Science and Technology Journal,
Vol. 3
Issue: II ISSN: 2321-3388
Cloning and Sequencing of Methyl-coenzyme
Reductase A (mcrA) Gene of Methanogenic
Archaea from Landϐill
2
Shailendra Yadav1, S.S. Maitra2 and Sankar K. Ghosh1*
Department of Biotechnology, Assam University, Silchar–788011
School of Biotechnology, Jawaharlal Nehru University, New Delhi–110067
E-mail: *drsankarghosh@gmail.com
1
Abstract—Methanogens are classi ied under domain Archaea, Phylum Euryarchaeota. They are responsible for 90% of
biogenic methane production on earth. Methanogenesis last step is catalysed by α subunit of enzyme methyl coenzyme
reducatse A gene and is used as a biomarker for identi ication of methaogens. In present study, we irst optimized the protocol
for extraction of metagenomic DNA from the leachate samples using “Fast DNA spin Kit for Soil” and ampli ication of mcrA
gene. The phylogenetic analysis of mcrA gene indicated presence of methanogens belonging to order Methanobacteriales
and Methanomicrobiales. Clone MCR2.KM041254 clustered with Uncultured archaeon clone and Methanotermobacter
thermoϔlexus. Clone BMCR2.KM041258 clustered with Methanoculleus marisngri JR1. Molecular characterization of
methanogens in land ill site may enrich our knowledge about relation between methanogens active population and methane
emission potentials.
Keywords: Land ill, Leachate, mcrA, K2P, Metagenomics
LIST OF ABBREVIATION
with two active sites, each containing one molecule of the
nickel hydrocorphinate F430 as the prosthetic group [5].
mcr A
MSW
GHG
K2P
MEGA
IPTG
BLAST
The highly conserved nature of mcrA gene makes it
suitable for phylogenetic analysis. The primer MLFWD
and MLREV designed by Luton et al 2002 is widely used
for identi ication and quanti ication of methanogens [6–8].
Methane is an important greenhouse gas and it is 25 times
more potent than CO2 in causing global warming [9–11].
Land ill sites are estimated to release 40 Tg CH4 per year,
which accounts for 6.7% of global methane emissions. Rice
paddy ields, land ills, livestock, fossils fuel production
and biomass burnings are considered as important
anthropogenic sources of CH4 [12–18]. Methane is third
most abundant gas in ecosystem and it contributes about
18% in total greenhouse effect. It has highest generation
(60%) than other gases [9]. Therefore, there is immense
concern for its abatement or utilization.
Methyl Coenzyme Reductase A
Municipal Solid Waste
Green House Gas
Kimura 2 Parameter
Molecular Evolutionary Genetic Analysis
Isopropy Thiogalactosidase
Basic Local Alignment Search Too
INTRODUCTION
Methanogens are strictly anaerobic microorganisms which
come under Kingdom Archaea, Phylum Euryarchaeota.
They are classi ied under six phylogenetic orders of
Euryarchaeota:
Methanobacteriales,
ethanopyrales,
Methanococcales, Methanomicrobiales, Methanosarcinales
and Methanocellales [1, 2]. All of these orders are highly
diverse and has the ability to produce methane metabolically.
Methyl-coenzyme M reductase (MCR) in methanogenic
archaea catalyses the rate limiting and inal step in methane
biosynthesis [3, 4]. MCR is a 280 kDa enzyme built from three
different protein chains in a C2 -symmetric α2β2γ2 assembly
Compared to the western countries, the composition
of municipal solid waste (MSW) in developing countries
like India has higher (40%–60%) organic waste [19].
These sites lack base liner to avoid percolation of leachate
to groundwater table and neither has biogas nor leachate
Cloning and Sequencing of Methyl-coenzyme Reductase
collection facility. City generates about 6000 tonnes of solid
waste per day and the expected quantity of solid waste
generation in Delhi would be about 12,750 tonnes per day
by 2015 [12]. Due to scarcity of land in big cities, municipal
authorities are using same land ill for nearly 10–20 years.
Hence, the possibility of anaerobic emission of GHG’s
further increases [19]. In other words, biogas and leachate
generation are inevitable consequences of the practice of
waste disposal in land ills. Microbial decomposition, climatic
conditions, refuse characteristics are amongst the many
factors that contribute to the gas and leachate generation
at land ill sites [20–22]. The migration of gas and leachate
away from the land ill boundaries and their release into the
surrounding environment pose potential health hazards,
unpleasant odours, ground water pollution, air pollution
and global warming [23, 24].
