DNA-DNA Hybridization of Methylotrophic Halophilic

INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,OCt. 1991, p. 558-562 Vol. 41, No. 4 0020-7713/91/040558-05$02.OO/O

DNA-DNA Hybridization of Methylotrophic Halophilic Methanogenic Bacteria and Transfer of Methanococcus halophilusvPto the Genus Methanuhalophilus as Methanohaluphilus halophilus comb. nov. T. WILHARM,l T. N. ZHILINA,’* AND P. HUMMEL’ Lehrstuhl fur Mikrobiologie der Universitat Regensburg, Regensburg, Germany, and Institute of Microbiology of the USSR Academy of Sciences, Moscow, USSR’

Twelve strains of halophilic, methylotrophic, methanogenic bacteria, including the type strains of the validly published species Methanohalophilus mahii, Methanohalophilus zhilinae, and Methanohalobium evestigatum, as well as the type strain of Methanococcus halophilus, were compared on the basis of DNA-DNA hybridization and phenotypic characteristics. Two groups (moderately halophilic Methanohalophilus strains and extremely halophilic Methanohalobium strains) were formed on the basis of G+ C contents and DNA-DNA hybridization data. Type strain SLP of Methanohalophilus mahii and type strain 2-7982 of Methanococcus halophilus exhibited 87% sequence similarity and belong to the same genus (Methanohalophilus), if not to the same species. A regulation of the International Code of Nomenclature of BacteriQ implies that the specific epithet halophilus has priority by validation and should be retained. Therefore, we propose that Methanococcus halophilus should be transferred to the genus Methanohalophilus as Methanohalophilus halophilus comb. nov. (type strain, strain 2-7982 [= DSM 3094 = OCM 1601); an emended description is given. Moderately halophilic strains TR-7,2-7404, and 2-7405, which were isolated from geographically separated sites, exhibited high levels of DNA-DNA hybridization with Methanohalophilus mahii and might be regarded as strains of the same Methanohalophilus species. The alkaliphilic organism Methanohalophilus zhilinae is a valid species in the genus Methanohalophilus. The extremely halophilic organism Methanohalobium evestigatum exhibited no significant DNA-DN A hybridization with moderately halophilic species and should be regarded as a member of an independent genus. Other extremely halophilic strains (strains 2-7408 and 2-7403) exhibited low levels of DNA-DNA hybridization with Methanohalobium evestigatum Z-7303T (= DSM 3721) (T = type strain).

The obligately methylotrophic halophilic methanogens are anococcus halophilus and Methanolobus tindarius, Metha- a coherent physiological group of microorganisms. They nococcoides methylutens, and Methanohalobium eves- inhabit hypersaline water and utilize noncompetitive path- tigaturn was more than 20 bases (4). Thus, Methanococcus ways of methanogenesis by using methylamines that are halophilus does not belong to any of these genera. produced in the course of decomposition of osmoregulatory Paterek and Smith (19) proposed a new genus, Methano- substances (7, 8, 29). After the first descriptions of marine halophilus, for the moderately halophilic methanogens; the methylotrophic methanogens (Methanolobus tindarius [lo] type species of this genus, Methanohalophilus mahii, was and Methanococcoides methylutens [20]), a moderately isolated from the Great Salt Lake in Utah (18). Two new halophilic strain was isolated and assigned to the genus alkaliphilic species, Methanohalophilus zhilinae (17) and Methanococcus under the name Methanococcus halophilus Methanohalophilus oregonensis (13), were assigned to the (24). same genus. The name Methanohalophilus was validly pub- Later, the type species of the genus Methanococcus was lished at the same time as the name Methanohalobium (6b, transferred to the genus Methanosarcina as Methanosarcina 19). mazei, and the description of the genus Methanococcus was The systematics of the weakly halophilic Methanolobus emended by Mah and Kuhn (15). The type species of the and Methanococcoides species was confirmed by Sowers et emended genus Methanococcus is Methanococcus vannielii, al. (21) on the basis of RNA-DNA and DNA-DNA hybrid- a marine bacterium which utilizes formate but not methyl- ization data. The aim of this work was to perform a compar- amines. Therefore, assignment of Methanococcus haloph- ative study of the moderately and extremely halophilic ilus to the genus Methanococcus became unjustifiable. methanogenic bacteria to clarify their taxonomic positions. Meanwhile, the following new species of extremely halophilic methanogens had been described: Methanohalobium evestigatum Halomethanococcus doii (28) and (23). The MATERIALS AND METHODS later taxon was not included in this study because the type strain was not available to us. The results of 5s rRNA analyses of strains of weakly, moderately, and extremely Bacterial strains. Strains 2-7301, 2-7302, 2-7305, 2-7405, halophilic methylotrophic methanogens demonstrated that 2-7401, 2-7404, and 2-7408 were isolated by T. N. Zhilina these organisms belong to the same cluster and are closely from saline lagoons in the Crimea, USSR (25). Strain TR-7 related to methanosarcinas (4); the difference between Meth- was isolated by T. Wilharm from salt soils near Trapani, Sicily (22). Reference strains Methanohalophilus mahii SLPT (= ATCC 35705) (T = type strain), Methanohalophilus zhilinae WeNST (= DSM 4017), Methanococcus halophilus * Corresponding author. Z-7982= (= DSM 3094), and Methanohalobium evestigatum

