uch Microbiol (1982) 132:31-36
Archives of
Microbiology
w» Springer-Verlag 19X2
Methanoplanus limicola, a Plate-Shaped Methanogen Representing a Novel Family, the Methanoplanaceae
G e r t r u d W i l d g r u b c r \ M i c h a e l T h o m m1, H e l m u t K ö n i g1, K u r t O b e r2, T h c o d o r o R i c c h i u t o3, and K a r l O . Stettcr1
1 Lehrstuhl für Mikrobiologie, Universität Regensburg, D-8400 Regensburg, Federal Republic of Germany
2 Lehrstuhl für Genetik, Universität Regensburg, D-8400 Regensburg, Federal Republic of Germany
3 Agip S.p.A., 20097 S. Donato Milanese (Milan), Italy
Abstract. A n angular plate-shaped weakly motile mesophilic methanogen was isolated from a swamp of drilling waste in Italy. G r o w t h occurs o n H2/ C 02 or on formate. Acetate is required in addition. T h e o p t i m a l d o u b l i n g time is 7 h at 40" C . T h e cell envelope is composed most likely of glyco- protein subunits in hexagonal arrangement. The G C - c o n t e n t of its D N A is 47.5 m o l % . O n the basis o f D N A - R N A hybridization it was found to represent a new family, the
Methanoplanaceae within the order M c t h a n o m i c r o b i a l e s . Key words: Methanogens — Archaebacteria — Cell division
- G l y c o p r o t e i n - Acetate — T a x o n o m y
Recently, a square-shaped flat bacterium was discovered in a saturated salt brine (Walsby 1980; Stoeckcnius 1981), w h i c h , however, cannot yet be cultivated in the laboratory. It was assumed (Walsby 1980) that the unusual shape may be explained by the absence o f cell turgor i n bacteria i n a high ionic strength environment. F r o m the c o m p o s i t i o n o f its envelope, Walsby (1980) speculates that this organism be- longs to the archaebacteria.
I lere, we report on the isolation and properties o f another Hat archaebaclerium, w h i c h , however, grows at much lower ionic strength and which belongs to the methanogens.
Materials and Methods Strains
Methanogenium marisnigri* D S M 1498, was obtained from the Deutsche S a m m l u n g von M i k r o o r g a n i s m e n , G ö t t i n g e n .
Cull arc Conditions
The isolate M 3 was cultivated by using the technique described by Halch and Wolfe (1976). If not mentioned otherwise, the isolate was grown in %* M C P medium, that is medium 3 of Halch el al. (1979), modified by the use of
" P e p t o n aus Casein, tryptisch verdaut" ( M e r c k ) instead o f Irypliease (HHl.) and by adjusting the p i I to (>.9 ( 1 12S 04) .
Twenty milliliter cultures were grown i n stoppered pres- surized UK) ml serum bottles ( H o r m i o l i . Italy) made of
"type I I P - g l a s s by incubation in water bath shakers (New Brunswick) at 140rpm and 37 "C.
.ihbrrvhitii'ns t i l t * . I itiauiik* I </>losinc; SPS: Sodium dodccylsul- falc (Sodium lauryl sulfate)
Methanogenium marisnigri, as a reference, was g r o w n in the same medium.
Plating
Polysilicate plates were prepared as described (Stettcr ct a l . 1981) except that they were equilibrated w i t h M G - m e d i u m containing penicillin, v a n c o m y c i n , k a n a m y c i n (each
150 ug/ml) and tetracycline (100 ug/ml) to prevent eubacterial contaminations.
Light Microscopy
The cells were viewed and photographed with a Leitz O r t h o l u x l l microscope, equipped with a v a r i o - o r l h o m a t camera system (Leitz). Fluorescence was observed in a Zeiss Standard fluorescence microscope with an excitation filler H 4 3 6 and a selection filler L P 470.
Electron Microscopy
F o r thin sectioning, cell sediments were fixed in M G - m e d i u m not containing organic components with 20 g glutar- aldehyde/l for 2 h and post fixed w i t h 10 g O s 04/ l for
1 h. D u r c u p a n ( F l u k a ) epoxy resin was used for embedding and thin sections were contrasted with lead citrate (5 min), uranylacetate (5 min) and again with lead citrate (3 min).
