Arch Mierobiol (1984) 139:388- 396 Arehivea of

Microbiology �9 Springer-Verlag 1984

Sporomusa, a new genus of gram-negative anaerobic bacteria including Sporomusa sphaeroides spec. nov. and Sporomusa ovata spec. nov. *

Bernhard MOller 1, Rolf OBmer z, Bernard H. Howard a, Gerhard Gottsehalk z, and Hans Hippe 1

1 Deutsche Sammlung von Mikroorganismen der Gesellschaft ffir Biotechnologische Forschung mbH, Grisebachstrage 8, D-3400 G6ttingen, Federal Republic of Germany

2 Institut fiir Mikrobiologie der Universit/it, GrisebachstraBe 8, D-3400 G6ttingen, Federal Republic of Germany 3 Department of Biochemistry, Lincoln College, Canterbury, New Zealand

Abstract. A new genus of strictly anaerobic, gram-negative, banana-shaped bacteria is described. Cells formed spores and were motile by means of up to 15 laterally inserted flagella. Nitrate or sulfate were not used as electron acceptor. Organic substrates that were fermented included N-methyl compounds, such as betaine, N,N-dimethylglycine and sar- cosine, primary alcohols, hydroxy fatty acids, and 2,3-butanediol. In addition, molecular hydrogen and carbon dioxide were fermented to acetate. The latter was the characteristic fermentation product in general. During growth on betaine, trimethylamine was formed in addition. The degradation of N,N-dimethylglycine yielded acetate, monomethylamine, and trimethylamine. The presence of cytochrome b and of ubiquinone in the cells was shown. The deoxyribonuleic acid base composition of the strains was between 41.3 and 47.4 tool% guanine plus cytosine. The name Sporomusa is proposed for this new genus. On the basis of the DNA-DNA homology values obtained, the shape of the spores and some other properties, the isolated strains were assigned to two species. Names proposed: Sporomusa sphaeroides and Sporomusa ovata. The type species is S. sphaeroides and the type strains are strain E, DSM 2875 (S. sphaeroides) and strain H1, DSM 2662 (S. ovata).

Key words: Sporomusa sphaeroides - Sporomusa ovata - Spore formation - N-methyl compounds - Degradation of betaine and N,N-dimethylglycine - Acetogenic anaerobes

A number of N-methyl compounds including choline, be- taine, and N,N-dimethylglycine have been shown to give rise to rapid production of methane in bacterial enrichments (Hippe et al. 1979). Choline was found to be degraded first to trimethylamine and acetate by a Desulfovibrio species; these products were then used by organisms such as Methanosarcina barkeri in methanogenesis (Fiebig and Gottschalk 1982). Anaerobic breakdown of betaine could also be demonstrated: Eubacterium limosum grew on betaine and produced N,N-dimethylglycine, acetate, and butyrate from it (Miiller et al. 1981). Clostridium sporogenes used

* Dedicated to Prof. H. G. Schlegel on the occasion of his 60th birthday

Offprint requests to: G. Gottschalk

betaine as oxidant in the Stickland reaction and reductively cleaved it into trimethylamine and acetate (Naumann et al. 1983).

In our efforts to detect additional organisms that grew on betaine or N,N-dimethylglycine, we isolated a number of banana-shaped strains that stained Gram-negatively but formed spores. These strains are so different from all the currently recognized genera that they are described here; the establishment of a new genus with two species is proposed.

Materials and methods

Bacterial strains

The origin and designation of the strains used in this study are listed in Table 1. For maintenance the strains were grown in betaine standard medium and subsequently stored at room temperature or at 4 ~ C.

Media and cultivation methods

The media were prepared anaerobically under a gas atmo- sphere of 80% N2 and 20% CO2 by the Hungate technique (Hungate 1969; Bryant 1972). The basal medium contained the following compounds per liter: KzHPO4, 0.348 g, KHzPO4, 0.227 g; NH4C1, 0.5 g; MgSOr 7 H 2 0 , 0.5 g; CaCI2 �9 2 H20, 0.025 g; NaC1, 2.25 g; FeSO4 �9 7 H20, 0.002 g; trace element solution SL10, 3 ml (Widdel et al. 1983); NaHSeO3, 15 ~tg; resazurin, I mg; NaHCO3, 4 g; vitamin solution (Wolin et al. 1964), 20 ml. The medium was boiled for 5 min and cooled in ice to room temperature under a gas stream of 80% N2 and 20% CO2. NaHCO3 dissolved in water (4 g/80 ml) and gassed with the N2/CO2 mixture for 20 min, and 2 ml of vitamin solution from a tenfold concentrated stock solution were added.

