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Novel molecular markers for the detection of methanogens and phylogenetic analyses of methanogenic communities.

Dziewit L, Pyzik A, Romaniuk K, Sobczak A, Szczesny P, Lipinski L, Bartosik D, Drewniak L - Front Microbiol (2015)

Bottom Line: Therefore, biodiversity studies have relied on the use of 16S rRNA and mcrA [encoding the α subunit of the methyl coenzyme M (methyl-CoM) reductase] genes as molecular markers for the detection and phylogenetic analysis of methanogens.The selectivity of the markers was analyzed using phylogenetic methods.Our results indicate that the selected markers and the PCR primer sets can be used as specific tools for in-depth diversity analyses of methanogenic consortia.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw Warsaw, Poland.

ABSTRACT
Methanogenic Archaea produce approximately one billion tons of methane annually, but their biology remains largely unknown. This is partially due to the large phylogenetic and phenotypic diversity of this group of organisms, which inhabit various anoxic environments including peatlands, freshwater sediments, landfills, anaerobic digesters and the intestinal tracts of ruminants. Research is also hampered by the inability to cultivate methanogenic Archaea. Therefore, biodiversity studies have relied on the use of 16S rRNA and mcrA [encoding the α subunit of the methyl coenzyme M (methyl-CoM) reductase] genes as molecular markers for the detection and phylogenetic analysis of methanogens. Here, we describe four novel molecular markers that should prove useful in the detailed analysis of methanogenic consortia, with a special focus on methylotrophic methanogens. We have developed and validated sets of degenerate PCR primers for the amplification of genes encoding key enzymes involved in methanogenesis: mcrB and mcrG (encoding β and γ subunits of the methyl-CoM reductase, involved in the conversion of methyl-CoM to methane), mtaB (encoding methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, catalyzing the conversion of methanol to methyl-CoM) and mtbA (encoding methylated [methylamine-specific corrinoid protein]:coenzyme M methyltransferase, involved in the conversion of mono-, di- and trimethylamine into methyl-CoM). The sensitivity of these primers was verified by high-throughput sequencing of PCR products amplified from DNA isolated from microorganisms present in anaerobic digesters. The selectivity of the markers was analyzed using phylogenetic methods. Our results indicate that the selected markers and the PCR primer sets can be used as specific tools for in-depth diversity analyses of methanogenic consortia.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic placement of mcrB amplicons from AD (A) and WD (B) samples. The width of the red branches corresponds to the number of unique mcrB amplicon sequence reads in that particular branch (this can be either a leaf or node). The leaf for Methanoculleus sp. MH98A was shortened, as indicated by two slashes.
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Figure 4: Phylogenetic placement of mcrB amplicons from AD (A) and WD (B) samples. The width of the red branches corresponds to the number of unique mcrB amplicon sequence reads in that particular branch (this can be either a leaf or node). The leaf for Methanoculleus sp. MH98A was shortened, as indicated by two slashes.

Mentions: This analysis revealed that LMCRB/RMCRB primers, designed to the mcrB gene, amplified DNA fragments comprising sequences representing four methanogenic orders: Methanobacteriales, Methanomassiliicoccales, Methanomicrobiales, and Methanosarcinales (Figure 4). The dominant genus in both digesters was Methanoculleus spp. (48% for AD and 67% for WD), with M. marisnigri as the most abundant species (37 and 53%, respectively). This finding remains in good agreement with previous observations showing that the predominant order in biogas-producing microbial communities in anaerobic digesters is usually Methanomicrobiales, and the most abundant species is hydrogenotrophic M. marisnigri (Wirth et al., 2012). Moreover, in AD, 27% of sequences were classified as uncultured Methanomassiliicoccales (with 4% described as Candidatus Methanoplasma termitum) and 17% as Methanosaeta concilli. The second and third most abundant methanogens in WD were Methanomethylovorans hollandica (19%) and Methanosaeta concilli (6%), respectively (Figure 4).