F o r shadowing, the cells were fixed o n p a r l o d i o n coated grids and shadowcasted (Edwards vacuum coaler 306) with a p l a t i n u m - i r i d i u m alloy (angle 7 ) for 15 s followed by c a r b o n coating of the p a r l o d i o n film for stabilization.
Electron micrographs were taken with a J E O L J E M 1 0 0 C electron microscope at 80 k V and with a 40 \im objective aperture.
Isolation of DNA
T w o grams cells (wet weight) were suspended i n 8 m l buffer ( 5 0 m M T r i s - H C l , p H 8; 5 0 m M N a C I ; l O m M H D T A ) .
Then, S D S ahd Na-deoxycholate were added up to final concentrations o f 0.5 % and 7 m M , respectively. This mixture was incubated for 15 min al 65 C . Then, K C l was added to a final concentration o f 0.5 M . After I h at 2 C the precipitated potassium lauryl sulfate was removed by c e n t r i f u g a r o n (20 m i n , 21,000 r p m , rotor J A 21, Heckman J2-21). In the next step solid C s C l (0.92 g/ml) and ethidium bromide (20 ug/ml) were added to the supernatant and ccntrifuged 48 h at 45,000 rpm (rotor 5 0 T i , 2 0 " C . Heckman L5-50). T h e Ü N A - band was isolated by suction with a syringe, e t h i d i u m
0302-X933/82/0132/0031 /SOI .20
bromide was removed by extraction 3 x with >i-butanol and the s o l u t i o n was dialyscd against 0.1 x S S C (0.015 M N a C l ; 0.0015 M N a3- c i t r a t e ) .
Analysis of the Cell Wall
T h e existence o f rigid cell wall sacculi was checked for as described by Stettcr ct a l . (1981). H y d r o l y s a t c s of whole cells were analysed for m u r a m i c acid w i t h an a m i n o acid analyzer ( K ö n i g and Stettcr 1982, i n preparation).
F o r the identification o f proteins a n d glycoproteins, cell envelopes were obtained after sonification (Sonifter B 1 2 ,
B r a n s o n S o n i c Power C o m p a n y ) o f the cells at 50 W for 20 s. The iysate was suspended in basal salt medium ( M G - medium without organic components), c o n t a i n i n g 1 mg Deoxribonuclease 1 (Boehringcr, M a n n h c i m ) / 1 , and was af- terwards ccntrifuged at 40,000 r p m (rotor 50 T i , 20° C , B c c k m a n L5-50). T h e pellet was washed twice with basal salt solution, solubilized in L a e m m l i ' s sample buffer ( L a e m m l i
1970) and applied onto exponential P o l y a c r y l a m i d e gels, which were prepared according to L a e m m l i (1970) a n d M i r a u l t and Scherrer (1971). T h e gradient ranged from 5 to 25 % Polyacrylamide. The volume o f the closed m i x i n g vessel was 18 ml for a 20 ml gel. T h e gel was stained for protein and carbohydrates with coomassic blue and pcriodatc-Schiff- reagent (Scgrest and Jackson 1972).
Temperature Measurement
Temperature i n the field was determined w i t h an electronic thermometer (Metratherm 1200 d, B B C M e t r a w a t t , G e r m a n y ) equipped with electrode T 1 2 6 .
Methane Detection
Methane was quantilatcd by gas chromatography using a Hewlett P a c k a r d gas Chromatograph, model 5880 A . It was determined on a 6 feet glass c o l u m n filled w i t h C a r b o s i c v c S (Supclco) at 7 0 " C isothermal.
Results
Collection of the Sample
T h e sample M l l / 3 was taken from a small swamp composed of drilling waste, w h i c h was left from d r i l l i n g the neighbour- i n g steam well i tM o f c t e l ^, near B a i a i n the N a p l e s area (Italy). The swamp is provided w i t h water by a streamlet tlowing out from the condensor o f the well.
