Post on 08-Mar-2023
R E S EA RCH L E T T E R
Analysis of [FeFe]-hydrogenase genes for the elucidation of ahydrogen-producing bacterial community in paddy field soil
Ryuko Baba, Makoto Kimura, Susumu Asakawa & Takeshi Watanabe
Laboratory of Soil Biology and Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan
Correspondence: Ryuko Baba, Laboratory of
Soil Biology and Chemistry, Graduate School
of Bioagricultural Sciences, Nagoya
University, Chikusa 464-8601, Nagoya,
Japan. Tel.: +81 52 789 5323;
fax: +81 52 789 4136;
e-mail: baba.ryuko@c.mbox.nagoya-u.ac.jp
Present address: Makoto Kimura, Food and
Agricultural Materials Inspection Center
(FAMIC), Saitama Shintoshin National
Government Building, Kensato Building 2-1,
Shintoshin, Chuo-ku, Saitama-shi, Saitama
330-9731, Japan
Received 28 July 2013; revised 3 September
2013; accepted 12 November 2013. Final
version published online 11 December 2013.
DOI: 10.1111/1574-6968.12335
Editor: Tim Daniell
Keywords
Firmicutes; Deltaproteobacteria; Chloroflexi;
rice paddy soil; functional gene analysis;
hydrogen producers.
Abstract
Hydrogen (H2) is one of the most important intermediates in the anaerobic
decomposition of organic matter. Although the microorganisms consuming
H2 in anaerobic environments have been well documented, those producing H2
are not well known. In this study, we elucidated potential members of
H2-producing bacteria in a paddy field soil using clone library analysis of
[FeFe]-hydrogenase genes. The [FeFe]-hydrogenase is an enzyme involved in
H2 metabolism, especially in H2 production. A suitable primer set was selected
based on the preliminary clone library analysis performed using three primer
sets designed for the [FeFe]-hydrogenase genes. Soil collected in flooded and
drained periods was used to examine the dominant [FeFe]-hydrogenase genes
in the paddy soil bacteria. In total, 115 and 108 clones were analyzed from the
flooded and drained paddy field soils, respectively. Homology and phylogenetic
analysis of the clones showed the presence of diverse [FeFe]-hydrogenase genes
mainly related to Firmicutes, Deltaproteobacteria, and Chloroflexi. Predominance
of Deltaproteobacteria and Chloroflexi suggests that the distinct bacterial
community possessed [FeFe]-hydrogenase genes in the paddy field soil. Our
study revealed the potential members of H2-producing bacteria in the
paddy field soil based on their genetic diversity and the distinctiveness of the
[FeFe]-hydrogenase genes.
Introduction
Anaerobic decomposition of organic matters is accom-
plished through complex pathways with diverse anaer-
obes, which utilize organic and/or inorganic substances
other than molecular oxygen as electron acceptors
(Schink, 1997). Molecular hydrogen (H2) is one of the
most important intermediates in the anaerobic decompo-
sition processes. As the production and competitive con-
sumption of H2 regulate the decomposition pathways, for
example syntrophy with interspecies electron transfer
between H2 producers and consumers (Schink, 1997;
McInerney et al., 2008) and competition among diverse
anaerobes such as iron reducers, sulfate reducers, and
methanogens (Robinson & Tiedje, 1984; Conrad, 1999),
elucidation of H2-producing and consuming processes
and the related microorganisms are essential to under-
stand anaerobic decomposition of organic matter.
Hydrogenases are the enzymes that catalyze the produc-
tion and consumption of H2 (Vignais & Billoud, 2007).
They are classified into three groups depending on the
metal composition in their active site: [FeFe]-, [NiFe]-, and
[Fe]- (formerly called ‘metal-free’) hydrogenases (Vignais
& Billoud, 2007). [FeFe]-hydrogenases have been found in
anaerobic bacteria and eukaryotes and are known to cata-
lyze H2-forming reaction in anaerobic environments,
although some types of [FeFe]-hydrogenases (i.e. periplas-
mic hydrogenases in Desulfovibrio) are considered to cata-
lyze oxidation of H2 (Meyer, 2007; Vignais & Billoud,
2007). Recently, three sets of primers have been designed
FEMS Microbiol Lett 350 (2014) 249–256 ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
MIC
ROBI
OLO
GY
LET
TER
SD
ownloaded from
https://academic.oup.com
/femsle/article/350/2/249/565945 by guest on 02 June 2022
based on the sequences of the gene encoding the H-cluster
domain of [FeFe]-hydrogenase ([FeFe]-hydrogenase gene)
and have been used to elucidate H2-producing bacterial
communities present in anaerobic environments, for exam-
ple hydF1/hydR1 in bioreactors (Xing et al., 2008), FeFe-
272F/FeFe-427R in saline microbial mat (Boyd et al., 2009)
and Yellowstone National Park (Boyd et al., 2010), and
HydH1f/HydH3r in a moderately acidic fen (Schmidt
et al., 2010) and earthworm gut (Schmidt et al., 2011). In
addition, there have been studies on [FeFe]-hydrogenase
genes in termite gut, which have used metagenomic and
genomic information (Ballor & Leadbetter, 2012; Ballor
et al., 2012). These analyses have provided novel findings
to elucidate the diversity and functions of H2-producing
bacterial communities in various environments.
