international Journal of Systematic Bacteriology (1999), 49, 247-255 Printed in Great Britain

Reclassification of tationis and Methanogenium liminatans as tationis gen. nov., comb. now and Methanofollis lirninatans comb. nov. and description of a new strain of Methanofollis liminatans

Gerhard Zellner,’ David R. Boone,’ Jyoti Ke~wani,~William B. Whitman,3 Carl R. W~ese,~Anja Hagelstein,’ B. J. Tindal15 and Erko Stackebrandt’

Author for correspondence : Gerhard Zellner. Tel: + 49 89 3 187 2579. Fax : + 49 89 3 187 3361. e-mail : [email protected]

1 Institute of Hydrology, Sequencing of 165 rRNA genes and phylogenetic analysis of Methanogenium GSF National Research - tationis DSM 2702T(OCM 433 (T = type strain) and Methanogenium liminatans Center for Environment and Health, Ingolstadter GKZPZT (= DSM 41403 as well as other members of the family LandstraBe 1, D-85764 revealed that both belong to a separate line of Neuherberg, descent within this family. In addition, a new strain of Methanogenium Germany liminatans, strain BM1 (= DSM 10196), was isolated from a butyrate- * Oregon Graduate Institute degrading, fluidized bed reactor and characterized. Cells of both species are of Science and Technology, Portland, OR 97291 -1000, mesophilic, highly irregular cocci that use HJCO, and formate for growth and USA methanogenesis. In addition, Methanogenium liminatans strains GKZPZT and

3 Department of BMI used 2-propanol/C02, 2-butanol/C02 and cyclopentanol/CO,. Both species Microbiology, University of contained diether and tetraether lipids. The polar lipids comprised amino- Georgia, 527 Biological phosphopentanetetrol derivatives, which appear to be characteristic Iipids Sciences Building, Athens, GA 30602-2605, USA within the family Methanomicrobiaceae. The pattern of glycolipids, phosphoglycolipids and amino-phosphoglycolipids was consistent with the 4 Department of M icro bioI ogy, U n iversity of assignment of these two species to a taxon within the family Illinois, Urbana, IL 61801, Methanomicrobiaceae, but also permitted them to be distinguished from USA other higher taxa within this family. The G+C contents of the DNA of 5 DSMZ - Deutsche Methanogenium tationis and Methanogenium liminatans were 54 and Sammlung von 60 mol0h (T, and HPLC), respectively. On the basis of the data presented, the Mikroorganismen und Zellkulturen, D-38124 transfer of Methanogenium tationis and Methanogenium liminatans to the Braunschweig, Germany Methanofollis gen. nov. as Methanofollis tationis comb. nov. and Methanofollis liminatans comb. nov., respectively, is proposed, with Methanofollis tationis as the type species.

Keywords : Methanogeniuni tationis, Methanogenium lirninatans, Methanofollis gen. nov., lipids, phylogeny

INTRODUCTION indicates a wide evolutionary diversity among these methanogenic . However, only a limited num- The DNA of irregularly coccoid has a ber of phenotypic characteristics of irregularly coccoid wide range of G+C contents (38-61 mol%), which methanogens are available for taxonomic purposes. In addition to morphology and substrate spectrum, et ,. , . , . . , , , , ., . , ...... , . . . . , ...... , ...... , , , ...... , , ...... , ...... , ...... characteristics such as polyamine content (Zellner Abbreviations: PAS, periodic acid-Schiff; UASB, upflow anaerobic sludge al., 1989a; Blotevogel et al., 1991 ; Boone et al., 1993), blanket. cellular lipids (Grant et al., 1985; Zellner et al., The CenBanWEMBL accession numbers for the 165 rDNA and rRNA se- 1989a, b, c; Blotevogel et al., 1991 ; Koga et al., 1993) quences reported in this paper are Y16428, Y16429, AF095266-AF095272. and whole-cell protein patterns (Maestrojuan et al.,

