Methanoculleus Marisnigri and Methanogenium, and Description of New Strains of Methanoculleus Bourgense and Met H a Noc U 11 E Us Ma Ris N Igri GLORIA M
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INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY , Apr. 1990, p. 117-122 Vol. 40, No. 2 OO20-7713/90/020117-06$02.OO/O Copyright 0 1990, International Union of Microbiological Societies Transfer of Methanogenium bourgense, Methanogenium marisnigri, Methanogenium olentangyi, and Methanogenium thermophilicum to the Genus Methanoculleus gen. nov., Emendation of Methanoculleus marisnigri and Methanogenium, and Description of New Strains of Methanoculleus bourgense and Met h a noc u 11 e us ma ris n igri GLORIA M. MAESTROJUAN,l DAVID R. BOONE,l* LUYING XUN,2t ROBERT A. MAH,, AND LANFANG ZHANG' Department of Environmental Science and Engineering, Oregon Graduate Center, Beaverton, Oregon 97006-1999, and School of Public Health, University of California, Los Angeles, California 9O01ij2 Two strains of Methanogenium bourgense, strains MS2T(T = type strain) and LX1, were characterized, and, based in part on previously published DNA hybridization data, this species was transferred to a new genus, Methanoculleus, as Methanoculleus bourgense comb. nov. Methanogenium marisnigri JRIT and a new strain of Methanogenium marisnigri, strain ANS, were also characterized. This species was also transferred to the genus Methanoculleus as MethanocuUeus marisnigri comb. nov. et emend., and its description was emended to indicate that the species has a temperature optimum near 40°C, is halotolerant, and is slightly alkaliphilic; in contrast, the previous description of this organism indicates that it has a temperature optimum of 20 to 25"C, is halophilic, and is slightly acidophilic. We also propose the transfer of two other phylogenetically related species, Methanogenium thermophilicum and Methanogenium olentangyi, to the genus Methanoculleus as Methanoculleus thermophilicum and Methanoculleus olentangyi, respectively. Methanogenium cariaci JRIT was also further characterized, and its description is emended. The results of studies of diffusion of H, and formate from (16), Methanogenium olentangyi (9,and Methanogenium bulk solutions to methanogenic cells suggest that the small organophilum (14). DNA hybridization data indicate deep size of many methanogens may be advantageous in scaveng- divisions among these species, with Methanogenium ing small concentrations of these substrates (1). However, marisnigri, Methanogenium thermophilicum, Methanoge- the isolation and characterization of small coccoid methano- nium bourgense, and Methanogenium olentangyi being gens from digestors and other natural methanogenic environ- distinct from other members of the genera Methanogenium ments are fairly recent developments (5, 9, 12, 15, 16, 18, and Methanocorpusculum (15). Sequences have been de- 21), perhaps because these methanogens grow slowly and termined for the 16s rRNAs of six of these organisms, are sensitive to detergents and physical and osmotic stress. Methanoplanus limicola, Methanogenium organophilum, Colonies may often be found in roll tube media inoculated Methanogenium cariaci, Methanomicrobium mobile, Meth- with high dilutions if a small amount of salt is included in the anogenium marisnigri, and Methanogenium thermophilicum media and the incubation period is sufficiently long (4). (P. E. Rouvi&re and C. R. Woese, manuscript in prepara- The genus Methanogenium is a genus of coccoid, meth- tion). As determined by both evolutionary distance and anogenic archaeobacteria which was originally described as maximum parsimony analyses, these six sequences (which marine (12). Since the original description, a number of new are all more than 90% related to one another) fall into two species of coccoid methanogens have been isolated and distinct, but specifically related, phylogenetic groups. One placed in the genus Methanogenium because they appeared comprises the first four organisms. The other includes Meth- to fit there better than in other genera. The inclusion of these anogenium marisnigri and Methanogenium thermophilicum organisms led to an informal broadening of the genus de- (Rouviere and Woese, in preparation). This finding is in scription. Recently, the genus Methanocorpusculum, a new accord with the DNA hybridization data (15), which indicate genus in the family Methanomicrobiaceae, was described that Methanogenium marisnigri, Methanogenium thermo- (18). Methanogenium aggregans was transferred to that philicum, Methanogenium bourgense, and Methanogenium genus (15), two new species, Methanocorpusculum bavari- olentangyi should be separated into a new genus. Therefore, cum and Methanocorpusculum sinense, were described, and we propose that this group of organisms should be trans- a new family for the genus was proposed (19). Currently, ferred