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Interspecific, intraspecific and interoperonic variability in the 16S rRNA gene of methanogens revealed by length and single-strand conformation polymorphism analysis

Daniele Daffonchio , Andrea De Biase , Aurora Rizzi , Claudia Sorlini
DOI: http://dx.doi.org/10.1111/j.1574-6968.1998.tb13116.x 403-410 First published online: 1 July 1998

Abstract

Thirty-seven strains of mesophilic and thermophilic methanogenic Archaea, belonging to 30 species, were analyzed by length polymorphism (LP) and single-strand conformation polymorphism (SSCP) of an amplified 300-bp fragment of the 16S rRNA gene (Escherichia coli positions 9–331) including the variable regions V1 and V2. LPs and SSCPs were detected between species and between strains of the same species (Methanobacterium formicicum). LPs were found in Mb. formicicum DSMZ 3637, Mb. ivanovii DSMZ 2611, Mb. wolfei DSMZ 2970, Methanosarcina barkeri DSMZ 800, and Methanosaeta concilii DSMZ 3671, suggesting the presence of polymorphic 16S rRNA genes in the genome. We propose that LP and SSCP analysis of the 16S rRNA gene could be of practical help for strain identification.

Key words
  • Methanogenic Archaea
  • 16S rDNA variable region
  • Interoperonic variability
  • Length variability
  • Polymerase chain reaction-single strand conformation polymorphism
  • Identification

1 Introduction

Methanogenic Archaea represent a unique group among microorganisms since they have the specific characteristic of producing methane from a few substrates. In anaerobic environments, methanogens represent the final link of the food chain and play an important role in the microbial community of many natural and man-made environments [14]. The identification of methanogenic species is currently based on chemotaxonomic and molecular data [5], since the traditional physiological methods are not sufficiently discriminative due to the limited number of substrates used by methanogens. Sequencing of 16S ribosomal RNA for isolated strains [610] is included in the minimal standards for methanogen identification as a recommended method [5]. With direct PCR sequencing and an automated sequencer, many samples can be processed at the same time. DNA sequencing is expensive and not available as routine technology in most laboratories. To overcome this problem, other methods, such as restriction fragment length polymorphism of the amplified 16S rRNA gene, have been proposed as suitable alternative tools for identification of methanogenic species [11].

In addition to interspecies polymorphisms of 16S rRNA genes, length and sequence differences have been observed between strains of the same species, as shown by the Ribosomal Database Project (RDP) [12]. To our knowledge, the molecular diversity among methanogenic strains of the same species has not been surveyed extensively. Nölling et al. [13] found sequence differences in the 16S rDNA gene between strains of Methanobacterium thermoautotrophicum and identified three groups of strains in accord with DNA/DNA hybridization data [14]. The development of rapid methods to detect interspecific and intraspecific polymorphisms could be of practical help in strain identification.

Here we report the use of length polymorphism (LP) and single-strand conformation polymorphism (SSCP) [15] analyses to characterize a 300-bp fragment including the V1 and V2 regions of the 16S rDNA, amplified from 30 species of mesophilic and thermophilic methanogens, to evaluate 16S rDNA polymorphisms and for identification purposes.

2 Materials and methods

2.1 Bacterial strains, culture conditions and DNA extraction

The list of methanogenic strains used in this study is given in Table 1. The media and the culture conditions were those of the DSMZ catalog [16]. All the methanogenic strains were cultivated in 120 ml serum bottles filled with 50 ml of the appropriate medium. Microorganism manipulations were performed in an anaerobic gas chamber (Forma Scientific, Marietta, OH, USA), under 85% N2, 10% H2 and 5% CO2. After manipulation, the gas phases of the cultures were modified according to the indications of the DSMZ catalog [16]. All the strains were incubated at 37°C, except for thermophilic strains, which were incubated at 57°C. The serum bottles with hydrogenotrophic Archaea were pressurized every 2–3 days with 80% H2 and 20% CO2 up to 1.2 bar. After growth (1 week on average), the cells were harvested for DNA extraction.