~ ~~ ~ 00846 0 1999 IUMS 247 G. Zellner and others

1990) have been used. The S-layer architecture and the cultures, isolation procedures and cultivation of Methano- apparent molecular mass of glycopro tein subunits genium liminatans strains. WHP medium was based on the have also been used (Zellner et al., 1989a, c, 1990; recipe described by Zellner & Jargon (1997) except that yeast Messner & Sleytr, 1992; Sleytr et al., 1993). Due to the extract, peptone and additional sodium sulfate were omitted. WHP medium contained 1 pM sodium tungstate and was fact that few physiological tests can be used to used for the cultivation of strains DSM 2702T,GKZPZT and differentiate taxa within the methanogenic Archaea, BM 1. Substrates (e.g. sodium formate, sodium acetate, 2- other phenotypic characters (e.g. cell wall structure, propanol, 2-butanol, cyclopentanol) were added from an- chemical composition, polyamines) and genotypic aerobic, sterile stock solutions (207'0, w/v or v/v) as characters (e.g. 16s rDNA sequence) must be used to indicated to give concentrations of 0.5 YO(w/v or v/v) in the delineate the higher taxonomic ranks. Recent serum bottles. Cells were grown under H,/CO, (4: 1, v/v, developments in the of both Archaea and 300 kPa) in 120ml serum bottles on a rotary shaker Bacteria indicate that this combination of phenotypic (150 r.p.m.). When other substrates were used instead of and genotypic methods is suitable for the delineation hydrogen, N,/CO, (4: 1, v/v, 300 kPa) served as the gas at- of taxa in a system which seeks to base these on the mosphere. For the preparation of large masses of cells, cells were grown in H,/CO, (4: 1, v/v) at a flow rate of 50 1 h-l in phyletic groupings detected by such data. a 12 1 fermenter (Biostat S; Braun). Analysis of the sequences of 16s rRNA or its genes Methanogenium tationis was grown as described previously revealed a high diversity of irregularly coccoid meth- (Zabel et al., 1984). olentangyi and anogenic strains, with some deeply rooted lines of Methanoculleus bourgensis were grown as described by descent in phylogenetic trees (Zellner et al., 1989a; Corder et al. (1983) and Ollivier et al. (1986), respectively. Rouviere et al., 1992; Boone et al., 1993). As a labreanum was grown as described by consequence, several coccoid methanogens were re- Zhao et al. (1989). Methanocorpusculum sinense and classified on the basis of DNA-DNA hybridization Methanocorpusculum bavaricum were grown as described by studies and/or 16s rDNA sequence analysis (Xun et Zellner et al. (1989a). al., 1989; Zellner et al., 1989c; Maestrojuan et al., Source and habitat of strain BMI. Strain BM1 (= DSM 1990; Boone et al., 1993). This resulted in the descrip- 10 196) was isolated from a butyrate-degrading, fluidized bed tion of the five current genera of hydrogenotrophic, reactor inoculated with a culture originally derived from the irregularly coccoid methanogens of the order upflow anaerobic sludge blanket (UASB) reactor from the Met hanom icrobiales 1 ium, Methanocul- wastewater treatment plant at a sugar refinery in Bruhl, leus, Methan olacin ia, Met hanoplan us and Met hanocor- Germany (Zellner et al., 1991). The predominant pusculum. methanogen in this bioreactor was an autofluorescent irregular coccus, similar in appearance to the new isolate Recently, the reclassification of Methanogenium (strain BM 1) described here, and several other methanogens tationis (Zabel et al., 1984) as Methanofollis tationis were identified immunologically (Zellner et al., 1991, 1997). was suggested (Boone et al., 1993) but not formally Enrichment and isolation of strain BM1. WHP medium with proposed. In this paper, the transfer of Methanogenium H,/CO, (4: 1, v/v, 300 kPa) as substrate was inoculated with tationis and Methanogenium liminatans as Methano- 1 YO(v/v) bioreactor liquid and incubated at 37 "C. The gas follis tationis gen. nov., comb. nov. and Methanofollis phase was regularly replaced and autofluorescent, irregularly liminatans comb. nov. is proposed on the basis of 16s coccoid cells increased in number simultaneously with the rDNA sequencing and chemotaxonomic and physio- formation of . This enriched culture was serially logical data. In addition, the isolation and charac- diluted into WHP medium and the highest dilution showing terization of a new Methanofollis liminatans strain, growth of the fluorescent, irregularly coccoid cells was again serially diluted. Aliquots (0.1 ml) of the dilutions were then BM1, is reported. streaked on agar plates with WHP medium plus 2.5 % (w/v) Oxoid agar (Unipath). All manipulations were performed in METHODS an anaerobic chamber (M. Braun) under a nitrogen at- mosphere. The plates were transferred into a stainless steel Reference strains. Methanogenium tationis strain (DSM anaerobic jar, pressurized to 300 kPa with H,/CO, (4: 1, 2702T) was obtained from the Deutsche Sammlung von v/v) and incubated at 37 "C. After 20 d incubation, the gas Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, pressure had dropped to 150 kPa, indicating gas consump- Germany). Methanogenium liminatans strain GKZPZ' (= tion. The anaerobic jar was transferred into the anaerobic DSM 4140T) was isolated and (after deposition in the chamber. Cells were picked with a syringe needle from a DSMZ) subcultured in this laboratory. The newly isolated well-isolated colony, injected into a 120 ml serum bottle strain BMl has been deposited at the DSMZ as DSM 10196. containing 20 ml WHP medium under H,/CO, (4: 1, v/v, The following strains were obtained from the Oregon : 300 kPa) and incubated at 37 "C. This procedure resulted in Collection of Methanogens (Beaverton, OR, USA) the isolation of strain BM 1. Methanoculleus olentangyi RC/ER' (OCM 52T), Methano- culleus bourgensis MS2' (OCM l 5T), Methanocorpusculum Microscopy. Phase-contrast and epifluorescence microscopy bavaricum SZSXXZT (OCM 127T), Methanocorpusculum were carried out by using a Zeiss Axiophot microscope as sinense China ZT (OCM 128') and Methanocorpusculum described previously (Zellner et al., 1991). Motility was labreanum ZT (OCM 1'). checked microscopically. Media, media preparation and cultivation. A synthetic basal Analyses. Volatile fatty acids, alcohols, hydrogen and medium (WHP medium) containing tungstate, with acetate methane were analysed by GC (Zellner &Winter, 1987). The and L-cysteine (the latter as a component of the reductant) as Colour Gram 2 staining set from bioMerieux was used for the sole organic carbon sources, was used for enrichment Gram staining according to the manufacturer's instructions.

