INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1990, p. 79-82 Vol. 40, No. 1 0020-7713/9O/010079-O4$02 .OO/O Copyright 0 1990, International Union of Microbiological Societies

Methanosaeta concilii gen. nov. sp. nov. (“Methanothrix concilii”) and Methanosaeta thermoacetophila nom. rev., comb. nov.?

GIRISHCHANDRA B. PATEL” AND G. DENNIS SPROTT Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada KIA OR6

Methanosaeta concilii gen. nov., sp. nov. (“Methanothrix concilii”) is described. Cells of species in the genus Methanosaeta are obligately anaerobic, gram-negative, nonmotile rods (length, 2.5 to 6.0 pm) with flat ends. The cells are enclosed within a proteinaceous, cross-striated sheath. Growth can occur as long filaments which represent chains of individual cells separated by spacer plugs and continuously enclosed by the tubular sheath. Acetic acid is used as the sole source of energy; its metabolism results in the production of about equimolar amounts of CH, and CO,. Acetic acid and CO, are carbon sources for growth. The description of Methanosaeta concilii, the type species, is based on type strain GP6 (= DSM 3671 = OGC 69 = NRC 2989 = ATCC 35969).

The genus Methanothrix was created by Huser et al. (9) to MATERIALS AND METHODS include rod-shaped, flat-ended, sheathed, methanogenic bac- teria that are capable of growth in long filaments and of using The methods used for isolation and characterization (18) acetic acid as a sole energy source. Neither H,-C02 nor and for studies of the ultrastructure (2), metabolic pathways formate can serve as a sole methanogenic substrate. The (3,nutritional requirements (19), sheath structure (20), and original description of Methanothrix soehngenii, the type lipid structures (7, 8) of strain GP6T have been described species, is based on the description of strain OpfikonT (= previously. DSM 2139T) (T = type strain) (9, 25). Strain OpfikonT was In this study the base composition of strain GP6T was assessed by the Deutsche Sammlung von Mikroorganismen determined by the thermal denaturation procedure of Man- (DSM) to be contaminated (as received) by two morpholog- del et al. (15), using DNA that was isolated and purified ically distinct bacteria, a vibrio (presumably a Desulfovibrio (ratio of to 1.669; ratio of A,,, to A230, 1.165) after sp. based on blackening of Postgate medium) and a rod- mild chemical lysis of cells with 2% (wthol) sodium dodecyl shaped sporeformer (H. Hippe, DSM, personal communica- sulfate and 32.0 mM dithiothreitol in 50 mM NH,HC03 tion). The presence of contaminating bacteria in strains buffer (pH 8.0) at 37°C for 1 h. The guanine-plus-cytosine OpfikonT and FE of Methanothrix soehngenii was confirmed content was calculated by using the method of De Ley (4). in our laboratory and by Touzel et al. (23). No axenic culture of either strain OpfikonT (= DSM 2139T) or FE (= DSM RESULTS AND DISCUSSION 3013) has been deposited with the DSM (Hippe, personal Strain GP6T was formerly named “Methanothrix concilii” communication). Methanothrix soehngenii strain VNBF has (11, 18). However, the genus “Methanothrix” is nomencla- been described (6), but its purity was not verified and it was turally invalid according to the International Code of No- not deposited in a recognized culture collection. menclature of Bacteria. This is because the description of According to Rules 18a and 31a of the International Code the type species, “Methanothrix soehngenii” (strain Op- of Nomenclature of Bacteria (13), Methanothrix soehngenii, fikonT) (9, lo), is based on a mixed culture of more than one the nomenclatural type species of the genus Methanothrix, is species (Rule 31a) and because the type strain has not been invalid. Consequently, the genus “Methanothrix” (9, 24) maintained in pure culture (Rule 18a) (13). Since the genus does not have nomenclatural standing and “Methanothrix “Methanothrix” is invalid, so are all of the species placed in concilii” GP6T (= DSM 3671T), the first and presently only the genus. Therefore, we propose that type strain GP6 of axenic mesophilic