W i t h i n the swamp, there were some places, were gas bubbles ascended continuously to the surface. F r o m one o f them, about 3 m away from the embankment, a sample ( M l l / 3 ) of the loose greyish m u d sediment was d r a w n i n a depth o f about 0.2 m with a 11 beaker mounted on a l o n g stick. T h e original temperature o f the sample was 1 9 " C , the p H was 7.0 and the conductivity was 5 mS, that is about 7 0 % of sea water. T h e sample was immediately filled into a sterile 100 ml storage bottle, which was sealed with a rubber stopper after the addition o f 0.1 m l o f tesa/urin (0.1 % w/v in water).
T h e n , i n order to lower the redox potential, 1 ml o f an aqueous solution o f each 1.2",; (w/v) o f i.-eystcin • MCI - H20 and N a2S • 9 H20 (pi I 7.0, adjusted w i t h N a O H ) and 1 m l o f a freshly prepared aqueous solution o f sodium dithionite (0.2 % w/v) were injected into the sample through the stopper with
1 m l syringes. T h e sample was then carried to the laboratory at r o o m temperature (around 20" C ) .
Enrichment
1 n a Freier type anaerobic chamber ( A r a n k i a n d Fretcr 1972), 100 m l serum bottles c o n t a i n i n g 20 ml M G - m c d i u m and 10 ug v a n c o m y c i n / m l were inoculated with 1 ml o f sample M l 1/3.
After scaling with stoppers the scrum bottles were pressurized (200 k P a H2: C 02 = 8 0 : 2 0 ; Batch a n d Wolfe 1976) a n d then incubated in a water bath shaker ( N e w B r u n s w i c k ) at 30 C . After 3 days, l o w but significant amounts o f methane c o u l d be detected in the gas atmosphere o f the culture vessels. In the UV-fluorescence microscope, some emerald green fluorescing almost crystal-plate shaped particles were observed a m o n g large amounts o f rods, spirilli, and, due to v a n c o m y c i n , atypical spheres. A strong enrichment o f this novel m c l h a - nogen, designated M 3 , was obtained by the simultaneous addition o f v a n c o m y c i n , penicillin, k a n a m y c i n (each 150ug/ml) and tetracycline (tOOug/ml) i n t o the M G - m e d i u m .
Isolation Procedure
T h e enriched methanogen M 3 c o u l d be isolated by serial dilutions in M G - m c d i u m in serum bottles. A t the 10 H d i - l u t i o n , no infection c o u l d be detected in the microscope even without antibiotics. T o obtain single colonies, the culture was streaked parallclly onto polysilicatc and o n t o agar plates, b o t h prepared with M G - m c d i u m . After 3 months i n c u b a t i o n at 3 0 " C , r o u n d , smooth, bright ochre-colored colonies about 2 m m in diameter became visible on the polysilicatc plates. N o growth occurred on agar. F r o m single colonies l i q u i d cultures c o u l d be obtained over night.
Culture and Storage
L i q u i d cultures are routinely transferred after 2 — 3 days into fresh medium ( 5 % inoculation). In order to preserve the strain for longer periods, it was grown for 2 days a n d then, after renewing the gas atmosphere, it was simply fro/en and stored at - 2 0MC .
Optimal Growth Temperature
T h e isolate M 3 grows between 17 C a n d 4 r C ( F i g . 1 ) w i t h a n o p t i m u m a r o u n d 40" C . A t 43 C and at 1 5 " C no g r o w t h occurs.
Growth Requirements
H2 and formate serve as substrates for growth. In a d d i t i o n , acetate is essentially required (Table 1). 0 . 1 % acetate is sufficient (data not shown). O p t i m a l growth is obtained by c o m b i n a t i o n o f acetate with yeast extract or with peptone a n d vitamins ( T a b i d ) . N o growth occurs o n acetate alone, methanol and mclhylamincs (data not shown) as substrates.
M 3 grows in the presence of 0.4 5.4°,, N a ( I ( F i g . 2). The optimal salt concentration is a r o u n d I % N a C l . The o p t i m a l p l l for growth is between p i 16.5 7.5.
