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Characterization of a Plasmid Carried by Methanobacterium thermoautotrophicum ZH3, a Methanogen Closely Related to Methanobacterium thermoautotrophicum Marburg
System. Appl. Microbial. 17,484-491 (1994) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Characterization of a Plasmid Carried by Methanobacterium thermoautotrophicum ZH3, a Methanogen Closely Related to Methanobacterium thermoautotrophicum Marburg ROLF STETTLER, PETER PFISTER, and THOMAS LEISINGER" Mikrobiologisches Institut, Eidgenossische Technische Hochschule, ETH-Zentrum, CH-8092 Zi.irich, Switzerland Received September 12, 1994
Summary Strain ZH3, a hydrogenotrophic, thermophilic methanogenic archaeon was isolated from a sewage sludge digester. Based on its phenotypic properties and its 16S rRNA sequence (99.9% similarity), strain ZH3 is closely related to Methanobacterium thermoautotrophicum Marburg. The newly isolated organism harbored pME2200, a cryptic 6.2 kb plasmid, which is related to, but not identical with the previously characterized cryptic 4.4 kb plasmid pME2001 of strain Marburg. Plasmid pME2200 was characterized by restriction mapping and by Southern blot and DNA sequence analysis and compared to plasmid pME2001. The two elements are composed of a presumably identical, 3.8 kb backbone structure. In addition to the common backbone, both plasmids carry a region of high interplasmid homology, which in pME2200 was interspersed with three segments not present in pME2001. These interspersed segments did not encode potential open reading frames. One of them contained a singular restriction site which may be of use for the construction of potential cloning vectors that are undamaged in essential plasmid functions.
Key words: Archaea - Methanogen - Thermophile - Plasmid - 16S rRNA
Introduction
Thermophilic representatives of the genus Methanobacterium are characterized by an optimum growth temperature between 55°C and 65 °C and by their ability to use either H 2 /C0 2 only or H 2 /C0 2 and formate as carbon and energy source. Several strains belonging to this group have been isolated and characterized, but the taxonomic and phylogenetic relationships among these organisms are not completely resolved. Recently, efforts have been made to redefine the phylogeny and taxonomy of some thermophilic strains of the genus Methanobacterium. Nailing et al. (1993) have used comparative analysis of 16S rRNA sequences to study the relationships between members of the two species Methanobacterium thermoautotrophicum and Methanobacterium thermoformicicum. They proposed that the members of these two species should be separated into at least three groups. A first group comprises M. thermoformicicum strains Z-245, FTF, THF, CSM3, FF1, FF3, and M. thermoautotrophicum i1H. A " Corresponding author
second group includes the M. thermoformicicum strains CB12, SF-4 and HN4, and the third group is represented by M. thermoautotrophicum Marburg only. The classification of these strains in three different groups is supported by the results of antigenic fingerprinting and DNADNA hybridization analysis (Touzel et al., 1992). Within a different set of thirteen thermophilic Methanobacterium strains the taxonomic relationships were recently studied by DNA-DNA hybridization, whole-cell protein analysis and antigenic fingerprinting (Kotelnikova et al., 1993). The authors reached the conclusion that these strains fall into three clusters within the genus Methanobacterium. M. thermoautotrophicum Marburg is the best studied thermophilic Methanobacterium strain with respect to biochemistry of methanogenesis (Thauer et al., 1993), gene organization (Palmer and Reeve, 1993), and genome structure. A combined physical and genetic map of its circular 1,623 kb chromosome has been prepared (Stettler and Leisinger, 1992). The organism harbors the cryptic multicopy plasmid pME2001, and it is infected by the
A Plasmid in M. thermoautotrophicum ZH3
virulent, generally transducing phage '\j!M1 (Leisinger and Meile, 1993). While these features would appear to make this organism attractive for genetic studies, progress in establishing a gene transfer system has been hampered by the apparent inefficiency of transformation using plasmid DNA, phage DNA or chromosomal DNA (Settler, R., unpublished). These limitations, and the fact that large differences with respect to transformability are known to exist between strains of the same species (Mercenier and Chassy, 1988), have promped us to isolate from nature Methanobacterium strains closely related to M. thermoautotrophicum Marburg for testing them as recipients in a transformation system. In the present communication we describe one of these isolates, M. thermoautotrophicum ZH3, and compare it with respect to phenotypic properties, 16S rRNA sequence and genome organization to the Marburg strain.
Materials and Methods Bacterial strains, plasmids and phages. The bacterial strains, plasmids and the phage used in this study are listed in Table 1. M. thermoautotrophicum Marburg was cultivated at 65 °C according to Schonheit et al. (1980). The media were reduced with 0.5 g of Na2S· 9H 20 and 0.5 g of L-cysteine-HCI per liter. E. coli strains were grown at 37"C in LB (Sambrook et aI., 1989). Ampicillin was used at a final concentration of 150 l1g1ml. Isolation and culture conditions of M. thermoautotrophicum ZH3. Samples were collected from an anaerobic sewage sludge digester (municipal sewage treatment plant in Opfikon, Switzerland) in a serum bottle which had been flushed previously with a N 2/C0 2 gas mixture; the serum bottle was filled to the top with sample. 0.1 ml of sewage sludge were used to inoculate a flask that contained a H 2 /C0 2 (80: 20) gas phase and 10 ml of the minimal medium described by Schonheit et al. (1980). After 7 days of incubation at 60 °C the medium was turbid and methane was detected in the gas phase. Cells were then transferred into fresh minimal medium and incubated at 60 °C for 24h . Diluted
485
culture samples were plated (plates contained minimal medium supplemented with 1.0 mM titanium [III] citrate as reducing agent) by the soft agar-overlay method described previously (Kiener and Leisinger, 1983 ), followed by incubation at 60 °C for 8 days in anaerobic jars containing the H 2 /C0 2 gas mixture at 2-2.5 bar. Single colonies were picked and transferred into liquid minimal medium. After a second purification step on agar plates, cultures were grown from single colonies. Microscopic analysis of the enrichment cultures revealed fluorescent rods of uniform shape. One of the isolates, M. thermoautotrophicum ZH3, was used for further study. M. thermoautotrophicum ZH3 was routinely cultivated in minimal medium (Schonheit et aI., 1980). To test formate as a substrate, minimal medium was supplemented with 40 mM, 50 mM, 75 mM, and 100 mM sodium formate. Propagation of archaeophage 'lJM1 was performed as described previously (Meile et aI., 1989). DNA isolation and manipulation. M. thermoautotrophicum ZH3 and M. thermoautotrophicum Marburg plasmid DNA was isolated from lysates obtained by treatment of cell suspensions with partially purified pseudomurein endopeptidase from Methanobacterium wolfei (Kiener et aI., 1989) and purified by CsCI-ethidium bromide density gradient centrifugation. Total DNA of strains ZH3 and Marburg was extracted by the same procedure. Plasmid isolation from E. coli strains, digestions of DNA, and cloning were performed by standard procedures (Sambrook et aI., 1989). All enzymes used in the manipulation of DNA were used according to the manufacturer's specifications. Southern transfers (Southern, 1975 ) of DNA from agarose gel to nylon membranes (Hybond-N, Amersham International) were performed according to the membrane manufacturer's protocol. DNA for Southern hybridization was labeled with [a- 32 P]dATP using the oligo-labelling technique of Feinberg and Vogelstein (1983 ). DNA sequencing and analysis. Subclones in phages M13mp18 and M13mp19 or in the pBluescript vector were sequenced by the dideoxy chain termination method of Sanger et al. (1977), using [a- 32P]dATP. Sequencing reactions with single-stranded DNA and double-stranded DNA were performed with Sequenase version 2.0 (United States Biochemicals, Cleveland, Ohio). Regions, in which no usable restriction sites were found for subcloning,
Table 1. Microbial strains and extrachromosomal elements used in this study Strain or element
Relevant characteristics
Reference or source
Methanobacterium thermoautotropicum Marburg (DSM 2133 )
wild type
Fuchs et al. (1978 )
Methanobacterium thermoautotropicum ZH3 (DSM 9446 )
wild type
this work
E. coli DH5a
F-
Hanahan (1983 )
E. coli JM109
recAl endAl gyrA96 "A.- hsdR17 supE44 relAl thi L'1(IacproAB ) F' [traD36 proAB + lacQZL'1M15]
Yanisch-Perron et al. (1985)
pME 2001
cryptic multicopy plasmid of M. thermoautotrophicum Marburg
Meile et al. (1983 )
pME2200
cryptic multicopy plasmid of M. thermoautotrophicum ZH3
this work
'lJ M1 pBluescript II SK+
virulent phage of M. thermoautotrophicum Marburg
Meile et al. (1989)
Cloning vector, Apr
Stratagene, La Jolla, CA
pME2106
1.92 kb BamHIIECORI fragment covering the 16S rRNA
this work
M13mp18/19
gene from M. thermoautotrophicum ZH3 in pBluescript
Yanisch-Perron et al. (1985 )
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R. Stettler, P. Pfister, and T. Leisinger
were spanned by sequencing with synthetic oligonucleotide primers. Computer analysis was carried out with the University of Wisconsin Genetics Computer Group software packages, version 7, on the VAX. Nucleotide sequence accession numbers. The nucleotide sequences of the 16S rRNA gene of M. thermoautotrophicum ZH3 and of the 3.3 kb fragment of plasmid pME2200 have been deposited in the GenBank data base under accession numbers Z37156 and Z37155, respectively.
