1983; Green et al., 1988). Methylobacterium sp. strain (CM1, CM2, CM4, CM9, CM29, CM30, CM34 and CM4T has been studied to determine a pathway for CM35) were isolated from soil samples near a petro- conversion of chloromethane to formate (Vannelli et chemical factory in Russia (Doronina et al., 1996). The al., 1998, 1999; Studer et al., 1999). It was shown that phenotypic characteristics of these strains indicated the protein CmuA first transfers the methyl group of that they were of the genera Hyphomicrobium (CM1, chloromethane to a corrinoid protein, from where it is CM2, CM9, CM29, CM35) and Methylobacterium transferred to tetrahydrofolate by the protein CmuB. (CM4, CM30, CM34) (Doronina et al., 1996). The Both CmuA and CmuB displayed sequence similarity Hyphomicrobium strains had characteristics typical of to methyltransferases of methanogenic members of the the Hyphomicrobium type species Hyphomicrobium domain Archaea. As well as growing on chloro- vulgare except for their growth on chloromethane. The methane, CM2T and CM4T will also oxidize, but not cells were rod-shaped, oval or bean-shaped, 0n3–1n2i grow on, bromomethane and iodomethane (C. 1–3 µm, having polar prosthecae. The bacteria were McAnulla and others, unpublished; Vannelli et al., appendaged, reproduced by budding (Hirsch, 1989), 1998). Here we report the phylogenetic characteri- and were restricted facultative methylotrophs capable zation based on the 16S rRNA gene sequences, and of growth on one-carbon compounds such as methanol propose names for strains CM2T and CM4T. and methylamine (Harder & Attwood, 1978). The DNA of strains CM2T, CM4T, CM29 and CM30 The Methylobacterium strains (CM4, CM30, CM34) was extracted (Marmur, 1961), and the 16S rRNA have characteristics typical of the type species Methyl- genes were amplified and sequenced (Lane, 1991). obacterium organophilum apart from their growth on Complete 16S rRNA sequences were obtained for T T ...... strains CM2 and CM4 , and partial sequences The GenBank accession numbers for the 16S rRNA gene sequence of strains (700 bp) were obtained for strains CM29 and CM30. CM2T and CM4T are AF198623 and AF198624, respectively. The four sequences were compared using the Genetics

01479 # 2001 IUMS 119 I. R. McDonald and others

...... Fig. 2. Phylogenetic analysis of the 16S rRNA sequences of Fig. 1. Phylogenetic analysis of the 16S rRNA sequences of T Hyphomicrobium sp. strain CM2T and other Hyphomicrobium Methylobacterium sp. strain CM4 and other Methylobacterium strains and related genera. The dendrogram shows the results strains and related genera. The dendrogram shows the results of an analysis in which DNADIST was used. Bootstrap values of an analysis in which DNADIST was used. Bootstrap values greater than 50% derived from 100 replicates are also shown. greater than 50% derived from 100 replicates are also shown. The bar represents 10% sequence divergence, as determined by The bar represents 10% sequence divergence, as determined by measuring the lengths of the horizontal lines connecting any measuring the lengths of the horizontal lines connecting any two species. two species.