isolate (18) placed in the genus “Metha- “Methanothrix concilii” be named Methanosaeta concilii nothrix,” is nomenclaturally invalid. In addition to the DSM, gen. nov., sp. nov. and that, for the reasons outlined below, strain GP6T has been deposited in the National Research the genus Methanosaeta be included in the family Metha- Council Culture Collection as strain NRC 2989T and in the nosarcinaceae (3). Oregon Graduate Center Collection of Methanogenic Ar- Although morphologically strain GP6T most closely re- chaeobacteria as strain OGC 69T, where its purity has been sembles the genus Methanospirillum, strains of which have a confirmed. The culture has also been deposited in the similar proteinaceous sheath enclosing cells within a fila- American Type Culture Collection as strain ATCC 35969T. ment, there are distinct structural and chemical daerences The purpose of this paper is to describe the characteristics of between the sheaths (20). Also, unlike the strains of the Methanosaeta concilii gen. nov., sp. nov. (“Methanothrix genus Methanospirillurn, strain GP6T is a nonmotile, aceti- concilii”). We also propose that the only other two axenic clastic methanogen which is unable to use H,-CO, as a isolates currently included in the genus “Methanothrix,” growth substrate. Comparative immunological analysis in strain CALS-1 (= DSM 3870) (26) and “Methanothrix ther- which an indirect immunofluorescence reaction was used moacetophila” strain Z517T (= DSM 4774T) (16), which are showed that strain GPGT is weakly related to the genus both thermophiles, should be included in the genus Metha- Methanosarcina and not related to the genus Methanospiril- nosae fa. lum (18). Characterization by oligonucleotide cataloging of 16s rRNA of “Methanothrix soehngenii” strain OpfikonT (22) has indicated that this organism is distantly related to * Corresponding author. the family Methanobacteriales (SAB[binary matching coef- t National Research Council of Canada paper 30952. ficient] = 0.27) and more closely related to Methanosarcina 79 80 PATEL AND SPROTT INT. J. SYST.BACTERIOL. barkeri (SAB, 0.44). Moreover, it has been shown (23) that the filamentous cells in the mixed culture of “Methanothrix soehngenii” strain OpfikonT are genetically related to those of “Methanothrix concilii” strain GP6T. Strain GP6T and Methanosarcina barkeri can utilize acetic acid as a methano- genic substrate, and both organisms utilize an incomplete tricarboxylic acid pathway operating in the oxidative direc- tion for the synthesis of glutamate and amino acids derived from glutamate (5). Other methanogens, including members of the genus Methanospirillurn (3,use an incomplete tricar- boxylic acid pathway operating in the reductive direction. The membrane lipids of both strain GP6T (5) and Methanosa- rcina barkeri consist predominantly of diphytanylglycerol diethers and not dibiphytanyldiglycerol tetraethers. On the basis of these observations, strain GP6T is more closely related to the family Methanosarcinaceae, and we propose that it be placed in this family. The cell morphology and the range of growth substrates of strain GP6T are distinct from those of the genera Methanosa- rcina, Methanococcus, and Methanolobus of the Metha- nosarcinaceae (3). Although strain GP6T is more closely related to the genus Methanosarcina than to the other genera in the family, the low SAB value of relatedness with Metha- 0 3 6 9 12 15 18 21 nosarcina barkeri, the higher guanine-plus-cytosine content Days (49 mol%) compared with the values of 36 to 44 for Metha- nosarcina species, and the inability to use H,-CO,, metha- FIG. 1. Methane production during growth of strain GP6= in the nol, or methylamines clearly distinguish strain GP6T from absence (0)and presence (A) of 0.05% (wtlvol) yeast extract (Difco Laboratories, Detroit, Mich.). The organism was cultured in 100 ml the species of the genus Methanosarcina. Therefore, we of Aa medium (18), with stationary incubation at 35°C. propose that strain GPtiT should be placed in a new genus, Methanosaeta. The type species of the genus