INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1988, p. 139-142 Vol. 38, No. 2 0020-7713/88/0201 39-04$02.00/0 Copyright 0 1988, International Union of Microbiological Societies

Methanohalophilus zhilinae sp. nov. , an Alkaliphilic, Halophilic, Methylotrophic INDRA M. MATHRAN1,l DAVID R. BOONE,'t* ROBERT A. MAH,l GEORGE E. FOX,, AND PAUL P. LAU2 Division of Environmental and Occupational Health Sciences, School of Public Health, University of California, Los Angeles, Calfornia, 90024l and Department of Biochemical and Biophysical Sciences, University of Houston, Houston, Texas 77004,

Methanohalophilus zhilinae, a new alkaliphilic, halophilic, methylotrophic species of methanogenic bacteria, is described. Strain WeNST (T = type strain) from Bosa Lake of the Wadi el Natrun in Egypt was designated the type strain and was further characterized. This strain was nonmotile, able to catabolize dimethylsulfide, and able to grow in medium with a methyl group-containing substrate (such as methanol or trimethylamine) as the sole organic compound added. Sulfide (21 mM) inhibited cultures growing on trimethylamine. The antibiotic susceptibility pattern of strain WeNST was typical of the pattern for archaeobacteria, and the guanine-plus-cytosine content of the deoxyribonucleic acid was 38 mol%. Characterization of the 16s ribosomal ribonucleic acid sequence indicated that strain WeNST is phylogenetically distinct from members of previously described genera other than Methanohalophilus and supported the partition of halophilic metha- nogens into their own genus.

Methanogenesis occurs in sediments of some alkaline, Na,S . 9H@, and no cysteine hydrochloride monohydrate. saline lakes, as demonstrated by enrichment and isolation The isolation medium (3) was the same as the enrichment techniques. Methanogenic enrichment cultures from Big medium except that the NaCl concentration was reduced to Soda Lake in Nevada catabolize methanol, trimethylamine, 120 g/liter and 15 g of purified agar per liter was added. dimethylsulfide, and methane thiol (14); from Culture techniques. Anaerobic culture techniques of Bosa Lake in the Wadi el Natrun, Egypt, catabolize tri- Hungate (7) were used. Inoculations were made by using methylamine (3); and enrichment cultures from Mono Lake syringes flushed with 0,-free gas. Enrichment cultures were in California catabolize dimethylsulfide, dimethyldisulfide, incubated at 37°C without shaking, and all other cultures and methane thiol (9). Only compounds which contain were incubated at 45°C; the inoculum volume was 1.7%. The methyl groups are used as substrates; H,-CO, is not used by effects of pH, NaC1, and temperature (reported previously isolates or in enrichment cultures. Some or all of these [3]) and the effects of sodium sulfide were measured by bacteria belong in a recently described genus, Methanoha- adjusting the normal culture medium as required and calcu- lophilus (16). In contrast, alkaliphilic isolates from nonsaline lating the growth rate early in the exponential phase. Specific environments use only H,-CO, for growth and methanoge- growth rates were determined by measuring the methane nesis (2, 3,21). Previously (3), we reported the isolation from accumulation rates in the culture vessels; the contribution of Bosa Lake of strain WeNST (T = type strain), the first inoculum was considered when the specific growth rate was methylotrophic, alkaliphilic methanogen isolated in axenic calculated (17). When the growth rates at various pH values culture. In this paper this organism, which uses trimethyla- were measured (3), a HC031--C032- buffer was used; media mine and methanol for growth and methanogenesis, is fur- were prepared with 5 g of Na,CO, per liter, and CO, was ther characterized and is designated the type strain of added to obtain the desired pH. Methanohalophilus zhilinae sp. nov. Determination of 16s rRNA partial sequences. We purified and partially sequenced 16s ribosomal ribonucleic acid MATERIALS AND METHODS (rRNA) by using the reverse transcriptase procedure of Lane Inocula and media. Strain WeNST was isolated (3) from et al. (11). The procedures of Lane et al. were followed Bosa Lake in the Wadi el Natrun, Egypt, by using the exactly. This approach employs 16s rRNA-specific oligo- techniques and media described below. The culture medium deoxynucleotide primers in conjunction with dideoxynucleo- had the following composition (per liter of Milli-Q-deionized tide sequencing by reverse transcriptase (Seikagaku Amer- water, 5.9 mS m-l [Millipore Corp., Bedford, Mass.]): 3.0 g ica). The deoxyribonucleic acid primers were made by of NaHCO,, 2.0 g of Na,CO,, 2.0 g of yeast extract (Difco beta-cyanoethyl phosphoramidite chemistry (18), using an Laboratories, Detroit, Mich.), 2.0 g of Trypticase peptone automated procedure (Bioresearch model 8700). The partic- (BBL Microbiology Systems, Cockeysville, Md.), 40 g of ular primers used in this study (International Union of NaC1, 1.0 g of NH,C1, 3.5 g of MgC1,. 6H20, 3.0 g of Biochemists nomenclature; Escherichia coli 16s rRNA po- MgSO, . 7H,O, 1.0 g of KC1, 0.4 g of K,HPO,, 1 mg of sitions are indicated in parentheses) were ATGCCCTCCG resazurin, 10 ml of a trace minerals solution (4), 0.5 g of TCGTC (357), TTTGARTTTMCTTAAC (926), GTTGCTC cysteine hydrochloride monohydrate, and 0.25 g of GCGTTGGGA (11lo), CRTGTGTGGCGGGCA (1406), and Na,S . 9H20. The gas phase was nitrogen, and the pH was TTCAGCATTGTTCCATTGG (1510). 9.3. The enrichment medium contained 0.5 g of yeast ex- Analytical techniques. Lysis by sodium dodecyl sulfate and tract, 0.5 g of Trypticase peptone, 250 g of NaCl, 0.5 g of susceptibility of cultures to antibiotics were tested as previ- ously described (13). The guanine-plus-cytosine content of * Corresponding author. the deoxyribonucleic acid was determined by the buoyant t Present address: Department of Environmental Science and density method, and methane was determined by gas chro- Engineering, Oregon Graduate Center, Beaverton, OR 97006-1999. matography (1).The total dissolved solids content of the lake