Irrigated paddy fields, which are flooded during most
periods of rice cultivation, are one of the well-investigated
methanogenic environments for methane emission (Le
Mer & Roger, 2001), soil reduction processes (Takai &
Kamura, 1966), carbon flow (Kimura et al., 2004), and
microbial community (Liesack et al., 2000; Asakawa &
Kimura, 2008). In previous studies, microbial communi-
ties that are involved in electron-accepting processes such
as sulfate reducers (Liu et al., 2009) and methanogenic
archaea (Watanabe et al., 2009) have been studied. How-
ever, information on H2-producing bacterial communities
in paddy field soil is lacking.
We aimed to evaluate the potential members of
H2-producing bacteria in paddy field soil using molecular
biology techniques with specific emphasis on studying the
[FeFe]-hydrogenase genes. We examined the applicability
of three available primer sets that amplify [FeFe]-hydrog-
enase genes. Next, we studied the diversity and character-
istics of [FeFe]-hydrogenase genes in the microorganisms
present in the paddy field soil by clone library analysis
using the short-listed primer set to estimate potential
H2-producing bacterial community.
Materials and methods
Soil samples
Soil samples were collected from the paddy field located
at the Aichi-ken Anjo Research and Extension Center,
central Japan (Anjo field; latitude 34°8′N, longitude
137°5′E), on April 14, 2011 and were used for examining
of three primer sets. To evaluate the diversity of [FeFe]-
hydrogenase genes in the paddy field soil, two soil sam-
ples were taken from the same Anjo field on April 11,
2003 under drained condition and on July 28, 2003 under
flooded condition, and these were used as a representative
soil sample for clone library analysis with the selected pri-
mer set. Bacterial (Kikuchi et al., 2007) and methanogen-
ic archaeal (Watanabe et al., 2006) communities in the
same field soil were investigated with DGGE analysis for
16S rRNA gene (16S rDNA). Chemical properties of the
Anjo soil were as follows: total C, 12.6 g kg�1; total N,
1.1 g kg�1; pH [H2O], 5.8; free iron content, 11.0 g kg�1.
The soil was classified as Oxyaquic Dystrudept (Soil Sur-
vey Staff, 1999) with light clay texture. The field has been
managed with double cropping of rice or soybean and
wheat as summer and winter cultivation, respectively.
Details of the field managements in 2003 were described
by Watanabe et al. (2006). About 1 kg of the composite
soil samples was taken from three or four spots from the
plow layer (0–10 cm) using a trowel and was transferred
into a polyethylene bag. The soil samples were then
passed through a 2-mm mesh sieve, mixed thoroughly,
and stored at 4 °C until use. The samples were normally
subjected to DNA extraction on the same or next day of
sampling.
DNA extraction
DNA extraction from the soil samples collected in 2003
was performed four times according to the beads-beating
method, and the DNA samples were purified with Sepha-
dex G-200 as described in the previous studies (Cahyani
et al., 2003; Watanabe et al., 2004). ISOIL for beads beat-
ing (Nippon Gene, Tokyo, Japan) was used for DNA
extraction, performed three times, from the soil sample
collected in 2011 according to the manufacturer’s instruc-
tion. The DNA samples were appropriately diluted with
TE (10 mM Tris-HCl, 1 mM EDTA, pH8.0) buffer for
the subsequent PCR assays depending on the DNA
concentration of the samples.
PCR amplification of [FeFe]-hydrogenase gene
Three degenerate primer sets, hydF1 (5′-GCCGACCTKACMATMATGGA-3′)/hydR1 (5′-ATRCARCCRCCSGGRCAGGCCAT-3′) (Xing et al., 2008), FeFe-272F (5′-GCHGAYM
TBACHATWATGGARGA-3′)/FeFe-427R (5′-GCNGCYTCCATDACDCCDCCNGT-3′) (Boyd et al., 2009), and
HydH1f (5′-TTIACITSITGYWSYCCIGSHTGG-3′)/HydH3r
(5′-CAICCIYMIGGRCAISNCAT-3′) (Schmidt et al., 2010),
which target the [FeFe]-hydrogenase gene, were examined.