Results
Properties of M. thermoautotrophicum ZH3
The phenotypic properties of M. thermoautotrophicum ZH3 are listed in Table 2. On the basis of its ability to utilize C0 2 /H 2 (but not formate) and to produce methane gas, its optimum growth temperature of 65°C, its cell morphology, and its sensitivity to partially purified pseudo murein endopeptidase from Methanobacterium wolfei (Kiener et aI., 1987), the organism was identified as a member of the species Methanobacterium thermoautotrophicum. It differed from strain Marburg only by its insensitivity to archaeophage 'ljJM1 (Meile et aI., 1989) and by the formation of comparatively short rods « 5 [lm) during growth under suboptimal conditions such as limitation by hydrogen. At the level of the genome, M. thermoautotrophicum ZH3 and M. thermoautotrophicum Marburg were similar with respect to the sizes of their chromosomes (Table 2). However, digestion of the M. thermoautotrophicum ZH3 chromosome with the restriction enzyme Notl yielded five fragments, and none of these were similar in size to one of the six fragments obtained upon NotI digestion of the M. thermoautotrophicum Marburg chromosome (Settler and Leisinger, 1992). This difference in the macrorestriction patterns of the two strains indicates divergence in chromosomal DNA sequences. Another difference at the genome level between the two strains concerned the size of the plasmid carried by each organism. The cryptic plasmid pME2001 harbored by M. thermoautotrophicum Marburg has a size of 4.439 kb (Bokranz et aI., 1990) while in M. thermoautotrophicum ZH3 we have detected a 6.2 kb plasmid which was named pME2200.
16S rRNA sequence of M. thermoautotrophicum ZH3
To explore the phylogenetic relationship between M. thermoautotrophicum ZH3 and M. thermoautotrophicum Marburg, we have determined the 16S rRNA sequence of the former organism and compared it to the 16S rRNA sequence of the Marburg strain (0stergaard et aI., 1987). An oligonucleotide probe (5'-GAA ACT GGG GAT AAA CC-3') complementary to nucleotide positions 137 through 153 of the 16S rRNA gene of M. thermoautotrophicum Marburg was designed and used to analyse Southern blots of digested chromosomal DNA of M. thermoautotrophicum ZH3. A single 1.95 kb BamHI/EcoRI
GGA A
Fig. 1. Secondary structure of variable region I of 16S rRNA from M. thermoautotrophicum ZH3 according to the model of 0stergaard et al. (1987). The cytidine-phosphate residue at position 196 (arrow) is deleted in M. thermoautotrophicum Marburg and in 10 other thermophilic Methanobacterium strains whose 16S rRNA sequences have been determined (Nailing et aI., 1993).
Morphology
rods, 0.5 x 5.0 to 20 f-tm 65°C
rods, 0.5 x 10 f-tm 65°C
Carbon and energy source
a
1,650 kb pME2200, 6.2 kb
Data from Stettler and Leisinger (1992).
U
U AU AA, A ACUGGGGAAAACCUGG CACCC UU I • I I I I I I I • • I I I I I I AGGGCCCCAAUAGGGUC GUGGG C AG - C G G-C \ G-C 200 U-A U-A 5' 3'
M. thermoautotrophicum Marburg
Chromosome size a Endogenous plasmid
U
A-U G-C U-A G-C G-C A GA
M. thermoautotrophicum ZH3
Sensitivity to pseudomurein endopeptidase Sensitivity to phage 1jJM1
U
C-G GC-G G·U C-G G-C
Characteristics
Optimum growth temperature
U
+ 1,623 kb pME2001, 4.4 kb
Table 2. Comparison of M. thermoautotrophicum ZH3 with M. thermoautotrophicum Marburg
A Plasmid in M. thermoautotrophicum ZH3
fragment hybridized to the probe. Cloning (plasmid pME2106, Table 1) and sequencing of this fragment provided the entire 165 rRNA sequence of M. thermoautotrophicum ZH3. It differed in a single base at position 196 (numbering according to 165 rRNA sequence of M. thermoautotrophicum Marburg [0stergaard et al., 1987]) from the sequence of the Marburg strain. As shown in Fig. 1, strain ZH3 165 rRNA carries a one-base (C) insertion in variable region I, one of the two highly variable regions of 165 rRNA from thermophilic Methanobacterium strains. This insertion increases the stability of the proposed secondary structure by generating an additional base pair in one of the loops of variable region I (0stergaard et al., 1987; Nolling et al., 1993). The very high sequence identity indicates that strains ZH3 and Marburg should be assigned to the same species.
M. thermoautotrophicum ZH3 harbors pME2200, a plasmid related to plasmid pME2001 Upon agarose gel electrophoresis of undigested genomic DNA of M. thermoautotrophicum ZH3 a distinct fraction was observed whose mobility corresponded to a linear DNA fragment of 6 kb. The same DNA was obtained in the ccc-fraction (covalently closed circular DNA) by equilibrium centrifugation of total DNA in CsCI in the presence of ethidium bromide. Restriction analysis of this DNA led to a circular map of a 6.2 kb plasmid which we designated pME2200. Plasmid pME2200 was not cut by the restriction endonucleases Alul, Dral, HindIII, Mlul, Pvul, Sad and SalI, and it contained single cleavage sites for Clal and XhoI. A large part of the restriction map of pME2200 was similar with respect to number and position of restriction
487
sites to the restriction map of plasmid pME2001 of M. thermoautotrophicum Marburg (compare Fig. 3). To examine the relation between the two plasmids further, plasmids pME2001 and pME2200 were labeled and used as probes to hybridize restriction fragments of pME2200 separated by agarose gel electrophoresis. As shown in Fig. 2, most restriction fragments of pME2200 yielded signals of identical intensity when probed with pME2001 or pME2200. However, some restriction fragments of pME2200 did not hybridize to pME2001. This suggested that the two plasmids are very similar, if not identical, but that pME2200 carries additional sequences that are not present in pME2001. Hybridization of the labeled plasmid pME2200 DNA to total DNA isolated from M. thermoautotrophicum ZH3 (Fig. 2) and M. thermoautotrophicum Marburg (data not shown) digested with Alul yielded no signals, besides those expected from the intact plasmids. Partial nucleotide sequence of plasmid pME2200
A 3305 bp fragment of plasmid pME2200, extending from coordinates 2.9 to 6.2 of the restriction map (Fig. 3), covering the region not present in pME2001, was sequenced. Comparison of this sequence with the published sequence of pME2001 (Bokranz et al., 1990) revealed that the pME2200-specific sequences were arranged in three distinct segments. The three DNA fragments are designated IF1, IF2, and IF3, and their positions on the restriction map of pME2200 as well as the locations of their insertion into pME2001 are shown in Fig. 3. A schematic comparison of the sequenced segment of pME2200 with the corresponding region of pME2001 is shown in Fig. 4. The nucleotide sequence shown is num-
A
B
C
1 2 3 4
123 4
3 4
(kb) 6.0 -
4.1 -
2.0 -
1.0 0.5 -
Fig. 2. Agarose gel electrophoresis of total DNA of strain ZH3 and plasmid pME2200 (A), and auto radiographs of corresponding Southern hybridizations using radiocatively labeled plasmid pME2200 DNA (B), and plasmid pME2001 DNA (C) as probes. Lane 1, Alul digested total DNA; lane 2, pME2200 undigested; lane 3, pME2200 BamHIIClal digested; lane 4, pME2200 SmaIlClal digested. Arrows indicate the positions of the pME2200 fragments which did not hybridize to labeled pME2001. Sizes of linear fragments are in kilobases.