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Table 1

Methanogen strainsa, environmental sources of isolationb and types of SSCP found

No.SpeciescStrainSourceSSCP type
1Mb. formicicum1535Anaerobic digester1
2Mb. formicicumCLH1Anaerobic digester1
3Mb. formicicum6299Anaerobic digester1
4Mb. formicicum2639Anaerobic digester1
5Mb. formicicum3636Endosymbiont of Metopus striatus2
6Mb. formicicum3722River mud3
7Mb. formicicum3637Endosymbiont of Pelomyxa palustris4
8Mb. ivanovii2611Oil-bearing sandstone5
9Mb. ulginosum2956Marshy soil6
10Mb. bryantii863Syntrophic culture7
11Mb. palustre3108Peat bog8
12Mb. wolfei2970River sediment and sewage sludge9
13Mb. thermoalcaliphilum3267Biogas plant10
14Mb. thermoautotrophicum1053Sewage sludge11
15Mb. thermoautotrophicum3720Manure12
16Mb. thermoaggregans3266Mud from cattle pasture13
17Mb. thermophilum6529Thermophilic methane tank11
18Mbb. ruminantium1093Bovine rumen14
19Mbb. arboriphilicus1125Eastern Cotton Wood tree, wetwood15
20Mbb. oralis7256Human oral cavity16
21Mbb. smithi861Primary sewage digester17
22Msh. stadtmanae3091Human feces18
23Mc. maripaludis2067Marine marsh sediment19
24Mc. voltae1537Mud20
25Mc. vannieli1224Marine mud21
26Mg. cariaci1497Marine sediment22
27Msp. hungatei864Sewage sludge23
28Mcp. parvum3823Mesophilic, anaerobic whey digester24
29Mcp. sinense4274Distillery wastewater24
30Mp. limicola2279Mud of drilling swamp25
31Msa. barkeri800Anaerobic sewage digester26
32Msa. vacuolata1232Sludge of a methane tank27
33Msa. mazei2053Sewage sludge plant28
34Msa. acetivorans2834Marine mud29
35Mt. thermophila6194Thermophilic anaerobic digester30
36Mst. concilii3671Pear waste digester31
  • aAll strains were purchased from DSMZ (Deutsche Sammlung von Mikrorganismen und Zellkulturen) except Mb. formicicum CLH1 which was isolated in our laboratory, and Mbb. oralis which was kindly provided by Dr. Tullio Brusa from the Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche of the University of Milan.

  • b [16] and references therein.

  • cAbbreviations of methanogen's generic names: Mb., Methanobacterium; Mbb., Methanobrevibacter; Msh., Methanosphaera; Mc., Methanococcus; Mg., Methanogenium; Mpl., Methanoplanus; Mcu., Methanoculleus; Msp., Methanospirillum; Mcp., Methanocorpusculum; Msa., Methanosarcina; Mst., Methanosaeta; Mt., Methanothrix.

To extract DNA, the grown cells were aseptically harvested from 50-ml volumes by centrifugation, the supernatant was decanted, and the recovered cells were washed twice with sterile phosphate-buffered saline (0.05 M, pH 7.4). The resultant cell pellets were stored at −20°C until they were used for nucleic acids extraction. The cells were resuspended in 0.2–1 ml of sterile lysis suspension, depending on the dimension of the pellet, and boiled for 15 min in a boiling water bath. After centrifugation (12 000×g for 10 min), the supernatant was recovered, and 0.1–3 μl were used for PCR. The cell lysis suspension was made of 20% (w/v) Chelex 100 sodium form beads (100–200 mesh) (Bio-Rad, Milan, Italy) suspended in a 0.1% (w/v) sodium dodecyl sulfate (SDS), 1% (v/v) Nonidet-P40, 1% (v/v) Tween-20 aqueous solution [17].