The PCR conditions and primer concentrations were mod-
ified slightly from the original program (Supporting Infor-
mation, Table S1). All reaction mixtures contained 5 lL of
109 Ex TaqTM buffer (20 mM Mg2+ plus, TaKaRa, Otsu,
Japan), 5 lL of dNTPs (2.5 mM each, TaKaRa), 0.25 lL of
Ex TaqTM polymerase (5 U lL�1, TaKaRa), and 4 lL of
template DNA, and the reaction volume was made up to
50 lL with sterilized ultrapure water. The amplicons
were checked by agarose gel electrophoresis followed by
FEMS Microbiol Lett 350 (2014) 249–256ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
250 R. Baba et al.
Dow
nloaded from https://academ
ic.oup.com/fem
sle/article/350/2/249/565945 by guest on 02 June 2022
ethidium bromide staining. The PCR amplifications were
performed with triplicate or quadruple DNA samples. The
amplified product from one replicate was used for the com-
parison of the three primer sets, and the amplicons of the
mixed four replications were used for the subsequent
analysis with the selected primer set.
Cloning and sequencing analysis
Each PCR product was purified using NucleoSpin Extract
II (Macherey-Nagel, D€uren, Germany) and cloned into
pT7-Blue-T-Vector (Novagen, Darmstadt, Germany) with
Ligation Solution I (Takara). Escherichia coli XL1-Blue
competent cells (Toyobo, Osaka, Japan) were transformed
with ligated vectors. Colonies carrying positive clones were
confirmed by blue/white selection and by colony PCR with
the same primer set used for [FeFe]-hydrogenase genes.
Plasmid DNA was extracted using the alkaline extraction
method or using the Zyppy Plasmid Miniprep Kit (Zymo
Research, CA). Sequencing analysis was carried out using
the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied
Biosystems, CA) and was carried out on ABI PRISM 3130
Genetic Analyzer (Applied Biosystems) or outsourced to
Hitachi Solutions (Yokohama, Japan) with Applied Biosys-
tems 3730 DNA Analyzer (Applied Biosystems).
Nucleotide sequences obtained from the clones were
translated to amino acid sequences using the European
Molecular Biology Open Software Suite (EMBOSS) Transeq
program (Rice et al., 2000). Closest relatives of the in
silico-translated [FeFe]-hydrogenase amino acid sequences
were searched using the Basic Local Alignment Search
Tool (BLAST) program (Altschul et al., 1990) on the
National Center for Biotechnology Information web site.
The sequences were aligned using the CLUSTALW 1.83
(Chenna et al., 2003) on the DNA Data Bank of Japan
(DDBJ) web site (http://clustalw.ddbj.nig.ac.jp) with default
parameters, and then, phylogenetic trees were constructed
by the neighbor-joining method on the DDBJ web site.
Unweighted UNIFRAC analysis (Lozupone & Knight, 2005)
was performed using the MOTHUR 1.23.0 (Schloss et al.,
2009). Criterion of operational taxonomic units (OTUs)
and Chao1 indices (Chao et al., 2009) was determined in
the threshold of 80% sequence similarity by the MOTHUR
according to the previous study (Schmidt et al., 2011).
Coverage was calculated according to this formula OTU/
Chao1 9 100.
Accession numbers
The nucleotide sequences of [FeFe]-hydrogenase genes
determined in the clone libraries have been submitted to
the DDBJ database under accession numbers AB760556–AB760947.
Results and discussion
Comparison between the three primer sets for
analysis of [FeFe]-hydrogenase genes
Detection ranges of the three different primer sets (hydF1/
hydR1, FeFe-272F/FeFe-427R, and HydH1f/HydH3r)
designed in the previous studies were compared. Either
blurred, nonspecific or no band was observed when the
original PCR conditions were applied to the paddy soil
sample, expected lengths of PCR amplicons (c. 700, 450,
and 600 bp for hydF1/hydR1, FeFe-272F/FeFe-427R, and
HydH1/HydH3r, respectively) were successfully obtained
after modification of the PCR conditions (data not
shown). We analyzed 52, 54, and 61 clones obtained from
the hydF1/hydR1, FeFe-272F/FeFe-427R, and HydH1f/
HydH3r libraries. Three sequences (two in the hydF1/
hydR1 library and one in the HydH1f/HydH3r library)
did not seem to associate with the [FeFe]-hydrogenases
and therefore were omitted for the subsequent analyses.
Almost all clones showed a preserved L2 sequence motif
(PCxxKxxE; Vignais & Billoud, 2007; Meyer, 2007),
although four clones showed a few substituted amino
acids (hydF1_1, 17, and 37 showed CCTAKKYE, and
HydH1f_10 showed PSTAKKFE; the substituted amino
acids are underlined). In this study, the criterion of OTU
was set at a threshold of 80% amino acid sequence simi-
larity (Schmidt et al., 2011). The numbers and values of
OTUs/Chao1/coverage (%) were 32/74/43 (hydF1/hydR),
20/25/80 (FeFe-272F/FeFe-427R), and 23/36/64 (HydH1f/
HydH3r). The UNIFRAC analysis showed significant differ-
ence (P < 0.0010) between the HydH1f/HydH3r library
and the other two libraries.