The medium was autoclaved at 121~ for 20 rain. Sub- strates (N-methyl compounds, 50 mM, or other substrates, 0.5 %) were added before autoclaving or from filter sterilized anaerobic stock solutions (neutralized) depending on the heat stability of the compounds. Prior to use, media were reduced by injecting concentrated solutions either of sodium sulfide or L-cysteine to a final concentration of 0.3 g per liter. Solutions of reducing agents were sterilized by autoclaving under nitrogen. The final pH of the medium was 7 . 0 - 7.2.

Strains were routinely cultured at 30~ in Hungate tubes inoculated with 2% cell suspension. Growth was fol-

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lowed by measuring the optical density at 545 or 600 nm with a Bausch & Lomb Spectronic 88 spectrophotometer.

Betaine standard medium contained 2 g of yeast extract and 2 g of casitone per liter and was reduced with sodium sulfide; agar medium was reduced with e-cysteine.

Isolation agar for use in the GasPak anaerobic jar (BBL, Cockeysville, USA) contained in addition to the substrate 0.5 g of yeast extract, 0.5 g of casitone and 15 g of agar per liter; NaHCO3 (1 g per liter) and e-cysteine were sterilized separately and added to the autoclaved agar medium after it had been cooled down to 50 ~ C.

Enrichment and isolation procedure

1 - 2 ml of mud or soil samples were added to 10 ml of sterile basal medium plus betaine or N,N-dimethylglycine and pasteurized by heating at 80~ for 15 min or incubated directly without pasteurization. A few samples were treated for 1 h with sterile ethanol at 50% (v/v) final concentration (Koransky et al. 1978), centrifuged, washed twice with distilled water and then added to the enrichment medium. Upon incubation at 30~ turbidity developed usually within 1 to 3 weeks. At intervals substrate utilization was assayed by TLC; betaine, solvent acetone:methanol: HC1 22.5:2.5:2.5 (Eneroth and Lindstedt 1965); N,N- dimethylglycine, solvent phenol:water 4:1, detection re- agent 0.4% orcinol in l-butanol:ethanol 1:1, made 0.08 M with HzSO 4 (Ingraham 1953). When substrate utilization proved to be positive, cultures were transferred into fresh medium. After another transfer and positive chromato- graphic result samples were streaked on isolation agar. Yellowish to brownish colonies of 1 to 3 mm in diameter developed within 1 week. Single colonies were purified by repeated streaking and finally transferred into standard medium with betaine or N,N-dimethylglycine.

Morphological characterization

The morphological properties of the strains were examined by phase contrast microscopy and by electron microscopy after negative staining according to Valentine et al. (1968). For thin sectioning, samples were fixed and dehydrated by the method of Behn and Arnold (1974), embedded according to Spurr (1969), and stained with lead citrate as described by Venable and Coggeshall (1965). Electron microphotographs were taken with a Philipps EM 301 electron microscope.

Characteristics of colonies were determined on betaine standard agar incubated for 4 and 14 days.

Physiological and biochemical tests

The temperature optimum and range was determined in a Temperatur Gradient Incubator Model TN-3 (Tokyo Kakagu Sangyo Ltd., Tokyo, Japan) using betaine standard medium. Optical density readings at 545 nm were used to calculate the doubling time to.

For the assessment of growth at different pH values the medium was prepared without bicarbonate and phosphate under 100% nitrogen. 0.5 ml of sterile 0.4 M potassium phosphate buffer or Tris-HC1 buffer of varying pH were injected into 10-ml medium tubes to reach various pH values.

To determine the Gram reaction, Bartholomew's standardized method (1962) using n-propanol as decoloriz-

ing agent was applied. The Gram reaction was compared with the lysis of cells in a solution of 30 g KOH/1 (Gregersen 1978) and with the aminopeptidase test (Cerny 1978) using Bactident Aminopeptidase strips from Merck (Darmstadt, FRG).

Biochemical tests were performed according to Holde- man et al. (1977). Media were prepared without vitamin K and heroin. Oxidase was checked after Collins and Lyne (1976) with cultures grown on betaine standard agar. Reduc- tion of sulfate, sulfite and thiosulfate was tested in Postgate's medium 3 (Postgate 1963).