Computer Group Sequence analysis software package microbium denitrificans (96n3%) and Hyphomicrobium methylovorum (96n0%), and as such is distinct enough version 8.0 (Genetics Computer Group, Madison, T Wisconsin). Hyphomicrobium strains CM2T and CM29 to define strain CM2 as a new species. were found to be identical, and Methylobacterium Analysis of the 16S rDNA of strain CM4T (Fig. 2) strains CM4T and CM30 were also identical to each showed it to cluster within the Methylobacterium other. The complete 16S rRNA gene sequences from species, as supported by bootstrap values, and is most T T strains CM2 and CM4 were aligned, using the  closely related to Methylobacterium extorquens (98n0% program for sequence alignment (O. Strunk and sequence similarity), Methylobacterium rhodesianum others; http:\\www.mikro.biologie.tu-muenchen.de), (97n6%), Methylobacterium zatmanii (97n8%) and to representative organisms from the same and related Methylobacterium rhodinum (96n4%). However, strain genera of bacteria, and their phylogenetic position was CM4T is distinct from the other Methylobacterium determined using the , ,  and species, being the only Methylobacterium species to  programs of the  package (Felsenstein, grow on chloromethane, and as such is being described 1993). Phylogenetic dendrograms were constructed as a new species based on DNA–DNA reassociation from the distance data using the Fitch–Margoliash data (Table 1). None of the eight type strains of method and the dendrograms were drawn using the Hyphomicrobium species tested [H. facile subsp. facile TreeView program version 1.5 (Page, 1996). Phylo- H-526T (DSM 1565), H. facile subsp. ureaphilum CO- genetic analysis of the 16S rDNA of strain CM2T (Fig. 582T (ATCC 27492), H. facile subsp. tolerans I-551T 1) showed it to be located within cluster II of the (ATCC 27489), H. vulgare MC-750T (ATCC 27500), Hyphomicrobium species (Rainey et al., 1998), as Hyphomicrobium aestuarii NQ-521T (NCIMB 11052), supported by bootstrap values. The 16S rDNA of H. denitrificans HA-905, Hyphomicrobium hollandicum strain CM2T has high sequence similarity with Hypho- KB 677T (ATCC 27498), Hyphomicrobium zavarzinii T T microbium facile subsp. facile H-526 (97n1%),Hypho- ZV 622 (ATCC 27496)] could grow on chloromethane