248 International Journal of Systematic Bacteriology 49 Methanofollis gen. nov.

Growth was measured as described previously (Zellner et al., (Rouviere et al., 1992 ; Maidak et al., 1989a). 1997). A greater degree of resolution among closely related Extraction of polar lipids and presumptive lipoquinones. taxa was obtained by using the ae2 editor (Maidak et al., Freeze-dried cells (100 mg) were subjected to the two-stage 1997). A phylogenetic dendrogram was reconstructed using extraction method described by Tindall (1990a, b). Respir- the treeing algorithm of De Soete (1983), based on similarity atory lipoquinones were recovered in the first stage and values which had been transformed into phylogenetic distance values that compensate for multiple substitutions at polar lipids were recovered in the second stage. The presence any given site in the sequence (Jukes & Cantor, 1969). The or absence of respiratory lipoquinones was confirmed by TLC of the first stage extract on silica gel thin layers (Art. nr. programs NEIGHBOR-JOINING and MAXIMUM-LIKELIHOOD contained in the PHYLIP package (Felsenstein, 1993) were 805 023, Macherey-Nagel) cut to 10 x 10 cm squares, with : used in the construction of phylogenetic dendrograms hexane: tert-butylmethylether (9 1, v/v) as the developing & solvent. (Saitou Nei, 1987). Bootstrap values were determined by using the programs NJFIND and NJBOOT of the PHYLIP package Polar lipids were separated by two-dimensional TLC on (Felsenstein, 1993). silica gel thin layers (Macherey-Nagel, Art. nr. 818 135, Nucleotide sequence accession numbers. which is a 10 x 10 cm format). The plates were developed in The sequences of chloroform : methanol :water (65 : 25 : 4, by vol.) in the first 16s rDNA of Methanogenium liminatans strain DSM 4140T direction, followed by chloroform :methanol : acetic and strain BM 1 were deposited in the EMBL database under acid :water (80 : 12 : 15 : 5, by vol.) in the second direction. the accession numbers Y 16428 and Y 16429, respectively. The 16s rRNA sequences of Methanogenium tationis DSM Total lipids were visualized with 5 O/O dodecamolybdato- phosphoric acid and specific functional groups were detected 2702', Methanoculleus bourgensis MS2T( = OCM 15T, DSM with periodate-Schiff (a-glycols), ninhydrin (free amino 3045T) and Methanoculleus olentangyi RC/ERT (= OCM groups), a-naphthol (sugars), Dragendorf (quaternary nitro- 52T, DSM 2772T), Methanocorpusculum bavaricum gen) and anisaldehyde-H,SO, (sugars and terpenoids). SZSXXZT, (= OCM 127', DSM 4179T), Methano- corpusculum sinense China ZT (= OCM 128T,DSM 4274T) Extraction of ether lipids and TLC analysis. Ether lipids were and Methanocorpusculum labreanum ZT (= OCM IT, DSM extracted from 100 mg freeze-dried cells by an acid hy- 4855T) were deposited in GenBank under the accession drolysis method that did not hydrolyse diether lipids numbers AF095266-AF095272, respectively. Other se- containing a hydroxyl group in the side chain. Diether lipids quences utilized were obtained from EMBL (Methanoculleus were subjected to double development in hexane: diethyl palmeoli, Y 16382) or the Ribosomal Database Project ether (4: 1) to resolve phytanyl and sesterpanyl diether lipids (Maidak et al., 1997). (i.e. C,, : C2,, C,, : C2,, C,, :C,,). Tetraethers and diethers were separated in the solvent hexane :diethyl ether : acetic acid (25 :25 : 1, by vol.). RESULTS AND DISCUSSION Determination of G + C content of DNA. DNA from strain Morphology of strains, S-layer architecture and BM 1 was isolated and purified and the mol YOG + C content composition of the DNA was determined by HPLC according to Zellner et al. (1989a). Cells of the newly isolated strain BM1 were highly irregular cocci. Some cells also had a ring-shaped Sequence analysis of 16s rDNA and phylogenetic analysis. For Methanogenium tationis DSM 2702T and Methano- appearance under phase-contrast microscopy. The genium liminatans strains GKZPZTand BM l, genomic DNA cells were 1-25-2-0 pm in diameter, stained Gram- was extracted, the 16s rDNA was amplified by PCR and the negative and were non-motile. The addition of 1 YO sequences of the purified PCR products were analysed as (w/v) SDS led to lysis of cells of Methanogenium described previously (Rainey et al., 1996). The sequencing liminatans GKZPZT and strain BM1, indicating a cell reaction products were electrophoresed with a model 373A envelope with a proteinaceous cell wall. With the automated DNA sequencer (Applied Biosystems). The exception of Methanogenium liminatans strains, which almost complete 16s rRNA genes of Methanogenium appear as ring-shaped cells under phase-contrast mi- liminatans GKZPZT( 1396 nucleotides) and Methanogenium croscopy (Zellner et d., 1990), the species described to liminatans BM 1 were determined. Approximately 1380 date of the genera Methanogenium and Methanoculleus nucleotides of the Methanogenium liminatans GKZPZT 16s are irregular cocci and are indistinguishable from each rRNA were also sequenced directly by the reverse trans- criptase method (Rouvikre et al., 1992). This sequence was other (Boone et al., 1993). However, some differences 99-7YO identical to the sequence obtained for the 16s rRNA in cell envelope composition have been observed gene. The 16s rRNA gene sequence was used for the (Table 1). All irregularly coccoid methanogens within phylogenetic analysis. In addition, the 16s rRNA of the order Methanomicrobiales possess hexagonal S- Methanogenium tationis DSM 2702T, Methanoculleus layer lattices consisting of glycoprotein subunits with olentangyi RC/ERT, Methanoculleus bourgensis MS2T, M, ranging from 90000 to 155000. However, only Methanocorpusculum bavaricum SZSXXZT, Methano- methanogens belonging to the family Methano- corpusculum sinense China ZT and Methanocorpusculum corpusculaceae have an S-layer glycoprotein with Mr labreanum ZT were sequenced directly by the reverse ranging from 90000 to 94000. The Mr of S-layer transcriptase method. About 1380 nucleotides were de- glycoprotein subunits of genera of the family termined for each of these sequences. Methanomicrobiaceae range from 101 000 to 155000. The global phylogenetic positions of the strains were analysed by using the ARB database (Strunk & Ludwig, During the course of compiling this paper, the in- 1997). 16s rRNA and rDNA sequences were compared with correct orthography of Methanoculleus thermophilicus the existing 16s rDNA database of members of the order was noticed which, according to Rule 61 of the