Growth in the presence of oxygen was tested by aerobic incubation of various inoculated liquid or agar media which supported anaerobic growth. Growth was also tested under conditions of reduced oxygen tension and increased carbon dioxide concentration as generated by the "Oxoid Gas Gen- erating Kit for Campylobacters" (Oxoid, Basingstoke, UK).

Cultures were usually incubated for 5 days before per- forming the various tests. For testing the utilization of substrates, cultures were incubated up to 6 weeks before recording them as negative. Prolonged incubation was re- quired especially for some of the N-methyl compounds, amino acids or alcohols.

Determination of DNA base composition and DNA-DNA hybridization

Cells were grown in betaine standard medium supplemented with 1% (v/v) methanol to increase the cell yield. Deoxyribo- nucleic acid (DNA) was prepared by the method of Marmur (1961) with additional proteinase K treatment (50 ~g/ml) for 15 -30 min at 37~ after the lysozyme and ribonuclease steps.

The tool% G + C content of DNA was determined opti- cally by thermal denaturation using a Gilford model 2600 spectrophotometer and the thermoprogrammer model 2527 (Mandel and Marmur 1968). All calculations have been made using the equation (G + C)= 2.44 (T in - 69.4) (De Ley 1970). DNA from E. coli K12 tool% G + C = 5 1 . 7 ) was used as a reference.

The degree of binding was determined spectro- photometrically from the renaturatlon rates of the indi- vidual DNA samples and their mixtures using the method of De Ley et al. (1970) as described by Claus et al. (1983).

Cytochromes and quinones

The membrane as well as the soluble fraction of betaine grown cells was investigated for the presence of cytochromes as described (Kfihn and Gottschalk 1983). Quinones were isolated from freeze dried cells and analyzed by the methods described by Kroppenstedt (1982).

Analytical procedures

Organic acids and alcohols were quantified by gas chro- matography as described recently (Bahl et al. 1982). The betaine concentration was measured as described by Miiller et al. (1981). N,N-dimethylglycine was quantitatively deter- mined by capillary tube isotachophoresis (Analyzer IP-2a, Shimadzu Siesakusko Ltd., Kyoto, Japan). The leading electrolyte consisted of 8 mM HC1 plus 0.2% Triton X-100 which was purified over a mixed-bed ion exchanger (Merck AG, Darmstadt, FRG). The pH was adjusted to 9.5 with

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Table 1. Designation and source of the isolated strains a

Strain Spore form Origin of sample Isolation substrate Treatment b designation

E round mud, Leine river, F R G betaine ethanol B 39 round mud, Leine river betaine heat B46 round sugar beet field, Hildesheim, FRG betaine heat B 50 round waste water, beet sugar factory, Kleve, FRG betaine heat DMG58 round or slightly oval mud, Leine river N,N-dimethylglycine heat H~ oval sugar beet leaf silage, G6ttingen, FRG N,N-dimethylglycine - H 6 never observed mud, Leine river N,N-dimethylglycine - Nile oval mud, Nile river, Egypt betaine ethanol

a Further strains not included in the table were isolated from mud samples of lakes and a ditch in northern Germany, soil samples from Yugoslavia, Mexico and Pakistan, and from horse and cattle dung

b Samples were pasteurized by heat treatment at 80~ for 15 rain or they were treated with 50% ethanol for I h as described by Koransky et al. 1978

Fig. l a -d . Phase contrast micrographs of betaine-fermenting sporeformers grown in standard medium, a Strain E, vegetative cells; b strain E, showing round spores; c strain Ill , vegetative cells; d strain Nile, showing oval spores; bar equals 10 ~tm

2-amino-2-methyl-l,3 propanediol. The terminating electro- lyte was 8 m M fl-alanine and Ba(OH)2 at pH 10.6. The electrolytes were prepared under an atmosphere of nitrogen and were stored under nitrogen. Separations were carried out in a 15-cm tube (inner diameter, 1.0 mm) and a 10-cm tube (inner diameter, 0.5 mm). The temperature was 20~ and the migration current was 300 gA for 6 rain and 50 laA until end of the analysis.

Five microliter samples containing up to 2.0 mM N,N- dimethylglycine were injected. Methylamines were deter- mined as described previously (Naumann et al. in press).

Chemicals

All chemicals were of the highest purity available, generally of analytical grade. Proteinase K and ribonuclease were from Merck, Darmstadt, lysozyme from Boehringer, Mann- heim, FRG.