The present study was aimed to detect presence of
methanogens using mcrA gene PCR ampli ication, cloning
and sequencing in the leachate samples of land ill site.
MATERIALS AND METHOD
SĆĒĕđĎēČ Ćēĉ DNA EĝęėĆĈęĎĔē
Leachate samples were collected from the land ill sites of
New Delhi India and stored at 4oC prior to DNA extraction.
DNA was extracted from leachate sample using “Fast DNA
Spin Kit for Soil” (MP Biomedical Santana Ana, CA) as per
manufacturer instructions including some pre-treatment
steps. Since, leachate samples are very rich in humic acid
and other contaminant which affect the purity of extracted
DNA and limit downstream processing like PCR, cloning etc.
Therefore, to overcome this problem, some modi ications
were done to the protocol. Initially, 10 ml of leachate sample
was taken from the reactor and centrifuged at 3000rpm
for 15 minutes. As centrifugation at this speed will not
cause precipitation of bacterial/ archaeal cells but heavier
particles and contaminants will be settle down. Then
supernatant was transferred to another tubes and were
vortexed for 1 minute and centrifuged at 13,000 rpm for 15
minutes. The supernatant was discarded and the pellet was
resuspended in 5ml nuclease free water by gentle tapping
and vortexing. Then the tube was centrifuged at 13,500
rpm for 15 minute. Again, the supernatant was discarded
and resulting pellet was used for DNA extraction following
protocol as per recommended by manufacturer. Before
elution of DNA, column was placed inside laminar air low
for drying about 30–45 minutes (2 minutes in kit protocol)
to remove residual ethanol of wash buffer which may also
199
inhibit PCR ampli ication. During elution step after addition
of 70μl DNase/ Pyrogen-Free Water, column was placed
inside waterbath at 56oC for 30 minutes (optional 5 minutes
in Kit protocol).
PCR AĒĕđĎċĎĈĆęĎĔē Ĕċ MĊęčĞđ CĔĊēğĞĒĊ
RĊĉĚĈęĆĘĊ A
Extracted DNA was ampli ied using primer MLFWD 5’GGT
GGT GTM GGA TTC ACA CAR TAY GCW ACA GC3’ and MLREV
5’TTC ATT GCR TAG TTW GGR TAG TT3’speci ic for mcrA
gene of methanogens [18]. PCR was performed in a thermocycler (Applied Biosystem–Gene amp 9700). A typical
reaction mixture of (50μl) for PCR of mcrA gene consisted of
Taq buffer 5μl, Taq polymerase 1μl,dNTP 5μl, fwd primer 2
μl rev primer 2μl, MgCl2 2μl and 32μl of nuclease free water
and template DNA 1μl. The ampli ication pro ile was initial
denaturation at 94oC for 5min, subsequent denaturation at
94oC for 30s for 30 cycles, annealing at 52oC for 1minute,
elongation at 72oC for 2 minute and inal extension at 72oC
for 10 minutes followed by a cooling step down to 4oC. The
annealing temperature was optimized by doing gradient PCR
and annealing at 52 oC for 1minute gave better ampli ication.
CđĔēĎēČ Ćēĉ SĊĖĚĊēĈĎēČ Ĕċ MĊęčĞđ
CĔĊēğĞĒĊ RĊĉĚĈęĆĘĊ A
The PCR products of 450–500 BP amplicons size were
puri ied using PCR puri ication kit (Fermentas, UK) as
recommended by manufacturer protocol. Puri ied mcrA
gene amplicons were cloned inside PTZ57R/T vector using
the Insta-T/ A cloning kit (Fermentas, UK) and transformed
into Escherichia coli DH5α. The positive clones were
selected using blue-white screening on Luria-Bertani plates
containing Ampicillin (100mg/ml), X-gal (20mg/ml), and
IPTG (100 mM). Then positive clones were sequenced using
M13 FWD primer. Clones are sequenced using automated
sequencer (ABI 3500) at Department of Biotechnology,
Assam University.
PčĞđĔČĊēĊęĎĈ AēĆđĞĘĎĘ ċĔė ĒĈėA ČĊēĊ
Methyl Coenzyme Reductase-A (mcrA) sequences obtained
from land ill site in this study were checked for vector
contamination using VecScreen tool/ NCBI. Then sequences
showing similarity with vector sequences from both ends
were trimmed and were searched for similarity using BLAST
[25]. After performing BLAST, sequences showing similarity
above 85–95% were used and aligned using CLUSTAL X 2.0.