International Journal of Systematic Bacteriology 49 249 G. Zellner and others

Table 1. S-layers of irregularly coccoid methanogens of the families Methanomicrobiaceae, Methanoplanaceae and

Strain DSM no. Lattice Glycoprotein Reference constant subunit c/c (nm)* M, (x

Genus Methanofollis M. tationis Chile 9T 2702T ND 120 Zabel et al. (1984); Zellner et al. (1998) M. liminatans GKZPZT 4 140T 15-4 118 Zellner et al. (1990, 1998) Genus Methanoculleus M. marisnigri JRIT 149ST ND 138 Zellner et al. (1990) M. olentangyi RC/ERT 2772T 15.4 132 Zellner et al. (1990) M. bourgensis MS2T 3045T 15.4 101 Zellner et al. (1990) M. thermophilus CR-1 2373T ND 130 Zabel et al. (1985) M. thermophilus UCLA 2624 ND 130 Zabel et al. (1985) M. thermophilus Ratisbona 2640 ND 130 Zabel et al. (1985) M. palmolei INSLUZT 4273T 15.2 120 Zellner et al. (1998) Genus Methanogenium M. cariaci JRIT 1497T 14.0 117 Zellner et al. (1990) M.frittonii FR-4T 2832T 15.4 106 Zellner et al. (1990) Genus M. paynteri G2000T 2545T 15.3 l55t Zellner et al. (1 989c) Genus M. limicola M3T 14.0 143 Zellner et al. (1989a) M. endosymbiosus MC 1 ND 110 Zellner et al. (1 989a) Genus Methanocorpusculum M. parvum XIIT 3823T 14.3 90 Zellner et al. (1989a) M. bavaricum SZSXXZT 41 79T 16.0 94 Zellner et al. (1989a) M. sinense CHINAZT 4274T 15.8 92 Zellner et al. (1989a) M. uggregans MStT 3027T 15.4 92 P. Messner & G. Zellner, unpublished

ND, Not determined. * Centre-to-centre spacing of hexagonally arranged protein subunits of S-layer. ?A second, weaker band with an M, of 135000 that reacted with PAS stain presumably represented a degradation product or a less glycosylated precursor.

