J O U R N A L O F B A C T E R I O L O G Y , F e b . 1 9 8 3 , p. 1 0 6 0 - 1 0 6 2 0 0 2 1 - 9 1 9 3 / 8 3 / 0 2 1 0 6 0 - 0 3 $ 0 2 . 0 0 / 0
C o p y r i g h t © 1 9 8 3 , A m e r i c a n S o c i e t y for M i c r o b i o l o g y
V o l . 1 5 3 , N o . 2
Evidence for a Plasmid in a Methanogenic Bacterium
MICHAEL THOMM,
1JOSEF ALTENBUCHNER,
2A N D KARL O. STETTER
1*
Lehrstuhl für Mikrobiologie1 and Lehrstuhl für Genetik,2 Universität Regensburg, D-8400 Regensburg, Federal Republic of Germany
Received 24 June 1982/Accepted 25 October 1982
Among 15 strains of methanogens, one plasmid, p M P l , was identified in the new coccoid isolate PL-12/M. It could not be detected in the cleared lysate, but it was detected in the viscous pellet. The plasmid had a molecular weight of ca. 4.6
x 10
6. A restriction enzyme cleavage map of the cloned plasmid was derived.
Methanogenic bacteria are a diverse group of strict anaerobes which share the ability to pro- duce methane. According to 16S r R N A analy- ses, the methanogens belong to the archaebac- teria, the third kingdom of life besides eubacteria and the eucaryotic cytoplasm (1, 5, 19). Recently, the DNA-dependent R N A poly- merases from representatives of different orders of archaebacteria have been isolated (20), in- cluding those from methanogenic bacteria (13), which all show striking similarities to eucaryotic R N A polymerases (20, 21). For studying archae- bacterial transcription systems in more detail, especially for establishing an in vitro transcrip- tion system, homologous templates are re- quired. In this kingdom, however, no phages or Plasmids are known, the only exception being the extreme halophiles (9,10,12,16,17), which, however, seem to be less appropriate for in vitro transcription studies because of their high intra- cellular salt concentrations (3). We therefore screened methanogenic bacteria for the exis- tence of plasmids. In this paper, we report on the isolation and first characterization of a cryp- tic plasmid from a novel methanogen.
A total of 15 strains of methanogens of the orders methanococcales and methanomicro- biales cultivates by the technique of Balch and Wolfe (2) were screened for the presence of plasmids..Included were strains obtained from culture collections and new isolates from our laboratory. Cells were lysed by a procedure described for Escherichia coli (4), but without the lysozyme treatment. In all of the methano- gens, however, the cleared lysate, which in E.
coli harbors the enriched plasmid D N A and fragments of chromosomal D N A (4), contained no plasmid D N A detectable by agarose gel elec- trophoresis (data not shown). Thus, the viscous pellet, obtained by clearing the lysate, which is known to contain the bulk of chromosomal D N A in E. coli (4), was analyzed for the presence of plasmids after alkaline treatment, phenol-chlo-
roform extraction, and concentration by ethanol precipitation. In one of our new isolates, the coccoid methanogen PL-12/M, this fraction ex- hibited in agarose gel electrophoresis two strong additional bands besides the chromosomal D N A band (Fig. 1, lane c); the faster one probably corresponded to the covalently closed circular (ccc) and the slower one to the open circular (oc) form of a plasmid. The methanogen plasmid was further purified by cesium chloride-ethidium bromide gradient centrifugation. After ultracen- trifugation, two clearly separated bands became visible, indicating the presence of ccc-DNA be- sides the chromosomal D N A . This result was confirmed by agarose gel electrophoresis. The heavier band consisted of the plasmid D N A (Fig. 1, lane b), and the lighter band contained the chromosomal D N A (data not shown). In control experiments, the known plasmids (9,18) of Halobacterium cutirubrum (Fig. 1, lane e), Halobacterium halobium (data not shown), and E. coli DS609 (Fig. 1, lane d) could be detected by the same procedure. In contrast to the meth- anogen, the plasmid D N A in halobacteria and E.
coli was found both in the viscous pellet and in the cleared lysate (data not shown).