120 International Journal of Systematic and Evolutionary Microbiology 51 Chloromethane-utilizing bacteria

Table 1. DNA–DNA hybridization of Methylobacterium Growth is strictly aerobic. Grows on chloromethane as chloromethanicum CM4T with representatives of the sole carbon and energy source. Methanol, methyl- genus Methylobacterium amine, succinate and fumarate are also growth sub- ...... strates. Assimilation of C" compounds is via the serine DNA–DNA hybridization was carried out according to the pathway, the major quinone is Q"! and the main fatty method of Denhardt (1966). acid is C"):". The optimum temperature for growth is 30 mC. The optimum pH for growth is 6n7–7n2. The Methylobacterium species DNA–DNA GjC content of DNA is 64n4 mol%. The GenBank hybridization accession number for the 16S rRNA gene sequence of (%) strain CM4T is AF198624. M. chloromethanicum strain CM4T was isolated from soil at the Nizhekamsk T M. radiotolerans DSM 1819 18n2 petrochemical factory, Tatarstan, Russia. The type T M. organophilum NCIMB 11278 14n6 strain is strain CM4T and is deposited in the All- T M. rhodinum DSM 2163 19n6 Russian Collection of Microorganisms as VKM B- T M. extorquens ATCC 43645 62n8 2223T and the National Collections of Industrial, Food T M. rhodesianum DSM 5687 21n8 and Marine Bacteria as NCIMB 13688T. M. zatmanii DSM 5688T 22n6 T M. aminovorans ATCC 51358 47n3 Acknowledgements M. mesophilicum ATCC 29983T 14n2 M. fujisawaense NCIMB 12417T 17n4 We acknowledge the financial support provided by the Natural Environment Research Council (GR9\2192) and INTAS (94-3122). We thank Don Kelly (University of Warwick) for useful comments on the manuscript and as sole carbon and energy source, nor could M. Christian Gliesche (Kiel University) for kindly supplying extorquens. Hyphomicrobium type species. References Description of Hyphomicrobium chloromethanicum sp. nov. Denhardt, D. T. (1966). A membrane filter technique for de- termination of complementary DNA. Biochem Biophys Res Hyphomicrobium chloromethanicum (chlo.ro.me. Commun 23, 641–646. thahni.cum. N.L. n. chloromethanicum chloromethane- Doronina, N. V., Sokolov, A. P. & Trotsenko, Y. A. (1996). utilizing). Isolation and initial characterisation of aerobic chloromethane- Gram-negative, monoprosthecate rods having buds on utilising bacteria. FEMS Microbiol Lett 142, 179–183. the prosthecal tips. Cells are 0n5–0n6 µm in diameter Felsenstein, J. (1993).  (Phylogeny Inference Package) and 1n3–1n8 µm long. Cells are motile and non- version 3.5c. Department of Genetics, University of Wash- pigmented. Growth is aerobic. Grows on chloro- ington, Seattle, USA. methane, methanol, methylamine and ethanol as sole Green, P. N. & Bousfield, I. J. (1983). Emendation of Methylo- carbon and energy sources. C"-compound assimilation bacterium Patt, Cole and Hanson 1976; Methylobacterium is via the serine pathway, the major quinone is Q* and rhodinum (Heumann 1962) comb. nov. corrig.; Methylobac- terium radiotolerans (Ito and Iizuka 1971) comb. nov. corrig.; the main fatty acid is C"):". The optimum temperature for growth is 28–30 C. The optimum pH for growth is and Methylobacterium mesophilicum (Austin and Goodfellow m 1979) comb. nov. Int J Syst Bacteriol 33, 875–877. 6n5–7n5. The GjC content of DNA is 60n0mol%.H. chloromethanicum has 29n4% DNA–DNA hybridi- Green, P. N., Bousfield, I. J. & Hood, D. (1988). Three new zation to H. zavarzinii ZV-622T (ATCC 27496). The Methylobacterium species: M. rhodesianum sp. nov., M. zatmanii sp. nov., and M. fujisawaense sp. nov. Int J Syst Bacteriol GenBank accession number for the 16S rRNA gene 38, 124–127. sequence of strain CM2T is AF198623. H. chloromethanicum strain CM2T was isolated from soil at the Harder, W. & Attwood, M. M. (1978). Biology, physiology and biochemistry of hyphomicrobia. Adv Microb Physiol 17, 303– Nizhekamsk petrochemical factory, Tatarstan, Russia. 359. The type strain is strain CM2T and is deposited in the All-Russian Collection of Microorganisms as VKM B- Hirsch, P. (1989). Genus Hyphomicrobium Stutzer and Hartleb 1898, 76AL.InBergey’s Manual of Systematic Bacteriology, 9th 2176T, and the National Collections of Industrial, T edn, vol. 3, pp. 1879–1904. Edited by J. T. Staley, M. P. Bryant, Food and Marine Bacteria as NCIMB 13687 . N. Pfenning & J. G. Holt. Baltimore: Williams & Wilkins. Khalil, M. A. K., Moore, R. M., Harper, D. B., Lobert, J. M., Description of Methylobacterium chloromethanicum Erickson, D. J., Koropalov, V., Sturges, W. T. & Keene, W. C. sp. nov. (1999). Natural emissions of chlorine-containing gases: reactive chlorine emissions inventory. J Geophys Res 104, 8333–8346. Methylobacterium chloromethanicum (chlo.ro.me. chloromethanicum Lane, D. J. (1991). 16S\23S rRNA sequencing. In Nucleic Acid thahni.cum. N.L. n. chloromethane- Techniques in Bacterial Systematics, pp. 115–175. Edited by E. utilizing). Stackebrandt & M. Goodfellow. Chichester: Wiley. Gram-negative rods 0n8–1n0 µm in diameter and 2n5– Marmur, J. (1961). A procedure for the isolation of deoxyribo- 3n5 µm long. Cells are motile and pink pigmented. nucleic acid from microorganisms. J Mol Biol 3, 208–218.

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Page, R. D. M. (1996). TreeView: an application to display chloromethane utilization by Methylobacterium sp. strain CM4. phylogenetic trees on personal computers. Comput Appl Biosci Eur J Biochem 264, 242–249. 12, 357–358. Vannelli, T., Studer, M., Kertesz, M. & Leisinger, T. (1998). Rainey, F. A., Ward-Rainey, N., Gliesche, C. G. & Stackebrandt, E. Chloromethane metabolism by Methylobacterium sp. strain (1998). Phylogenetic analysis and intrageneric structure of the CM4. Appl Environ Microbiol 64, 1933–1936. genus Hyphomicrobium and the related genus Filomicrobium. Vannelli, T., Messmer, M., Studer, A., Vuilleumier, S. & Leisinger, Int J Syst Bacteriol 48, 635–639. T. (1999). A corrinoid-dependent catabolic pathway for growth Studer, A., Vuilleumier, S. & Leisinger, T. (1999). Properties of of a Methylobacterium strain with chloromethane. Proc Natl the methylcobalamin:H%folate methyltransferase involved in Acad Sci U S A 96, 4615–4620.

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