INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1991, p. 301-305 Vol. 41, No. 2 0020-7713/91/020301-05$02.00/0 Copyright 0 1991, International Union of Microbiological Societies Contribution of Genome Characteristics to Assessment of Taxonomy of Obligate Methanotrophs J. P. BOWMAN,” L. I. SLY, AND A. C. HAYWARD Department of Microbiology, University of Queensland, Brisbane, Queensland, Australia 4072 The DNAs of a variety of obligate methanotrophic bacteria were analyzed for base composition and nucleotide distribution. Genome molecular weights were determined for representative strains. Similarity ‘\, maps attained by plotting DNA base composition versus nucleotide distribution and genome molecular weight showed that related species formed distinct clusters. Group I methanotrophs were found to form three clusters in both nucleotide distribution and genome size analyses. The first cluster consisted of five Methylomonas species, Methylomonas methanica, Methylomonas fodinarum, Methylomonas aurantiaca, “Methylomonas ru- bra,” and “Methylomonas agile.” The other clusters included both Methylomonas species and Methylococcus species, indicating the heterogeneity within these genera. One cluster contained low-G+ C-content Methylo- coccus strains and included Methylococcus whittenburyi, Methylococcus bovis, Methylococcus vinelandii, Methylococcus luteus, and ‘4Methylococcus ucrainicus. ” The type strains of Methylomonas pelagica and “Methylomonas alba” and a marine methanotrophic strain also clustered with the low-G+C-content Methylococcus group rather than with the genus Methylomonas. “Methylomonas gracilis” also appeared to be genetically distinct from the true Methylomonas species and clustered with the high-G+ C-content Methylococ- cus strains. This cluster included Methylococcus capsulatus, Methylococcus thermophilus, and other moderately thermophilic group I methanotrophic strains. The group I1 methanotrophs belonging to the genera “Methy- losinus” and “Methylocystis” formed separate generic clusters according to genome molecular weight data but not according to nucleotide distribution data. Methanotrophic bacteria are a diverse group of gram- ing the shape of DNA melting profiles. Two measures can be negative bacteria which obligately utilize C, compounds, obtained. The first measure is the DNA melting transition including methane, methanol, and methylamine, as sole width, which is indicative of the nucleotide distribution in a sources of carbon and energy (3, 5, 11, 27). They are unable bacterial genome. The nucleotide distribution (al+ a,) is to utilize as sole energy sources any compounds with car- determined from the time interval required for DNA to bon-carbon bonds. The taxonomy of methanotrophs is based denature one standard deviation to the left (a,)and right (a,) mainly on morphological characters, and the systematics of of the melting temperature and is expressed as G+C content. this group remains confused and incomplete (26). Studies on The second measure is the ratio (a,/a,), which is a measure biochemical pathways, ultrastructure of intracytoplasmic of the skewness or asymmetry of the melting curve. The membranes (3, ll),16s rRNA sequences (25), and fatty acid results of De Ley (7) showed that nucleotide distributions contents (1, 2, 20) have shown that methanotrophs form two within bacterial genera appear to be consistent and thus can major groups (groups I and 11). The group I methanotrophs be used effectively in taxonomic studies. Likewise, the use include the genera Methylomonas and Methy Eococcus and of DNA renaturation techniques has proved to be a useful are characterized by the presence of stacked vesicular disks means to accurately determine genome molecular weight of intracytoplasmic membrane, the ribulose monophosphate (10). These properties of DNA were used in this study in an pathway for C, compound incorporation, and mainly 16:l attempt to observe the inter- and intrageneric relationships and 16:O fatty acids; these organisms appear to belong to the of various representatives of methane-utilizing bacteria and y subdivision of the proteobacteria. The group I1 methano- to assess the taxonomy of these organisms as a guide to trophs (the genera “Methylosinus” and “Methylocystis”) further study. are characterized by intracytoplasmic membranes that are arranged peripherally in the cell parallel to the cytoplasmic MATERIALS AND METHODS membrane, the serine pathway for C, compounds, and mainly 18:l fatty acid and belong to the 01 subdivision of the Bacterial strains. The strains used in this study are listed in proteobacteria. Currently only the group I genera Methy- Table 1. lomonas and Methylococcus have valid taxonomic status Cultivation. Methane-utilizing strains were grown on so- (17, 22). The other genera are not on the Approved Lists of lidified NMS medium (5) under a methane-air-CO, (5:4:1) Bacterial Names (17, 