DIVERSITY OF MCRA GENE OF METHANOGENS
Wannaporn Muangsuwan11,*, Pattarawan Ruangsuj21, Pichai Chaichanachaicharn32, Kosum
Chansiri42 and Montri Yasawong51, #
1
Department of Biochemistry, Faculty of Pharmacy, Mahidol University
2
Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University
*e-mail: [email protected], #e-mail: [email protected]
Abstract
Methanogens have a specific characteristic function in the methane formation as
producers of the forceful greenhouse gas (CH4). Methyl-coenzyme M reductase (MCR) is the
key enzyme for methanogenesis. The mcrA gene, which encoding for α-subunit of MCR, has
been applied as a specific marker for distinction assay of methanogens from dissimilar
habitats. The study was based on the collection of mcrA gene sequences, evolutionary model
selection and reconstruction of mcrA phylogenetic tree. The mcrA gene sequences were
collected from the genome database of GenBank. Phylogenetic tree was analyzed based on
Bayesian inference method using parallel version of MrBayes. The genes were distributed
into four classes of the methanogenic archaea, which were Methanobacteria, Methanococci,
Methanomicrobia and Methanopyri. The mcrA gene sequence of Methanopyrus kandleri
(AE009439) was selected for an out group for the Bayesian analysis. The mcrA gene of
methanogen was clearly separated in two groups, which were group A and B. The mcrA
group A was able to classify into two subgroups (subgroup A1 and A2). The first linage,
mcrA group A possesses three pathways of methanogesis that were hydrogenotropic,
acetoclastic and C1-pathway. The second lineage, mcrA group B, possessed only
hydogenotropic pathway for the methane production. This study shows the evolutionary and
diversity of mcrA genes among the methanogens genome. The information that gains from
this study will help the researcher for environmental research that related with the methane
production.
Keywords: mcrA gene, methyl-coenzyme M reductase, methanogens
Introduction
Methane is a forceful greenhouse gas, progressive forceful than carbon dioxide unit
per unit. Electrical generation used methane fundamentally flaming it as fuel. Other
hydrocarbon fuels, burning creates carbon dioxide for each unit of heat discharged more than
methane. Methane creates heat more than any other complex hydrocarbons. Compressed
natural gas from methane is used as conveyance fuel and it’s more environmentally friendly
than other complex hydrocarbons. Natural gas from methane is mostly created by
microorganisms and called process methanogenesis. Methanogenesis is a form of anaerobic
conditions used by microorganisms. It involves in landfill, marshland, digestive tracts of
animals such as ruminants and humans, in marine sediments, biomethanation is generally
confined to where sulfates are depleted, in anaerobic wastewater treatments and the guts of
termites. Methanogens are constituted by methyl coenzyme M-reductase (MCR), an enzyme
is extensively and wholly being in methanogens [11]. One enzyme is held in the terminal step
of methane biosynthesis. Accordingly mcrA, the gene encoding the α-subunit of MCR has
been applied as a specific marker for distinction assay of methanogens from dissimilar
habitats. Methanogens can be calculated correctly using a speciality ‘‘functional” marker
gene mcrA coding a-subunit of methyl-coenzyme M reductase (MCR), the key enzyme of
methanogenesis. Distinct else enzymes in methanogenic metabolism, MCR present to be
characteristic to methanogens and is interlacing in the last stage of methanogenesis begetting
reduction of methyl group absorbed to coenzyme M [18]. The owning of MCR enzyme
determine a cell as a methanogen so mcrA sequences can be used for methanogen
phylogenies [11]. Therefore, the dissimilar methanogenic pathways are prominent by the
method of getting the methyl group for chang to methane. The pathways are classified within
three general groups based on methyl group acquisition; the first is hydrolysis. Second are
acidogenesis and acetogenesis. The last is methanogenesis.
This research was focus on microbial diversity of the methanogens. The study was
based on the collection of mcrA gene sequences, evolutionary model selection and
reconstruction of mcrA phylogenetic tree.
Methodology
Construction of mcrA gene database
The mcrA gene sequences were collected from the genome database of GenBank.
Quality of the gene sequences was manually justified. Only high quality of the gene
sequences was kept for further analysis.
Phylogenetic analysis
Multiple sequences alignment was performed based on iterative refinement method
using MUSCLE version 3.8.31. Evolutionary model of mcrA gene was selected based on AIC
and hLRTs criteria using MrModeltest version 2.3. Phylogenetic tree was analyzed based on
Bayesian inference method using parallel version of MrBayes. The phylogenetic analysis was
performed on computer cluster (KIRI cluster). The cluster was assembled from four IBM
servers (x3250 M4), which were connected by a gigabit switch (HP ProCurve 1410-16G).
The Bayesian posterior probabilities were obtained by performing two separate runs with
twelve Markov chains. Each run was conducted with 1×107 generations and sampled every
100 generations. A consensus tree was calculated after discarding the first 25% of the
iterations as burn-in. Phylogenetic tree drawing was performed using TreeView version 1.6.6.