Bacteriological Code (Lapage et al., 1992), may be and methanogenesis. The secondary and cyclic corrected to Methanoculleus thermophilus (corrig.). alcohols were oxidized to the respective ketones. Strain BMl did not grow or produce methane in media with acetate, methanol, methylamines, ethanol or 1-pro- Culture conditions and substrates for growth and panol as substrate. In contrast, Methanogenium methanogenesis tationis DSM 2702T did not grow or produce methane on 2-propanol/C02 or 2-butanol/C02 (Zellner & The optimal growth temperature of strain BM1 was Winter, 1987) or cyclopentanol/CO,. Acetate was about 37-40 "C. No growth was obtained below 20 "C required for growth of strain BM1 on formate. Thus, or above 45 "C. The optimal pH was about 7. Growth while strain BM 1 resembled Methanogenium limi- and methane production were observed on H,/CO,, natans more closely in terms of substrate utilization, it formate, 2-propanol/C02 and 2-butanol/C02. could not be distinguished from other species of Growth and methane production by strain BM1 on the genera Methanoculleus and Methanogenium on this potential substrates was as follows (maximal OD,,, basis (Table 2). obtained/total methane produced per 20 ml culture in a 120 ml serum bottle) : H,/CO, (0.86/3285 pmol), formate (0.14/172 pmol), 2-propanol/C02 Cell physiology (0.06/52 pmol), 2-butanol/C02 (0.04/44 pmol) and cyclopentanol/CO, (0.00 1/2 1 pmol). Thus, except for The original descriptions of Methanogenium tationis cyclopentanol/CO,, which supported only methano- and Methanogenium liminatans were supplemented by genesis, all these substrates supported both growth material on the utilization of secondary and cyclic

250 In ternation a I Jo urnaI of Systematic Bacteriology 49 Methanofollis gen. nov.

Table 2. Phenotypic features of irregularly coccoid strains of the families Methanornicrobiaceae and Methanoplanaceae

Strain DSM no. Cell size (pm) Flagella Substrates* Min. $, (h) Growth temp. ("C)? G + C content Reference (mol%)S H F 2P ZB