558 VOL. 41, 1991 METHANOHALOPHILUS HALOPHILUS COMB. NOV. 559

TABLE 1. Differential characteristics of the obligately methylotrophic methanogens"

~ ~ ~~ ~ Cell diam Lysis by Optimal Optimum Species or strain sodium salinity f;zi:: Source (Fm) dodecyl sulfate (M NaCI) (M NaCI) temp ("'I Moderate halophiles Methanohalophilus mahii 0.8-1.8 + 2.0 0.5-3.5 35 Great Salt Lake, Utah Methanohalophilus zhilinae 0.8-1.5 + 0.7 0.2-2.1 45 Wadi el Natrun, Egypt Methanococcus halophilus 0.5-2 .O + 1.2-1.5 0.3-2.6 26-36 Shark Bay, Australia TR-7 0.4-0.8 + 0.6-1.0 0.3-3.4 33 Trapani, Sicily 2-7404 0.5-1.3 + 1.7 0.8-2.5 37 Chokrack Lake, Crimea, USSR 2-7405 0.2-1.1 + 1.7 0.8-1.7 37 Kalmykia, USSR 2-7302 0.3-1 .O + 0.8-1.7 0.8-2.5 ND~ Chokrack Lake, Crimea, USSR 2-7301 0.3-0.5 + 1.7-2.5 1.7-4.2 ND Sivash Lake, Crimea, USSR 2-7401 0.3-1.4 + 0.8-1.7 0.8-3.4 ND Arabat Lagoon, Crimea, USSR Extreme halophiles Methanohalobium evestigatum 0.2-2 .o - 4.3 2.6-5.1 50 Arabat Lagoon, Crimea, USSR 2-7408 0.1-1.6 - 2.5-3.4 1.7-5.1 ND Sivash Lake, Crimea, USSR 2-7403 0.2-1.6 - 2.5-3.4 1.7-4.2 ND Kalmykia, USSR

a Data from references 17-19, 22, 24, 25, 26, and 28. ND. not determined.