Results
Construction of mcrA gene database
Thirty six sequences of mcrA gene were obtained from GenBank database. The size of
the complete mcrA gene sequences were ranged from 1,653 to 1,722 bases. The genes were
distributed into four classes of the methanogenic archaea, which were Methanobacteria,
Methanococci, Methanomicrobia and Methanopyri.
Diversity of mcrA gene
Of the mcrA gene database, the largest group of mcrA gene was belonging to the
methanogen in class Methanomicrobia (63.89%) (Figure 1). The mcrA gene of the
methanogens, which were the member of class Methanobacteria and Methanococci, were
shared the same propotion (16.67%) (Figure 1.). There was only 2.78% of mcrA gene
sequences were detected from the methanogen in class Methanopyri (Figure 1.).
25
20
OTUs
15
10
5
0
MB
MC
MM
MP
Methanogen
Figure 1. The proportion of methane producing from different Class. Bar chart showing the relationship
between operational taxonomic units (OTUs) and class of methanogenic archaea. Order names has been
abbreviated in agreement with as follows: MB, Methanomicrobia; MC, Methanococci;
MM, Methanomicrobia; MP, Methanopyri.
Phylogenetic analysis
The mcrA gene sequence of Methanopyrus kandleri (AE009439) was selected for an
out group for the Bayesian analysis. The mcrA gene tree was shown in Figure 2. The mcrA
gene of methanogen was clearly separated in two groups, which were group A and B. The
mcrA group A was able to classify into two subgroups (group A1 and A2) based on the
Bayesian tree (Figure 2). The group A of mcrA gene consisted of 23 species of methanogen.
The mcrA gene group B contained 12 species of methanogen (Figure 2.).
Figure 2. Bayesian tree of mcrA gene. The tree was constructed using Bayesian inference method with the
GTR+I+G model of nucleotide substitution. The values associated with nodes correspond to the clade
credibility support in %.
Characteristics and properties of methanogen
Methane producing archaea is classified into three distinctions, which were mcrA
group A1, mcrA group A2 and mcrA group B. The members of mcrA group A1 were able to
produce methane using three different pathways that were hydrogenotrophic, acetoclastic and
C1 pathway (Table 1.). The methanogens, which were the member of mcrA group A2,
produced methane by hydrogenotrophic and acetoclastic pathway (Table 2.).
Hydrogenotrophic pathway was the main pathway for methane production of the mcrA group
B (Table 3.).
Table 1. Differential characteristics properties and habitat of mcrA group A1
Accesion
Organism
Class
Habitat
Condition
Pathway
Ref.
Methanococcoides
Methanomicrobia
Ace Lake
T 23°C
C1-
[1]
no.
CP000300
burtonii
in
Pathways
Antarctica
CP001994
Methanohalophilus
Methanomicrobia
mahii
Sediments
pH 7.5,T
C1-
,Great
35°C
Pathways
Sediments
pH 7-8,T
C1-
of saline
30-40°C
Pathways
Bosa Lake
high pH,
C1-
of the Wadi
high T
Pathways
[3]
Salt Lake.
CP002069
Methanohalobium
Methanomicrobia
evestigatum
[5]
lagoons
CP002101
Methanosalsum
Methanomicrobia
zhilinae
[7]
el Natrun
CP003083
Methanolobus
Methanomicrobia
psychrophilus
Zoige
wet
pH 6-8,T
C1-
land
soil,
18°C
Pathways
[8]
china
CP003362
Methanomethylovorans
Methanomicrobia
hollandica
Eutrophic
pH
lake
7.0,T
6.5-
C1-
[9]
Pathways
37°C
CP000099
Methanosarcina
Methanomicrobia
barkeri
Marine
or
brackish
pH
5,T
Hydro-
37-42°C
genotrophic
Marine and
pH
Aceto
freshwater
7.5,T 37-
sediments
40°C
Sediments
pH
of a marine
7.0,T 35-
canyon
40°C
[14]
mud
AE008384
AE010299
Methanosarcina mazei
Methanosarcina
Methanomicrobia
Methanomicrobia
acetivorans
AM114193
Methanocella
Methanomicrobia
arvoryzae
AP011532
Methanocella
Methanomicrobia
paludicola
CP003243
Methanocella conradii
Methanomicrobia
Rice
field
pH
7.0-
6.5-
7.0,
-trophic
C1-
Hydro-
T 45°C
genotrophic
Rice paddy
pH 7.0, T
Hydro-
soil
35-37°C
genotrophic
Chinese
pH 6.8, T
Hydro-
Rice
55°C
genotrophic
Soil
[20]
Pathways
soil
Field
[19]
[23]
[25]
[29]
Table 2. Differential characteristics properties and habitat of mcrA group A2
Accesion
Organism
Class
Habitat
Condition
Pathway
Ref.
Methanosaeta
Methanomicrobia
Waste
water
pH
Aceto-
[2]
Beijing,
7.6,
no.
CP003117
harundinacea
in
China.