2.216 S rRNA amplification and single-strand conformation polymorphism analysis

The sequence of the forward primer, named S-F-Mbac-0009-a-A-22 according to the Oligonucleotide Database Project OPD [18], is reported in Table 2. As reverse primer, the oligonucleotide S-F-Mbac-0310-a-A-22 initially reported by Raskin et al. [19] as MB310, was used (Table 2). PCR amplifications of the 300-bp fragment from 16S rDNA of methanogens were performed in a PTC100 thermal cycler (MJ Research, Watertown, MA, USA), in a 50-μl final volume consisting of 2 μl of methanogen DNA solution, 1.25 U of Dynazyme thermostable DNA polymerase (from Thermus brockianus, Finnzymes OY, Espoo, Finland), 5 μl of the provided 10×Mg-free PCR reaction buffer, 0.2 mM of each dNTP (Pharmacia Biotech, Milan, Italy), 1.5 mM MgCl2, and 0.25 μM of each primer. The PCR mixture was overlaid with 35 μl of mineral oil (Sigma, Milan, Italy). Thirty-five cycles of amplification were performed with the denaturation step at 94°C for 1 min; the annealing step was performed at different temperatures for 1 min depending on the strains: 70°C for Methanobacteriaceae and 55°C for the other methanogens. The elongation step was at 72°C for 1 min.

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Table 2

Sequences of the forward (S-G-Mbac-0009-a-S-20) and reverse (S-F-Mbac-0310-a-A-22) primers used for the amplification of the 300-bp fragment of the 16S rDNA including the V1 and V2 regions and mismatches with the target sequences in different methanogens species. The nucleotide differences between the sequences complementary to the primers are shown in boldface.

SpeciesaStrainS-G-Mbac-0009-a-S-20MismatchesS-F-Mbac-0310-a-A-22Mismatches
3′-GGAGGCGGTCCTAGTTTGCC-5′3′-GCCTCTACCTTGGACTCTGTTC-5′
Mb. formicicum12325′-CCTCCGCCAGGATCAAACGG-3′05′-CGGAGATGGAACCTGAGACAAG-3′0
Mb. bryantii8635′-CCTCCGCCAGGATCAAACGG-3′05′-CGGAGATGGAACCTGAGACAAG-3′0
Mb. thermoautotrophicumMarburg5′-CCTCCGCCAGGATCAAACGG-3′05′-CGGAGATGGAACCTGAGACAAG-3′0
Mbb. ruminantium10935′-CTTCTGCCAGGATCAAACAG-3′35′-CGGAGATGGAACCTGAGACAAG-3′0
Mbb. arboriphilicus15365′-CCTCTGCCAGGATCAAACAG-3′25′-CGGAGATGGAACCTGAGACAAG-3′0
Msh. stadtmanae30915′-CTTCCGCCAGGATCAAACGG-3′15′-CGGAGATGGAACCTGAGACAAG-3′0
Mc. maripaludis20675′-CCTCCGGCGGGATCAACCGG-3′35′-CGGAGATGGGGACTGAGACACG-3′4
Mc. voltae15375′-CCTCCGGCGGGATCAACCGG-3′35′-CGGAGATGGGGACTGAGACACG-3′4
Mc. vannieli12245′-CCTCCGGCGGGATCAACCGG-3′35′-CGGAGATGGGGACTGAGACACG-3′4
Mg. cariaci14975′-CCTCTGGCAGGATCAACCAG-3′45′-CGGAGATGGAATCTGAGACACG-3′2
Mpl. limicola22795′-CCTCTGGCAGGATCAACCAG-3′45′-CGGAGATGGAATCTGAGACACG-3′2
Mcu. marisnigri14985′-CCTCTGGCAGGATCAACCAG-3′45′-CGGAGTTGGATTCTGAGACACG-3′4
Msp. hungatei8645′-CCTCTGGCAGGATCAAnCAG-3′45′-CGGAGATGGATTCTGAGACACG-3′3
Mcp. parvum38235′-CCTCTGGCAGGATCAACCAG-3′45′-CGGAGATGGATTCTGAGACATG-3′3
Msa. acetivorans28345′-CCTCTGGCAGGATCAACCAG-3′45′-CGGAGATGGATTCTGAGACATG-3′3
Msa. barkeri15385′-CCTCTGGCAGGATCAACCAG-3′45′-CGGAGATGGATTCTGAGACATG-3′3
Msa. vacuolata12325′-CCTCTGGCAGGATCAACCAG-3′45′-CGGAGATGGATTCTGAGACATG-3′3
Mst. concilii36715′-CCTCTGGCAGGATCAACCAG-3′45′-CGGAGATGGATTCTGAGACACG-3′3
Mt. thermophila61945′-CCTCTGGCAGGATCAACCAG-3′45′-CGGAGATGGATTCTGAGACACG-3′3
  • aAbbreviations of the generic names of methanogens are as in Table 1.