Protein BLAST analysis of the deduced amino acid
sequences of the clones showed similar detection ranges of
[FeFe]-hydrogenase genes among the primer sets (Table
S2–S4). Most of the clones in the same OTU aligned with
the same taxonomic group, suggesting 80% similarity level
is an acceptable range in this study. The clones which were
closely related to the [FeFe]-hydrogenase genes in Firmi-
cutes, Chloroflexi, Proteobacteria, Spirochetes, Bacteroidetes,
and Caldithrix were amplified using the same three primer
sets. A few clones were closely related to the [FeFe]-
hydrogenase genes in Acidobacteria, Elusimicrobia, and
Verrucomicrobia. In all the primer sets, the clones related
to the [FeFe]-hydrogenase genes in Firmicutes, Chloroflexi,
Proteobacteria, and Bacteroidetes accounted for more than
half of the clones.
All the primer sets showed similar detection ranges and,
in this sense, were applicable for analyzing H2-producing
bacterial communities in paddy field soil. However, the
UNIFRAC analysis showed a significant difference for the pri-
mer set HydH1f/HydH3r from the rest. In addition, this
FEMS Microbiol Lett 350 (2014) 249–256 ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
H2 producers in paddy soil estimated by [FeFe]-H2ase gene 251
Dow
nloaded from https://academ
ic.oup.com/fem
sle/article/350/2/249/565945 by guest on 02 June 2022
primer set was designed to detect the widest range of
[FeFe]-hydrogenase genes from the largest number of
[FeFe]-hydrogenase genes (Schmidt et al., 2010). Thus, for
further analysis, we decided to use the primer set HydH1f/
HydH3r.
Genetic diversity and characteristics of [FeFe]-
hydrogenase genes in paddy field soil
Clone library analysis of [FeFe]-hydrogenase genes in the
Anjo paddy field soil showed that diverse microorganisms
harboring the [FeFe]-hydrogenase genes could be retrieved
from the soil. In this study, two soil samples collected in
both flooded and drained periods were used as the repre-
sentative soil conditions to evaluate the diversity and
characteristics of [FeFe]-hydrogenase genes in the paddy
field soil. In total, 115 and 108 clones were obtained from
the soil samples collected under flooded and drained
conditions, respectively. All obtained sequences showed
similarity with the [FeFe]-hydrogenase sequence. The L2
sequence motif (see above) of each clone was found, and
only two clones had a single amino acid substitution
(e20411052 had CCTCKKAE, and e20411057 had
PCMAMKFE; substituted amino acids are underlined). The
indices of OTUs/Chao1/coverage (%) were 41/72/67 under
the flooded condition, 34/64/53 under the drained condi-
tion, and 57/99/58 in total. The UNIFRAC analysis showed no
significant difference between these two libraries. The num-
ber of unique OTUs, which consisted of single clone, was
24 and 21. The numbers of total OTUs, unique OTUs, and
Chao1 richness suggest that diverse [FeFe]-hydrogenase-
producing bacteria exist in both flooded and drained paddy
soils. The protein BLAST analysis (Tables S5 and S6) and
phylogenetic tree (Fig. 1) showed most clones obtained in
the present study were closely related to Firmicutes, Chloro-
flexi, and Proteobacteria (Table 1 and Fig. 1). Most clones
affiliated with Firmicutes and Proteobacteria were closely
related to Clostridia and Deltaproteobacteria (Fig. 1 and
Table 1). All clones affiliated with Chloroflexi were closely
related to Dehalococcoides (Fig. 1 and Table 1). Therefore,
these bacteria are speculated to be possible H2 producers in
the paddy field soil, although some [FeFe]-hydrogenases
catalyze H2 consumption rather than H2 production in
some occasions (see below), and these results should be
interpreted with caution as already mentioned in the previ-
ous study (Schmidt et al., 2010) as the phylogeny of
[FeFe]-hydrogenase gene is slightly different from
that of 16S rRNA gene. The top hits of the BLAST search of
the 93 and 94 clones obtained from the flooded and
drained paddy soils were uncultured environmental clones
(Table S5 and Table S6), indicating the presence of
diverse unknown [FeFe]-hydrogenase genes in the paddy
field soil.
Phylogenetic affiliation of [FeFe]-hydrogenase
genes
Most of [FeFe]-hydrogenase clones in Firmicutes were
related to the orders of Clostridiales. The Clostridiales
group consisted of clones related to various genera such
as Clostridium, Desulfotomaculum, and Pelotomaculum,
members having an ability of growing syntrophically with
methanogens (McInerney et al., 2008).