Morphology
In the light microscope, slowly w o b b l i n g plates with sharp crystal-like edges 1 — 3 urn l o n g and 1 — 2 urn wide can be seen
O 15 20 25 30 35 40 45 t c e ) — .
FIR. t. Optimal growth temperature. Growth was determined several limes during the exponential phase by O.D.5 7 f rmeasurement. The hours/doubling were calculated from the slopes of the growth curves (not shown)
T a b i d . Influence of organic components on growth. The basal salt medium (medium 3 of Balch et al. 1979, but without organic ingredients) was supplemented with organic components in the following con- centrations: Acetate 0.1 %; peptone 0.2%; yeast extract 0.2%; vitamins (trace vitamins according to Balch ct al. 1979) 0.2 ml/20 ml medium. Gas phase: H2/ C ( )2. The samples (20ml) were inoculated with 1 ml of a culture grown in a modified MG-mcdium, in which all organic com- ponents were reduced to 1/10 of the normal concentration. The initial ( ) . D .5 7 H was around 0.03
Organic components Maximal absorbance
( O . P, , 7 „ ) - (Control)
Peptone — Yeast extract — Vitamins — Acetate 0.21 Peptone + yeast extract -
Peptone + vitamins -
Yeast extract + vitamins — Acetate 4- yeast extract 0.62 Acetate + peptone 0.45 Acetate 4-vitamins 0.18 Peptone -f- yeast extract -1 vitamins
Acetate I ye<tst extract ~\ peptone 0.37 Acetate \ peptone 4- vitamins 0.60 Acetate \ vitamins -f yeast extract 0.49 Acetate \- yeast extract \ peptone t vitamins 0.47
( F i g . 3a). In the profile (see arrows), they appear as rods about 0.2 0.3 [tin in diameter. The cells showed a negative gram reaction. In the electron microscope angular plates
i.o 2.8urn l o n g and 1.5urn wide are visible (Figs. 3b, 4b), K>1
~c , , , , ,—
0 1 2 3 4 5 NaCt [•/.] fr-
Fig. 2. Effect of NaCl on growth. The NaCl was added to a MG-mcdium prepared without NaCl supplement. This basal medium already contains 0.33% NaCl, which are not considered in the diagram
s h o w i n g large indentations in the center ( F i g . 3b). In cross- sections, often "bone-shaped" ( F i g . 3c) profiles with a d i a - meter o f only 0.07 urn in the center, a n d 0.1 —0.25 urn on the ends are seen besides n o r m a l rod-shaped profiles. Sometimes also j>-shapcd cross-sections ( F i g . 3e) and profiles with c o n - vexities, possibly buds, can be delected. Septa or d i a p h r a g m - like indentations were never seen. T h e cells often c o n t a i n electron dense r o u n d inclusions, possibly granules o f reserve material (Figs. 3b, 3 d , 4b). In thin sections the granules frequently seem to be shrunken ( F i g . 3 d) or often to be b r o k e n out, leaving behind a less electron dense hole ( F i g . 3c, 3d).
A polar tuft o f flagclla can be observed ( F i g . 4a). Mach flagcllum is about 13.3nm i n diameter a n d up to 32 urn l o n g (not shown).
Cell Envelope
T h e cell envelope shows a hexagonal surface pattern ( F i g . 4a).
T h e distance o f the subunits — center to center — was determined from E M - p h o t o g r a p h s to be 14.0nm.
Preparations o f the cell envelope show one d o m i n a t i n g protein band ( F i g . 5, lane 3) w i t h an apparent molecular weight o f 143,000 as determined by co-electrophorcsis with molecular weight standards i n the S D S - g c l ( F i g . 5, l a n c l ) . T h i s band is also stained with the periodate-Schiff-rcagcnt ( F i g . 5, lane 5), and therefore seems to be a glycoprotein. W e believe this band to consist o f the m a i n envelope protein. N o rigid cell wall sacculi c o u l d be isolated. In accord, the cells were completely lysed with 2 % S D S at r o o m temperature. N o m u r a m i c acid c o u l d be detected.
