Analysis of [FeFe]hydrogenase genes for the elucidation of a ...

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: [email protected]

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


H2 producers in paddy soil estimated by [FeFe]-H2ase gene 251

Dow


ic.oup.com/fem


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


252 R. Baba et al.

Dow


ic.oup.com/fem


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.



Dow


ic.oup.com/fem


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.


254 R. Baba et al.

Dow


ic.oup.com/fem


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.



Dow


ic.oup.com/fem


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).


with the primer set FeFe-272F/FeFe-427r (Boyd et al.,

2009).


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).


256 R. Baba et al.

Dow


ic.oup.com/fem


Analysis of [FeFe]hydrogenase genes for the elucidation of a ...

Documents

Transcript of Analysis of [FeFe]hydrogenase genes for the elucidation of a ...