All clones that aligned with Deltaproteobacteria were
related to sulfate reducers except one clone that aligned
with Syntrophus. These clones were affiliated with the fami-
lies of Pelobacteriaceae and Desulfovibrionaceae which
include bacteria that are able to grow syntrophically with
H2-consuming bacteria (McInerney et al., 2008). Only one
clone in our study was closely related to Syntrophus acidi-
trophicus in Syntrophobacteriales, which exhibits obligate
syntrophic growth by interspecies H2 transfer (Jackson
et al., 1999). Although the periplasmic [FeFe]-hydrogenase
of Desulfovibrio catalyzes H2 consumption during the sul-
fate reduction process (Vignais & Billoud, 2007), some of
those [FeFe]-hydrogenases have a function to produce H2
under syntrophic conditions (Meyer et al., 2013), suggest-
ing that [FeFe]-hydrogenases of those sulfate reducers may
be bifunctional depending on the growth conditions. Delta-
proteobacteria was not a dominant group in the other envi-
ronments tested (Boyd et al., 2009, 2010; Schmidt et al.,
2010, 2011). As sulfate reducers are one of the key players
in the carbon cycles in paddy field soil, they may have
important functions for H2 metabolism in paddy field soil.
Clones belonging to Chloroflexi, Deltaproteobacteria, and
Firmicutes were also retrieved from the DGGE analysis of
bacterial 16S rRNA gene (Kikuchi et al., 2007). Therefore,
bacteria belonging to these three groups may be one of
the major groups of the bacterial community and play an
important role for H2 production in the paddy field soil.
In addition, Firmicutes and Deltaproteobacteria, especially
Firmicutes, were frequently detected in nutrient-rich spots,
such as rice straw (Weber et al., 2001), rice straw compost
(Tanahashi et al., 2005), plant residues (Matsuyama et al.,
2007; Rui et al., 2009), rhizospheric soil and roots (Shres-
tha et al., 2011). These findings suggest that Firmicutes
and Deltaproteobacteria actively produce H2 in nutrient-
rich sites in paddy field soil.
Dehaloccoides, which was the sole group belonging to
Chloroflexi detected in the present study, is able to cata-
lyze reductive dehalogenation using H2 as electron donors
(He et al., 2003), while syntrophic growth with hydro-
genotrophic methanogens was reported for the species in
Chloroflexi isolated from paddy field soil (Yamada et al.,
2007). No [FeFe]-hydrogenase gene related to Chloroflexi
was detected in the other environments tested (Boyd
et al., 2009, 2010; Schmidt et al., 2010, 2011) except in a
FEMS Microbiol Lett 350 (2014) 249–256ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
252 R. Baba et al.
Dow
nloaded from https://academ
ic.oup.com/fem
sle/article/350/2/249/565945 by guest on 02 June 2022
reductive dechlorinating soil column (Marshall et al.,
2012). Therefore, Chloroflexi may be unique among the
H2-producing bacteria in the paddy field soil, and eluci-
dation of the roles of the members in Chloroflexi in H2
production and consumption in paddy field soil needs
further investigations.
Small numbers of clones affiliated with Verrucomicro-
biae were also detected in the present study and DGGE
analysis of bacterial 16S rRNA gene not only in the bulk
soil (Kikuchi et al., 2007) but also in rice straw (Sugano
et al., 2005), rice straw compost (Tanahashi et al., 2005),
and plant residue (Rui et al., 2009). Opitutus terrae of
Verrucomicrobia was abundant in paddy field soil (Chin
et al., 1999) and was found to produce H2 (Chin et al.,
2001), indicating that this group may also participate in
H2 production in paddy fields.
We examined [FeFe]-hydrogenase genes in the paddy
field soil using molecular biology techniques. Members of
0 5 10
69
1520
Fig. 1. Neighbor-joining tree of [FeFe]-hydrogenase gene sequences obtained from two clone libraries (drained and flooded paddy field soil) and
reference sequences. The primer set HydH1f/HydH3r (Schmidt et al., 2010) was used to obtain two libraries. GenBank accession numbers are
indicated in parentheses. The number of resampling is 1000 for the bootstrap analysis, and the number of bootstrap values above 500 (closed
circles) is shown. Sequences were grouped into 57 different OTUs based on an amino acid sequence with the threshold sequence similarity of
80% (Schmidt et al., 2011). Representative sequences selected by MOTHUR (Schloss et al., 2009) are shown for each OTU. The bar indicates a 0.1
change per amino acid. The bar graph on the right side displays the number of clones included in each OTU. Phylogenetic assignment
represented besides brackets are based on the topology of the tree and the results of BLAST search.
FEMS Microbiol Lett 350 (2014) 249–256 ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
H2 producers in paddy soil estimated by [FeFe]-H2ase gene 253
Dow
nloaded from https://academ
ic.oup.com/fem
sle/article/350/2/249/565945 by guest on 02 June 2022
Firmicutes, Deltaproteobacteria, and Chloroflexi are pre-
sumably predominant H2 producers in the paddy field
soil, although some [FeFe]-hydrogenases, especially delta-
proteobacterial ones, may also be involved in H2 con-
sumption. Clones of Firmicutes were also detected in
other anaerobic environments, and Chloroflexi and Delta-
proteobacteria seem to be distinctive groups in the paddy
field soil. These results indicate that paddy field soil has a
unique H2-producing bacterial community. Further stud-
ies such as analyzing the temporal change and spatial dis-
tribution of the community, isolating the H2-producing
bacteria, and determining the roles of the [FeFe]-hydrog-
enases in sulfate reducers and Chloroflexi will be needed
to elucidate the dynamics of the microbial community
and its role in the anaerobic decomposition of organic
matters in paddy field soil.