to a new genus, Methanoculleus, with Methanocul- there are the following seven valid species of the genus leus bourgense as the type species. Methanogenium: Methanogenium cariaci (12), Methanoge- nium marisnigri (12), Methanogenium thermophilicum (ll), MATERIALS AND METHODS Methanogenium bourgense (9), Methanogenium tationis Media and culture techniques. We used the anaerobic techniques of Hungate (6), with syringes and serum tubes. * Corresponding author. MG medium was similar to MS medium (2) but also con- t Present address: Department of Biochemistry and Bacteriology, tained 2.5 g of NaCl per liter and 5 mmol of sodium acetate University of Idaho, Moscow, ID 83843. per liter, and 0.5 g of 2-mercaptoethanesulfonic acid re- 117 118 MAESTROJUAN ET AL. INT.J. SYST.BACTERIOL. placed the cysteine. This medium also contained 7 g of nium olentangyi RC/ERT (= OGC 52T), Methanogenium NaHCO, per liter, which was added after boiling to remove tationis DSM 2702T (= OGC 43T), Methanocorpusculum O,, or it contained 4 g of NaOH per liter and was equili- aggregans MSt (= OGC 21), and Methanocorpusculum brated with an N,-CO, mixture (7:3) without boiling to parvum XI1 (= OGC 63) were obtained from the Collection remove dissolved 0, (8). The final pH was 7.2 to 7.3. of Methanogenic Archaeobacteria at the Oregon Graduate Enrichment medium was the same as MG medium but Center. The authenticities of our cultures of Methanogenium without cysteine or 2-mercaptoethanesulfonate and with the marisnigri JRIT and Methanogenium cariaci were confirmed peptone and yeast extract concentrations reduced to 0.5 by electrophoretic comparison with additional cultures ghiter. Routinely, the catabolic substrate was 100 mM for- kindly supplied by Hans Hippe of the Deutsche Sammlung mate, and cultures were incubated at 37°C without shaking. von Mikroorganismen. For growth on H,-C02, media (with an N,-CO, gas phase) The inoculum from which we isolated strain AN8 (= OGC were inoculated and then pressurized with pure H, to a 51 = DSM 4552) was effluent from an anaerobic pig manure partial pressure of 101 kPa. The resulting cultures were digestor in Spain, and the inoculum from which we isolated incubated with orbital shaking (120 rpm with a 27-mm stroke strain LX1 was effluent from a sewage sludge digestor at the radius) and were frequently repressurized to 101 kPa above Hyperion Wastewater Treatment Plant in Los Angeles, atmospheric pressure with an H,-C02 mixture (3:l). The Calif. ability to use soluble catabolic substrates was tested by G+C content of the DNA. To determine the guanine- inoculating the following media: media supplemented with plus-cytosine (G+C) content of DNA, late-log-phase cul- 20 mM formate, media supplemented with 20 mM formate tures were cooled to 4°C and were harvested by centrifuga- plus the substrate to be tested at a concentration of 10 mM, tion at 5,000 X g for 15 min. DNA was isolated by a media supplemented with the substrate to be tested at a modification of the Marmur method (7), and the density was concentration of 10 mM, and controls without a catabolic determined by ultracentrifugation in CsCl gradients (10). substrate. Measurement of the methane produced by these DNAs from Micrococcus lysodeikticus (density assumed to cultures indicated whether the substrate was inhibitory and be 1.731 g/cm3; Sigma Chemical Co., St. Louis, Mo.) and whether it could be catabolized. Media at various pH values Clostridium perfringens (density assumed to be 1.691 g/cm3; were prepared by equilibrating medium with gas at various Sigma) were used as standards. The G+C content of the CO? partial pressures. For pH values higher than 8.1, we DNA was calculated by using the formula of Schildkraut et equilibrated the medium with 100% N, and added a sterile, al. (13). anoxic NaOH solution to adjust the pH. For pH values lower Analytical techniques. Proteins were analyzed by poly- than 6.5 we equilibrated the medium with 100% CO, and acrylamide gel electrophoresis as described previously (8). added sterile, anoxic HC1 to adjust the pH. The pH was Specific growth rates were determined from an analysis of checked after autoclaving and equilibration at the growth methane production, and methane was quantified by gas temperature and again after the growth of cultures for chromatography (8). We used a type 02filter set (Carl Zeiss, determination of the optimal pH for growth, During mea- Inc., Thornwood, N.Y .) for epifluorescence microscopy to surement of specific growth rates at various pH values, the distinguish colonies likely to be methanogenic based on their pH did not change more than 0.2 pH unit during growth; the blue-green fluorescence, pH values indicated in Fig. 2 are the initial pH values. Media with various Na+ concentrations were prepared, or the Na+ RESULTS AND DISCUSSION content of MG medium was adjusted by adding a sterile, anoxic