Genus Methanofollis M. tationis Chile gT 2702T 1.53.0 + ++-- 12 3740/2S45 54 Zabel et al. (1984) M. liminatans GKZPZT 4 140T 1.2s2.0 + ++++ 7.5 40/2S45 59.3 Zellner et al. (1 990) M. liminatans BMl 10196 1.252.0 ND ++++ ND 40/2&45 600b This paper Genus Methanoculleus M. marisnigri JRIT 1498T 1.3/1-2 + ++++ 10 2&25/ lW5 61-62" Romesser et al. (1979) M. olentangyi$ RC/ERT 2772T 1.G1.5 - + + (+I (+I 10.9 37/3@45 544" Corder et 01. (1983) M. bourgensi4 MS2T 304ST la-2.0 + ++++ 18.0 37/25-55 59", 5657 Ollivier et al. (1986) M. thermophilus CR-IT 2373T 0.8- 1'3 + ++NDND 2.5 55/37-65 599, 57-59 Rivard & Smith (1982) M. thermophilus UCLA 2624 07-1.8 + ++-- 1.8 5-01 < 70 56, 59.7 Ferguson & Mah (1983) M. thermophilus Ratisbona 2640 0.9-1.5 + ++-- 3.4 58/3MO 57 Zabel et al. (1985) M. thermophilus TCI 3915 0.61.5 NR ++++ NR 55/30-60 54.7 Widdel et al. (1988) M. oldenburgensis 6216T 1.0 - ++-- 3.8 45/2&50 48.6 Blotevogel et al. (1991) M. palmolei INSLUZT 4273T 1.252.0 + ++++ 13.5 40/21-51 59.0 Zellner et al. (1998) Genus Methanogenium M. cariaci JRIT <2.6, 1-3 - ++++ 11.0 37-45/1550 51.6" Romesser et al. (1979) M. organophilum CF 0.s1.5 - +++- ND 3&35/ 15-39 46.7 Widdel eta!. (1988) M.frittonii FR-4T 1-2.5 - +--- 1-2 57/2&62 49.2 Harris et al. (1984) M. frigidurn Ace-2 1.2-23 - ++NDNlJ 69.6 15/&17 ND Franzmann et al. (1997) Genus Methanolacinia M. paynteri G-2000T 254ST + +-++ ND 40/2&45 45". 38 Zellner et al. (1989~) Genus Methanoplanus M. limicola M3T 1-3 x 0.1-0.25 + ++-- ND 40/17-41 47.5 Wildgruber et al. (1982) M. endosymbiosus MCIT 163.4 + ++NDND 7 32/1&36 38.7 van Bruggen et al. (1986) NR, Not reported; ND, not determined. * H, H,/CO,; F, formate; 2P, 2-propanol/C02; 2B, 2-butanol/C02.None of the strains utilized acetate, methanol or methylamines. Methanogenium organophilum utilized ethanol, 1-propanol and 1-butanol in addition. Data on alcohol utilization partly taken from Zellner & Winter (1987). In addition, both Methanofollis liminatans strains, GKZPZ and BM1, Methanoculleus palmolei and Methanolacinia paynteri utilized cyclopentanol/CO, with concomitant production of cyclopentanone and CH,, while Methanofollis tationis could not utilize cyclopentanol/CO,. ( + ), Weakly positive. t Growth temperature is given as optimum temperature/temperature range. $ G + C content of DNA was determined by T, unless otherwise indicated, in which case it was determined by buoyant density (a)or HPLC (b). 9 Methanoculleus olentangyi and Methanoculleus bourgensis are apparently subjective synonyms (Xun et al., 1989 ; Boone et al., 1993). acids from Zellner & Winter (1987) and Bleicher et al. Lipids of Methanogenium tationis and (1 989). Methanogenium liminatans contains a second- Methanogenium liminatans ary alcohol dehydrogenase activity that is devoid of Both diether- and tetraether-linked glycerol lipids were F420 & zinc and is dependent on factor (Bleicher present in Methanogenium tationis DSM 2702T and Winter, 199 1). Methanogenium liminatans GKZPZT (data not The pterin of Methanogenium tationis has a structural shown). The diether lipids were diphytanyl glycerol modification not found in other methanogens and is diethers, while the tetraether lipids showed no evidence called tatiopterin. Tatiopterin lacks a 7-methyl sub- of cyclization and only acyclic C,, : C40 dibiphytanyl stituent but has both a glutamyl and an aspartyl tetraethers were present. These results are consistent residue conjugated to the phosphoglutaryl moiety with the results from other members of the family (Raemakers-Franken et al., 1989) and can be dis- Methanomicrobiaceae examined to date (Grant et al., tinguished from methanopterin and sarcinapterin 1985; Zellner et al., 1989c; Blotevogel et al., 1991; (Keltjens et al., 1983; DiMarco et al., 1990). B. J. Tindall & G. Zellner, unpublished data).

Presumptive respiratory lipoquinones of The major polar lipids present in Methanogenium tationis Methanogenium liminatans Methanogenium tationis and Methanogenium DSM 2702T and liminatans GKZPZT were glycolipids, phospholipids and phosphoglycolipids (Fig. 1). The major phospholipids Examination of Methanogenium tationis DSM 2702T present were ether derivatives of phosphatidyl glycerol, and Methanogenium liminatans GKZPZT for the pres- phosphopentanetetrol amine and phosphopenta- ence of respiratory lipoquinones indicated that none netetrol trimethylamine. The major glycolipid present were present, a finding consistent with results obtained was a diglycosyl ether lipid, the nature of the sugars for all other members of the methanogenic Archaea and their mode of linkage not being determined. A examined to date (B. J. Tindall, unpublished data). single major phosphoglycolipid was also detected. The

International Journal of Systematic Bacteriology 49 251 G. Zellner and others

Melhanospirillumhungalei Melhanocorpusculum lnbreanum Mefhanocorpusculumparvirm -85 Methanocorpsculuni bavuricuni/M sinense Melhanogeniirm cariaci Methanogenium organoplrilum mobile Melhanoplanus limicola Methanofallis liminatans DSM 10196 Methanofollis lirninalans DSM 4140 T Methanofallis tationis DSM UOLT

Mefhanocullarspalmolei

10%

...... Fig. 2. Dendrogram showing the phylogenetic positions of Methanofollis tationis DSM 2702T, Methanofollis liminatans GKZPZT (= DSM 41403 and Methanofollis liminatans BM1 (= DSM 10196) within the family Methanomicrobiaceae. Numbers at branch points represent percentage bootstrap support. Bar, 10 % sequence divergence.