The molecular weight of the methanogen plas- mid was determined by agarose gel electropho- resis. Comparison of its electrophoretic mobility (Fig. 1, lane b) with that of marker plasmids (Fig. 1, lane a) in a calibration curve led to a molecular weight of ca. 4.6 x 10
6.
Analysis of the plasmid band by electron microscopy showed that it contained a single species of D N A molecule existing as a monomer in the ccc- and oc-forms (Fig. 2). The molecular weight determined by contour length measure- ment was 4.57 ( ± 0 . 1 1 ) x 10
6.
Repeated attempts to isolate the methanogen
plasmid from transferred cultures of strain P L -
12/M led to highly variable plasmid yields, indi-
cating that the plasmid is occasionally not de-
tectable by the isolation procedure. The reason
V O L . 153,1983
a b c d e ehr-
Ir 3-
1 -
FIG. 1. Plasmids of the methanogen PL-12/M, / / . cutirubrum and £ . co/i resolved by agarose gel electro- phoresis. Electrophoresis was performed in 0.7%
(wt/vol) vertical agarose gels in Tris-phosphate buffer (6) at 4 V c m "
1for 16 h at 5°C. (a) Marker plasmids of E. coli: 1, pACYC184 (2.65 x 10
6); 3, pJOE106 (6.05 x 10
6); 4, pJOE229 (9 x 10
6); 6, pRIT10003 (12.2 x 10
6).
(b) Methanogen plasmid p M P l purified by cesium chloride-ethidium bromide centrifugation: 2, ccc form;
5, oc form, (c) D N A of PL-12/M isolated without gradient centrifugation, showing chromosomal D N A (ehr) and the ccc (band 2) and oc (band 5) forms of the plasmid p M P l . (d) D N A of E. coli DS609 and (e) H.
cutirubrum isolated without gradient centrifugation, showing chromosomal (chr) and plasmid D N A .
for this unusual phenomenon is unknown at this point.
F o r further analysis of the plasmid, it was cloned into the vector plasmid pBR322. A s the methanogen plasmid p M P l was cleaved once by the restriction endonuclease Pstl (data not shown), /\sfl-cleaved linear p M P l D N A was ligated into the Pstl site of pBR322 (14) by a standard technique (15) and amplified in E. coli K 1 2 strain H B 1 0 1 (data not shown). Analysis of three recombinant plasmids by restriction en- zyme cleavage revealed that the p M P l plasmid was inserted in both orientations (data not shown). One of them, pPF1260-3, was used for detailed mapping of the restriction sites. The plasmid was digested by using various restric- tion enzymes either individually or in combina- tion (see legend to F i g . 3). The positions of restriction sites within the plasmid pBR322 are taken from Sutcliffe (14). The restriction enzyme cleavage map shown i n F i g . 3 is based on the sizes of the restriction fragments obtained from single and double digests.
N o n e of the methanogens from culture collec- tions investigated i n the present study did pos- sess a detectable plasmid. Therefore, we as-
NOTES 1061 sumed that possible plasmids in methanogens coding for additional functions during life in nature had been lost during the serial transfers in the laboratory on artificial media. A s a conse- quence, we isolated strains from natural habitats and screened them immediately for the presence of plasmids. One isolate from a submarine fuma- role close to Vulcano Island, Italy, named P L - 12/M, was found to bear a plasmid w h i c h could be isolated. T o our knowledge, it is the first methanogen plasmid described.
During its enrichment, the plasmid could not be detected in the supernatant of the centrifuged cell lysate where plasmids are usually found, but it was detected almost quantitatively in the pellet, together with the chromosomal D N A . This uncommon behavior could be explained by a very close association of the plasmid with the chromosomal D N A or with the membrane.