T
7.2-
trophic
34-
37°C
CP000477
Methanosaeta
Methanomicrobia
thermophila
CP002565
Methanosaeta
Methanomicrobia
Thermal lake
pH 6.7,
Aceto-
mud
T 55°C
trophic
Mat form
pH
Aceto-
concilii
7.1-
7.5,
T
[4]
[10]
trophic
35-
40°C
CP003167
Methanoregula
Methanomicrobia
formicicum
Granular
pH 7.4,
Hydro-
sludge
T
genotrophic
30-
[12]
33°C
CP002117
Methanoplanus
Methanomicrobia
petrolearius
African
pH 7.0,
Hydro-
T 37°C
genotrophic
pH 8,
Hydro-
T 40°C
genotrophic
Digested
pH 6.7,
Aceto-
sewage
T 37°C
trophic
pH 7.5,
Hydro-
T 30°C
genotrophic
Surface
pH 7,
Aceto-
sediments of
T 37°C
trophic
Minerotrophic
pH 5.5,
Hydro-
fen peatland
T 28–30
genotrophic
offshore
oil
[15]
field
CP000562
Methanoculleus
Methanomicrobia
marisnigri
pig
manure
digestor
in
[10]
Spain
HE964772
Methanoculleus
Methanomicrobia
bourgensis
[22]
sludge
CP000254
Methanospirillum
Methanomicrobia
Soil.
hungatei
CP000559
Methanocorpusculum
Methanomicrobia
labreanum
[27]
[30]
Tar Pit Lake
CP001338
Methanosphaerula
Methanomicrobia
palustris
[31]
°C
CP000780
Methanoregula
boonei
Methanomicrobia
Oil wells,
pH 5.1,
Hydro-
T 53°C
genotrophic
[32]
Table 3. Differential characteristics properties and habitat of mcrA group B
Accesion
Organism
Class
Habitat
Methanobacterium sp.
Methanobacteria
French
Condition
Pathway
Ref.
T 60 °C
Hydro-
[6]
no.
CP002551
oil
field
CP002278
Methanothermus
Methanobacteria
fervidus
CP002737
CP001901
Methanotorris igneus
Methanocaldococcus
Methanococci
Methanococci
sp.
genotrophic
Icelandic hot
pH 6.5,
Hydro-
springs
T 83 °C
genotrophic
Coastal
pH 5.7,
Hydro-
hydrothermal
T 88°C
genotrophic
Deep-sea
T 92°C
Hydro-
hydrothermal
[13]
[16]
[17]
genotrophic
vent fluid
L77117
Methanocaldococcus
Methanococci
jannaschii
Sea
floor
pH 6.0,
Hydro-
surface at the
T 85°C
genotrophic
Deep-sea
pH 6.5,
Hydro-
hydrothermal
T 80°C
genotrophic
Salt-marsh
pH
Hydro-
sediment
7.2,
base
of
[17]
a
2600 m-deep
CP001787
Methanocaldococcus
Methanococci
vulcanius
[21]
chimney
BX950229
Methanococcus
Methanococci
maripaludis
T
6.8-
[24]
genotrophic
35-39
°C
CP002792
Methanothermococcus
Methanococci
okinawensis
Deep-sea
pH 6-7,
Hydro-
hydrothermal
T 60–65
genotrophic
vent
°C
[26]
chimney
CP001719
Methanobrevibacter
Methanobacteria
ruminantium
CP004050
Methanobrevibacter
Methanobacteria
sp.
AE000666
Methanothermobacter
Rumen fluid
pH 7.0,
Hydro-
of animal
T 37°C
genotrophic
T 38°C
Hydro-
Rumen
of
animal
Methanobacteria
Hot springs
thermautotrophicus
[28]
[33]
genotrophic
pH
7.2-
7.6,
T
Hydro-
[34]
genotrophic
65-70
°C
CP001710
Methanothermobacter
marburgensis
Methanobacteria
Hot springs
pH
6.8-
7.4,
T 65 °C
Hydrogenotrophic
[35]
Discussion and Conclusion
Discussion and Conclusion
The phylogenetic analysis represented two lineages of methanogen. The first linage,
mcrA group A possesses three pathways of methanogesis that were hydrogenotropic,
acetoclastic and C1-pathway. The second lineage, mcrA group B, possessed only
hydogenotropic pathway for the methane production. Class Methanomicrobia was the major
member of mcrA group A. Class Methanomicrobia and Methanococci shared the same
proportion of mcrA group B. Most of methanogens, which belonged to mcrA group A1, were
mesophiles and isolated from sediments and rice paddy fields (Table 1.). The mcrA group A2
was mostly mesophiles that were able to grow on acidic to neutral pH. The mcrA group A2
was isolated from several habitats such as soil, sludge, wetland and etc (Table 2.). The mcrA
group B2 was mostly thermophiles, which isolated from hot spring, deep-sea hydrothermal
vent, rumen fluid, oil field and etc.
This study shows the evolutionary and diversity of mcrA genes among the
methanogens genome. The information that gains from this study will help the researcher for
environmental research that related with the methane production.
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Acknowledgements:
This work was financially supported by the National Research Council of Thailand (NRCT).