488
R. Stettler, P. Pfister, and T. Leisinger
bered in accordance with the sequence of pME2001 and starts at position 2879. Moving in this scheme from left to right, there is a 747-bp strech which exhibits sequence identity in the two plasmids. Identity ends at position 3626 of the maps, where a CT dinucleotide of pME2001 is exchanged for a GG dinucleotide in pME2200. This leads to an additional SmaI recognition site in the latter element. Between this SmaI site and IF1, pME2200 carries a 142 bp region with slightly different nucleotide sequence to the corresponding region in pME2001. The next element on the pME2200 map, IF1, extends over 1058 bp and is the largest insert exclusively present in pME2200. It is followed by a 232 bp region with extended homology with pME2001. As shown in Fig.4B, the complete sequence of this region can be found in plasmid pME2001 but is arranged there in a different order. The following element, IF2, comprises 183 bp. It is succeeded by a 379 bp region with an interplasmid similarity of 99%. IF3, the third pME2200-specific segment, extends over 364 bp. It is flanked by 25 bp direct repeats (DR 7 in Fig.4A). Only one copy of the DR7 sequence is present at the corresponding site of pME2001. Finally, at the end of the sequenced segment of pME2200 is a 200 bp region with identical nucleotide sequence to plasmid pME2001. The sequenced segment of plasmid pME2200 was screened in all six reading frames for regions potentially encoding proteins. Besides the open reading frames which are also present on pME2001 (Fig. 3), there were no convincing open reading frames. Several repetitive sequences
were identified in the sequenced region. Most conspicuous among these direct repeats are three AT-rich 41 bp sequences in pME2200 which also occur two times on pME2001 (Table 3). A seven base-pair sequence (TGTGACA) occurs 12 times in the IF1 region between nucleotide residues 3839 and 4013. However, the functional significance of this repeated sequence remains obscure. Discussion M. thermoautotrophicum ZH3, the methanogen described in the present communication, is phenotypically closely related to M. thermoautotrophicum Marburg, and the 99.9% similarity of their 16S rRNA sequences clearly demonstrates that strains Marburg and ZH3 are members of the same phylogenetic group of thermophilic Methanobacterium strains. It has previously been shown, that the species Methanobacterium thermoautotrophicum is phylogenetic ally heterogeneous (Brandis et aI., 1981; Nailing et aI., 1993; Touzel et aI., 1993) and that the members of this species should be assigned to at least two separate groups. Strain ZH3 thus expands the M. thermoautotrophicum Marburg group which so far consisted of a single representative. At the level of the genome we have observed two major differences between the new isolate and M. thermoautotrophicum Marburg, namely different NotI restriction pat-
HindU HildU
Sma I Kpnl
IF 2 IF 1 pME2200 6,2kb
Stu I
Hind II
4,439 kb Stu I
Et:o RI Hind II
Fig. 3. Physical maps of plasmids pME2200 and pME2001. Open reading frames ORF A through F are indicated by solid arrows and the mapped transcript of pME2001 (Meile et aI., 1988) by a broken arrow. Sequenced regions of pME2200 are marked by solid lines close to the center of the map. Bars on the map of pME2200 show the location of insertion fragments IF1, IF2 and IF3. Their positions on the map of pME2001 are indicated by arrowheads.
A Plasmid in M. thermoautotrophicum ZH3
DF\1
Sma I
I
>-
D~ DR3...
..
OR7~
D~~
~~
489
DR7~
- - -.. . . . _' s..',.....,.----
V I
~---'-V'-471 Jl _'Ll
/
---
pME2200
A pME2001
DR~~
a
~
A
I
B
A
I
B
DRS
OR~D~
OR6~
~
I0
C
~I
F •
0
~I
G
C
0
~I
G
l~
pME2200
B b
~
F
B
pME2001
Fig.4. Comparison of plasmid pME2200 harboured by strain ZH3 with pME2001 harboured by strain Marburg. (A) Schematic arrangement of the sequenced region of pME2200 (nucleotide positions 2879 to 6183, numbered according to the sequence of pME2001 [Bokranz et aI., 1990]) compared to the corresponding region of pME2001 (Bokranz et aI., 1990). The three pME2200specific segments are indicated by hatched boxes. Regions which in the two plasmids are identical or almost identical (99%) are shown by darkly shaded areas whereas regions exhibiting less sequence homology are indicated by a weaker shading. Direct repeats in the sequence of pME2200 are indicated by arrows. (B) Comparison of the genetic organization of the region located between IF1 and IF2 with the corresponding region of pME2001. Segments of identical nucleotide sequences are indicated by the letters A through G. Further explanations are given in the text.
Table 3. Repeated AT-rich sequences in pME2200 and pME2001
Position a
Sequence b
pME2200 3580 4778 4873
AAAAAAAtctAAaAAAAt ct AAAAAAAt caAAAt AAAAa IT AAAAAAAcccAAaAAAAcccAAAAAAAcct AAAgAAAAa IT AAAAAAAtacAAgAAAAtgcAAAAAAAtgcAAAtAAAAtIT
pME2001
a
b
3580
AAAAAAAtctAAaAAAAtctAAAAAAAtcaAAAtAAAAaIT
3734
AAAAAAAt acAAgAAAAtgcAAAAAAAzgcAAAtAAAAt IT
The nucleotide position indicated relates to the first nucleotide of the sequence. Numbering in accordance with the sequence of pME2001 (Bokranz et aI., 1990). Capital letters indicate consensus nucleotides.