139 H. r-olcanii ALLCCGGLLG ALCCLGCCGG AGGLCALL JC LAbLGGGGLC CGAbLLAGCC N. zhi 1 inae

H. v. XLGCbAG'UUG . CACG.-\GL. L CXLXCLCGLG GCGAAAAGC UCAGUAAC 4C GLlGGCCAXAC M.z.

H.V. LXCCCUACAG XGAALGALAA CCLCGGGAAA C'VGAGGCLAA LAGL LCAL AC GGGAGL( XCG M.Z.

H.v. CLtitiAALGCC GACLCCCCGA AACGCLCAGG CGCLGI AGGA LGLG(rCLGCG LCCGALCAGG P1.Z. GCCCAA,bl?A lIGG4UC ti(;CG CrCCLAbCAGG

H.v. P1.z.

H.v. CCGGAGACGG AALCLGAGAC AXGALL-CCGG (X.CCbXCGGG GCGCAGCAtiG CGCGA LACCL P1.z. CCGGAGALGG AUIJCbGACAC AUGAALICCAC GcCCLACtiGG GCGCAGCAGG CGLGAAAACI'

H.v. UUAC ACUGCA CGCAAGUCXG ALAAGGGGAC CCCAAGLGC G AGGGC Ab.4iiA CCCC LCGCC L N.Z. ubACAAUGCG GGAAACCGCG ALAAGGGGAC .4CCGAtiUnnC .AGCAU.ACA.. AL'nLL'nnnnL

H.v. LLCLCGACCG LiAXGCCGtiUC GAGG-4A.UA.A GAGLUGGGLA AGACLCGCGC CAGCLGCCLC ?l.z. C.CGCAGGLG UAAACGGCAI tiCGUIAGCAA GGGCCGGGCA AGACCG

H.v. GGLAAUACCG GCAGCUCAAG LGALGACCGA LALUALLGGG CLL 4.AAGCGL CVGCAtiC CGG Y.Z. aLLGGG LCL SAAGGGL CC GnAGCC'GG

H.v. CCACCAAGGL LC:ILCC;C;GAA ALCCGCCAGC L CAACLGGCG GGCGLCCGGL tiAAA.-\CC .iCG M.Z. LLLGALCAGb C CbC CGGGAA AUC,lJGAC?GC CCBACL'nLL -iGGCc,l,CnAGG GGALACLGUC

H.V. LGGCLLGGGA CCGGXAGGCU CGAGGGGL'AC GLCCGGGGLX LG ~G~,GAAALJCCLGLAACCC F1.Z. AGACLLCGGA C CGGGAGkGG LnAGAGGl AC nACXGGGGLA GGAGLnAAAL CLbGtlaACCL

H.v. LGGACGGXCC ACCGAUGGCG A,\AGCXCCL;C GAGAAGALdG ALCCGACGGL GAG3GA( G.A.4 ?1.z, nnnLnGGAC C ACCAGUGGCti A.LZGGC GCk UG ACLaG.4AiGG GI CCGAnGGl GAGGGACLAA

H.v. Pl. 1..