Due to its small size, the methanogen plasmid may be well suited for studies of gene expression in methanogens, e.g., transcription studies and promoter analyses. Experiments to determine
FIG. 2. Electron micrograph of ccc (top) and oc D N A (below) molecules of the methanogen plasmid.
D N A molecules were prepared for electron microsco- py by the cytochrome c spreading technique of Kleinschmidt (7). D N A was stained with 50 |xg of uranyl acetate in 90% ethanol and rotary shadowed with platinum-iridium at an angle of 5°. Microscope magnifications were calibrated by using RSF2124 D N A (molecular weight, 7.35 x 10
6) as an internal standard. From the contour length of the p M P l plas- mid molecule (10 independent molecules measured), a molecular weight of 4.57 (±0.11) x 10
6was calculated.
Bar equals 0.2 u,m.
1062 NOTES
J . B A C T E R I O L .FIG. 3. Restriction endonuclease cleavage map of the recombinant plasmid pPFl260-3. The heavy line marks the extent of the methanogen plasmid, pMPl, and the thin line represents the vector pBR322 with the location and polarity of the tetracycline resistance determinant (Te
1) indicated by an arrow (11). One scaling unit corresponds to one kilobase pair. The map is based on the sizes of restriction fragments from single and double digests obtained after electrophore- sis of these fragments in 0.7% (wt/vol) horizontal agarose gels in Tris-borate (8). Electrophoresis was performed for 15 h at 4 V c m
- 1.
the reason for the varying plasmid yield and its possible functions are in progress.
T h a n k s are due to G e r t a G e b h a r d and Petra F r i s c h e i s e n for excellent technical assistance.
T h i s w o r k was supported b y grants o f the Deutsche F o r - schungsgemeinschaft.
L I T E R A T U R E C I T E D
1. B a l c h , W . E . , G . E . F o x , L . J . M a g r u m , C . R . Woese, and R . S. Wolfe. 1979. Methanogens: reevaluation o f a unique biological group. M i c r o b i o l . R e v . 43:260-296.
2. B a l c h , W . E . , and R . S. Wolfe. 1976. N e w approach to the c u l t i v a t i o n o f methanogenic bacteria: 2-mercaptoethane- sulfonio» a c i d ( H S - C o M ) - d e p e n d e n t g r o w t h o f Methano- bacterium ruminantium i n a pressurized atmosphere.
A p p l . E n v i r o n . M i c r o b i o l . 32:781-791.
3. C h r i s t i a n , J . H . B . , a n d J . A . W a l t h o . 1962. Solute concentrations w i t h i n cells o f halophilic and non-halophil- ic bacteria. B i o c h i m . B i o p h y s . A c t a 65:506-508.
4. C l e w e l l , D . B . , and D . R . H e l i n s k i . 1969. S u p e r c o i l e d c i r c u l a r D N A - p r o t e i n c o m p l e x i n Escherichia coli: purifi-
cation and induced c o n v e r s i o n to an open c i r c u l a r D N A form. P r o c . N a t l . A c a d . S e i . U . S . A . 62:1159-1169.
5. F o x , G . E . , E . Stackebrandt, R . B . Hespell, J . G i b s o n , J . Maniloff, T . A . D y e r , R . S. Wolfe, W . E . B a l c h , R . S.
Tanner, L . J . M a g r u m , L . B . Z a h l e n , R . B l a k e m o r e , R . G u p t a , L . B ö n e n , B . J . L e w i s , D . A . S t a h l , K . R . L u e h r - sen, K . N . C h e n , and C . R . Woese. T h e phylogeny o f prokaryotes. 1980. Science 209:457-463.
6. H a y w o r d , G . G . 1972. G e l electrophoretic separation o f the complementary strands o f bacteriophage D N A . V i r o l - ogy 49:342-344.