490
R. Stettler, P. Pfister, and T. Leisinger
terns of their chromosomes (Stettler and Leisinger, 1992) the chromosome of strain Marburg. It therefore seems and the occurrence of different variants of the same cryptic likely that pME2200 carries DNA originating from a common ancestor of pME2200 and pME2001 or acquired multi copy plasmid. Plasmid pME2200 harbored by M. thermoautotrophi- from a host other than strains ZH3 and Marburg. The close relationship of two plasmids implies a similar cum ZH3 was compared by restriction mapping and hybridization to the previously characterized plasmid replication mechanism for both replicons. Since IF1 pME2001 of M. thermoautotrophicum Marburg (Bok- through IF3 are not present on pME2001, it is expected ranz et al., 1990; Meile et al., 1983). Extended homology that functions necessary for plasmid replication and was observed between these two plasm ids. It became evi- maintenance are not located in these regions. IF1 through dent that the two elements are identical with the exception IF3 thus appear to be dispensable for replication, and in of a region with interplasmid similarity which is inter- the course of constructing potential shuttle vectors for M. rupted by three segments (IF1, IF2, and IF3) not present in thermoautotrophicum Marburg, are attractive targets for pME2001. Evidence for a common backbone in the two the insertion of selective markers and bacterial replicons. plasmids is based on 1200 bp of sequenced DNA, which Previous efforts towards this goal have suffered from the we show to be identical in both elements and by the strict lack of information on those regions of pME2001 that are conservation of restriction sites in the remaining part of dispensable for replication (Meile and Reeve, 1985). the common backbone (Fig. 3). Most of the differences pME2200 with its singular Clal site in IF1 (Fig. 3) may between the two plasmids are in the IF1 flanking regions, help to overcome these shortcomings. where a substantial sequence variability was found (Fig.4). Acknowledgement. We are greatly indebted to L. Meile for The comparison between plasm ids pME2001 and stimulating discussions and to R. Hermann for electron microspME2200 raises a number of questions. The finding of an copy. This work was supported by grant 31-25177.88 from the identical backbone structure is intriguing. It indicates sub- Swiss National Foundation for Scientific Research. stantial selective pressure for conservation of the 3.86 kb backbone segment. However, in the absence of informaReferences tion on the function of the putative ORFs encoded by the backbone, the nature of this pressure can not be identified. Brandis, A., Thauer, R. K., Stetter, K. 0.: Relatedness of strain Homologous plasmids have also been detected in the boH and Marburg of Methanobacterium thermoautotroMethanobacterium thermoformicicum strains THF and Zphicum. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. 245. While these strains are 99.9% homologous with reAbt. 1 Orig. Reihe C 2,311-317 (1981) spect to their 16S rRNA sequences, the sequence blocks Bokranz, M., Klein, A., Meile, L.: Complete nucleotide sequence common to the two related plasmids, each carried by one of plasmid pME2001 of Methanobacterium thermoautotrophicum Marburg. Nucleic Acids Res. 18, 363 (1990) of the organisms, exhibited an overall similarity of 91 % Feinberg, A. P., Vogelstein, B.: A technique for radio labeling (Nolling et al., 1992). DNA restriction endonuclease fragments to high specific activIt is open to speculation whether pME2001 is an anteceity. Anal. Biochem. 132, 6-13 (1983) dent of pME2200 or vice versa, or whether these plasmids Fuchs, G., Stupperich, E., Thauer, R. K.: Acetate assimilation are co ancestral. We favor the latter possibility since we and the synthesis of alanine, aspartate and glutamate in have not been able to find a model which could explain the Methanobacterium thermoautotrophicum. Arch. Microbiol. origin of one plasmid from the other by deletion/insertion 117,61-66 (1978) events only. Hanahan, D.: Studies on transformation of Escherichia coli with An argument for the acquisition of pME2001 and plasmids. J. Mol. BioI. 166,557-563 (1983) pME2200 by their respective hosts from an unknown Kiener, A., Leisinger, T.: Oxygen sensitivity of methanogenic bacteria. System. Appl. Microbiol. 4, 305-312 (1983) donor strain is also provided by the absence of Alul (recognition sequence AGCT) restriction sites in both ele- . Kiener, A., Konig, H., Winter, J., Leisinger, T.: Purification and use of Methanobacterium wolfei pseudomurein endopeptidase ments. Assuming a mol% G+C of about 50% (pME2001: for lysis of Methanobacterium thermoautotrophicum. J. Bac45.5%), one would expect to encounter the Alul site four teriol. 169, 1010-1016 (1987) times per kilobase of DNA (Sam brook et al., 1989 ). In Kotelnikova, S. V., Obraztsova, A. Y., Blotevoge/, K.-H., Popov, contrasts to plasmids pME2001 and pME2200, this seems I. N.: Taxonomic analysis of thermophilic strains of the genus to be the case for the chromosomes of both M. thermoMethanobacterium: Reclassification of Methanobacterium autotrophicum ZH3 and M. thermoautotrophicum Marthermoalcaliphilum as a synonym of Methanobacterium thermoautotrophicum. Int. J. Syst. Bacteriol. 43, 591-596 (1993) burg, which were extensively digested with Alul into predominantly small-sized fragments, as shown for strain Leisinger, T., Meile L.: Plasmids, phages and gene transfer in methanogenic bacteria, pp. 1-12. In : Genetics and molecular ZH3 in Fig. 3A (lane 1). This observation suggests that the biology of anaerobic bacteria (M. Sebald, ed.). Springer Verplasmids or their common ancestor originally resided in a lag, New York 1993 host with an Alul restriction/modification system from Meile, L., Kiener, A., Leisinger, T.: A plasmid in the archaebacwhich they were transferred into strains ZH3 and Marterium Methanobacterium thermoautotrophicum. Mol. Gen. burg. Genet. 191,480-484 (1983) Hybridization experiments indicated that the pME Meile, L., Reeve, J. N.: Potential shuttle vectors based on the 2200-specific elements share no detectable homology methanogen plasmid pME2001. Bio Technology 3, 69-72 (1985) neither with the chromosome of the host strain nor with
A Plasmid in M. thermoautotrophicum ZH3
Meile, L., Madon, j., Leisinger, T.: Identification of a transcript and ist promoter region on the archaebacterial plasmid pME2001. J. Bacteriol. 170,478-481 (1988 ) Meile, L., Jenal, U., Studer, D., Jordan, M., Leisinger, T. : Characterization of 1jJM1, a virulent phage of Methanobacterium thermoautotrophicum Marburg. Arch. Microbiol. 152, 105-110 (1989) Mercenier, A., Chassy, M.: Strategies for the development of bacterial transformation systems. Biochimie 70, 503-517 (1988) Nailing, j., de Vos, W. M.: Characterization of the archaeal, plasmid-encoded restriction-modification system MthTI from Methanohacterium thermoformicicum THF: homology to the bacterial NgoPII system from Neisseria gonorrhoeae. J. Bacteriol. 174, 5719-5726 (1992) NOlling, j., Hahn, D., Ludwig, W., de Vos, W. M.: Phylogenetic analysis of thermophilic Methanobacterium sp.: evidence for a formate-utilizing ancestor. System. Appl. Microbial. 16, 208-215 (1993) 0stergaard, L., Larsen, N., Leffers, H., Kjems, j., Garrett, R.: A ribosomal RNA operon and its flanking region from the archaebacterium Methanohacterium thermoautotrophicum, Marburg strain: transcription signals, RNA structure and evolutionary implications. System. Appl. Microbial. 9, 199-209 (1987) Palmer, j. R., Reeve, j. N.: Methanogen genes and the molecular biology of methane biosynthesis, pp. 13-35 . In: Genetics and molecular biology of anaerobic bacteria (M. Sebald, ed. ). Springer Verlag, New York 1993
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Sambrook, j., Fritsch, E. F., Maniatis, T.: Molecular cloning: a laboratory manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press 1989 Sanger, F., Nicklen, S., Coulson, A. R.: DNA sequencing with chain-terminating inhibitors. Proe. Nat!. Acad. Sci. USA 84, 5463-5467 (1977) Schanheit, P., Moll, j., Thauer, R. K.: Growth parameters (K" Ilmax, Ys) of Methanohacterium thermoautotrophicum. Arch. Microbiol. 127, 59-65 (1980) Southern, E. M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioI. 98, 503-517 (1975) Stettler, R., Leisinger, T.: Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome. J. Bacteriol. 174, 7227-7234 (1992) Thauer, R. K., Schwarer, B., Zirngibl, c.: Enzymes involved in methanogenesis from CO 2 , pp. 151-162. In: Microbial growth on C 1 compounds U. C. Murrell and D. P. Kelly, eds.). Intercept Ltd., Andover 1993 Touzel, J. P., Conway de Macario, E., NOlling, j., de Vos, W. M ., Zhilina, T., Lysenko, A. M.: DNA relatedness among some thermophilic members of the genus Methanohacterium': emendation of the species Methanohacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum. Int. J. Syst. Bacteriol. 42, 408-411 (1992) Yanisch-Perron, c., Vieira, j., Messing, j.: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp 18 and pUC19 vectors. Gene 33, 103-119 (1985)
Professor Dr. Thomas Leisinger, Mikrobiologisches Institut, ETH-Zentrum, CH-8092 Zurich, Switzerland. Phone: +41-1-632-3324 Fax: +41-1-632-1148 Elecronic Mail address : [email protected].