DNA Base Composition
T h e D N A contains 47.5 m o l % G C as determined by the melting point in 0.1 x S S C ( M a r m u r and D o t y 1962), using calf thymus D N A ( 4 2 m o l " , , G C ) as a reference.
Discussion
T h e new isolate M 3 occurs as plates with sharp edges, strongly reminding o f fiat crystals, which i n the electron microscope show an almost "pneumatic b o a f ' - l i k c ap- pcarence: between a surrounding puffed up r o l l , 0.1 — 0.25 urn in diameter, there is an inner plate, only 0.05 —
Fig. 3a—c
Light and electron micrographs of Mctlumoplamis Iim kola, a Phase contrast of exponentially growing cells. Arrows indicate bacteria appearing in profile;
b platinum shadowed (7 '); c, d, c thin sections. Slatcmenls of size in urn
0.07 um thick, usually showing some humps. A l t h o u g h not quadratic, the flat shape with the sharp edges strongly reminds o f the square halophilic bacterium discovered by Walsby (19K0). T h e novel methanogen, however, grows at much lower ionic strength. Even after a transfer i n dcslilled water, ils Hat shape is perfectly stable al least for hours (data not shown). The shape-maintaining principle remains unclear al the moment. One could speculate, that the organism may cither possess an efficient osmoregulation or, more l i k e l y , contain internal structures supporting the envelope in order to obtain the flat shape.
N o septa formation c o u l d be detected, indicating that cell division does not occur by the usual binary fission. T h e y-
shaped profiles and the profiles w i t h swellings in thin sections point to an unusual b u d d i n g mechanism.
F r o m its ability to form methane from H2 and C ( )2 a n d from its green fluorescence the new organism is clearly defined to be a methanogen. T h e obligate requirement'for acetate in the medium, w h i c h , however, by itself cannot serve as a substrate, was already reported for Mcihaiutgcnium variad (Romesser et a l . 1979), Other organic c o m p o u n d s , such as a mixture of peptone and vitamins or yeast extract are not essential, but stimulate g r o w t h . N o methanol or methyl- amines can be used, indicating that this bacterium does not
belong to Ihe Mcthanosarcinaccae. T h i s is proven in a d d i t i o n by the lack o f a rigid cell w a l l , the possession o f flagclla a n d
Fig. 4a and b
Electron micrographs of MvtlumopUmus limitóla, a HnUny shail'*;*od O b thin section. Statements of size >n urn
the negative gram reaction o f the isolate. A close relationship to the Mcthanococcales, which similar to the isolate can grow on formate, was very unlikely because o f a much higher G C - contcnt o f the isolate. D u e to the latter feature and based o n
the polyaminc c o m p o s i t i o n (S. Schoberth, personal c o m - munication), however, the isolate seems to belong to the
M c t h a n o m i c r o b i a l c s . D N A - R N A hybridization experiments (Tu ct al. 1982) with D N A and ^ P - l a b e l l e d 16s r - R N A substantiated this assumption: the hybrid of the M 3 D N A with the Methanogenium R N A showed a higher thermosta- bility (fs - fractional stability « 0.59) than that with the M c l h a u o c o c c u s R N A (fs - 0.43), further indicating that M 3
belongs to the M e t h a n o m i c r o b i a l c s . Vvi'hin litis or'1. Methanogenium R N A yielded an even more ^tab!¿ h y l v i d with the M c t h a n o s a r c i n a D N A (Is ~ 0.62V fhitn with .Á.K D N A , demonstrating that the two families arc closer - to each other than to M 3 . Therefore, we believe the new isolate to be a member o f at least a new family, which \\v I M P the Melhanoplanaceae.
T h e distribution and the biotope o f the isolate is not i A « r a l the moment. F r o m Í I N original habitat, its >:rowl!,
the mcsophilic temperature range and its salt re tuirem%"< " d tolerance, the organism can be assumed to h« cnxn v . swamps of freshwater and scawatcr. There ti may nr.
