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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 mcrA amplicons from AD (A) and WD (B) samples. The width of the red branches corresponds to the number of unique mcrA amplicon sequence reads in that particular branch (this can be either a leaf or node). The collapse of some branches (mapped to uncultured archaeons) to increase the clarity of the trees is indicated by a black triangle. Numbers next to the entries “uncultured archeon” indicate the same microorganism on both trees.
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Figure 3: Phylogenetic placement of mcrA amplicons from AD (A) and WD (B) samples. The width of the red branches corresponds to the number of unique mcrA amplicon sequence reads in that particular branch (this can be either a leaf or node). The collapse of some branches (mapped to uncultured archaeons) to increase the clarity of the trees is indicated by a black triangle. Numbers next to the entries “uncultured archeon” indicate the same microorganism on both trees.

Mentions: In addition to the 16S rRNA marker, the mcrA gene was used for taxonomic profiling of methanogenic communities in both digesters. The mcrA gene fragments amplified using primers MLf/MLr (Luton et al., 2002) were sequenced and analyzed. More than half of the sequences (57%) amplified from the AD sample were assigned to uncultured Archaea, belonging to the Methanomassiliicoccales (23%), Methanomicrobiales (13%), Methanobacteriales (11%) and Methanosarcinales (10%) orders (Figure 3), suggesting dominance of hydrogenotrophic methanogens over acetoclastic Archaea. The most abundant genera in AD were Methanobacterium sp. Maddingley MBC34 (11%) followed by Methanosaeta concilli (9%) and Methanoculleus spp. (4%) (Figure 3). Similarly in WD, the majority of the mcrA amplicons were classified as uncultured Archaea belonging to orders Methanomicrobiales (27%) and Methanomassiliicoccales (7%) (Figure 3), while at the genus level most of the methanogens were identified as Methanometylovorans hollandica (21%), Methanosaeta concilli (16%), Methonoculleus spp. (12%), or Methanoplanus petrolearius (3%) (Figure 3).


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 mcrA amplicons from AD (A) and WD (B) samples. The width of the red branches corresponds to the number of unique mcrA amplicon sequence reads in that particular branch (this can be either a leaf or node). The collapse of some branches (mapped to uncultured archaeons) to increase the clarity of the trees is indicated by a black triangle. Numbers next to the entries “uncultured archeon” indicate the same microorganism on both trees.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Phylogenetic placement of mcrA amplicons from AD (A) and WD (B) samples. The width of the red branches corresponds to the number of unique mcrA amplicon sequence reads in that particular branch (this can be either a leaf or node). The collapse of some branches (mapped to uncultured archaeons) to increase the clarity of the trees is indicated by a black triangle. Numbers next to the entries “uncultured archeon” indicate the same microorganism on both trees.
Mentions: In addition to the 16S rRNA marker, the mcrA gene was used for taxonomic profiling of methanogenic communities in both digesters. The mcrA gene fragments amplified using primers MLf/MLr (Luton et al., 2002) were sequenced and analyzed. More than half of the sequences (57%) amplified from the AD sample were assigned to uncultured Archaea, belonging to the Methanomassiliicoccales (23%), Methanomicrobiales (13%), Methanobacteriales (11%) and Methanosarcinales (10%) orders (Figure 3), suggesting dominance of hydrogenotrophic methanogens over acetoclastic Archaea. The most abundant genera in AD were Methanobacterium sp. Maddingley MBC34 (11%) followed by Methanosaeta concilli (9%) and Methanoculleus spp. (4%) (Figure 3). Similarly in WD, the majority of the mcrA amplicons were classified as uncultured Archaea belonging to orders Methanomicrobiales (27%) and Methanomassiliicoccales (7%) (Figure 3), while at the genus level most of the methanogens were identified as Methanometylovorans hollandica (21%), Methanosaeta concilli (16%), Methonoculleus spp. (12%), or Methanoplanus petrolearius (3%) (Figure 3).

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