FEMS Microbiology Ecology, 91, 2015
doi: 10.1093/femsec/fiu004
Advance Access Publication Date: 5 December 2014
Research Article
RESEARCH ARTICLE
Detection and diversity of copper containing nitrite
reductase genes (nirK) in prokaryotic and fungal
communities of agricultural soils
Andrew Long1 , Bongkeun Song1,2,3,∗ , Kelly Fridey1 and Amy Silva1
1
Department of Biology and Marine Biology, University of North Carolina, Wilmington, NC 28403, 2 Department
of Biological Sciences, Virginia Institute of Marine Sciences, Gloucester Point, VA 23062, USA and 3 Department
of Life Science, Dongguk University-Seoul, Seoul 100-715, South Korea
∗ Corresponding author: Department of Biological Sciences, Virginia Institute of Marine Science, College of William & Mary, PO Box 1346, Gloucester
Point, VA 23062, USA. Tel: +804-684-7411; Fax: +804-684-7399; E-mail: [email protected]
One Sentence Summary: With a new set of nirK specific PCR primers the diversity of denitrifying bacteria and fungi in agricultural soils has examined in
this study.
Editor: Dr Cindy Nakatsu
ABSTRACT
Microorganisms are capable of producing N2 and N2 O gases as the end products of denitrification. Copper-containing nitrite
reductase (NirK), a key enzyme in the microbial N-cycle, has been found in bacteria, archaea and fungi. This study seeks to
assess the diversity of nirK genes in the prokaryotic and fungal communities of agricultural soils in the United States. New
primers targeting the nirK genes in fungi were developed, while nirK genes in archaea and bacteria were detected using
previously published methods. The new primers were able to detect fungal nirK genes as well as bacterial nirK genes from a
group that could not be observed with previously published primers. Based on the sequence analyses from three different
primer sets, five clades of nirK genes were identified, which were associated with soil archaea, ammonium-oxidizing
bacteria, denitrifying bacteria and fungi. The diversity of nirK genes in the two denitrifying bacteria clades was higher than
the diversity found in other clades. Using a newly designed primer set, this study showed the detection of fungal nirK genes
from environmental samples. The newly designed PCR primers in this study enhance the ability to detect the diversity of
nirK-encoding microorganisms in soils.
Key words: nirK; denitrification; nitrification; archaea; fungi
INTRODUCTION
Nitrous oxide (N2 O) is a powerful greenhouse gas, which contributes to the destruction of stratospheric ozone in Earth’s atmosphere (Crutzen 1970). As the global use of nitrogen fertilizer increases, the sources and regulation of N2 O emission are of
particular interest. Denitrification and nitrification are the major microbial pathways that generate N2 O through the reduction
of nitric oxide (NO), although dissimilatory nitrate reduction to
ammonia and NO detoxification are also capable of producing
N2 O (Stein 2011; Schreiber et al., 2012). Denitrification is the stepwise reduction of nitrate (NO3 − ) or nitrite (NO2 − ) to NO, N2 O or
dinitrogen gas (N2 ) with organic carbon as an electron donor. It
is a process that has been described in bacteria, fungi and archaea (Zumft 1997). Denitrification is found throughout many
ecosystems and is often the dominant nitrogen loss process in
agricultural soil (Philippot, Hallin and Schloter 2007). Both bacterial and fungal denitrification can utilize copper-containing nitrite reductases (NirK) in their nitrogen metabolism pathway to
Received: 24 June 2014; Accepted: 22 October 2014
C FEMS 2014. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Microbiology Ecology, 2015, Vol. 91, No. 2
reduce NO2 − to NO, which is then used to produce N2 O and/or
N2 (Zumft 1997; Shoun et al., 2012). The diversity of nirK genes in
denitrifying bacteria is well documented in many ecosystems
such as native and agricultural soils (e.g. Sharma et al., 2005;
Wertz et al., 2009; Kelly et al., 2013).
Ammonium-oxidizing bacteria (AOB) and ammoniumoxidizing archaea (AOA) convert NH3 to NO2 − as a part of
nitrification. AOB can produce N2 O via two different pathways,
hydroxylamine (NH2 OH) oxidation and nitrifier denitrification
(Stein 2011; Schreiber et al., 2012). NH2 OH can be oxidized to NO
by hydroxylamine oxidoreductase instead of converting to NO2 −
during NH3 oxidation (Hooper and Terry 1979). Alternatively,
nitrifier denitrification generates N2 O by reducing NO2 − to NO
by nitrite reductase which is then converted to N2 O by NO
reductase (Abeliovich and Vonshak 1992). AOB was thought to
utilize NirK for NO2 − reduction during nitrifier denitrification
(Abeliovich and Vonshak 1992; Bock et al., 1995; Dundee and
Hopkins 2001; Shaw et al., 2006; Jason, Cantera and Stein 2007).