The PCR products were electrophoresed in native 6% polyacrylamide gel (acrylamide/bis acrylamide 29/1) electrophoresis in 1×Tris borate EDTA (TBE) buffer [20]. For SSCP analysis, the PCR products were mixed with the gel loading buffer (40% sucrose, 0.1 mM EDTA, 0.5% SDS, 0.05% Bromophenol blue), and denatured by adding a NaOH solution to a 0.3 M final concentration. Strand separation was performed in different conditions by using 6% polyacrylamide gels, native or with glycerol (10%) or formamide (10, 20, or 30%) in 1×TBE buffer on a minislab gel (size 80×73×1 mm, Bio-Rad) at room temperature [21]. Both for LP and SSCP, analysis the electrophoresis was performed at 50 V for 2 h. After electrophoresis, the gel was stained with ethidium bromide and photographed on a UV transilluminator [20].

3 Results and discussion

Methanogenic Archaea harbor rDNA transcriptional units of the eubacterial type, including in the following order the genes of the 16S, 23S and 5S subunits [2226]. In some strains, a 5S rRNA gene can be displayed far from the rRNA operon [26]. tDNA genes may be present in the 16S–23S rRNA spacer [2226]. It has been shown that methanogens have at least two ribosomal operons, such as Mb. formicicum[24], Mb. thermoautotrophicum[23], or Mc. jannaschii[26], or more, such as Mc. vannielii, which harbors four rRNA operons [25].

The primers for amplification of the selected fragment of 16S rDNA, including V1 and V2 regions, were chosen by aligning the sequences of the methanogenic Archaea 16S rRNAs available at the RDP. Since we did not find sequence traits conserved among all the methanogens and useful to design primers yielding amplicons with size suitable for SSCP analysis (not longer than 300–400 bp [15]), we used the following criteria in the primer selection, such that strains not specifically targeted by the primers could be amplified by simply lowering the annealing temperature during the PCR: (1) selection of primers specific for a defined group of methanogens; (2) selection of relatively long primers (at least 20 nt) which could amplify specifically the target methanogens at relatively high annealing temperature; (3) presence in each primer of not more than 20% mismatches with the sequences of the non-target methanogens; (4) selection of primers with the 3′-end matching the sequences of all the methanogens reported in the RDP.

Table 2 reports the primer sequences and the alignment of the primer complementary regions of the different methanogenic species as reported in the RDP. The forward primer was specific for the 16S rDNA of the members of the genus Methanobacterium and the reverse primer was specific for the 16S rDNA of the members of the family Methanobacteriaceae. For both primers, the highest number of mismatches with the sequences of the outgroup methanogens were four (Table 2). All the strains tested could be amplified with the selected primers by lowering the annealing temperature to 55°C during the PCR. The amplified products separated on a 6% polyacrylamide gel are shown in Fig. 1. LP were observed among the families, (for example Methanomicrobiaceae relative to Methanocorpusculaceae and Methanoplanaceae, Fig. 1D), genera (for example Methanosarcina relative to Methanosaeta/Methanothrix, Fig. 1C) and species (for example in the genus Methanobrevibacter, Fig. 1B). LP were also observed among strains of the same species (Fig. 1A). On the basis of the LP, Mb. formicicum strains could be classified in four different groups, the first including strains 1535, 2639, 6299 and CLH1, the second including strain 3636, the third including strain 3722, and the fourth including strain 3637. The latter strain showed two distinct bands, indicating the presence in the genome of at least two distinct types 16S rDNA alleles. Strains Mb. ivanovii 2611, Mb. wolfei 2970, Ms. barkeri 800 and Mst. concilii 3671 also showed polymorphic 16S rDNA in the genome (Fig. 2).