Acknowledgements
The present study was supported by the Grant-in-Aid for
Young Scientists from the Japan Society for the Promo-
tion of Science. We thank H. Honjo and N. Saka of the
Anjo Research and Extension Station, Aichi-ken Agricul-
tural Research Center, Japan, for their help in collecting
the soil sample.
References
Altschul SF, GishW,Miller W, Myers EW& Lipman DJ (1990)
Basic local alignment search tool. J Mol Biol 215: 403–410.Asakawa S & Kimura M (2008) Comparison of bacterial
community structures at main habitats in paddy field
ecosystem based on DGGE analysis. Soil Biol Biochem 40:
1322–1329.Ballor NR & Leadbetter JR (2012) Patterns of [FeFe]
hydrogenase diversity in the gut microbial communities of
lignocellulose-feeding higher termites. Appl Environ
Microbiol 78: 5368–5374.Ballor NR, Paulsen I & Leadbetter JR (2012) Genomic analysis
reveals multiple [FeFe]-hydrogenases and hydrogen sensors
encoded by Treponemes from the H2-rich termite gut.
Microb Ecol 63: 282–294.Boyd ES, Spear JR & Peters JW (2009) [FeFe] hydrogenase
genetic diversity provides insight into molecular adaptation
in a saline microbial mat community. Appl Environ
Microbiol 75: 4620–4623.Boyd ES, Hamilton TL, Spear JR, Lavin M & Peters JW (2010)
[FeFe]-hydrogenase in Yellowstone National Park: evidence
for dispersal limitation and phylogenetic niche conservatism.
ISME J 4: 1485–1495.Cahyani VR, Matsuya K, Asakawa S & Kimura M (2003)
Succession and phylogenetic composition of bacterial
Table 1. Top three representative species in each class of closest relatives in clone libraries obtained by PCR with HydH1f/HydH3r (Schmidt et al.,
2010) and the number of clones affiliated with them
Phylum Class Species with strain name
Accession
no.
Identity, %
(similarity, %)
range
Number of clones
Flooded
paddy
soil
Drained
paddy
soil
Firmicutes 23 16
Clostridia Moorella thermoacetica ATCC 39073 ABC20019 45–74 (67–89) 6 1
Pelotomaculum thermopropionicum SI BAF60191 53–69 (71–84) 4 1
Acetivibrio cellulolyticus CD2 ZP_09466277* 50–78 (70–89) 2 2
Proteobacteria 51 33
Alphaproteobacteria Rhodopseudomonas palustris BisA53 ABJ07787 68 (81) 1 0
Deltaproteobacteria Desulfovibrio fructosovorans JJ EFL52165 56–73 (74–84) 28 13
Desulfovibrio magneticus RS-1 BAH74274 55–74 (69–86) 10 5
Pelobacter carbinolicus DSM 2380 ABA88877 82–83 (91–93) 3 4
Gammaproteobacteria Thiorhodococcus drewsii AZ1 EGV33414 70–71 (86) 2 1
Chloroflexi 31 49
Dehalococcoidetes Dehalococcoides sp. BAV1 ABQ16813 67–71 (81–86) 17 26
Dehalococcoides sp. VS ACZ61328 65–67 (83–85) 5 6
Dehalococcoides sp. CBDB1 CAI82422 68–69 (84–85) 3 6
Bacteroidetes Bacteroidia Odoribacter splanchnicus DSM 20712 ADY31293 70–73 (87–88) 3 1
Anaerophaga thermohalophila
DSM 12881
ZP_08845393* 70–74 (86–88) 2 2
Elusimicrobia Elusimicrobia Elusimicrobium minutum Pei191 ACC98088 62–68 (81–85) 3 3
Lentisphaerae – Victivallis vadensis ATCC BAA-548 EFA99820 72 (85) 0 1
Spirochetes Spirochaetia Spirochaeta smaragdinae DSM 11293 ADK79621 65–67 (79–80) 0 2
Spirochaeta thermophila DSM 6578 AEJ60920 39 (57) 0 1
Verrucomicrobia Opitutae Opitutus terrae PB90-1 ACB74828 81–83 (89) 2 0
*NCBI reference sequence accession number.