Novel molecular markers for the detection of methanogens and phylogenetic analyses of methanogenic communities.

Dziewit L, Pyzik A, Romaniuk K, Sobczak A, Szczesny P, Lipinski L, Bartosik D, Drewniak L - Front Microbiol (2015)

Phylogenetic placement of mcrB amplicons from AD (A) and WD (B) samples. The width of the red branches corresponds to the number of unique mcrB amplicon sequence reads in that particular branch (this can be either a leaf or node). The leaf for Methanoculleus sp. MH98A was shortened, as indicated by two slashes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493836&req=5

Figure 4: Phylogenetic placement of mcrB amplicons from AD (A) and WD (B) samples. The width of the red branches corresponds to the number of unique mcrB amplicon sequence reads in that particular branch (this can be either a leaf or node). The leaf for Methanoculleus sp. MH98A was shortened, as indicated by two slashes.
Mentions: This analysis revealed that LMCRB/RMCRB primers, designed to the mcrB gene, amplified DNA fragments comprising sequences representing four methanogenic orders: Methanobacteriales, Methanomassiliicoccales, Methanomicrobiales, and Methanosarcinales (Figure 4). The dominant genus in both digesters was Methanoculleus spp. (48% for AD and 67% for WD), with M. marisnigri as the most abundant species (37 and 53%, respectively). This finding remains in good agreement with previous observations showing that the predominant order in biogas-producing microbial communities in anaerobic digesters is usually Methanomicrobiales, and the most abundant species is hydrogenotrophic M. marisnigri (Wirth et al., 2012). Moreover, in AD, 27% of sequences were classified as uncultured Methanomassiliicoccales (with 4% described as Candidatus Methanoplasma termitum) and 17% as Methanosaeta concilli. The second and third most abundant methanogens in WD were Methanomethylovorans hollandica (19%) and Methanosaeta concilli (6%), respectively (Figure 4).

Bottom Line: Therefore, biodiversity studies have relied on the use of 16S rRNA and mcrA [encoding the α subunit of the methyl coenzyme M (methyl-CoM) reductase] genes as molecular markers for the detection and phylogenetic analysis of methanogens.The selectivity of the markers was analyzed using phylogenetic methods.Our results indicate that the selected markers and the PCR primer sets can be used as specific tools for in-depth diversity analyses of methanogenic consortia.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw Warsaw, Poland.

ABSTRACT
Methanogenic Archaea produce approximately one billion tons of methane annually, but their biology remains largely unknown. This is partially due to the large phylogenetic and phenotypic diversity of this group of organisms, which inhabit various anoxic environments including peatlands, freshwater sediments, landfills, anaerobic digesters and the intestinal tracts of ruminants. Research is also hampered by the inability to cultivate methanogenic Archaea. Therefore, biodiversity studies have relied on the use of 16S rRNA and mcrA [encoding the α subunit of the methyl coenzyme M (methyl-CoM) reductase] genes as molecular markers for the detection and phylogenetic analysis of methanogens. Here, we describe four novel molecular markers that should prove useful in the detailed analysis of methanogenic consortia, with a special focus on methylotrophic methanogens. We have developed and validated sets of degenerate PCR primers for the amplification of genes encoding key enzymes involved in methanogenesis: mcrB and mcrG (encoding β and γ subunits of the methyl-CoM reductase, involved in the conversion of methyl-CoM to methane), mtaB (encoding methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, catalyzing the conversion of methanol to methyl-CoM) and mtbA (encoding methylated [methylamine-specific corrinoid protein]:coenzyme M methyltransferase, involved in the conversion of mono-, di- and trimethylamine into methyl-CoM). The sensitivity of these primers was verified by high-throughput sequencing of PCR products amplified from DNA isolated from microorganisms present in anaerobic digesters. The selectivity of the markers was analyzed using phylogenetic methods. Our results indicate that the selected markers and the PCR primer sets can be used as specific tools for in-depth diversity analyses of methanogenic consortia.

No MeSH data available.


Related in: MedlinePlus