Results

Isolation

A total o f 8 strains was isolated from anoxie mud samples, waste water and soil applying the procedure described under

Methods. The strains and the substrates used for isolation as well as the conditions of pasteurization are summarized in Table 1. These strains exhibited a number of unusual properties that justified their closer investigation: they con- tained spores, they stained Gram-negatively at all stages of growth, they were able to grow on betaine or N,N-dimethyl- glycine as sole energy source.

Cellular and colonial features

Strain E and strain H1 were chosen for the morphological characterization. Cells o f these strains were slightly curved, banana-shaped rods with tapered ends occurring single or in pairs (Fig. 1). S-shaped or helical cells were sometimes formed. Vegetative cells of strain H1 growing in betaine medium measured 0 . 7 - 0.9 • 3 - 5 ~tm those of strain E measured 0 . 5 - 0 . 8 x 2 - 4 lam. Sporulating cells were o f al- most double size and swollen.

Strain H1 and E differed in that spores of the former were oval-shaped measuring 0.9 - 1.0 • 1.3 - 1.4 lam whereas the latter formed round spores of 1 . 0 -1 . 2 ~m in diameter. The spore position was subterminal to terminal in both strains (Fig. 1). Spores survived heating to 80~ for 10 min.

Strain E as well as all other strains isolated from pasteurized samples sporulated readily in betaine-containing

391

Fig. 2a-e. Electron micrographs of negatively stained cells and of ultrathin sections of strains E and HI. a Strain E, vegetative cells and enlarged sporulating cells exhibiting an altered surface structure; b strain E, showing the insertion points of flagella and three ring pairs (marked by arrows) of the basal body; e strain H1, showing flagella on the concave side of the cell; d strain E, demonstraing a multilayered, typically Gram-negative cell wall; OM outer membrane; ML murein layer; CMcytoplasmic membrane; e strain E, showing the double layered structure of the outer membrane OM; a - d bar equals 0.5 pm; e bar equals 0.1 pm

Table 2. G + C contents of representative strains and DNA-DNA homologies among them

Strain Mol% % Homology with reference DNA G + C from strain

E H1 Nile

E 46.7 100 - 24 B 50 47.4 82 - 22 B46 47.4 75 - 35 H1 42.2 22 100 -- H6 41.3 - 104 - Nile 42.2 24 92 - DMG58 43.3 30 - 55

liquid media and on agar media, whereas strain H1 sporu- lated only sporadically. In strain H6 spores have never been detected. Cells of strain H1 a n d E were motile by means of unilateral ly inserted flagella (Fig. 2). Up to 15 flagella per

cell were observed which were distr ibuted at the concave side; their length was approximate ly 12 lam and their width 20 nm. Cells exhibited usually a tumbling movement and a rota t ion a round the transverse axis typical of selenomonads. A quick, directed movement was most ly observed in expo- nential phase cultures.

Colonies of both strains formed on betaine agar were 1 - 3 m m in diameter and of very similar appearance. They were circular, slightly convex, entire, yellowish to amber or brownish colored, opaque, smooth, and butyrous to mucoid. Pigments were not excreted into the surrounding agar.

Composition of cell walls

As already mentioned, cells of the isolated strains stained Gram-negatively. Cells behaved also Gram-negat ive in the K O H lysis test but did not react positively in the aminopep- tidase test. Further , cells were rapidly lysed when 0.1 - 1% (w/v) sodium dodecylsulfate was added to exponential phase

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Table 3. Characteristics of the betaine-fermenting strains

Parameter Strain

E B 39 B 49 B 50 Hi H 6 Nile DMG 58

Spore form rd rd rd rd ov ov rd to ov position ST/T ST/T ST/T ST/T ST/T ST/T ST/T

Gram staining . . . . . . . .

Catalase +~ + + + ( - ) ( - ) ( - ) ( - )

Indole formation a - + . . . . . .

Substrate utilization b Betaine + + + + + + + + N,N-dimethylglycine + + + + + + + + Sarcosine + - + + + + + - N,N-dimethylethanolamine + + + + - - - + Trimethylamine + + + + - - - + Fructose . . . . + + + - Glycerol + + + + . . . . DL-lactate + + + + -t- + + -- Pyruvate + + + + + + + + nL-glycerate + + + + . . . . . 3-Hydroxybutyrate + + + + . . . . Formate +__ +_ • +_ + • _+ • L-alanine + -- -- + + + -- -- L-serine + + + + . . . . Methanol + + + + + + + + Ethanol + + + + + + + + n-Propauol + + + + + + + + 2-Propanol - nd nd nd nd nd nd nd n-Butanol + + + _+ + + + +

1,2-Propanediol + + + + + - + -

1,2/1,3/1,4-Butanediol - nd nd nd nd nd nd nd

2, 3-Butanediol + + + + + + + +

Ethylene glycol + + + + . . . .