Methanosaeta is Methanosaeta concilii. The optimum growth temperature is 35 to 40°C in acetate Description of Methanosaeta gen. nov. Methanosaeta (Me. medium at pH 7.2 (18). Growth does not occur at 10 or 45°C. tha. no. sae’ta. N.L.n. rnethanum, methane; Gr.n. saeta, The optimum growth pH at 35°C is between 7.1 and 7.5, with bristle; N.L. fem. n. Methanosaeta, methane [-producing] growth occurring at least between pH 6.6 and 7.8 (18). The bristle). The cells of members of this genus are obligately mass doubling time is 65 to 70 h based on either CH, anaerobic, gram-negative, nonmotile, nonsporeforming rods production (Fig. 1) or turbidity of the culture between 3 and with flat ends. Single cells are 2 to 6 pm long and 0.8 to 1.3 9 days of incubation. If a small inoculum is used, then a pm wide and are enclosed inside a tubular, proteinaceouS requirement is demonstrable for sodium carbonate or CO, sheath. Growth can occur as long flexible filaments. Each for initiation of growth in acetate medium (18). Inorganic filament consists of a chain of cells which are separated by nitrogen serves as a sole nitrogen source for growth, but spacer plugs and are enclosed by a continuous sheath. organic nitrogen, including cysteine, alanine, phenylalanine, Acetic acid is used as the sole source of energy, with the glutamic acid, aspartic acid, and methylamine, does not (19). production of about equimolar amounts of CH, and CO, as Cysteine hydrochloride, sodium sulfide, or glutathione the metabolic end products. None of the other common serves as a sole sulfur source, and biotin, thiamine hydro- methanogenic substrates, such as H,-CO,, formate, metha- chloride, and p-aminobenzoic acid stimulate growth (19). nol, or methylamines, can serve as sole sources of energy. Iron, nickel, manganese, zinc, and cobalt enhance growth, Acetate and CO, are sole carbon sources. whereas both calcium and magnesium are essential (19). Description of Methanosaeta concilii sp. nov. Methanosaeta Sludge fluid is stimulatory (18). Organic growth factors are concilii (con.ci‘1i.i. L. gen.n. concilii, of a council, named not required for culture maintenance in mineral-salts-vitamin after the National Research Council of Canada) is the type medium Aa (18). species of the genus Methanosaeta. Growth is almost completely inhibited by either methyl or The cells are gram-negative, nonmotile, nonsporeforming, benzyl viologen (5 bm), by chloroform (50 pM), and by strictly anaerobic rods with flat ends (18). The cells are 0.8 potassium cyanide (100 pM) (18). Inhibition by 2-bromoet- pm wide by 2.5 to 6.0 pm long and are enclosed in a hanesulfonic acid is reversed by a five-fold excess of 2- proteinaceous sheath. Chains of cells grow as long filaments mercaptoethanesulfonic acid (18). Severe inhibition of CH, which aggregate into bundles. Filaments do not easily break production that is caused by 40 mM NH,Cl during short- into smaller fragments when they are subjected to vigorous term (6-h) incubation of cells in anaerobic, 0.1 M HEPES shaking. Cultures incubated without shaking grow as a mat (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid) buff- or as a sediment (18). The sheath consists of annular hoops er is completely reversed in the presence of 20 to 40 mM stacked to form the sheath tube (2). In-growth of spacer CaCl, (21). In longer-term (21-d) incubations in growth plugs and filament splitting at the site of a nonreplicating unit medium, 120 mM NH,Cl is required to cause about 80% of the protoplasm are part of the cell division process (1). inhibition of growth and methanogenesis (21). Yeast extract The sheath consists of a matrix with an outer paracrystalline (0.05%, wthol) results in a 50% inhibition of CH, production surface array composed of small subunits exhibiting p2 (Fig. 1) and growth. An equivalent amount of ashed yeast symmetry, and it resists dissociation by various chemical extract (SSOOC, 4 h) does not affect growth or methanogen- treatments (20). esis. Vancomycin (1 mg/ml), kanamycin (1 mg/ml), and VOL. 40, 1990 METHANOSAETA GEN. NOV. 81