Z-7303T (= DSM 3721) were obtained from the Deutsche RESULTS AND DISCUSSION Sammlung von Mi kroorganismen. Media and growth conditions. Biomass for DNA studies Morphology and other phenotypic features. Differential was obtained by anaerobic cultivation in 0.5-liter serum characteristics of the strains which we studied, as well as flasks containing 350 ml of medium. The yield was in the reference strains, are shown in Tables 1 and 2. All of the range 0.1 to 0.2 g per flask. The medium used for Methano- strains which we studied could be divided into two groups on halophilus zhilinae and Methanococcus halophilus has been the basis of the substrates used, the optimum salinities, and described previously (17, 24). All of the other strains were the ranges of salinity tolerated. cultivated in medium containing (per liter) 0.34 g of KCl, The cells of the moderately halophilic methanogens are 2.75 g of MgC12 * 2H20, 3.45 g of MgSO, * 7H,O, 0.25 g of irregular cocci (diameter, 0.2 to 2 km) that aggregate in slimy NH,Cl, 0.14 g of CaC1, . 2H,O, 0.14 g of K,HPO,, 0.1 g of groups and are easily lysed by 0.05% sodium dodecyl NaHCO,, 0.5 g of Na2S . 9H,O, 5 g of trimethylamine, 1 mg sulfate. Only strain 2-7401 is monotrichous; the other strains of resazurin, 10 ml of a vitamin solution (l),and 10 ml of a are not motile. All of the strains are morphologically similar trace element solution (1); the NaCl content varied (100,150, and microscopically indistinguishable. All of the strains or 250 g/liter). For strain TR-7 1 g of sodium acetate per liter isolated from marine hypersaline lagoons grow optimally at was added. The gas phase was nitrogen, and the pH was 7.0. near-neutral pH values. The alkaliphilic organism Methano- Each strain was cultivated at the optimal salinity and tem- halophilus zhilinae WeN5 was isolated from terrestrial saline perature (Table 1). Strains 2-7302, 2-7301, 2-7401, 2-7408, waters (17). The strains differ in the range of salinities and 2-7403 were cultivated at 37°C. tolerated, the optimum salinity, and cell size. All of the DNA isolation. DNA was isolated as described previously strains utilize methylamines and to some extent methanol. (12). However, Methanococcus halophilus, which has been DNA base composition. The G+C content of the DNA was grown on methylamine for a long time, has lost its ability to determined from the melting point in 0.1 X SSC (1 X SSC is utilize methanol. 0.15 M NaCl plus 0.015 M sodium citrate) (16) by using calf The Methanohalobium group includes extremely halo- thymus DNA (42 mol% G+C) as a reference. Each G+C philic strains that grow most rapidly on trimethylamine; content was determined by at least two independent mea- these organisms do not utilize methanol. Like Methanohalo- surements. bium evestigatum, these strains look like flat methanosarci- DNA-DNA hybridization. DNA-DNA hybridization was nas. Methanohalobium evestigatum forms macrocystlike performed (9) after radioactive labeling of the DNA (5) with microcolonies with thick glycocalyxes. These macrocysts of a oligolabeling kit (Pharmacia) by using the filter technique the Methanosarcina rnazei type all disintegrate into irregular (3, 6). flat cells that look like broken glass. The morphology de-

TABLE 2. Comparative data for Methanohalophilus speciesa % G+C Optimum DNA-DNA hybridization with: NaC1 Optimum PH Species content NaCl concn concn range PH range Methanohalophilus Methanohalophilus (mol%) (molAiter) (mol/liter) mahii zhilinae

Methanohalophilus mahii 45 .O 2.0 0.5-3.5 7.5 6.5-8.5 20 Methanohalophilus zhilinae 41 .O 0.7 0.2-2.1 9.2 8.2-10.3 Methanohalophilus halophilus 44.0 1.5 0.3-2.6 7.4 6.3-8.0 87.0 8.0

a Data from references 17-19 and 24 and this study. 560 WILHARM ET AL. INT. J. SYST.BACTERIOL.

TABLE 3. Levels of DNA-DNA hybridization and G+C contents of the halophilic methanogens % Hybridization with 32P-labeled DNA from: G+C Filter-bound Methano- Methano- Methano- Methanococcus Strain content DNA from: halophilus halophilus halophilus halobium TR-7 (mol%) mahii zhilinae evestigatum Methanohalophilus mahii 100 ND" ND ND ND 45 )Wethanococcus halophilus 87 15 100 17 63 44 'TR-7 86 ND ND ND 100 48 12-7404 86 6 42 13 62 43 :Z-7405 50 7 25 11 80 44 .Methanohalophilus sp. strains 2-7401 48 5 30 6 57 44 2-7301 47 11 23 3 68 45 2-7302 37 7 23 4 39 42 Methanohalobiurn evestigatum 6 7 3 100 6 37 2-7403 7 7 5 15 7 40 2-7408 11 5 4 7 6 39 Methanohalophilus zhilinae 20 100 8 13 6 41 Calf thymus DNA 12 5 5 12 5 '' ND, not determined.