Triethylene glycol - nd nd nd nd nd nd nd

Polyethylene glycol - nd nd nd nd nd nd nd

H2 + CO2 + nd nd + + + + +

Temperature (~ C) optimum 35/37 35/39 35/39 35/39 34 37 33/37 35/39 Maximal growth temp. d 45 45 45 45 43 43 45 37

pH-optimum 6.5 7.1 7.4 6.9 6.3 6.5 6.3 6.2

range 5 . 7 - 8.7 5 . 0 - 8.1

All strains were negative in KOH lysis test alld aminopeptidase test (see under methods) as well as for oxidase, nitrate reduction, reduction of sulfate, thiosulfate and sulfite to H2S, for lipase, lecithinase, gelatin hydrolysis, meat digestion and hemolysis; milk was not changed

b The following substrates were tested but not utilized by any of the strains examined: Amygdalin, esculin, glucose, inulin, lactose, maltose, mannitol, mannose, melibiose, ribose, starch, sucrose, xylose, acetate, butyrate, citrate, fumarate, succinate, L-tartrate, glycolate, glycine, D-alanine, choline, ethanolamine. The following substrates were tested only with strains HI and H6 but were not utilized: Arabinose, cellobiose, galactose, glycogen, melezitose, raffinose, rhamnose, sorbose, trehalose, salicin, adonitol, dulcitol, erythritol, inositol, sorbitol; H2 plus acetate; H2 plus fumarate; formate plus fumarate; oxalate, malate, ketoglutarate; uric acid, hypoxanthine, xanthine; dimethylamine, methylethanolamine, dimethylformamide, dimethyl urea, trimethyl phenylamine, creatine + , Positive or utilized; - , negative or not utilized; (-), negative or very weak reaction; _+, weak growth; rd, round; ov, oval; ST/T, subterminal to terminal; nd, not determined

d Minimal growth temperature was in the range of 15 ~ C

cultures. Analys is o f cell walls o f s trains H1 and E showed tha t the pep t idog lycan was o f the m - d i a m i n o p i m e l a t e direct ly l inked type (O. Kandler , pe rsona l communica t ion ) . U l t r a - th in sections revealed tha t these o rgan isms were su r rounded by a mul t i l ayered cell wall as typical o f G r a m - negat ive bac ter ia (Fig. 2). The presence o f this type o f cell wall was conf i rmed by chemical analysis o f the isolated

l ipopolysacchar ide layer (H. -M. K u h n and H. Mayer , pe rsona l communica t ion ) .

DNA base composition and DNA-DNA homology

The m o l % G + C con ten t o f the D N A o f s train H1 was 42.2 and of strain E 46.6. O the r strains examined cou ld be

attributed to either of them indicating the separation into two genotypically different groups. This separation was supported by the results of DNA-DNA homology determi- nations. Low homology was found between strains belong- ing to the two different G + C groups whereas high homol- ogy was found between strains within each group (Table 2). The only exception was strain DMG 58 which could not be assigned to either groups.

g

(D

i J 0

E 0

E i_

o .g

10

E -? Z z" 5

c_ 0

zx

6 ' 16 ' ~ ' ~o ' ,.b Timelh)

Fig. 3. Growth of strain Ht on betaine and time course of substrate degradation and product formation. The organism was grown at 37~ in standard medium in a 2-1 bottle; gas atmosphere: 100% nitrogen. Symbols: O, betaine; II, trimethylamine; A, acetate; A, N,N-dimethylglycine; �9 CO2

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Nutrition and physiological characterization

The strains grew only under anaerobic conditions and can, therefore, be designated as strict anaerobes. Other physio- logical characteristics are summarized in Table 3. It should be emphasized that neither strain wag able to-carry out a dissimilatory reduction of nitrate or sulfate. The substrate spectrum of the strains was rather atypical of anaerobes. Characteristic substrates of all strains were N-methyl compounds, hydroxy fatty acids, and primary alcohols. In addition, a mixture of H2 and COz served for growth. The temperature optima given in Table 3 were determined with betaine as substrate. All strains showed growth with betaine between 15~ and 43~ Growth of the organisms was stimulated by CO2/bicarbonate. The doubling time of strain H1 in betaine standard medium was 3.1 h and that of strain E was 1.8 h.