Characterization of a Plasmid Carried by Methanobacterium thermoautotrophicum ZH3, a Methanogen Closely Related to Methanobacterium thermoautotrophicum Marburg ROLF STETTLER, PETER PFISTER, and THOMAS LEISINGER" Mikrobiologisches Institut, Eidgenossische Technische Hochschule, ETH-Zentrum, CH-8092 Zi.irich, Switzerland Received September 12, 1994
Summary Strain ZH3, a hydrogenotrophic, thermophilic methanogenic archaeon was isolated from a sewage sludge digester. Based on its phenotypic properties and its 16S rRNA sequence (99.9% similarity), strain ZH3 is closely related to Methanobacterium thermoautotrophicum Marburg. The newly isolated organism harbored pME2200, a cryptic 6.2 kb plasmid, which is related to, but not identical with the previously characterized cryptic 4.4 kb plasmid pME2001 of strain Marburg. Plasmid pME2200 was characterized by restriction mapping and by Southern blot and DNA sequence analysis and compared to plasmid pME2001. The two elements are composed of a presumably identical, 3.8 kb backbone structure. In addition to the common backbone, both plasmids carry a region of high interplasmid homology, which in pME2200 was interspersed with three segments not present in pME2001. These interspersed segments did not encode potential open reading frames. One of them contained a singular restriction site which may be of use for the construction of potential cloning vectors that are undamaged in essential plasmid functions.
Key words: Archaea - Methanogen - Thermophile - Plasmid - 16S rRNA
Introduction
Thermophilic representatives of the genus Methanobacterium are characterized by an optimum growth temperature between 55°C and 65 °C and by their ability to use either H 2 /C0 2 only or H 2 /C0 2 and formate as carbon and energy source. Several strains belonging to this group have been isolated and characterized, but the taxonomic and phylogenetic relationships among these organisms are not completely resolved. Recently, efforts have been made to redefine the phylogeny and taxonomy of some thermophilic strains of the genus Methanobacterium. Nailing et al. (1993) have used comparative analysis of 16S rRNA sequences to study the relationships between members of the two species Methanobacterium thermoautotrophicum and Methanobacterium thermoformicicum. They proposed that the members of these two species should be separated into at least three groups. A first group comprises M. thermoformicicum strains Z-245, FTF, THF, CSM3, FF1, FF3, and M. thermoautotrophicum i1H. A " Corresponding author
second group includes the M. thermoformicicum strains CB12, SF-4 and HN4, and the third group is represented by M. thermoautotrophicum Marburg only. The classification of these strains in three different groups is supported by the results of antigenic fingerprinting and DNADNA hybridization analysis (Touzel et al., 1992). Within a different set of thirteen thermophilic Methanobacterium strains the taxonomic relationships were recently studied by DNA-DNA hybridization, whole-cell protein analysis and antigenic fingerprinting (Kotelnikova et al., 1993). The authors reached the conclusion that these strains fall into three clusters within the genus Methanobacterium. M. thermoautotrophicum Marburg is the best studied thermophilic Methanobacterium strain with respect to biochemistry of methanogenesis (Thauer et al., 1993), gene organization (Palmer and Reeve, 1993), and genome structure. A combined physical and genetic map of its circular 1,623 kb chromosome has been prepared (Stettler and Leisinger, 1992). The organism harbors the cryptic multicopy plasmid pME2001, and it is infected by the
A Plasmid in M. thermoautotrophicum ZH3
virulent, generally transducing phage '\j!M1 (Leisinger and Meile, 1993). While these features would appear to make this organism attractive for genetic studies, progress in establishing a gene transfer system has been hampered by the apparent inefficiency of transformation using plasmid DNA, phage DNA or chromosomal DNA (Settler, R., unpublished). These limitations, and the fact that large differences with respect to transformability are known to exist between strains of the same species (Mercenier and Chassy, 1988), have promped us to isolate from nature Methanobacterium strains closely related to M. thermoautotrophicum Marburg for testing them as recipients in a transformation system. In the present communication we describe one of these isolates, M. thermoautotrophicum ZH3, and compare it with respect to phenotypic properties, 16S rRNA sequence and genome organization to the Marburg strain.
Materials and Methods Bacterial strains, plasmids and phages. The bacterial strains, plasmids and the phage used in this study are listed in Table 1. M. thermoautotrophicum Marburg was cultivated at 65 °C according to Schonheit et al. (1980). The media were reduced with 0.5 g of Na2S· 9H 20 and 0.5 g of L-cysteine-HCI per liter. E. coli strains were grown at 37"C in LB (Sambrook et aI., 1989). Ampicillin was used at a final concentration of 150 l1g1ml. Isolation and culture conditions of M. thermoautotrophicum ZH3. Samples were collected from an anaerobic sewage sludge digester (municipal sewage treatment plant in Opfikon, Switzerland) in a serum bottle which had been flushed previously with a N 2/C0 2 gas mixture; the serum bottle was filled to the top with sample. 0.1 ml of sewage sludge were used to inoculate a flask that contained a H 2 /C0 2 (80: 20) gas phase and 10 ml of the minimal medium described by Schonheit et al. (1980). After 7 days of incubation at 60 °C the medium was turbid and methane was detected in the gas phase. Cells were then transferred into fresh minimal medium and incubated at 60 °C for 24h . Diluted
485
culture samples were plated (plates contained minimal medium supplemented with 1.0 mM titanium [III] citrate as reducing agent) by the soft agar-overlay method described previously (Kiener and Leisinger, 1983 ), followed by incubation at 60 °C for 8 days in anaerobic jars containing the H 2 /C0 2 gas mixture at 2-2.5 bar. Single colonies were picked and transferred into liquid minimal medium. After a second purification step on agar plates, cultures were grown from single colonies. Microscopic analysis of the enrichment cultures revealed fluorescent rods of uniform shape. One of the isolates, M. thermoautotrophicum ZH3, was used for further study. M. thermoautotrophicum ZH3 was routinely cultivated in minimal medium (Schonheit et aI., 1980). To test formate as a substrate, minimal medium was supplemented with 40 mM, 50 mM, 75 mM, and 100 mM sodium formate. Propagation of archaeophage 'lJM1 was performed as described previously (Meile et aI., 1989). DNA isolation and manipulation. M. thermoautotrophicum ZH3 and M. thermoautotrophicum Marburg plasmid DNA was isolated from lysates obtained by treatment of cell suspensions with partially purified pseudomurein endopeptidase from Methanobacterium wolfei (Kiener et aI., 1989) and purified by CsCI-ethidium bromide density gradient centrifugation. Total DNA of strains ZH3 and Marburg was extracted by the same procedure. Plasmid isolation from E. coli strains, digestions of DNA, and cloning were performed by standard procedures (Sambrook et aI., 1989). All enzymes used in the manipulation of DNA were used according to the manufacturer's specifications. Southern transfers (Southern, 1975 ) of DNA from agarose gel to nylon membranes (Hybond-N, Amersham International) were performed according to the membrane manufacturer's protocol. DNA for Southern hybridization was labeled with [a- 32 P]dATP using the oligo-labelling technique of Feinberg and Vogelstein (1983 ). DNA sequencing and analysis. Subclones in phages M13mp18 and M13mp19 or in the pBluescript vector were sequenced by the dideoxy chain termination method of Sanger et al. (1977), using [a- 32P]dATP. Sequencing reactions with single-stranded DNA and double-stranded DNA were performed with Sequenase version 2.0 (United States Biochemicals, Cleveland, Ohio). Regions, in which no usable restriction sites were found for subcloning,
Table 1. Microbial strains and extrachromosomal elements used in this study Strain or element
Relevant characteristics
Reference or source
Methanobacterium thermoautotropicum Marburg (DSM 2133 )
wild type
Fuchs et al. (1978 )
Methanobacterium thermoautotropicum ZH3 (DSM 9446 )
wild type
this work
E. coli DH5a
F-
Hanahan (1983 )
E. coli JM109
recAl endAl gyrA96 "A.- hsdR17 supE44 relAl thi L'1(IacproAB ) F' [traD36 proAB + lacQZL'1M15]
Yanisch-Perron et al. (1985)
pME 2001
cryptic multicopy plasmid of M. thermoautotrophicum Marburg
Meile et al. (1983 )
pME2200
cryptic multicopy plasmid of M. thermoautotrophicum ZH3
this work
'lJ M1 pBluescript II SK+
virulent phage of M. thermoautotrophicum Marburg
Meile et al. (1989)
Cloning vector, Apr
Stratagene, La Jolla, CA
pME2106
1.92 kb BamHIIECORI fragment covering the 16S rRNA
this work
M13mp18/19
gene from M. thermoautotrophicum ZH3 in pBluescript
Yanisch-Perron et al. (1985 )
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R. Stettler, P. Pfister, and T. Leisinger
were spanned by sequencing with synthetic oligonucleotide primers. Computer analysis was carried out with the University of Wisconsin Genetics Computer Group software packages, version 7, on the VAX. Nucleotide sequence accession numbers. The nucleotide sequences of the 16S rRNA gene of M. thermoautotrophicum ZH3 and of the 3.3 kb fragment of plasmid pME2200 have been deposited in the GenBank data base under accession numbers Z37156 and Z37155, respectively.