sufficiently different to distinguish them from the members of the family Methanomicrobiaceae (Zellner et al., 1989a). The fact that members of the family Methanocorpusculaceae have a distinct polar lipid pattern would appear to contradict the 16s rDNA sequence data, since this family appears to branch within the family Met hanom icr o b iaceae. However, the branching order of some groupings within the members of the order Methanomicrobiales is uncertain. Such uncertainties in branching order, particularly where the pendant edges of the dendrogram are long in ...... relation to the length of internal branches, has been Figrn7. Thin-layer chromatograms of the polar lipids of (a) already documented. Methanogenium tationis DSM 2702* and (b) Methanogenium liminatans GKZPZT. Spots are identified as: 1, phosphatidyl The presence of identical polar lipids in both glycerol; 2, phosphatidyl pentanetetrol amine; 3, diglycosyl Methanogenium tat ion is and Methanogenium diether; 4, phosphatidyl pentanetetrol trimethylamine; 5, liminatans suggests that these features are not charac- phosphoglycolipid; 6, phosphoglycolipid; 7, amino-phospho- teristic of the individual species but indicative of a glycolipid; 8, amino-phospholipid. higher taxonomic group. Although certain aspects of their polar lipid pattern are similar to other members of the family Methanomicrobiaceae, there are differences in the details of the polar lipid patterns presence of aminopentanetetrol-containing phospho- which may be regarded as genus-specific markers. lipids is characteristic of members of the family Methanomicrobiaceae. Moreover, a detailed analysis of the total polar lipid composition of the various taxa Phylogenetic position within this group indicates that there are clear differences between the various taxa currently de- Sequencing of the 16s rRNA and rDNA was per- scribed (Grant et al., 1985; Zellner et al., 1989a, c; formed to determine the phylogenetic relationship of Blotevogel et al., 1991; B. J. Tindall & G. Zellner, Methanogenium tationis strain DSM 2702T and unpublished data). Thus, the chemical composition of Methanogenium liminatans strains GKZPZTand BM 1 the cell may be a useful way of differentiating genera in to other members of the family Methanomicrobiaceae. this phyletic group. Analysis of these sequences indicated that the three strains form a phylogenetically coherent group almost Although it is not known whether members of the equidistant from the genus Methanoculleus and other Methanocorpusculaceae also contain these unusual Methanogenium species (Fig. 2). The G + C content of aminopentanetetrol-containing phospholipids, the the DNA of strain BMl was determined to be polar lipid composition of members of this family is 60.0 mol %, i.e. nearly identical to that of Methano-