pends on the age of the culture and the concentration of rRNA of this organism differs from Methanococcus haloph- Ca2+ and Mg2+ in the medium. Flat cells of Methanohalo- ilus 5s rRNA by 26 bases, a value which is within the range bium evestigatum easily strip out the outer layers of cell wall of values found in the other genera of halophilic methano- (27). The cell walls resemble those of Methanosarcina gens (Methanohalophilus, Methanococcoides , and Metha- species in not being lysed by sodium dodecyl sulfate. How- nolobus) (4). In its morphology and in a number of pheno- ever, despite the similarities in morphology and substrates typic features (i.e., high temperature optimum, salinity utilized, Methanohalobium evestigatum actually differs from range, optimum salinity, and lower G+C content), Metha- freshwater methanosarcinas by 38 bases in their 5s rRNAs nohalobium evestigatum differs distinctly from members of and cannot be regarded as a member of the genus Metha- the genus Methanohalophilus, and new data have confirmed nosarcina (4). its taxonomic status. Two other extremely halophilic strains, Table 1 shows that closely related strains were isolated strains 2-7408 and 2-7403, appeared to be closely related to from geographically separated sites, eliminating the possibil- Methanohalobium evestigatum phenotypically and on the ity of geographical varieties. basis of G+C contents, but exhibited very low levels of DNA-DNA hybridization studies. The results of our DNA- DNA-DNA hybridization with the type strain, strain 2-7303 DNA hybridization studies are summarized in Table 3. The (= DSM 3721). G+C contents of the moderately halophilic strains range The alkaliphilic organism Methanohalophilus zhilinae ex- from 42 to 48 mol%. The three extremely halophilic strains hibited only 20% DNA homology with Methanohalophilus have lower G+C contents (37 to 40 mol%). The G+C mahii and still lower levels of DNA homology with Metha- contents of the reference strains in this study were 3 mol% nohalobium evestigatum. Methanohalophilus zhilinae dif- lower or higher than the values reported previously (14, 17, fers from Methanohalophilus mahii in G+C content, in its 19); the reason for these discrepancies is not known. The higher temperature optimum, in its high pH range, and in its differences in G+C content data have been discussed previ- lower salinity range (Table 2). There is no doubt that both ously (14). genotypically and phenotypically this organism forms a We observed high levels of DNA-DNA hybridization separate taxon, either at the species level or at the genus among the moderately halophilic strains, including Metha- level. nohalophilus mahii, Methanococcus halophilus, strain Nomenclature considerations. On the basis of phenotypic 2-7404, and strain TR-7. Thus, all of these strains belong to characteristics and DNA-DNA hybridization data (Table 2), the genus Methanohalophilus and may belong to the same Methanococcus halophilus Z-7982T and Methanohalophilus species. Although strain 2-7405 exhibited only 25% DNA mahii SLPT seem to be closely related and might be consid- homology with Methanococcus halophilus, these two organ- ered strains of the same species. isms were immunologically identical (2). Because of its high According to Rules 23a, 38, and 42 of the International levels of DNA homology with other strains of this group, Code of Nomenclature of Bacteria (ll), the specific epithet strain 2-7405 should be considered a member of the same halophilus has priority by validation (6a) over the specific species. epithet mahii (6b, 19) and should be retained. Another group included strains 2-7401, 2-7301, and According to Rule 37a of the International Code (ll),the 2-7302, which exhibited 37 to 48% DNA homology with genus name Methanohalophilus should be invalidated unless Methanohalophilus mahii. Although these organisms might an Opinion of the Judicial Commission of the International be considered members of separate species of the genus Committee on Systematic Bacteriology rules to the contrary. Methanohalophilus, we did not find enough significant phe- If both moderately and extremely halophilic methanogens notypic differences to create a new species. Therefore, we are united in the same genus, then the name Methanohalo- consider them to be Methanohalophilus sp. bium, which was validly published with the type species Methanohalobium evestigatum exhibited no DNA-DNA Methanohalobium evestigatum (type strain, strain 2-7303 hybridization with the moderately halophilic strains. The 5s [= DSM 3721]), should have priority over the invalid genus VOL. 41, 1991 METHANOHALOPHILUS HALOPHILUS COMB. NOV. 561

name Methanohalophilus. However, we prefer to conserve ACKNOWLEDGMENTS the genus Methanohalophilus (Paterek and Smith 1988) with The valuable comments of K. 0. Stetter, G. A. Zavarzin, D. R. its current taxonomic conception for the moderately halo- Boone, and anonymous reviewers on the taxonomy of the halophilic philic methylotrophic methanogens, with Methanohalo- methanogens are greatly appreciated. philus halophilus as the type species. There is no need to emend the description of the genus Methanohalophilus since REFERENCES the subsequently isolated type strain Methanohalophilus 1. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. mahii Wolfe. 1979. Methanogens: reevaluation of a unique biological ATCC 35705 was found to be very similar to type group. Microbiol. Rev. 43:260-296. strain 2-7982 (= DSM 3094). Until more data are available, 2. Bezrukova, L. V., A. Y. Obraztsova, and T. N. 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