Results
Properties of M. thermoautotrophicum ZH3
The phenotypic properties of M. thermoautotrophicum ZH3 are listed in Table 2. On the basis of its ability to utilize C0 2 /H 2 (but not formate) and to produce methane gas, its optimum growth temperature of 65°C, its cell morphology, and its sensitivity to partially purified pseudo murein endopeptidase from Methanobacterium wolfei (Kiener et aI., 1987), the organism was identified as a member of the species Methanobacterium thermoautotrophicum. It differed from strain Marburg only by its insensitivity to archaeophage 'ljJM1 (Meile et aI., 1989) and by the formation of comparatively short rods « 5 [lm) during growth under suboptimal conditions such as limitation by hydrogen. At the level of the genome, M. thermoautotrophicum ZH3 and M. thermoautotrophicum Marburg were similar with respect to the sizes of their chromosomes (Table 2). However, digestion of the M. thermoautotrophicum ZH3 chromosome with the restriction enzyme Notl yielded five fragments, and none of these were similar in size to one of the six fragments obtained upon NotI digestion of the M. thermoautotrophicum Marburg chromosome (Settler and Leisinger, 1992). This difference in the macrorestriction patterns of the two strains indicates divergence in chromosomal DNA sequences. Another difference at the genome level between the two strains concerned the size of the plasmid carried by each organism. The cryptic plasmid pME2001 harbored by M. thermoautotrophicum Marburg has a size of 4.439 kb (Bokranz et aI., 1990) while in M. thermoautotrophicum ZH3 we have detected a 6.2 kb plasmid which was named pME2200.
16S rRNA sequence of M. thermoautotrophicum ZH3
To explore the phylogenetic relationship between M. thermoautotrophicum ZH3 and M. thermoautotrophicum Marburg, we have determined the 16S rRNA sequence of the former organism and compared it to the 16S rRNA sequence of the Marburg strain (0stergaard et aI., 1987). An oligonucleotide probe (5'-GAA ACT GGG GAT AAA CC-3') complementary to nucleotide positions 137 through 153 of the 16S rRNA gene of M. thermoautotrophicum Marburg was designed and used to analyse Southern blots of digested chromosomal DNA of M. thermoautotrophicum ZH3. A single 1.95 kb BamHI/EcoRI
GGA A
Fig. 1. Secondary structure of variable region I of 16S rRNA from M. thermoautotrophicum ZH3 according to the model of 0stergaard et al. (1987). The cytidine-phosphate residue at position 196 (arrow) is deleted in M. thermoautotrophicum Marburg and in 10 other thermophilic Methanobacterium strains whose 16S rRNA sequences have been determined (Nailing et aI., 1993).
Morphology
rods, 0.5 x 5.0 to 20 f-tm 65°C
rods, 0.5 x 10 f-tm 65°C
Carbon and energy source
a
1,650 kb pME2200, 6.2 kb
Data from Stettler and Leisinger (1992).
U
U AU AA, A ACUGGGGAAAACCUGG CACCC UU I • I I I I I I I • • I I I I I I AGGGCCCCAAUAGGGUC GUGGG C AG - C G G-C \ G-C 200 U-A U-A 5' 3'
M. thermoautotrophicum Marburg
Chromosome size a Endogenous plasmid
U
A-U G-C U-A G-C G-C A GA
M. thermoautotrophicum ZH3
Sensitivity to pseudomurein endopeptidase Sensitivity to phage 1jJM1
U
C-G GC-G G·U C-G G-C
Characteristics
Optimum growth temperature
U
+ 1,623 kb pME2001, 4.4 kb
Table 2. Comparison of M. thermoautotrophicum ZH3 with M. thermoautotrophicum Marburg
A Plasmid in M. thermoautotrophicum ZH3
fragment hybridized to the probe. Cloning (plasmid pME2106, Table 1) and sequencing of this fragment provided the entire 165 rRNA sequence of M. thermoautotrophicum ZH3. It differed in a single base at position 196 (numbering according to 165 rRNA sequence of M. thermoautotrophicum Marburg [0stergaard et al., 1987]) from the sequence of the Marburg strain. As shown in Fig. 1, strain ZH3 165 rRNA carries a one-base (C) insertion in variable region I, one of the two highly variable regions of 165 rRNA from thermophilic Methanobacterium strains. This insertion increases the stability of the proposed secondary structure by generating an additional base pair in one of the loops of variable region I (0stergaard et al., 1987; Nolling et al., 1993). The very high sequence identity indicates that strains ZH3 and Marburg should be assigned to the same species.
M. thermoautotrophicum ZH3 harbors pME2200, a plasmid related to plasmid pME2001 Upon agarose gel electrophoresis of undigested genomic DNA of M. thermoautotrophicum ZH3 a distinct fraction was observed whose mobility corresponded to a linear DNA fragment of 6 kb. The same DNA was obtained in the ccc-fraction (covalently closed circular DNA) by equilibrium centrifugation of total DNA in CsCI in the presence of ethidium bromide. Restriction analysis of this DNA led to a circular map of a 6.2 kb plasmid which we designated pME2200. Plasmid pME2200 was not cut by the restriction endonucleases Alul, Dral, HindIII, Mlul, Pvul, Sad and SalI, and it contained single cleavage sites for Clal and XhoI. A large part of the restriction map of pME2200 was similar with respect to number and position of restriction
487
sites to the restriction map of plasmid pME2001 of M. thermoautotrophicum Marburg (compare Fig. 3). To examine the relation between the two plasmids further, plasmids pME2001 and pME2200 were labeled and used as probes to hybridize restriction fragments of pME2200 separated by agarose gel electrophoresis. As shown in Fig. 2, most restriction fragments of pME2200 yielded signals of identical intensity when probed with pME2001 or pME2200. However, some restriction fragments of pME2200 did not hybridize to pME2001. This suggested that the two plasmids are very similar, if not identical, but that pME2200 carries additional sequences that are not present in pME2001. Hybridization of the labeled plasmid pME2200 DNA to total DNA isolated from M. thermoautotrophicum ZH3 (Fig. 2) and M. thermoautotrophicum Marburg (data not shown) digested with Alul yielded no signals, besides those expected from the intact plasmids. Partial nucleotide sequence of plasmid pME2200
A 3305 bp fragment of plasmid pME2200, extending from coordinates 2.9 to 6.2 of the restriction map (Fig. 3), covering the region not present in pME2001, was sequenced. Comparison of this sequence with the published sequence of pME2001 (Bokranz et al., 1990) revealed that the pME2200-specific sequences were arranged in three distinct segments. The three DNA fragments are designated IF1, IF2, and IF3, and their positions on the restriction map of pME2200 as well as the locations of their insertion into pME2001 are shown in Fig. 3. A schematic comparison of the sequenced segment of pME2200 with the corresponding region of pME2001 is shown in Fig. 4. The nucleotide sequence shown is num-
A
B
C
1 2 3 4
123 4
3 4
(kb) 6.0 -
4.1 -
2.0 -
1.0 0.5 -
Fig. 2. Agarose gel electrophoresis of total DNA of strain ZH3 and plasmid pME2200 (A), and auto radiographs of corresponding Southern hybridizations using radiocatively labeled plasmid pME2200 DNA (B), and plasmid pME2001 DNA (C) as probes. Lane 1, Alul digested total DNA; lane 2, pME2200 undigested; lane 3, pME2200 BamHIIClal digested; lane 4, pME2200 SmaIlClal digested. Arrows indicate the positions of the pME2200 fragments which did not hybridize to labeled pME2001. Sizes of linear fragments are in kilobases.