Yadav, Maitra and Ghosh
The phylogenetic relatedness among clones was estimated
using the Maximum Likelihood Tree using T92+I model with
2000 bootstrap value. For model selection Bayesian analysis
was performed and the model with lowest BIC value (i.e.
3090.594) was chosen for tree construction. All positions
containing gaps and missing data were eliminated from
the dataset (complete deletion option). The Phylogenetic
analysis was carried out using MEGA software version
6.0 [26].
RESULTS AND DISCUSSION
DNA ĎĘĔđĆęĎĔē Ćēĉ PCR
DNA was extracted from leachate samples collected
from eight different locations of Delhi land ill site
Fig. 1. Metagenomic DNA was extracted on the same day of
sampling. The purity of extracted DNA (i.e.260/280) was
in range of 1.6-1.9 and concentration from 124.49ng/μl to
166.80ng/μl.
IDENTIFICATION OF METHANOGENS
FROM LANDFILL BY MCRA GENE CLONING
AND SEQUENCING
Nine mcrA partial cloned sequences were developed in this
study, out of which two sequences were used for phylogenetic
analysis in present study. The accession numbers of partial
nucleotide sequences of mcrA genes were KM041254
and KM041258 respectively. Two major clusters were
observed in the phylogenetic tree i.e. Fig. 3. In irst cluster,
clone BMCR2.KM041258 clustered with Methanoculleus
marisngri JR1 and Uncultured archaeon clones belonging to
order Methanomicrobiales. In second cluster, clone MCR2.
KM041254 clustered with uncultured archaeon clones and
Uncultured Methanobacteriales archaeon clones belonging
to order Methanobacteriales. Methanogens belonging to
order Methanobacteriales and Methanomicrobiales carry
out hydrogenotrophic pathway for methanogenesis. From
this we can conclude that hydrogenotrophic methanogens
are abundant in the land ill site and it may be the main
pathway for methane emission from land ill site.
Fig. 1: Showing Metagenomic DNA Isolated from the
Landϐill Site
Fig. 2: mcrA Gene Ampliϐication of Genomic DNA
Isolated from Landϐill Sites
Out of eight samples from which Metagenomic DNA
was extracted, seven of them gave positive mcrA gene
ampli ication using primer Luton speci ic for mcrA gene as
shown in Fig. 2. Lane 1 and 7 contain 100bp DNA ladder. L2
to L5 contain mcrA gene amplicons from leachate samples
G2 to G5 and Lane 8,9 and 11 contain mcrA gene amplicons
from leachate samples G6-G8.
200
Fig. 3: Shows Maximum Likelihood Tree of mcrA
Sequences Obtained from MSW Leachate Samples of
Landϐill Site, Delhi using Mega6.0
The earth harbors a huge prokaryotic diversity, and they
act as key functional drivers of our planet’s ecosystems [27].
Yet the diversity and interdependencies of these microscopic
organisms (such as methanogens) remain largely unknown
and our understanding of the functional potential of most
individual microbial taxa residing within any ecosystem is
extremely limited [28, 29].
Metagenomics (culture independent) holds enormous
potential for discovering novel organisms that are
drivers of processes relevant to disease, industry and
the environment [30, 31]. The term metagenomics was
coined by Jo Handelsman [32, 33]. Metagenomics can be
de ined as a molecular and computational approach for
Cloning and Sequencing of Methyl-coenzyme Reductase
understanding the microbial community structure, and its
functional potential associated with an ecosystem [34]. As
metagenomics seeks to understand microbial ecosystem
by studying the genome content of constituent microbes in
their natural habitat. It provides a relatively unbiased view
not only of the community structure (species richness and
distribution) but also of the functional (metabolic) potential
in a community [28] [30].
But, even culture independent/ metagenomic approach
can be also biased by the choice of method of DNA or RNA
extraction used which in turn can bias microbial diversity
studies. Since, these approaches are based upon both
quality and quantity of metagenomic (environmental
DNA) extracted. Harsh extraction methods, such as bead
beating, can shear the nucleic acids, leading to problems in
subsequent PCR detection. Different methods of nucleic acid
extractions may result in different yields of product. With
environmental samples, it is necessary to remove inhibitory
substances such as humic acids, which can be coextracted
and interfere with PCR analysis. Subsequent puri ication
steps can also lead to loss of DNA or RNA, again potentially
biasing molecular diversity analysis. Therefore, in present
study considering above mentioned facts, we irst optimized
the method of metagenomic DNA isolation from the leachate
samples of land ill site, Delhi.