7. K l e i n s c h m i d t , A . K . 1968. M o n o l a y e r techniques i n elec- tron m i c r o s c o p y o f nucleic a c i d molecules. M e t h o d s E n - z y m o l . 12:361-377.
8. M e y e r s , J . A . , D . Sanchez, L . P . E l w e l l , and S. F a l k o w . 1976. S i m p l e agarose gel electrophoretic method for the identification and characterization o f plasmid d e o x y r i b o - nucleic a c i d . J . B a c t e r i o l . 127:1529-1537.
9. Pfeifer, F . , G . Weidinger, and W . Goebel. 1981. Character- i z a t i o n o f plasmids i n halobacteria. J . B a c t e r i o l . 145:369- 374.
10. Schnabel, H . , W . Z i l l i g , M . Pfäffle, R . Schnabel, H . M i c h e l , and H . Delius. 1982. Halobacterium halobium phage (J>H. E M B O J . 1:87-92.
11. Schöffl, F . , W . A r n o l d , A . P u n i e r , J . Altenbuchner, and R . Schmitt. 1981. T h e tetracycline resistance transposons T n / 7 2 / and Tn/771 have three 38-base-pair repeats and generate five-base-pair direct repeats. M o l . G e n . Genet.
181:87-94.
12. S i m o n , R . D . 1978. H a l o b a c t e r i u m strain 5 contains a p l a s m i d w h i c h is correlated w i t h the presence o f gas vacuoles. N a t u r e ( L o n d o n ) 273:314-317.
13. Stetter, K . O . , J . W i n t e r , and R . H a r t l i e b . 1980. D N A - dependent R N A polymerase o f the archaebacterium Methanobacterium thermoautotrophicum. Z e n t r a l b l . B a k t e r i o l . Parasitenkd. Infektionskr. H y g . A b t . 1 O r i g . R e i h e C 1:201-214.
14. Sutcliffe, J . G . 1978. p B R 3 2 2 restriction map derived from the D N A sequence: accurate D N A size markers up to 4361 nucleotide pairs l o n g . N u c l e i c A c i d s R e s . 5:2721- 2728.
15. T a n a k a , T . , and D . W e i s b l u m . 1975. C o n s t r u c t i o n o f a c o l i c i n E l - R factor composite plasmid i n vitro: means for amplification o f d e o x y r i b o n u c l e i c a c i d . J . B a c t e r i o l . 121:354-362.
16. T o r s v i k , T . , and J . D . Dundas. 1980. Persisting phage infection i n H a l o b a c t e r i u m salinarium str. 1. J . G e n . V i r o l . 47:29-36.
17. W a i s , A . C , M . K o n , R . E . M c D o n a l d , and B . D . Stellar.
1975. Salt-dependent bacteriophage infecting Halobacte- rium cutirubrum and H. halobium. N a t u r e ( L o n d o n ) 256:314-315.
18. W i e b a u e r , K . , S. S c h r a m l , S. W . Shales, and R . Schmitt.
1981. T e t r a c y c l i n e resistance transposon T n / 7 2 / : recA- dependent gene amplification and expression o f tetracy- cline resistance. J . B a c t e r i o l . 147:851-859.
19. Woese; C . R . , L . J . M a g r u m , and G . E . F o x . 1978.
A r c h a e b a c t e r i a . J . M o l . E v o l . 11:245-252.
20. Z i l l i g , W . , K . O . Stetter, R . Schnabel, J . M a d o n , and A . G i e r l . 1982. T r a n s c r i p t i o n i n archaebacteria. Z e n t r a l b l . B a k t e r i o l . Parasitenkd. Infektionskr. H y g . A b t . 1 O r i g . R e i h e C 3:218-227.
21. Z i l l i g , W . , R . Schnabel, J . T u , and K . O . Stetter. 1982.
T h e phylogeny o f archaebacteria i n c l u d i n g novel anaero- bic thermoacidophiles i n the light o f R N A polymerase structure. Naturwissenschaften 69:197-204.