252 International Journal of Systematic Bacteriology 49 Methanofollis gen. nov.

genium liminatans GKZPZT (Zellner et al., 1990). HPLC). Habitats are anaerobic high-rate wastewater Thus, the G + C content of DNA of this group ranges bioreactors or solfataric fields. The type species of the from 54 to 60 mol YO. genus is Methanofollis tationis comb. nov. On the basis of the similarity in G+C content of DNA, substrate utilization patterns and other pheno- Description of Methanofollis tationis comb. nov. typic properties, strain BMl is assigned to the same Irregularly coccoid cells, 1.5-3 pm in diameter, with a species as Methanogenium liminatans GKZPZT. On proteinaceous, SDS-sensitive S-layer. Cells stain the basis of the 16s rDNA sequence and chemo- Gram-negative. Cells are motile or non-motile. taxonomic data, which indicate that Methanogenium Obligately anaerobic, no microaerophilic or aerobic tationis and Methanogeniurn liminatans represent a Methano- growth. Strains are mesophilic (range > 15 to distinct phyletic group within the family <45 "C). Substrates for growth and methane pro- microbiaceae, it is proposed that they be placed in a duction are H,/CO, and formate. Methanol, methyl- new genus, for which the name Methanofollis gen. amines, ethanol, 2-propanol/CO,, 2-butanol/C02 and nov., is proposed. The strains are described as cyclopentanol/CO, are not utilized. Cells contain Methanofollis tationis DSM 2702T gen. nov., comb. tatiopterin, a modified pterin distinct from methanop- nov., Methanofollis liminatans GKZPZT comb. nov. terin and sarcinapterin. Lipid composition as that and Methanofollis liminatans BM 1. described for the genus. The G + C content of DNA of The 16s rRNA sequences of Methanocorpusculurn the only strain known so far is 54 mol% (T,). The bavaricurn and Methanocorpusculurn sinense have a type strain is DSM 2702T and was isolated from a missing stretch of 40 bp. The remainders of their solfataric field on Mount Tatio in the Atacama desert sequences are 100% and about 99.6% identical to in northern Chile. Methanocorpusculum parvurn and Methano- corpusculum labreanurn, respectively, which are them- Description of Methanofollis liminatans com b. nov. selves 99.8 % identical. Therefore, the four Methano- corpusculum species are highly related. About l 100 Irregularly coccoid or ring-shaped cells, 1.25-2.0 pm in nucleotides were used for the alignment of Methano- diameter, with a proteinaceous, SDS-sensitive S-layer. corpusculurn bavaricum and Methanocorpusculurn Cells stain Gram-negative. Some strains may be sinense 16s rRNA sequences, while 1150 nucleotides motile. Obligately anaerobic, no microaerophilic or were used for the Methanocorpusculum parvurn and aerobic growth. Cells are mesophilic (range 2 15 "C to Methanocorpusculum labreanurn sequences. <45 "C) with an optimum at 40 "C. Substrates for growth and methane production are H,/CO,, formate, 2-propanol/C02, 2-butanol/C02 and cyclopentanol/ Description of Methanofollis gen. nov. CO,. Secondary and cyclic alcohols are oxidized to the Methanofollis (Me.tha.no.fol'lis. M.L. n. rnethanurn respective ketones. Methanol, methylamines, ethanol methane; M.L. n. follis a bag; Methanofollis a and acetate are not utilized as catabolic substrates. methane-producing bag). Cells of GKZPZT contain a secondary alcohol de- hydrogenase dependent on F,,, and devoid of Zn. Irregular cocci, 1.5-3-0 pm in diameter, with a pro- Lipid composition as that described for the genus. The teinaceous, SDS-sensitive S-layer. Cells stain Gram- G + C content of DNA of the type strain is 60 mol YO negative. Some strains are motile. Obligately anaer- (T,). The type strain is GKZPZT (= DSM 4140T), obic, no microaerophilic or aerobic growth. Meso- isolated from an industrial wastewater bioreactor philic (range > 15 to <45 "C). Substrates for growth (Biohochreaktor, Hoechst, Kelsterbach, Frankfurt, and methane production are H,/CO, or formate; Germany). Strain BMl (= DSM 10196) is a reference some species may use 2-propanol/C02, 2-butanol/ strain, isolated from a methane-producing fluidized CO, and cyclopentanol/CO,. Sulfate is not reduced. bed reactor operated with synthetic wastewater con- The ether lipids comprise diphytanyl diether and taining butyrate and present in a full-scale UASB dibiphytanyl tetraethers. No evidence for significant reactor treating sugar refinery wastewater anaerobic- quantities of other derivatives, including the presence ally in Bruhl, Germany. of pentacyclic rings. The major polar lipids are phospholipids, glycolipids and phosphoglycolipids. ACKNOWLEDGEMENTS Among these the predominant compounds are the ether derivatives of phosphatidyl glycerol, diglycosyl This investigation was supported in part by grants of the glycolipid, phosphopentanetetrol amine, phospho- Deutsche Forschungsgemeinschaft. Part of the practical pentanetetrol trimethylamine and a phospho- work of G.Z. was performed in the Institute of Micro- glycolipid. Minor components include a glycolipid, a biology, University of Hannover, Germany, and was com- piled during a period as a Senior Research Associate of the phospholipid, an amino-phospholipid, a phospho- Wageningen Agricultural University, financially supported glycolipid and an amino-phosphoglycolipid. This pat- by WIMEK (Wageningen Institute for Environment and tern differs in detail from other members of the family Climate, Wageningen, The Netherlands). W. B. W. received Methanornicrobiaceae. The G + C content of DNA of support from the US Environmental Protection Agency, known strains ranges from 54 to 60 mol% (T, and contract AERL-9003. C. R. W. received support from the

International Journal of Systematic Bacteriology 49 253 G. Zellner and others

National Science Foundation, grant DEB-9306 17 1. The Lapage, S. P., Sneath, P. H. A., Lessel, E. F., Skerman, V. B. D., G + C content of DNA of strain BM1 was determined by Seeliger, H. P. R. & Clark, W. A. (editors) (1992). International Mrs Inge Reupke. We wish to thank Mrs Ulrike Mendrock Code of Nomenclature of Bacteria, (1 990 Revision). Bacterio- for her help with the molecular work. logical Code. Washington, DC : American Society for Micro- biology. Maestrojudn, G. M., Boone, D. R., Xun, L., Mah, R. A. & Zhang, L. REFERENCES (1 990). Transfer of Methanogenium bourgense, Methanogenium Bleicher, K. & Winter, J. (1991). Purification and properties of mar isnigr i, Methanogen ium o lentangy i, and Methanogen ium F,,,- and NADP+-dependent alcohol dehydrogenases of thermophilicum to the genus Methanoculleus gen. nov., emen- Methanogenium liminatans and Methanobacterium palustre, dation of Methanoculleus marisnigri and Methanogenium, and specific for secondary alcohols. 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