22) and have no standing in bacterial atmosphere. Marine methanotrophic strain A4 was culti- nomenclature. Chemotaxonomic and immunological analy- vated on NMS medium supplemented with 0.5% NaCl and 5 ses (4, 6, 8, 15, 16) have clearly shown that the group I ml of Staley vitamin solution (24) per liter. Methylomonas methanotrophs can be separated into three groups. pelagica was grown on NMS medium prepared with artificial The physicochemical properties of DNA have proved to seawater (derived from Difco marine agar 2216 [catalog no. be useful as a means of characterizing and identifying 0979]), supplemented with 5 ml of vitamin solution per liter, budding and hyphal bacteria (9, 13). DNA compositional and solidified with 1% agarose (type V) (21). Most strains nucleotide distribution studies (7) basically involve measur- were incubated in the dark at 28°C; the exceptions were Methylococcus capsulatus strains, “Methylomonas graci- lis” strains, and Methylococcus sp. strains JB87, JB137, * Corresponding author. JB146, and JB173, which were incubated at 45°C. 301 302 BOWMAN ET AL. INT. J. SYST.BACTERIOL. TABLE 1. Physicochemical properties of DNAs isolated from obligate methanotrophs ~ ~-~~~ Nucleotide Laboratory G+C content distribution: Asymmetry: Genome size Strain (source)n no. (mol%) Ul + ur Ubr (lo9 Da) (mol% G+C) 1 Marine methanotrophic strain A4 (M. E. Lidstrom)b 55.4 14.4 1.5 1.6 2 Methylococcus bovis UQM 3504 (IMET 10593) 51.9 14.2 1.4 1.7 3 Methylococcus luteus UQM 3304T (NCIB 11914T)‘ 50.8 13.5 1.3 1.8 4 Methylococcus luteus IMV 3098T (Methylococcus bovis VKM-6T) 49.8 13.6 1.5 ND~ 5 “Methylococcus ucrainicus” UQM 3305* (NCIB 11915T) 48.7 13.9 1.3 1.8 6 Methylococcus vinelandii UQM 3586= (IMV 3030T) 52.8 13.3 1.3 1.5 7 Methylococcus whittenburyi UQM 3310 (NCIB 11128) 53.7 13.1 1.4 1.6 8 Methylococcus capsulatus UQM 3302 (NCIB 11132) 62.5 10.9 1.1 2.8 9 Methylococcus capsulatus JB175 62.3 10.9 1.1 2.4 10 Methylococcus sp. strain JB140 63.4 11.4 1.1 2.7 11 Methylococcus sp. strain JB87 65.1 10.3 1.0 ND 12 Methylococcus sp. strain JB137 65.2 10.7 1.1 3.0 13 Methylococcus sp. strain JB146 64.9 10.2 1.1 2.7 14 Methylococcus sp. strain JB173 65.6 11.5 1.1 ND 15 Methylococcus thermophilus UQM 35ST (IMV 3037T) 60.7 10.2 1.1 2.5 16 “Methylocystis parvus” UQM 3309T (NCIB 11129T) 66.0 9.4 1.2 2.5 17 “Methylocystis minimus” UQM 3497 (IMET 10578) 63.4 8.6 1.1 2.3 18 “Methylocystis echinoides” UQM 3495 (IMET 10491) 62.0 9.7 1.1 2.2 19 “Methylocystis” sp. strain JB170 64.4 9.4 1.1 2.0 20 “Methylocystis” sp. strain JB190 63.6 8.6 1.o ND 21 “Methylocystis” sp. strain JB196 66.9 9.7 1.0 2.5 22 “Methylomonas agile” UQM 3308 (NCIB 11124) 59.6 12.1 1.1 2.6 23 “Methylomonas alba” UQM 3314T (NCIB 11123T) 54.4 14.5 1.4 2.0 24 Methylomonas aurantiaca UQM 3406T 56.2 11.8 1.3 2.3 25 Methylomonas aurantiaca JB104 56.0 11.9 1.3 2.1 26 Methylomonas aurantiaca JB177B 56.9 11.8 1.3 ND 27 Methylomonas fodinarum UQM 3268T 57.8 11.2 1.3 1.8 28 Methylomonas fodinarum JB2 59.0 12.0 1.4 ND 29 Methylomonas fodinarum JB3 58.7 11.6 1.4 1.9 30 Methylomonas fodinarum JB4 58.6 12.0 1.4 ND 31 Methylomonas fodinarum JB6 58.1 11.3 1.3 ND 32 Methylomonas fodinarum JB7 58.2 11.8 1.3 2.1 33 Methylomonas fodinarum JB70 59.1 11.5 1.4 ND 34 “Methylomonas gracilis” UQM 3315T (NCIB 11912T) 60.1 11.1 1.0 2.8 35 “Methylomonas gracilis” JB185 59.5 10.5 1.o ND 36 “Methylomonas gracilis” JB187 59.3 10.8 1.0 ND 37 Methylomonas methanica UQM 3307T (NCIB 11130T) 53.4 10.6 1.2 2.0 38 Methylomonas methanica JB5 51.4 10.5 1.1 ND 39 Methylomonas methanica JB9 51.3 10.7 1.2 ND 40 Methylomonas methanica JB199 53.0 10.5 1.1 ND 41 Methylomonas methanica JB228 51.4 11.0 1.2 2.4 42 Methylomonas pelagica UQM 3505T (NCIMB 2265T) 48.5 13.8 1.4 1.5 43 “Methylomonas rubra” UQM 3303 (NCIB 11913) 52.8 10.6 1.2 2.2 44 “Methylosinus trichosporiurn” UQM 3311T (NCIB 11131T) 62.9 8.8 1.2 1.3 45 “Methylosinus sporiuin” UQM 3306T (NCIB 11126T) 67.1 9.2 1.2 1.5 46 “Methylosinus sporium” JB206 66.4 9.0 1.2 ND 47 “Methylosinus” sp. strain JB120 64.5 9.2 1.1 1.5 48 “Methylosinus” sp. strain HB-D1C 67.0 7.5 1.1 1.4 Abbreviations: UQM, Culture Collection, Department of Microbiology, University of Queensland, Brisbane, Australia; ATCC, American Type Culture Collection, Rockville, Md.; NCIB and NCIMB, National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland; DSM, German Collection of Microorganisms, Braunschweig, Federal Republic of Germany; IMET, National Collection of Microorganisms, Institute for Microbiology, Jena, German Democratic Republic; IMV, Institute of Microbiology and Virology, Academy of Sciences, Kiev, USSR; VKM, All-Union Collection of Microorganisms, Academy of Science, Moscow, USSR; NCTC, National Collection of Type Cultures, Public Health Laboratory Service, London, United Kingdom; HB, Helen Byers, Department of Microbiology, University of Queensland, Brisbane, Australia (environmental isolates); JB, J.