Figure 1

Polyacrylamide gel electropherograms of the amplified fragment of the 16S rDNA of methanogens showing length polymorphisms. Lanes M: 50-bp ladder. (A) Genus Methanobacterium: lanes 1–7, Mb. formicicum strains 1535, 2639, 6299, CLH1, 3636, 3722, 3637; lanes 8–10, Mb. bryantii, 863; Mb. ulginosum, 2956; Mb. palustre, 3108; lanes 11–15, Mb. thermoautotrophicum, 1053, 3720; Mb. thermoaggregans, 3266; Mb. thermoalcaliphilum, 3267; Mb. thermophilum, 6529. (B) Genera Methanosphaera and Methanobrevibacter; lanes 16–20, Msh. stadtmanae, 3091; Mbb. ruminantium, 1093; Mbb. arboriphilicus, 1125; Mbb. oralis, 7256; Mbb. smithi, 861. Genus Methanobacterium: lane 21, Mb. formicicum 1535. (C) Methanosarcinaceae. Genus Methanosarcina: lanes 22, 23, Msa. vacuolata, 1232; Msa. acetivorans, 2834. Genus Methanosaeta/Methanotrix: lanes 24, 25, Mst. concilii, 3671; Mt. thermophila, 6194. (D) Methanococcaceae, Methanomicrobiaceae, Methanocorpusculaceae, and Methanoplanaceae. Methanococcaceae: lanes 26–28, Mc. maripaludis, 2067; Mc. voltae, 1537; Mc. vannieli, 1224. Methanomicrobiaceae: lanes 29, 30, Mg. cariaci, 1497; Msp. hungatei, 864. Methanocorpusculaceae: lanes 31, 32, Mcp. sinense, 4274; Mcp. parvum, 3823. Methanoplanaceae: lane 33, Mp. limicola, 2279.

Figure 2

Interoperonic 16S rDNA length polymorphisms in different strains of methanogens. The amplified products were obtained from the first 330 bp trait of the gene including the V1 and the V2 regions and separated on 6% polyacrylamide gel electrophoresis. Lane 1, single band of Mb. formicicum 1535 as reference. Lanes 2–6, double bands found in Mb. formicicum 3637, Mb. ivanovii 2611, Mb. wolfei 2970, Msa. barkeri 800, Mst concilii 3671.

Length and sequence polymorphism in the 16S rDNA of different strains of the same methanogen species has been characterized by DNA sequencing [12]. In the first 300-bp region of the gene, two sequences differing in length for 5 nucleotides are reported for Mb. formicicum in the RDP database (Table 3) [12]. The two sequences are from strains DSMZ 1312 (a strain proposed in the DSMZ catalog as identical to the type strain 1535T[16]) and DSMZ 3636 and are consistent with the LP shown in this study. The sequence traits including insertions or deletions determining the LP between the two strains of Mb. formicicum reported in the RDP occur in the V1 and V2 regions (Table 3).

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Table 3

Alignment of the V1 (E. coli nucleotide positions 67–103) and V2 (E. coli nucleotide positions 190–223) regions of the 16S rDNA sequences of Mb. formicicum strains reported in the RDP database

StrainV1 (E. coli positions 67–103)V2 (E. coli positions 190–223)
DSMZ 13125′-GAUCCUUUCGGGGGUCGU-3′5′-AUUGGUACUCUAUUGAAAUGUAUUCGUGCGCCU-3′
DSMZ 36365′-GAUCCUU-CG-GGGUCGU-3′5′-AU-GGUUCCCUAUUGAAAUGU-UCCG-ACGCCU-3′
  • The nucleotide differences between the sequences are marked in boldface.