Products and fermentation balances

Strains H1 and E were grown on pyruvate, lactate, meth- anol, ethanol or H2 + CO2, strain H1 in addition on fructose and the products formed were determined in the culture fluid. The only product formed in large amounts was acetate. Small amounts of ethanol (final concentration below 1 raM) could be detected in addition. Thus, the organisms exhibited fermentative properties as typical of acetogenic bacteria such as C. aceticum or C. thermoautotrophicum (Braun et al. 1981 ; Wiegel et al. 1981).

The growth curve of strain H1, the course of betaine degradation and of product formation is depicted in Fig. 3. The products detected were acetate, trimethylamine, carbon dioxide and N,N-dimethylglycine. The latter was excreted in the logarithmic growth phase and partially consumed thereafter. The amounts of these compounds found accounted for the amount of substrate consumed (Table 4). Acetate and trimethylamine were apparently formed by re- ductive cleavage of betaine, and the reducing power required was provided by oxidation of methyl groups. Analogous experiments were carried out with N,N-dimethylglycine as growth substrate. The products formed were acetate,

Table 4. Balance of betaine fermentation and of N,N-dimethylglycine fermentation by strain HI"

Substrate and products Mol/t00 tool Mol C/100 tool Oxidation Oxidation degree of substrate of substrate degree (tool/100 mol)

Substrate Betaine 100 500 - 7 - 700

Products N,N-dimethylglyeine 16.1 64.4 - 5 - 80.5 Trimethylamine 82.3 246.9 --9 - 740.7 Acetate 85.5 17t 0 0 CO2 28.6 28.6 +4 + 114.4

Substrate N~N-dimethylglycine 100 400 - 5 - 500

Products Acetate 131.7 263.4 0 0 Monomethylamine 93.2 93.2 -- 5 - 466 Trimethylamine t2.7 38.1 -- 9 -- 114.3 COg 15.5 15.5 +4 + 62

" Carbon balance, 102.2%; redox balance, 1.01 (betaine); carbon balance, 102.5%; redox balance, 1.04 (N,N-dimethylglycine)

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monomethylamine, trimethylamine and carbon dioxide (Table 4).

The appearance of methylamine among the products of N,N-dimethylglycine degradation could be explained by a reductive cleavage reaction acting on sarcosine. This was supported by the finding that growth of strain H1 on sarcosine resulted in the production of acetate, methylamine and carbon dioxide. Glycine was not detectable among the products.

Cultures grown on betaine without the addition of selenite did not produce trimethylamine, and reached lower pH values. Reductive cleavage reactions were apparently not feasible in the absence of selenite.

Presence of cytochromes and ubiquinones

Membrane fractions of strain H1 and E were analyzed for the presence of cytochromes. The reduced minus oxidized difference spectra showed an e-band at 560 nm, a ?-band at 432 nm, and a fl-band at 530 nm indicating the presence of cytochrome b. Another fl-band at 523 nm and a shoulder at 550 nm indicated the presence of a c-type cytochrome. The presence of a cytochrome b was confirmed by analysis of an acetone-HC1 extract of the membrane fraction which clearly showed the presence of protoheme. Cytochromes were not detectable in the cytoplasmic fraction.

The analysis of cells of both strains for quinones gave a surprising result. Ubiquinones could be detected which hitherto have not been reported to occur in non-photosyn- thetic obligate anaerobes (Collins and Jones 1981). Menaquinones were not present.

Discussion

The obligately anaerobic sporeformers which have been iso- lated and characterized during this study show a unique combination of features and properties that is not found in established genera of anaerobic rod-shaped sporeformers. They are true Gram-negative organisms as could be demon- strated by the multilayered cell wall profile and the presence of lipopolysaccharide, and they are not able to carry out a dissimilatory sulfate reduction. These features exclude them from the Gram-positive clostridia as well as from the genus Desulfotomaculum. This is strongly supported by a rather low phylogenetic relationship of the new spore- formers to members of the anaerobic and aerobic spore- forming genera on the basis of their 16 S rRNA relatedness (E. Stackebrandt, personal communication). Interestingly, the highest degree of relatedness could be found to Seleno- monas as indicated by a SAB value of 0.47, which is, however, on or even above the level of families.