488
R. Stettler, P. Pfister, and T. Leisinger
bered in accordance with the sequence of pME2001 and starts at position 2879. Moving in this scheme from left to right, there is a 747-bp strech which exhibits sequence identity in the two plasmids. Identity ends at position 3626 of the maps, where a CT dinucleotide of pME2001 is exchanged for a GG dinucleotide in pME2200. This leads to an additional SmaI recognition site in the latter element. Between this SmaI site and IF1, pME2200 carries a 142 bp region with slightly different nucleotide sequence to the corresponding region in pME2001. The next element on the pME2200 map, IF1, extends over 1058 bp and is the largest insert exclusively present in pME2200. It is followed by a 232 bp region with extended homology with pME2001. As shown in Fig.4B, the complete sequence of this region can be found in plasmid pME2001 but is arranged there in a different order. The following element, IF2, comprises 183 bp. It is succeeded by a 379 bp region with an interplasmid similarity of 99%. IF3, the third pME2200-specific segment, extends over 364 bp. It is flanked by 25 bp direct repeats (DR 7 in Fig.4A). Only one copy of the DR7 sequence is present at the corresponding site of pME2001. Finally, at the end of the sequenced segment of pME2200 is a 200 bp region with identical nucleotide sequence to plasmid pME2001. The sequenced segment of plasmid pME2200 was screened in all six reading frames for regions potentially encoding proteins. Besides the open reading frames which are also present on pME2001 (Fig. 3), there were no convincing open reading frames. Several repetitive sequences
were identified in the sequenced region. Most conspicuous among these direct repeats are three AT-rich 41 bp sequences in pME2200 which also occur two times on pME2001 (Table 3). A seven base-pair sequence (TGTGACA) occurs 12 times in the IF1 region between nucleotide residues 3839 and 4013. However, the functional significance of this repeated sequence remains obscure. Discussion M. thermoautotrophicum ZH3, the methanogen described in the present communication, is phenotypically closely related to M. thermoautotrophicum Marburg, and the 99.9% similarity of their 16S rRNA sequences clearly demonstrates that strains Marburg and ZH3 are members of the same phylogenetic group of thermophilic Methanobacterium strains. It has previously been shown, that the species Methanobacterium thermoautotrophicum is phylogenetic ally heterogeneous (Brandis et aI., 1981; Nailing et aI., 1993; Touzel et aI., 1993) and that the members of this species should be assigned to at least two separate groups. Strain ZH3 thus expands the M. thermoautotrophicum Marburg group which so far consisted of a single representative. At the level of the genome we have observed two major differences between the new isolate and M. thermoautotrophicum Marburg, namely different NotI restriction pat-
HindU HildU
Sma I Kpnl
IF 2 IF 1 pME2200 6,2kb
Stu I
Hind II
4,439 kb Stu I
Et:o RI Hind II
Fig. 3. Physical maps of plasmids pME2200 and pME2001. Open reading frames ORF A through F are indicated by solid arrows and the mapped transcript of pME2001 (Meile et aI., 1988) by a broken arrow. Sequenced regions of pME2200 are marked by solid lines close to the center of the map. Bars on the map of pME2200 show the location of insertion fragments IF1, IF2 and IF3. Their positions on the map of pME2001 are indicated by arrowheads.
A Plasmid in M. thermoautotrophicum ZH3
DF\1
Sma I
I
>-
D~ DR3...
..
OR7~
D~~
~~
489
DR7~
- - -.. . . . _' s..',.....,.----
V I
~---'-V'-471 Jl _'Ll
/
---
pME2200
A pME2001
DR~~
a
~
A
I
B
A
I
B
DRS
OR~D~
OR6~
~
I0
C
~I
F •
0
~I
G
C
0
~I
G
l~
pME2200
B b
~
F
B
pME2001
Fig.4. Comparison of plasmid pME2200 harboured by strain ZH3 with pME2001 harboured by strain Marburg. (A) Schematic arrangement of the sequenced region of pME2200 (nucleotide positions 2879 to 6183, numbered according to the sequence of pME2001 [Bokranz et aI., 1990]) compared to the corresponding region of pME2001 (Bokranz et aI., 1990). The three pME2200specific segments are indicated by hatched boxes. Regions which in the two plasmids are identical or almost identical (99%) are shown by darkly shaded areas whereas regions exhibiting less sequence homology are indicated by a weaker shading. Direct repeats in the sequence of pME2200 are indicated by arrows. (B) Comparison of the genetic organization of the region located between IF1 and IF2 with the corresponding region of pME2001. Segments of identical nucleotide sequences are indicated by the letters A through G. Further explanations are given in the text.
Table 3. Repeated AT-rich sequences in pME2200 and pME2001
Position a
Sequence b
pME2200 3580 4778 4873
AAAAAAAtctAAaAAAAt ct AAAAAAAt caAAAt AAAAa IT AAAAAAAcccAAaAAAAcccAAAAAAAcct AAAgAAAAa IT AAAAAAAtacAAgAAAAtgcAAAAAAAtgcAAAtAAAAtIT
pME2001
a
b
3580
AAAAAAAtctAAaAAAAtctAAAAAAAtcaAAAtAAAAaIT
3734
AAAAAAAt acAAgAAAAtgcAAAAAAAzgcAAAtAAAAt IT
The nucleotide position indicated relates to the first nucleotide of the sequence. Numbering in accordance with the sequence of pME2001 (Bokranz et aI., 1990). Capital letters indicate consensus nucleotides.