H.V. PI. z.

H.V. CCGGtiXXGLA CGLCCGCXAG GAUGAAACGL AA.-\GGXALLG GCGGGCGAGC AI L AC,iA( CG ?I. z . CI GGGAAGU

H.V. Y.Z.

H.x.. M.2.

iI . 1- , ?i.z.

H.x-.

?I. 2 9

Ii . \- . ?1.2.

H.v. Y. t

H.v. b1.z.

H.v. Y.Z.

H.v.

Y. z I

H.V. M.Z. FIG. 1. Extensive partial sequences. Extensive partial sequences of strain WeNST are shown aligned with the known complete sequence of H. volcanii (5). The sequences were aligned according to primary sequence homology, with minor alignment adjustments to ensure conformity with known secondary structure features of 16s rRNA (6). A, Adenine; U, uracil; C, cytosine; G, guanine. Lowercase letters indicate uncertainty. The following other designations indicate ambiguity: n, unknown base; x, purine; y, pyrimidine; k, guanine or uracil; s, guanine or cytosine; m, adenine or cytosine. Ambiguities may be due to heterogeneity in the rRNA population or to inherent limitations of the method. Dots are used to indicate positions known to be deleted in the sequence. Unsequenced regions are left blank.

140 VOL. 38, 1988 METHANOHALOPHILUS ZHILINAE SP. NOV. 141 water was determined by drying a sample of filtered lake [16] has not been characterized.) The levels of sequence water to constant weight at 105°C. similarity were 78.8% with Methanobacterium formicicum, 81.8% with Methanospirillum hungatei, and 78.8% with RESULTS AND DISCUSSION Haloferax volcanii. . Halophilic, methylotrophic, coccoid methano- The isolation and partial characterization of strain WeNST gens are consistent with the descriptions of the following (= DSM 4017T) have been described previously (3). four currently recognized genera: Methanolobus (lo), Me- Lake and sediment characteristics. The sediments of the thanococcoides (20), Methanosarcina (Methanosarcina ace- lake were black and smelled of hydrogen sulfide; the lake tivorans) (19), and Methanohalophilus (16). A fifth genus, water was reddish. The pH of the lake water was 9.7. Its Methanococcus, contains a halophilic, methylotrophic, coc- specific conductance was greater than 0.50 mS m-l, and the coid species (22), but the description of this genus specifi- total dissolved solids content was 245 g/liter. The reddish cally precludes the inclusion of methylotrophic organisms color may have been due to the presence of phototrophic, (12). The genera Methanosarcina, Methanolobus, and Me- halophilic, sulfur-oxidizing bacteria, such as Ectothiorhodo- thanococcoides contain species which are only weakly ha- spira, which have been isolated previously from lakes in the lophilic. Methanohalophilus is the only genus whose de- Wadi El Natrun (8). scription (16) includes moderate halophiles. Thus, strain Characteristics of WeNST. In addition to trimethylamine WeNST appears to belong in the genus Methanohalophilus. and methanol (3), strain WeNST used dimethylamine and At least nine other methanogenic isolates have been monomethylamine as methanogenic substrates when they described, which, as a group, differ significantly from other were provided at concentrations of 20 mM. Dimethylsulfide genera and fall within the description of Methanohalophilus (5 mM) supported growth and methanogenesis as a sole (16). These isolates may be separated from less halophilic catabolic substrate, but in medium containing 20 mM di- genera by the requirement of the former for much higher methylsulfide methane production was inhibited. In such concentrations of salt for most rapid growth and generally medium, little methane was produced even when 20 mM higher temperature optima. These isolates are Methano- trimethylamine was also present. coccus halophilus 2-7982 (= DSM 3094) (22), Methanoha- Wet-mount slides of cells were prepared in an anaerobic lophilus mahii SLPT (= ATCC 35705T) (15, 16), strain SF1 chamber and were sealed with petroleum jelly-paraffin (1: 1, (= DSM 3243) (13), five strains described by Zhilina (23) vol/vol). When these slides were viewed microscopically (strains 2-7301, 2-7302, 2-7303, 2-7305, and 2-7403), and outside the chamber, no motility