However, Kozlowski, Price and Stein (2014) recently demonstrated that NirK does not have an essential role in nitrifier
denitrification of Nitrosomonas europaea, but requires NirK for
efficient oxidation of NH3 to NO2 − . In addition, AOA was shown
to be unable to perform nitrifer denitrification (Stieglmeier et al.,
2014), although AOA contain putative nirK genes (Bartossek
et al., 2010) and AOA cultures have been shown to produce
N2 O (Santoro et al., 2011; Loescher et al., 2012). The AOA nirK
genes might be involved in N2 O production through a biotic
N-nitrosation (Spott, Russow and Stange 2011; Stieglmeier et al.,
2014). Despite of the new findings, NirK is still considered to
be important in the nitrogen metabolism of both AOA and
AOB. The diversity of nirK genes in AOB has been explored
through bacterial isolates from soils, marine environments and
wastewater as well as from the environmental detection of nirK
genes in soils, groundwater and marine ecosystems (Casciotti
and Ward 2001; Avrahami, Conrad and Braker 2002; Yan et al.,
2003; Cantera and Stein 2007; Garbera et al., 2006; Oakley et al.,
2007). AOA nirK genes were detected in marine environments
with a number of methods, including metagenomics (Venter
et al., 2004), metatranscriptomics (Hollibaugh et al., 2011), a
clone library from the Tyrrhenian Sea (Yakimov et al., 2011),
clone libraries from the California Current, Monterey Bay and
San Francisco Bay (Lund, Smith and Francis 2012), and in
various soil types through the use of clone libraries (Bartossek
et al., 2010). The diversity of nirK genes in prokaryotes has
been well documented using many different methodological
approaches, but eukaryotic nirK gene diversity has not been
thoroughly investigated in soils and sediments. Likewise, the
diversity of nirK genes across the three domains of life has yet
to be examined simultaneously in any ecosystem.
This study seeks to fill some of the gaps in the knowledge of
nirK gene diversity throughout all three domains of life in agricultural soils. To achieve this goal, four agricultural fields in the
United States known to have microbial communities that produce N2 and N2 O were selected to examine the diversity of nirK
genes in bacteria, archaea and fungi (Long et al., 2013).
METHODS AND MATERIALS
Cultivation of Bacteria and Fungi
Two fungal species, Chaetomium globosum ATCC6205 and Penicillium chrysogenum ATCC28593, were obtained from the American Type Culture Collection (ATCC) and were grown in a R2A
medium supplemented with dextrose (10 g L−1 ). The denitrify-
ing isolates carrying nirK genes were cultured with R2A medium.
The isolates included Ochrobacterum spp. 3CB4, 4FB14, Ensifer sp.
2FB8 and Mesorhizobium sp. 4FB11, in which the detection of nirK
genes was previously reported (Song and Ward 2003). Cell pellets of a methane-oxidizing bacterium, Methylocystis sp. Rockwell
and four AOB species, including N. eutropha, N. ureae, N. europaea
and Nitrosospira multifromis, were obtained from the University
of Alberta.
Soil sampling
Soil from above 10 cm in the soil horizon was collected in three
replicate samples across four agricultural fields in the United
States during the summer of 2009: Beaufort County, NC (N35◦
27.681 , W076◦ 55.0926 ); Currituck County, NC (N36◦ 23 09.77 ,
W76◦ 07 18.82); Jewell County, KS (N39◦ 56.1008 , W098◦ 2.1027)
and Boone County, IA (N41◦ 55.186 ,W 93◦ 44.891 ). The fields had
a rotation of maize, soybean and/or wheat production and were
not subjected to organic fertilizer, but had inputs of inorganic
fertilizer in the previous five or more years prior to the study.
Soil samples from each core were homogenized separately and
stored in a −80◦ C freezer for DNA analysis.
Primer design for fungal nirK gene detection
Degenerate primers for fungal nirK genes were designed on the
basis of a comparison of putative NirK amino acid sequences
from seven fungal species genomes, which include Aspergillus
fumigatus (XP˙754129.1), A. terreus (XP˙001209749.1), Ajellomyces
capsulatus (XP˙001543228.1), C. globosum (XP˙001227922.1), Fusarium oxysporum (ABU88100.1), Neosartorya fisheri (XP˙001262952.1)
and P. chrysogenum (XP˙002564428.1). The Codehop program
(http://bioinformatics.weizmann.ac.il/blocks/codehop.html)
was used to recognize highly conserved regions of the selected
NirK sequences for primer development (fnirK2F and fnirK1R in
Table 1).