FEMS Microbiol Lett 350 (2014) 249–256ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
254 R. Baba et al.
Dow
nloaded from https://academ
ic.oup.com/fem
sle/article/350/2/249/565945 by guest on 02 June 2022
communities responsible for the composting process of rice
straw estimated by PCR-DGGE Analysis. Soil Sci Plant Nutr
49: 619–630.Chao A, Colwell RK, Lin CW & Gotelli NJ (2009) Sufficient
sampling for asymptotic minimum species richness
estimators. Ecology 90: 1125–1133.Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins
DG & Thompson JD (2003) Multiple sequence alignment
with the CLUSTAL series of programs. Nucleic Acids Res 31:
3497–3500.Chin K-J, Hahn D, Hengstmann U, Liesack W & Janssen PH
(1999) Characterization and identification of numerically
abundant culturable bacteria from the anoxic bulk soil of rice
paddy microcosms. Appl Environ Microbiol 65: 5042–5049.Chin K-J, Liesack W & Janssen PH (2001) Opitutus terrae gen.
nov., sp. nov., to accommodate novel strains of the division
‘Verrucomicrobia’ isolated from rice paddy soil. Int J Syst
Evol Microbiol 51: 1965–1968.Conrad R (1999) Contribution of hydrogen to methane
production and control of hydrogen concentrations in
methanogenic soils and sediments. FEMS Microbiol Ecol 28:
193–202.He J, Ritalahti KM, Yang K-L, Koenigsberg SS & L€offler FE
(2003) Detoxification of vinyl chloride to ethene coupled to
growth of an anaerobic bacterium. Nature 424: 62–65.Jackson BE, Bhupathiraju VK, Tanner RS, Woese CR &
McInerney MJ (1999) Syntrophus aciditrophicus sp. nov., a
new anaerobic bacterium that degrades fatty acids and
benzoate in syntrophic association with hydrogen-using
microorganisms. Arch Microbiol 171: 107–114.Kikuchi H, Watanabe T, Jia Z, Kimura M & Asakawa S (2007)
Molecular analyses reveal stability of bacterial communities
in bulk soil of a Japanese paddy field: estimation by
denaturing gradient gel electrophoresis of 16S rRNA genes
amplified from DNA accompanied with RNA. Soil Sci Plant
Nutr 53: 448–458.Kimura M, Murase J & Lu Y (2004) Carbon cycling in rice
field ecosystems in the context of input decomposition and
translocation of organic materials and the fates of their end
products (CO2 and CH4). Soil Biol Biochem 36: 1399–1416.Le Mer J & Roger P (2001) Production, oxidation, emission
and consumption of methane by soils: a review. Eur J Soil
Biol 37: 25–50.Liesack W, Schnell S & Revsbech NP (2000) Microbiology of
flooded rice paddies. FEMS Microbiol Rev 24: 625–645.Liu X-Z, Zhang L-M, Prosser JI & He J-Z (2009) Abundance
and community structure of sulfate reducing prokaryotes in
a paddy soil of southern China under different fertilization
regimes. Soil Biol Biochem 41: 687–694.Lozupone C & Knight R (2005) UNIFRAC: a new phylogenetic
method for comparing microbial communities. Appl Environ
Microbiol 71: 8228–8235.Marshall IPG, Berggren DRV, Azizian MF, Burow LC,
Semprini L & Spormann AM (2012) The hydrogenase chip:
a tiling oligonucleotide DNA microarray technique for
characterizing hydrogen-producing and -consuming
microbes in microbial communities. ISME J 6: 814–826.Matsuyama T, Nakajima Y, Matsuya K, Ikenaga M, Asakawa S
& Kimura M (2007) Bacterial community in plant residues
in a Japanese paddy field estimated by RFLP and DGGE
analyses. Soil Biol Biochem 39: 463–472.McInerney MJ, Struchtemeyer CG, Sieber J, Mouttaki H,
Stams AJM, Schink B, Rohlin L & Gunsalus RP (2008)
Physiology, ecology, phylogeny, and genomics of
microorganisms capable of syntrophic metabolism. Ann NY
Acad Sci 1125: 58–72.Meyer J (2007) [FeFe] hydrogenase and their evolution: a
genomic perspective. Cell Mol Life Sci 64: 1063–1084.Meyer B, Kuehl J, Deutschbauer AM, Price MN, Arkin AP &
Stahl DA (2013) Variation among Desulfovibrio species in
electron transfer systems used for syntrophic Growth. J
Bacteriol 195: 990–1004.Rice P, Longden I & Bleasby A (2000) EMBOSS: the european
molecular biology open software suite. Trends Genet 16:
276–277.Robinson JA & Tiedje JM (1984) Competition between
sulfate-reducing and methanogenic bacteria for H2 under
resting and growing conditions. Arch Microbiol 137: 26–32.Rui J, Peng J & Lu Y (2009) Succession of bacterial
populations during plant residue decomposition in rice field
soil. Appl Environ Microbiol 75: 4879–4886.Schink B (1997) Energetics of syntrophic cooperation in
methanogenic degradation. Microbiol Mol Biol Rev 61:
262–280.Schloss PD, Westcott SL, Ryabin T et al. (2009) Introducing
MOTHUR: open-source, platform-independent,
community-supported software for describing and
comparing microbial communities. Appl Environ Microbiol
75: 7537–7541.Schmidt O, Drake HL & Horn MA (2010) Hitherto unknown
[Fe-Fe]-hydrogenase gene diversity in anaerobes and anoxic
enrichments from a moderately acidic fen. Appl Environ
Microbiol 76: 2027–2031.Schmidt O, W€ust PK, Hellmuth S, Borst K, Horn MA &
Drake HL (2011) Novel [NiFe]- and [FeFe]-hydrogenase
gene transcripts indicative of active facultative aerobes and
obligate anaerobes in earthworm gut contents. Appl Environ
Microbiol 77: 5842–5850.Shrestha M, Shrestha PM & Conrad R (2011) Bacterial and
archaeal communities involved in the in situ degradation of13C-labelled straw in the rice rhizosphere. Environ Microbiol
Rep 3: 587–596.Soil Survey Staff (1999) Soil Taxonomy, a Basic System of Soil
Classification for Making and Interpreting Soil Surveys. 2nd
edn. United States Department of Agriculture Natural
Resources Conservation Service, Washington, DC.