With selenomonads our sporeformers have several features in common such as a similar cellular morphology, a lateral flagellation, a Gram-negative cell wall including the presence of lipopolysaccharide (Kamio et al. 1972), the occurrence of cytochromes (De Vries et al. 1974), and ubiquinones (R. Kroppenstedt, personal communication). However, selenomonads do not produce endospores just as some recently described Gram-negative anaerobic genera, e.g. Roseburia, Centipeda, Pectinatus, Acetivibrio (A. ethanolgignens), which all have been characterized as laterally flagellated, curved rods (Kingsley and Hoeninger 1973; Bryant 1974; Lee et al. 1978; Robinson and Ritchie

1981; Lai et al. 1983; Stanton and Savage 1983). Members of these genera including the selenomonads differ from our sporeformers not only by lacking sporeforming capability but also in that the former preferentially utilize various sugars and produce a different pattern of major and minor fermentation products.

Further, as far as reported they are unable to grow with H2-t-CO2 and do not have metabolic properties of acetogens.

On the basis of striking differences to currently recog- nized genera of anaerobic sporeformers a new genus and two new species are proposed for our strains. No affiliation to higher taxa is made at the present stage. We consider this could be usefully done only when phylogenetic data of other anaerobic Gram-negative genera become available.

A few remarkable catabolic properties of the isolated strains have to be mentioned. They degrade N-methyl compounds such as betaine, N,N-dimethylglycine or sarcosine in such a way that methyl groups are oxidized to COz; the reducing power generated is then used to carry out reductive cleavage reactions of betaine or sarcosine. This explains the occurrence of trimethylamine and monomethyl- amine among the products. N,N-dimethylglycine could, apparently, not be cleaved, because dimethylamine was never found. To formulate fermentation equations is difficult. The betaine fermentation can be described as follows:

1.5 betaine + 3 HzO ~ 1.5 N,N-dimethylglycine + 1.5 CO2 + 9 H (I)

0.5 betaine + 3 H20 ~ 0.5 acetate + 0.5 NHa -}- 1.5 CO 2 ~- 7 H (2)

8 betaine + 16 H ~ 8 acetate + 8 trimethylamine. (3)

The amount of N,N-dimethylglycine excreted as compared to the amount of betaine consumed differs from fer- mentation to fermentation, and so does the relative contribu- tion of Eqs. (1) and (2) to the generation of reducing power. The fermentation of N,N-dimethylglycine is even more complex. This substrate is not only demethylated to sarcosine but is partly methylated to betaine. Otherwise it is not understandable that trimethylamine was reproducibly formed in addition to monomethylamine. On the basis of the balance found, this fermentation can be described by the following equations:

2 N,N-dimethylglycine --, 1 betaine + 1 sarcosine (4)

8 N,N-dimethylglycine + 16 H20 --+ 8 sarcosine + 8 CO2 + 48 H (5)

1 betaine + 2 H ~ 1 acetate + 1 trimethylamine (6)

9 sarcosine + 18 H ~ 9 acetate + 9 monomethylamine (7)

7 CO2 + 28 H --+ 3.5 acetate. (8)

Sporomusa gen. nov. (Spo.ro.mu'sa. L. n. sporus a spore, M.L.n. musa a banana; M.L.f.n. Sporomusa meaning spore bearing banana).

Curved rods with tapered ends. Motile with unilateral flagella. Gram-negative by staining and cell wall profile. Cell wall contains lipopolysaccharide. Heat resistant endospores are formed. Strict anaerobes. Oxidase negative. Catalase may be formed.

Chemoorganotrophs; metabolism fermentative. Certain N-methyl compounds, amino acids, alcohols, diol

395

compounds, hydroxy fatty acids and pyruvate or glycerol may be utilized. Sugars are not the preferred substrate. Chemolithotrophic using hydrogen plus carbon dioxide as growth substrate. Acetate is the major fermentation product. Butyrate, propionate, succinate or lactate are not formed.

Do not reduce sulfate. Cytochromes (b; possibly c) and ubiquinones present.

Mesophiles. G § C content of the DNA ranges from 41.3 - 47.4 mol% (thermal melting).

Type species: Sporomusa sphaeroides. Sporomusa sphaeroides sp. nov. (sphae.roi'des Gr.f.adj.

sphaeroides spherical related to spore shape). Curved rods with tapered ends, 0 . 5 - 0 . 8 x 2 - 4 gm.

Cells single or in pairs; chains usually not formed; S-shaped, spirillum-like and helical cells occasionally produced. Spores are round, about 1 - 1 . 3 gm in diameter, subterminal to terminal, swelling the cell; resist heating to 80~ for 10 min. Cells are motile by active tumbling or directed movement; up to 15 flagella distributed at the concave side. Cell wall contains mesodiaminopimelic acid.