TABLE 1. Comparative characteristics of Methanosaeta concilii GP6T and strains Z517T and CALS-1“ Guanine- PH Temp (“C) Strain Broth culture Gas doublingMass cytosineplus- Inhibited Inhibition Vitamins growth width Vesicles by : by H, (i4 Optimum Range Optimum Range time (h) ‘Ontent (mol%)

~ ~ GP6= Mat form or 0.8 Absent 7.1-7.5 6.6-7.8 35-40 >10-<45 65 49.0b Yeast extract No Stimulatory loose sediment (0.05%) Z517T Smallflocs or 1.0-1.3 Present NR‘ NR 65d 50-70 NR NR NR Yes Stimulatory loose sediment CALS-1 Opalescent tur- 1.0-1.2 Present 6.5 6.0-8.0 60 >37-<70 24 NR Phosphate No Biotin re- bidity (20 mM) quired The data for strain Z517= are from reference 16, and the data for strain CALS-1 are from reference 26. Determined by the thermal denaturation method. NR, Not reported. An optimum temperature of 55°C and a growth temperature range of 50 to 65°C have been reported recently (17).

D-cycloserine (0.1 mg/ml) cause 90 to 100% inhibition of facility (18). This isolate represents a key methanogenic growth and methanogenesis (18). organism in mesophilic anaerobic digestion processes where Growth and CH4 production do not occur with methanol, acetic acid is a major intermediary metabolite of organic trimethylamine, formic acid, propionic acid, butyric acid, or waste fermentation (18). pyruvic acid as the sole source of carbon and energy (18). In There are only two other pure cultures that have been the presence of acetate, formic acid is split into H, and CO,. described in the genus “Methanothrix,” both of which are H,-CO, does not support growth, and 80% H, in the head- thermophiles. The culture purity of ‘‘Methanothrix” sp. space gas does not inhibit growth on acetate (18). Acetic acid strain CALS-1 (= DSM 3870) (26) and the culture purity of serves as the sole energy source, and both acetic acid and “Methanothrix themoacetophita” strain Z517T (= DSM CO, serve as carbon sources for growth (18). The metabo- 4774T) have been verified by H. Hippe of the DSM (personal lism of 1 mol of acetic acid results in the production of 1.0 communication). Until recently, strain Z517T was cultivated mol of CH,, 0.95 mol of COZYand 1.13 to 1.16 g (dry weight) only in monoculture and not in pure culture (16, 24). The of cells, and the apparent K, for acetate is 1.2 mM (18). The description of both of these isolates matches that of the CH, carbon is nearly all derived from the methyl carbon of genus Methanosaeta as given above. Although the charac- acetate, the carboxyl carbon of acetate is predominantly terizations of thermophilic strains Z517T and CALS-1 are not converted to CO,, and both carbons of acetate, as well as as complete as that of strain GP6T, there are distinct carbon from NaHCO,, are incorporated into cell biomass (5, differences (Table l), most notably thermophily and the 18). Acetate is carboxylated to form pyruvate, which is then presence of gas vesicles. Therefore, we propose that “Meth- converted to amino acids (5). anothrix thermoacetophila” (12, 16) be reclassified as Meth- About 10% of the cell dry weight of strain GP6T is lipid; of anosaeta thermoacetophila nom. rev., comb. nov., with this about 93% is polar lipids and the remainder is neutral strain 2517 (= DSM 4774) as the type strain. Further lipids (7, 8). The lipid structures are based on standard information is needed to determine whether strain CALS-1 is C,,,,,-diether core lipids or a hydroxylated analog (7, 8). a species distinct from Methanosaeta thermoacetophila. Strain GP6T does not react in the quantitative slide immu- noenzymatic assay with any of the 29 heterologous S probes ACKNOWLEDGMENT representing the genera Methanobrevibacter, Methano- We are grateful to K. Jarrell (Queen’s University, Kingston, bacterium, Methanosarcina, Methanocoecus, Methanoge- Ontario, Canada) for determining the base compositions of purified nium, Methanomicrobium , Methanothermus, Methanolo- DNA samples of strain GP6T. bus, Methanoplanus, and Methanosphaera (14). The indi- rect immunofluorescence reaction of strain GP6T with LITERATURE CITED antiserum S probes of Methanospirillum hungatei JF1, 1. Beveridge, T. J., B. J. Harris, G. B. Patel, and G. D. Sprott. Methanosarcina mazei MC6, Methanosarcina barkeri MS, 1986. 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