490
R. Stettler, P. Pfister, and T. Leisinger
terns of their chromosomes (Stettler and Leisinger, 1992) the chromosome of strain Marburg. It therefore seems and the occurrence of different variants of the same cryptic likely that pME2200 carries DNA originating from a common ancestor of pME2200 and pME2001 or acquired multi copy plasmid. Plasmid pME2200 harbored by M. thermoautotrophi- from a host other than strains ZH3 and Marburg. The close relationship of two plasmids implies a similar cum ZH3 was compared by restriction mapping and hybridization to the previously characterized plasmid replication mechanism for both replicons. Since IF1 pME2001 of M. thermoautotrophicum Marburg (Bok- through IF3 are not present on pME2001, it is expected ranz et al., 1990; Meile et al., 1983). Extended homology that functions necessary for plasmid replication and was observed between these two plasm ids. It became evi- maintenance are not located in these regions. IF1 through dent that the two elements are identical with the exception IF3 thus appear to be dispensable for replication, and in of a region with interplasmid similarity which is inter- the course of constructing potential shuttle vectors for M. rupted by three segments (IF1, IF2, and IF3) not present in thermoautotrophicum Marburg, are attractive targets for pME2001. Evidence for a common backbone in the two the insertion of selective markers and bacterial replicons. plasmids is based on 1200 bp of sequenced DNA, which Previous efforts towards this goal have suffered from the we show to be identical in both elements and by the strict lack of information on those regions of pME2001 that are conservation of restriction sites in the remaining part of dispensable for replication (Meile and Reeve, 1985). the common backbone (Fig. 3). Most of the differences pME2200 with its singular Clal site in IF1 (Fig. 3) may between the two plasmids are in the IF1 flanking regions, help to overcome these shortcomings. where a substantial sequence variability was found (Fig.4). Acknowledgement. We are greatly indebted to L. Meile for The comparison between plasm ids pME2001 and stimulating discussions and to R. Hermann for electron microspME2200 raises a number of questions. The finding of an copy. This work was supported by grant 31-25177.88 from the identical backbone structure is intriguing. It indicates sub- Swiss National Foundation for Scientific Research. stantial selective pressure for conservation of the 3.86 kb backbone segment. However, in the absence of informaReferences tion on the function of the putative ORFs encoded by the backbone, the nature of this pressure can not be identified. Brandis, A., Thauer, R. K., Stetter, K. 0.: Relatedness of strain Homologous plasmids have also been detected in the boH and Marburg of Methanobacterium thermoautotroMethanobacterium thermoformicicum strains THF and Zphicum. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. 245. While these strains are 99.9% homologous with reAbt. 1 Orig. Reihe C 2,311-317 (1981) spect to their 16S rRNA sequences, the sequence blocks Bokranz, M., Klein, A., Meile, L.: Complete nucleotide sequence common to the two related plasmids, each carried by one of plasmid pME2001 of Methanobacterium thermoautotrophicum Marburg. Nucleic Acids Res. 18, 363 (1990) of the organisms, exhibited an overall similarity of 91 % Feinberg, A. P., Vogelstein, B.: A technique for radio labeling (Nolling et al., 1992). DNA restriction endonuclease fragments to high specific activIt is open to speculation whether pME2001 is an anteceity. Anal. Biochem. 132, 6-13 (1983) dent of pME2200 or vice versa, or whether these plasmids Fuchs, G., Stupperich, E., Thauer, R. K.: Acetate assimilation are co ancestral. We favor the latter possibility since we and the synthesis of alanine, aspartate and glutamate in have not been able to find a model which could explain the Methanobacterium thermoautotrophicum. Arch. Microbiol. origin of one plasmid from the other by deletion/insertion 117,61-66 (1978) events only. Hanahan, D.: Studies on transformation of Escherichia coli with An argument for the acquisition of pME2001 and plasmids. J. Mol. BioI. 166,557-563 (1983) pME2200 by their respective hosts from an unknown Kiener, A., Leisinger, T.: Oxygen sensitivity of methanogenic bacteria. System. Appl. Microbiol. 4, 305-312 (1983) donor strain is also provided by the absence of Alul (recognition sequence AGCT) restriction sites in both ele- . Kiener, A., Konig, H., Winter, J., Leisinger, T.: Purification and use of Methanobacterium wolfei pseudomurein endopeptidase ments. Assuming a mol% G+C of about 50% (pME2001: for lysis of Methanobacterium thermoautotrophicum. J. Bac45.5%), one would expect to encounter the Alul site four teriol. 169, 1010-1016 (1987) times per kilobase of DNA (Sam brook et al., 1989 ). In Kotelnikova, S. V., Obraztsova, A. Y., Blotevoge/, K.-H., Popov, contrasts to plasmids pME2001 and pME2200, this seems I. N.: Taxonomic analysis of thermophilic strains of the genus to be the case for the chromosomes of both M. thermoMethanobacterium: Reclassification of Methanobacterium autotrophicum ZH3 and M. thermoautotrophicum Marthermoalcaliphilum as a synonym of Methanobacterium thermoautotrophicum. Int. J. Syst. Bacteriol. 43, 591-596 (1993) burg, which were extensively digested with Alul into predominantly small-sized fragments, as shown for strain Leisinger, T., Meile L.: Plasmids, phages and gene transfer in methanogenic bacteria, pp. 1-12. In : Genetics and molecular ZH3 in Fig. 3A (lane 1). This observation suggests that the biology of anaerobic bacteria (M. Sebald, ed.). Springer Verplasmids or their common ancestor originally resided in a lag, New York 1993 host with an Alul restriction/modification system from Meile, L., Kiener, A., Leisinger, T.: A plasmid in the archaebacwhich they were transferred into strains ZH3 and Marterium Methanobacterium thermoautotrophicum. Mol. Gen. burg. Genet. 191,480-484 (1983) Hybridization experiments indicated that the pME Meile, L., Reeve, J. N.: Potential shuttle vectors based on the 2200-specific elements share no detectable homology methanogen plasmid pME2001. Bio Technology 3, 69-72 (1985) neither with the chromosome of the host strain nor with
A Plasmid in M. thermoautotrophicum ZH3
Meile, L., Madon, j., Leisinger, T.: Identification of a transcript and ist promoter region on the archaebacterial plasmid pME2001. J. Bacteriol. 170,478-481 (1988 ) Meile, L., Jenal, U., Studer, D., Jordan, M., Leisinger, T. : Characterization of 1jJM1, a virulent phage of Methanobacterium thermoautotrophicum Marburg. Arch. Microbiol. 152, 105-110 (1989) Mercenier, A., Chassy, M.: Strategies for the development of bacterial transformation systems. Biochimie 70, 503-517 (1988) Nailing, j., de Vos, W. M.: Characterization of the archaeal, plasmid-encoded restriction-modification system MthTI from Methanohacterium thermoformicicum THF: homology to the bacterial NgoPII system from Neisseria gonorrhoeae. J. Bacteriol. 174, 5719-5726 (1992) NOlling, j., Hahn, D., Ludwig, W., de Vos, W. M.: Phylogenetic analysis of thermophilic Methanobacterium sp.: evidence for a formate-utilizing ancestor. System. Appl. Microbial. 16, 208-215 (1993) 0stergaard, L., Larsen, N., Leffers, H., Kjems, j., Garrett, R.: A ribosomal RNA operon and its flanking region from the archaebacterium Methanohacterium thermoautotrophicum, Marburg strain: transcription signals, RNA structure and evolutionary implications. System. Appl. Microbial. 9, 199-209 (1987) Palmer, j. R., Reeve, j. N.: Methanogen genes and the molecular biology of methane biosynthesis, pp. 13-35 . In: Genetics and molecular biology of anaerobic bacteria (M. Sebald, ed. ). Springer Verlag, New York 1993
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Sambrook, j., Fritsch, E. F., Maniatis, T.: Molecular cloning: a laboratory manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press 1989 Sanger, F., Nicklen, S., Coulson, A. R.: DNA sequencing with chain-terminating inhibitors. Proe. Nat!. Acad. Sci. USA 84, 5463-5467 (1977) Schanheit, P., Moll, j., Thauer, R. K.: Growth parameters (K" Ilmax, Ys) of Methanohacterium thermoautotrophicum. Arch. Microbiol. 127, 59-65 (1980) Southern, E. M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioI. 98, 503-517 (1975) Stettler, R., Leisinger, T.: Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome. J. Bacteriol. 174, 7227-7234 (1992) Thauer, R. K., Schwarer, B., Zirngibl, c.: Enzymes involved in methanogenesis from CO 2 , pp. 151-162. In: Microbial growth on C 1 compounds U. C. Murrell and D. P. Kelly, eds.). Intercept Ltd., Andover 1993 Touzel, J. P., Conway de Macario, E., NOlling, j., de Vos, W. M ., Zhilina, T., Lysenko, A. M.: DNA relatedness among some thermophilic members of the genus Methanohacterium': emendation of the species Methanohacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum. Int. J. Syst. Bacteriol. 42, 408-411 (1992) Yanisch-Perron, c., Vieira, j., Messing, j.: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp 18 and pUC19 vectors. Gene 33, 103-119 (1985)
Professor Dr. Thomas Leisinger, Mikrobiologisches Institut, ETH-Zentrum, CH-8092 Zurich, Switzerland. Phone: +41-1-632-3324 Fax: +41-1-632-1148 Elecronic Mail address : [email protected].