Fig. 3 reports the band patterns obtained by SSCP analysis of the amplified fragments resolved in glycerol-added polyacrylamide gels. We tested different electrophoresis conditions with native and glycerol or formamide-added gel, but the best band resolution was obtained with glycerol-added gels (data not shown). Almost all the different species examined were clearly distinguished, indicating that the method can be used for species identifications (Table 1). SSCP analysis showed sequence polymorphisms, not only among the different species but also among strains of the same species, as in the case of Mb. formicicum. In some cases, the SSCP differences between the species were not observed, such as between Mb. thermoautotrophicum 1053 and Mb. thermophilum 6529, or were doubtful, as between Mb. thermoautotrophicum 1053 and Mb thermoaggregans 3266. Other strains that showed practically identical SSCP patterns were Methanocorpusculum sinense 4274 and Mcp. parvum 3823.

Figure 3

Electropherograms showing the SSCPs in the 16S rDNA region examined between the strains of methanogens. Lanes M: 50-bp ladder. (A) Genus Methanobacterium: lanes 1–7, Mb. formicicum strains 1535, 2639, 6299, CLH1, 3636, 3722, 3637; lanes 8–11, Mb. bryantii, 863; Mb. ivanovii, 2611; Mb. ulginosum, 2956; Mb. palustre, 3108; lanes 12–17, Mb. thermoautotrophicum, 1053; Mb. wolfei, 2970; Mb. thermoaggregans, 3266; Mb. thermoalcaliphilum, 3267; Mb. thermoautotrophicum, 3720; Mb. thermophilum, 6529. (B) Genera Methanosphaera and Methanobrevibacter: lanes 18–22, Msh. stadtmanae, 3091; Mbb. ruminantium, 1093; Mbb. arboriphilicus, 1125; Mbb. oralis, 7256; Mbb. smithi, 861. (C) Methanosarcinaceae. Genus Methanosarcina: lanes 23–26, Msa. mazei, 2053; Msa. barkeri, 800; Msa. vacuolata, 1232; Msa. acetivorans, 2834. Genus Methanosaeta/Methanotrix: lanes 27, 28, Mst. concilii, 3671; Mt. thermophila, 6194. (D) Methanococcaceae, Methanomicrobiaceae, Methanocorpusculaceae, and Methanoplanaceae. Methanococcaceae: lanes 29–31, Mc. maripaludis, 2067; Mc. voltae, 1537; Mc. vannieli, 1224. Methanomicrobiaceae; lanes 32, 33, Mg. cariaci, 1497; Msp. hungatei, 864. Methanocorpusculaceae: lanes 34, 35, Mcp. sinense, 4274; Mcp. parvum, 3823. Methanoplanaceae: lane 36: Mp. limicola, 2279.

The evolutive meaning of the length and sequence variations observed in this survey is unclear. The LP could be the prodromes of the development of different genomic lines corresponding to different ‘ecological species’[27] evolved in different environments. In the case of Mb. formicicum strains, the four LP and SSCP types found correspond to strains isolated from very different environments: strains CLH1, 1535T, 6299 and 2639 (group 1) were isolated from anaerobic digestors; strain 3636 (group 2) is an endosymbiont of the microciliate Metopus striatus; strain 3722 (group 3) was isolated from river mud; and strain 3637 (group 4) is an endosymbiont of the anaerobic amoeba Pelomyxa palustris (Table 1; [16] and references therein).

In conclusion, LP and SSCP analysis of the V1–V2 region of the 16S rDNA gene can be proficiently used to address strain identification and to evaluate 16S rDNA polymorphisms. Using a simple boiling lysis to obtain DNA for amplification, the analysis can be completed in less than 8 h.

Acknowledgements

The study was performed in the ambit of the project BIOWARE (Development of a Biological Integrated Process for Purifying Olive Oil Wastewater Recovering Energy and Producing Alcohol) financed by the European Community (EC Contract AIR3-CT94-1987). We thank Dr. Tullio Brusa from DISTAM, University of Milan, who provided us with the original strain of Methanobrevibacter oralis.

References

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