was observed. The pres- strain WeNST. The major physiological distinction between ence of flagella was not directly determined, and the possi- strain WeNST and all of these other strains is its extremely bility of motility too weak to be observed was not excluded. high pH requirement for optimum growth. Growth occurred in mineral medium at about one-third Description of Methanohalophilus zhilinae sp. nov. Metha- (specific growth rate, 0.04 h-l) the rate of growth in complex nohalophilus zhilinae (zhilh’ae. n. L. gen. n. zhilinae of medium. Cultures in mineral medium supplemented with Zhilina, named for Tatjana N. Zhilina in recognition of her yeast extract (0.2%, wt/vol), Trypticase peptone (0.2%, many contributions to the biology and ecology of methano- wt/vol), or rumen fluid (5%, vol/vol) grew as well as cultures gens, especially methanogenesis in saline and hypersaline in normal medium; acetate (5 mM) was not stimulatory. environments. Irregular cocci, 0.75 to 1.5 pm in diameter, Strain WeNST produced methane in medium containing occurring individually and occasionally in clumps and tet- elevated sulfide concentrations, but this may have been rads. Very susceptible to lysis by sodium dodecyl sulfate. uncoupled from growth. When strain WeNST was inoculated Gram stain results are negative. Colonies in roll tubes are 0.2 into medium containing 21 or 42 mM sulfide (HZSaq+ HS1- to 0.4 mm within 7 days, yellowish tan, smooth, convex, + S2-), the methanogenic rate shortly after inoculation was opaque, and circular with entire margins. Methylamines, similar to the rate for controls containing 1.0 mM sulfide (the methanol and other methyl group-containing substrates (but normal medium concentration). However, the methanogenic not acetate) are used for growth and methanogenesis; H,- rate did not increase logarithmically, and after subsequent CO, and formate are not used. Yeast extract, Trypticase transfer into identical media (with elevated sulfide concen- peptone, and rumen fluid are stimulatory but not required. trations) cultures took longer to complete methanogenesis Trimethylamine may serve as a sole carbon and energy (twice as long at a concentration of 21 mM and four times as source. long at a concentration of 42 mM). After the next transfer the Grows in medium containing 0.2 to 2.1 M NaCl; optimal cultures containing 21 and 42 mM sulfide did not grow or growth in the presence of 0.7 M NaC1. produce methane. Most rapid growth occurs near pH 9.2 and 45°C. The susceptibility of strain WeNST to various antibiotics The guanine-plus-cytosine content of the deoxyribonu- (each at a concentration of 100 mg/liter) was typical for cleic acid is 38 mol%. methanogenic bacteria. Inhibitors of cell wall synthesis Habitat: alkaline, saline lake sediments. (penicillin G,ampicillin, carbenicillin, and cycloserine) were Type strain: WeN5 (= DSM 4017). not inhibitory; ribosome-affecting compounds (chloram- phenicol and tetracycline) completely inhibited growth. The ACKNOWLEDGMENTS guanine-plus-cytosine content of the deoxyribonucleic acid, We thank M.-E. Abdel-Samie and M. M. El-Halwagi, National measured by the buoyant density method, was 38 mol%. Research Centre, Cairo, Egypt, for help in collecting the sample and 16s rRNA sequence characterization. Sequence data were measuring the sediment pH and conductance. obtained for 918 of the estimated 1,450 nucleotide positions This research was supported in part by grants DE-AT03- in 16s rRNA from strain WeNST (Fig. 1). These data 80ER10684 from the U.S. Department of Energy and NSG-7440 from the National Aeronautics and Space Administration. convincingly support the separation of strain WeNST from all other methanogens and extreme halophiles whose 16s LITERATURE CITED rRNA sequences or oligonucleotide catalogs have been 1. Baresi, L., R. A. Mah, D. M. Ward, and I. R. Kaplan. 1978. determined. (The rRNA of Methanohalophilus mahii SLPT Methanogenesis from acetate: enrichment studies. Appl. Envi- 142 MATHRANI ET AL. INT. J. SYST.BACTERIOL.