ACKNOWLEDGEMENT
We are grateful to Department of Biotechnology (DBT) of
the government of India for providing a grant (BT/14NE/
TBP/2010).
REFERENCES
1.
2.
3.
4.
5.
6.
CONCLUSION
7.
In short, global warming and water pollution is among one
of the major causes of concern to humanity in this modern
era. Water vapour, carbon dioxide (CO2), methane (CH4) and
nitrous oxide (N2O) are the most important greenhouse
gases, and human activities such as industry, livestock and
agriculture, contribute to the production of these gases.
Land ill cover soils plays an important role in the CH4 cycle
as methanotrophy (oxidation of CH4) and methanogenesis
(production of CH4) take place in them. The information
about methanogenesis and microbes associated will enrich
our knowledge about how land ill and human activity
contribute to greenhouse gas emission and global warming.
It will also be useful in establishing new agriculture
techniques and industrial processes that will be ecofreindly
and may provide a better balance of greenhouse gas. Culture
independent PCR based molecular studies was performed
for detection of methanogens presence. Our future objective
will be to do NGS based metagenomic study for elucidation
of methanogenic diversity present in the land ill site,
effect of seasonal variations on methanogens and methane
emission potential.
8.
201
9.
10.
11.
12.
13.
14.
Garrity, G.M., Bell, J.A., Lilburn, T.G. 2004. Taxonomic outline of
the prokaryotes. Bergey’s manual of systematic bacteriology.
Springer, New York, Berlin, Heidelberg.
Bapteste, É., Brochier, C., Boucher, Y. 2005. Higher-level
classi ication of the Archaea: evolution of methanogenesis
and methanogens. Archaea 1:353-363.
Morris, R., Schauer-Gimenez, A., Bhattad, U., Kearney, C.,
Struble, C.A., Zitomer, D., Maki, J.S. 2014. Methyl coenzyme
M reductase (mcrA) gene abundance correlates with activity
measurements of methanogenic H2/CO2-enriched anaerobic
biomass. Microb. biotech. 7:77-84.
Alvarado, A., Montañez-Hernández, L.E., Palacio-Molina,
S.L., Oropeza-Navarro, R., Luévanos-Escareño, M.P.,
Balagurusamy, N. 2014. Microbial trophic interactions and
mcrA gene expression in monitoring of anaerobic digesters.
Front. microbiol. 5.
Wongnate, T., Ragsdale, S.W. 2015. The Reaction Mechanism
of Methyl-Coenzyme M Reductase How an enzyme enforces
strict binding order. J. Biol. Chem. 290:9322-9334.
Luton, P.E., Wayne, J.M., Sharp, R.J., Riley, P.W. 2002. The
mcrA gene as an alternative to 16S rRNA in the phylogenetic
analysis of methanogen populations in land ill. Microbiology
148:3521-3530.
Juottonen, H., Galand, P.E., Yrjälä, K. 2006. Detection of
methanogenic Archaea in peat: comparison of PCR primers
targeting the mcrA gene. Res. Microbiol. 157:914-921.
Steinberg, L.M., Regan, J.M. 2008. Phylogenetic comparison of
the methanogenic communities from an acidic, oligotrophic
fen and an anaerobic digester treating municipal wastewater
sludge. Appl.Enviro.Micro. 74:6663-6671.
Lelieveld, J., Crutzen, P.J., Dentener, F.J. 1998. Changing
concentration, lifetime and climate forcing of atmospheric
methane. Tellus B 50:128-150.
Houghton, J.T., Ding, Y., Griggs, D.J., Noguer, M., van der
LINDEN, P.J., Dai, X., Maskell, K., Johnson, C. 2001. Climate
change 2001: the scienti ic basis, vol 881. Cambridge
university press Cambridge.
Staley, B.F., Francis, L., Barlaz, M.A. 2011. Effect of spatial
differences in microbial activity, pH, and substrate levels on
methanogenesis initiation in refuse. Appl Environ Microbiol
77:2381-2391.
Gupta, S., Choudhary, N., Alappat, B.J. Bioreactor land ill for
MSW Disposal in Delhi, p 474-481. In (ed),
Hansen, L.B., Finster, K., Fossing, H., Iversen, N. 1998.
Anaerobic methane oxidation in sulfate depleted sediments:
effects of sulfate and molybdate additions. Aquat. Microb.
Ecol. 14:195-204.
Mayumi, D., Dol ing, J., Sakata, S., Maeda, H., Miyagawa,
Y., Ikarashi, M., Tamaki, H., Takeuchi, M., Nakatsu, C.H.,
Kamagata, Y. 2013. Carbon dioxide concentration dictates
alternative methanogenic pathways in oil reservoirs. Nat.
comm. 4.
