Limits...
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

Schematic diagram of the superpathway of methanogenesis. E.C. numbers for particular enzymes are shown in parentheses. The red star indicates the mcrA gene encoding subunit α of a methyl-coenzyme M reductase I, which is commonly used as a molecular marker for the detection of methanogens. The yellow stars denote molecular markers developed in this study, for which sets of PCR primers were designed.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493836&req=5

Figure 1: Schematic diagram of the superpathway of methanogenesis. E.C. numbers for particular enzymes are shown in parentheses. The red star indicates the mcrA gene encoding subunit α of a methyl-coenzyme M reductase I, which is commonly used as a molecular marker for the detection of methanogens. The yellow stars denote molecular markers developed in this study, for which sets of PCR primers were designed.

Mentions: Methanogenesis is a metabolic process driven by obligate anaerobic Archaea. It is responsible for the production of over 90% of methane on Earth (Costa and Leigh, 2014). There are three main methanogenic pathways: (i) hydrogenotrophic methanogenesis using H2/CO2 for methane synthesis, (ii) acetoclastic methanogenesis, in which the methyl group from acetate is transferred to tetrahydrosarcinapterin and then to coenzyme M (CoM), and (iii) methylotrophic methanogenesis, using methyl groups from methanol and methylamines (mono-, di-, and trimethylamine) for the production of methyl-coenzyme M (Figure 1). The final step in all these pathways is common and involves the conversion of methyl-CoM into methane by methyl-coenzyme M reductase, an enzymatic complex that is present in all methanogens (Borrel et al., 2013) (Figure 1).


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)

Schematic diagram of the superpathway of methanogenesis. E.C. numbers for particular enzymes are shown in parentheses. The red star indicates the mcrA gene encoding subunit α of a methyl-coenzyme M reductase I, which is commonly used as a molecular marker for the detection of methanogens. The yellow stars denote molecular markers developed in this study, for which sets of PCR primers were designed.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Schematic diagram of the superpathway of methanogenesis. E.C. numbers for particular enzymes are shown in parentheses. The red star indicates the mcrA gene encoding subunit α of a methyl-coenzyme M reductase I, which is commonly used as a molecular marker for the detection of methanogens. The yellow stars denote molecular markers developed in this study, for which sets of PCR primers were designed.
Mentions: Methanogenesis is a metabolic process driven by obligate anaerobic Archaea. It is responsible for the production of over 90% of methane on Earth (Costa and Leigh, 2014). There are three main methanogenic pathways: (i) hydrogenotrophic methanogenesis using H2/CO2 for methane synthesis, (ii) acetoclastic methanogenesis, in which the methyl group from acetate is transferred to tetrahydrosarcinapterin and then to coenzyme M (CoM), and (iii) methylotrophic methanogenesis, using methyl groups from methanol and methylamines (mono-, di-, and trimethylamine) for the production of methyl-coenzyme M (Figure 1). The final step in all these pathways is common and involves the conversion of methyl-CoM into methane by methyl-coenzyme M reductase, an enzymatic complex that is present in all methanogens (Borrel et al., 2013) (Figure 1).

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