SuganoA, TsuchimotoH, TunCC,Asakawa S&KimuraM (2005)
Succession and phylogenetic profile of eubacterial communities
in rice straw incorporated into a rice field: estimation by
PCR-DGGE analysis. Soil Sci PlantNutr 51: 51–60.
FEMS Microbiol Lett 350 (2014) 249–256 ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
H2 producers in paddy soil estimated by [FeFe]-H2ase gene 255
Dow
nloaded from https://academ
ic.oup.com/fem
sle/article/350/2/249/565945 by guest on 02 June 2022
Takai Y & Kamura T (1966) The mechanism of reduction in
waterlogged paddy soil. Folia Microbiol 11: 304–313.Tanahashi T, Murase J, Matsuya K, Hayashi M, Kimura M &
Asakawa S (2005) Bacterial communities responsible for the
decomposition of rice straw compost in a Japanese rice
paddy field estimated by DGGE analysis of amplified 16S
rDNA and 16S rRNA fragments. Soil Sci Plant Nutr 51:
351–360.Vignais PM & Billoud B (2007) Occurrence, classification, and
biological function of hydrogenases: an overview. Chem Rev
107: 4206–4272.Watanabe T, Asakawa S, Nakamura A, Nagaoka K & Kimura
M (2004) DGGE method for analyzing 16S rDNA of
methanogenic archaeal community in paddy field soil.
FEMS Microbiol Lett 232: 153–163.Watanabe T, Kimura M & Asakawa S (2006) Community
structure of methanogenic archaea in paddy field soil under
double cropping (rice–wheat). Soil Biol Biochem 38:
1264–1274.Watanabe T, Kimura M & Asakawa S (2009) Distinct
members of a stable methanogenic archaeal community
transcribe mcrA genes under flooded and drained conditions
in Japanese paddy field soil. Soil Biol Biochem 41: 276–285.Weber S, Stubner S & Conrad R (2001) Bacterial populations
colonizing and degrading rice straw in anoxic paddy soil.
Appl Environ Microbiol 67: 1318–1327.Xing D, Ren N & Rittmann BE (2008) Genetic diversity of
hydrogen-producing bacteria in an acidophilic
ethanol-H2-coproducing system, analyzed using
the [Fe]-hydrogenase gene. Appl Environ Microbiol 74:
1232–1239.
Yamada T, Imachi H, Ohashi A, Harada H, Hanada S,
Kamagata Y & Sekiguchi Y (2007) Bellilinea caldifistulae gen.
nov., sp. nov. and Longilinea arvoryzae gen. nov., sp. nov.,
strictly anaerobic, filamentous bacteria of the phylum
Chloroflexi isolated from methanogenic
propionate-degrading consortia. Int J Syst Evol Microbiol 57:
2299–2306.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Optimized PCR programs and primer concen-
trations with three primer sets.
Table S2. BLAST results of each clone obtained by PCR
with the primer set hydF1/hydR1 (Xing et al., 2008).
Table S3. BLAST results of each clone obtained by PCR
with the primer set FeFe-272F/FeFe-427r (Boyd et al.,
2009).
Table S4. BLAST results of each clone obtained by PCR
with the primer set HydH1f/HydH3r (Schmidt et al.,
2010).
Table S5. BLAST results of each clone obtained from the
flooded paddy soil by PCR with the primer set HydH1f/
HydH3r (Schmidt et al., 2010).
Table S6. BLAST results of each clone obtained from the
drained paddy soil by PCR with the primer set HydH1f/
HydH3r (Schmidt et al., 2010).
FEMS Microbiol Lett 350 (2014) 249–256ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
256 R. Baba et al.
Dow
nloaded from https://academ
ic.oup.com/fem
sle/article/350/2/249/565945 by guest on 02 June 2022