1 2 3 4 5
**|:
- I S
* i
•v»Fij».5. SDS-polyacrylamide gel. Flcctrophoresis was performed al 110 V for 14h. (/) Molecular weight standard: R N A polymerase summits of Escherichia coli (from top to the bottom): /F (160,000), ß (155,000), a (92,000), a (37,000); (2) and (4) envelope preparation of Methanogenium marisnigri: [3) and (5) envelope preparation of Metlianoplanus limicola:
( / ) ( . ? ) : coomassic staining: {4) and (.5): periodatc-Schiff staining
previously overlooked because o f its transparence and its unusual shape. Due to m o r p h o l o g y and habitat, we name the new isolate M 3 Metlianoplanus limicola.
Description and Classification of the Methanoplanaceae Order M c t h a n o m i c r o b i a l e s , B a l c h a n d Wolfe (1976).
F a m i l y I, Mcthanomierobiaceac, Balch and Wolfe (1976).
F a m i l y II, M c l h a n o s a r c i n a c e a c , Balch and Wolfc(1976).
F a m i l y 111, Methanoplanaceae, W i l d g r u b e r , T h o m m a n d Stetter (fam. nov.) Methanoplanaceae, M e . tha. no. p l a . na.
ee' ae. M . L . n c u l . n. Metlianoplanus type genus o f the f a m i l y ; -aceae e n d i n g to denote a f a m i l y ; M . L . fern. p i . n.
Methanoplanaceae the Metlianoplanus family. T h e Methanoplanaceae belong to the order M c t h a n o m i c r o b i a l e s , Balch and Wolfe (1976). T h e family Methanoplanaceae contains one genus.
Gram-negative cells, occurring as thin plates with sharp edges. T h e cell envelope shows a hexagonal surface pattern.
Cells oxidize H2 or formate as the sole energy source for growth and methane p r o d u c t i o n .
G e n u s 1 Metlianoplanus, Wildgruber, Thomm and Stettcr (gen. nov.) Me^ tha. no. pla'nus. M . L . n. methanum methane;
M . L . adj. planus Hat; M . L . masc. n. Metlianoplanus the meth- ane (-producing) plate. T h e description o f the genus is the same as that o f the family. Metlianoplanus limicola,
W i l d g t u h c r , Thomm and Stettcr (sp. nov.) Ii. m i V o . l a . I,.
limicola, I . masc. n. inhabitant o f a swamp on account o f its habitat.
A n g u l a r , crystal-like plates 0.07 0.30 urn thick and 1.6 — 2.S urn long and 1.5 m n wide, occurring singly. The cells are
sometimes branched, without septa. T h e cell envelope shows a hexagonal surface pattern a n d contains a d o m i n a t i n g glycoprotein. N o sacculus is present. A p o l a r tuft o f flagclla can be seen, each flagellum about 13.3 n m in diameter and up to 32 urn long. T h e cells c o n t a i n electron dense r o u n d inclusions*, about 0.1— 0.2 urn i n diameter. O n polysilicatc plates r o u n d , s m o o t h , bright, o c h r e c o l o r c d , flat colonics, about 2 m m i n diameter are formed. W e a k l y motile.
A n a e r o b i c . Gram-negative. G r o w t h between 1 7 ' C a n d 4 1 " C , o p t i m u m 4 0 ° C . O p t i m a l salt concentration is 1 % N a C l , o p t i m a l p H is 7. H2 and formate serve as substrates for g r o w t h a n d methane p r o d u c t i o n . In a d d i t i o n , acetate is strictly required. N o growth occurs o n acetate alone, o n methanol, a n d o n methylamines. Cells are resistent against v a n c o m y c i n , penicillin, k a n a m y c i n , a n d tetracycline.
T h e D N A forms complexes w i t h 16s r R N A from Methanogenium marisnigri w i t h a fractional stability o f 0.59.
T h e G + C content o f the D N A is 47.5 m o l °0. Lives possibly i n seawater a n d freshwater swamps.
T y p e s t r a i n : D S M 2279.
Acknowledgements. We wish to thank Gesa Thtes and Davoi in Janecovie for highly valuable hints in electron microscopy. Furthermore, thanks arc due to Tetra Frischeisen and Lisa Kleemeier for excellent technical assistence. This work was supported by a grant from the Deutsche , Forschungsgemeinschaft to K . O. Stettcr.
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