Surface colonies on betaine agar are circular, 1 - 3 mm in diameter, slightly convex, entire, yellowish to amber or brownish, opaque, smooth, butyrous to mucoid.

Growth in broth containing a fermentable substrate starts usually from the bottom. A homogenous turbidity is usually produced; small floccules or clumps may also be formed and sticking at the wall of culture vessels may occur depending on strain and growth conditions. Stationary phase cultures may form a heavy flocculent sediment.

During growth no gas formation is visible. Substrates fermented are betaine, N,N-dimethylglycine,

N,N-dimethylethanolamine, trimethylamine, glycerol, DL- lactate, pyruvate, DL-glycerate, 3-hydroxybutyrate, formate, L-serine, methanol, ethanol, n-propanol, 1,2-propanediol, 2,3-butanediol, ethylene glycol, and H2 + CO2. Some strains use sarcosine, L-alanine or n-butanol. Sugars are not utilized, neither are several other compounds.

Acetate is the major fermentation product; small amounts of ethanol may be formed.

Oxidase negative. Strong catalase reaction is given by cells grown on betaine agar. Gelatine not hydrolyzed. Meat not digested. Milk not changed. Nitrate, sulfate, thiosulfate, and sulfite not reduced. Lipase of lecithinase not produced. Not hemolytic. Indole rarely formed.

Grows in chemically defined medium containing min- erals, betaine, vitamines (vitamin B12, folic and nicotinic acid), and bicarbonate. Growth is stimulated by low concen- trations of yeast extract or peptone.

Mesophilic; optimal growth temperature 35-39~ range below 15 up to around 45 ~ C. Optimal pH between 6.4 and 7.6 depending on the strain; pH-range between 5.7 and 8.7. G + C content of the DNA is 46.7-47.4 mol%. Has been found in anoxic fresh water and sewage digester mud and in soil.

Type strain: Strain E, deposited with the Deutsche Sammlung von Mikroorganismen, G6ttingen, FRG, under the collection number DSM2875. Additional strains: B39, B46, B50.

Sporomusa ovata sp. nov. (o.va'ta. L.fem.adj. ovata egg- shaped, related to shape of spore).

The cellular and colonial characteristics as well as growth in betaine broth are the same as for S. sphaeroides. Cells measure 0 . 7 - 1 . 0 x l - 5 g m . Spores are oval, 0 . 9 - 1.0 x 1 . 3 - 1.4 gm, and heat resistant.

Substrates fermented are betaine, N,N-dimethylglycine, sarcosine, fructose, lactate, pyruvate, formate, methanol, ethanol, n-propanol, n-butanol, 2,3-butanediol, and Hz + CO2. L-Alanine and 1,2-propanediol may be utilized by some strains. Fructose is the only sugar fermented. No activity towards other compounds observed. Acetate is the major fermentation product. Small amounts of ethanol may be formed. No visible gas is produced.

Oxidase negative. Occasionally very weak catalase activ- ity found in betaine agar grown cells.

Grows in chemically defined medium containing min- erals, betaine, and bicarbonate without vitamins; low con- centrations of yeast extract or peptones stimulate.

Mesophilic; optimal growth temperature 34 -39~ range below 15~ up to about 45 ~ C. Optimal pH between 5.3 and 7.2 depending on the strain, pH range 5.0-8.1. G + C content of the DNA is 41.3-42.2 tool%. Has been found in anoxic fresh water mud and in sugar beet leaf silage.

Type strain: strain H1, deposited with the Deutsche Sammlung von Mikroorganismen, G6ttingen, FRG, under the collection number DSM2662. Additional strains: H6, Nile.

The taxonomic position of strain DMG 58 is uncertain.

Acknowledgement. We thank Dr. O. Kandler, Mfinchen, for the determination of the peptidoglycan type, Dr. R. Kroppenstedt, Darmstadt, for analyzing the quinones, Dr. E. Stackebrandt, Mtin- chen, for providing the 16S rRNA/SAB data, Dr. H. Mayer and Dr. H. M. Kuhn, Max-Planck-Institut ffir Immunbiologie, Freiburg, for their support in the LPS analyses, Dr. F. Mayer, Dr. W. Jo- hannssen and G. Kohring, G6ttingen, for their advice on electron microscopy.

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Received April 25, 1984/Accepted June 5, 1984