ron. Microbiol. 36:18&197. (Approved Lists 1980) as the type species, and emendation of 2. Blotevogel, K.-H., U. Fischer, M. Mocha, and S. Jannsen. 1985. the genus Methanosarcina. Int. J. Syst. Bacteriol. 34:26&267. Methanobacterium thermoalcaliphilicum spec. nov., a new 13. Mathrani, I. M., and D. R. Boone. 1985. Isolation and charac- moderately alkaliphilic and thermophilic autotrophic methano- terization of a moderately halophilic methanogen from a solar gen. Arch. Microbiol. 142:211-217. saltern. Appl. Environ. Microbiol. 50:140-143. 3. Boone, D. R., S. Worakit, 1. M. Mathrani, and R. A. Mah. 1986. 14. Oremland, R. S., L. M. Marsh, and D. J. DesMarais. 1982. Alkaliphilic methanogens from high-pH lake sediments. Syst. Methanogenesis in Big Soda Lake, Nevada: an alkaline, mod- Appl. Microbiol. 7:230-234. erately hypersaline desert lake. Appl. Environ. Microbiol. 43: 4. Ferguson, T. J., and R. A. Mah. 1983. Isolation and character- 462-468. ization of an H,-oxidizing thermophilic methanogen. Appl. 15. Paterek, J. R., and P. H. Smith. 1985. Isolation and character- Environ. Microbiol. 45:265-274. ization of a halophilic methanogen from Great Salt Lake. Appl. 5. Gupta, R., J. M. Lanter, and C. R. Woese. 1983. Sequence of Environ. Microbiol. 50:877-881. the 16s ribosomal RNA from Halobacterium volcanii, an ar- 16. Paterek, J. R., and P. H. Smith. 1987. Description of Methano- chaebacterium. Science 221:656-659. halophilus mahii gen. nov., sp. nov., a halophilic methanogen 6. Gutell, R. R., B. Weiser, C. R. Woese, and H. F. Noller. 1985. from the Great Salt Lake. Int. J. Syst. Bacteriol. 38:OOO-OOO. Comparative anatomy of the 16s like ribosomal RNA. Prog. 17. Powell, G. E. 1983. Interpreting gas kinetics of batch cultures. Nucleic Acid Res. Mol. Biol. 32:155-215. Biotechnol. Lett. 5:437-440. 7. Hungate, R. E. 1969. A roll tube method for cultivation of strict 18. Sinha, N. D., J. Biernat, J. McManus, and H. Koster. 1984. anaerobes, p. 117-132. In R. Norris and D. W. Ribbons (ed.), Polymer support oligonucleotide synthesis. XVIII. Use of beta- Methods in microbiology, vol. 3B. Academic Press, Inc., New cyanoethyl-N,N-dialkylamino-/N-morpholinophosphoramidite York. of deoxynucleosides for the synthesis of DNA fragments sim- 8. Imhoff, J. F., F. Hashwa, and H. G. Triiper. 1978. Isolation of plifying deprotection and isolation of the final product. Nucleic extremely halophilic phototrophic bacteria from the alkaline Acids Res. 12:45394557. Wadi Natrun. Arch. Hydrobiol. 84:381-388. 19. Sowers, K. R., S. F. Baron, and J. G. Ferry. 1984. Methnnosar- 9. Kiene, R. P., R. S. Oremland, A. Catena, L. G. Miller, and D. G. cina acetivorans sp. nov., an acetotrophic methane-producing Capbne. 1986. Metabolism of reduced methylated sulfur com- bacterium isolated from marine sediments. Appl. Environ. pounds in anaerobic sediments and by a pure culture of an Microbiol. 47:97 1-978. estuarine methanogen. Appl. Environ. Microbiol. 52:1037-1045. 20. Sowers, K. R., and J. G. Ferry. 1983. Isolation and characteri- 110. Konig, H., and K. 0. Stetter. 1982. Isolation and characteriza- zation of a methylotrophic marine methanogen, Methanococ- tion of Methanolobus tindarius, sp. nov., a coccoid methanogen coides methylutens gen. nov., sp. nov. Appl. Environ. Micro- growing only on methanol and methylamines. Zentralbl. Bakte- biol. 45:684-690. riol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe C 3: 21. Worakit, S., D. R. Boone, R. A. Mah, M.-E. Abdel-Samie, and 478490. M. M. El-Halwagi. 1986, Methanobacterium alcaliphilum sp. 1!1. Lane, D. J., B. Pace, G. J. Olsen, D. A. Stahl, J. L. Sogin, and nov., an H,-utilizing methanogen that grows at high pH values. N. R. Pace. 1985. Rapid determination of 16s ribosomal RNA Syst. Appl. Microbiol. 36:380-382. sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. 22. Zhilina, T. N. 1983. New obligate halophilic methane-producing USA 82:6955-6959. bacterium. Microbiology (Engl. Transl. Mikrobiologiya) 52: 1.2. Mah, R. A., and D. A. Kuhn. 1983. Rejection of the type species 290-297. Methanosarcina methanica (Approved Lists 1980), conserva- 23. Zhilina, T. N. 1986. Methanogenic bacteria from hypersaline tion of the genus Methanosarcina with Methanosarcina barkeri environments. Syst. Appl. Microbiol, 7:216-222.