Yadav, Maitra and Ghosh
15.
16.
17.
18.
19.
20.
21.
22.
23.
Gründger, F., Jiménez, N., Thielemann, T., Straaten, N., Lüders,
T., Richnow, H.-H., Krüger, M. 2015. Microbial methane
formation in deep aquifers of a coal-bearing sedimentary
basin, Germany. Front. microbiol. 6.
Watanabe, T., Kimura, M., Asakawa, S. 2006. Community
structure of methanogenic archaea in paddy ield soil
under double cropping (rice–wheat). Soil Biol. Biochem. 38:
1264-1274.
Watanabe, T., Kimura, M., Asakawa, S. 2007. Dynamics of
methanogenic archaeal communities based on rRNA analysis
and their relation to methanogenic activity in Japanese
paddy ield soils. Soil Biol. Biochem. 39:2877-2887.
Watanabe, T., Kimura, M., Asakawa, S. 2010. Diversity of
methanogenic archaeal communities in Japanese paddy
ield ecosystem, estimated by denaturing gradient gel
electrophoresis. Biol. Fertility Soils 46:343-353.
Rawat, M., Ramanathan, A. 2011. Assessment of methane lux
from municipal solid waste (MSW) land ill areas of Delhi,
India. Journ.Environ. Prot. 2:399.
Bridgham, S.D., Cadillo-Quiroz, H., Keller, J.K., Zhuang, Q.
2013. Methane emissions from wetlands: biogeochemical,
microbial, and modeling perspectives from local to global
scales. Global Change Biol. 19:1325-1346.
Yadav, S., Maitra, S., Pal, S., Singh, N., Gupta, S., Ghosh, S.,
Sreekishnan, T. 2014. Accumulation of Lactic Acid during
Biodigestion of Municipal Solid Waste Leachate and
Identi ication of Indigenous Lactic Acid Bacteria in Leachate.
Journal of Hazardous, Toxic, and Radioactive Waste.
Talyan, V., Dahiya, R., Anand, S., Sreekrishnan, T. 2007.
Quanti ication of methane emission from municipal solid waste
disposal in Delhi. Resources, conservation and recycling 50:
240-259.
Bloom, A.A., Palmer, P.I., Fraser, A., Reay, D.S., Frankenberg, C.
2010. Large-scale controls of methanogenesis inferred from
methane and gravity spaceborne data. Science 327:322-325.
202
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
Singh, V., Mittal, A. Toxicity Analysis and Public Health
Aspects of Municipal Land ill Leachate: A Case Study of Okhla
Land ill, Delhi, p. In (ed),
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J.
1990. Basic local alignment search tool. J. Mol. Biol. 215:
403-410.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S.
2013. MEGA6: Molecular Evolutionary Genetics Analysis
Version 6.0. Mol. Biol. Evol. 30:2725-2729.
Tringe, S.G., Rubin, E.M. 2005. Metagenomics: DNA
sequencing of environmental samples. Nature reviews
genetics 6:805-814.
Hugenholtz, P., Tyson, G.W. 2008. Microbiology:
metagenomics. Nature 455:481-483.
Bardgett, R.D., van der Putten, W.H. 2014. Belowground
biodiversity and ecosystem functioning. Nature 515:
505-511.
Knight, R., Jansson, J., Field, D., Fierer, N., Desai, N., Fuhrman,
J.A., Hugenholtz, P., van der Lelie, D., Meyer, F., Stevens, R.
2012. Unlocking the potential of metagenomics through
replicated experimental design. Nat. Biotechnol. 30:513-520.
Nesme, J., Cécillon, S., Delmont, T.O., Monier, J.-M., Vogel, T.M.,
Simonet, P. 2014. Large-Scale Metagenomic-Based Study
of Antibiotic Resistance in the Environment. Curr. Biol.
24:1096-1100.
Handelsman, J. 2004. Metagenomics: application of genomics
to uncultured microorganisms. Microbiol. Mol. Biol. Rev. 68:
669-685.
Handelsman, J., Rondon, M.R., Brady, S.F., Clardy, J., Goodman,
R.M. 1998. Molecular biological access to the chemistry of
unknown soil microbes: a new frontier for natural products.
Chem. Biol. 5:245-249.
Handelsman, J. 2007. Metagenomics and microbial
communities.
eLS.
DOI:
10.1002/9780470015902.
a0020367.