DNA extraction and PCR of nirK genes
Genomic DNA from two fungal species and nine bacterial isolates was extracted using the ArchivePure DNA Cell/Tissue kit
(Fisher Scientific, Waltham, MA) following the manufacturer’s
instructions. The genomic DNA was used to test the PCR conditions and specificity of fnirK2F and fnirK1R primers.
Environmental DNA was extracted from the soil samples using a ZR Soil Microbe DNA kit (Zymo Research Corporation. Orange, CA) following the manufacturer’s instructions. The concentration of the resulting DNA was calculated using a QuantR dsDNA Assay Kit (Invitrogen, Carlsbad, CA) usItTM PicoGreen
ing the manufacturer’s instructions.
The primers designed for this study as well as the primers
used from previous studies are listed in Table 1. Primers nirk1F
and nirk5R were used to amplify bacterial nirK genes (Braker, Fesefeldt and Witzel 1998), while qAnirk1F and qAnirk1R were utilized for archaeal nirK genes (Bartossek et al., 2010). A newly designed primer set (fnirK2F and fnirK1R) was used to detect fungal nirK genes. For all the primer combinations, the PCR mixR Green Master Mix (Promega,
ture consisted of 12.5 μl GoTaq
Madison, WI), 1 μl of each primer (10 μM stock solution), 1μl of
DNA (10–100 ng) and 9.5μl of sterile milliQ H2 O for a total volume of 25 μl. The PCR conditions for bacterial and archaeal nirK
genes were followed as described by Braker et al., (1998) and Bartossek et al., (2010), respectively. The conditions for fnirK2F and
fnirK1R were as follows: a single minute, 95◦ C denaturation step,
proceeded by 35 cycles of denaturation at 95◦ C for 30 s,
Long et al.
3
Table 1. PCR primers for nirK gene detection.
Target organism
Primer
Sequence (5 -3 )
Reference
Bacteria
nirk1F
nirk5R
fnirK2F
fnirK1R
qAnirK1F
qAnirK1R
GGMATGGTKCCSTGGCA
GCCTCGATCAGRTTRTGGTT
GTYCAYATYGCYAACGGSATGTACGG
GCRTGRTCNACMAGNGTRCGTCCC
TTCAAGGTAAAACCAGGTG
CTGGAGGWATCCACCATGTTTC
Braker et al. (1998)
Braker et al. (1998)
this study
this study
Bartossek et al. (2010)
Bartossek et al. (2010)
Fungi
Archaea
an annealing step for 1 min at 50◦ C and concluded with a 1
min extension step at 72◦ C. The PCR products were examined
using gel electrophoresis on 1.0% agarose gel. The amplicons
R SV Gel and PCR Clean-Up Syswere gel purified with Wizard
tem kits (Promega, Madison, WI) following the manufacturer’s
instructions.
positive results. PCR with either fungal or bacterial primers generated the expected 468 bp products when the soil DNA was used
a template. PCR with qAnirk1F and qAnirk1R primers yielded 171
bp products in soil samples.
Cloning and sequence analysis of nirK genes detected
in soil samples
Taxonomic identities of the sequences as defined by their closest match on NCBI BLAST are shown in Table S1 (Supporting Information). Sequences obtained with the primers fnirK2F
and fnirK1R were aligned with known and putative coppercontaining nitrite reductases from bacteria and fungi as well as
with a multi-copper oxidase (Fig. 1). Two amino acid residues
(His and Met) in type 1 copper binding ligands were found at the
beginning of the detected NirK sequences (Fig. 1). The sequences
denoted CS FNIRK 1 and KS FNIRK 4 share closest homology with
the putative NirK sequences from the fungi P. chrysogenum (82%
and 85% sequence identities, respectively) and F. oxysporum (76%
and 77%), the next closest homology with the crystal structure
of the copper-containing nitrite reductase AniA from Neisseria
Gonorrhoeae (48% and 50%), putative NirK sequences from Ralstonia solanacearum (48% and 48%) and Winogradskyella psychrotolerans RS3 (48% and 48%), and has the least homology with
the multi-copper oxidase from Methylacidiphilum infernorum V4
(34% and 34%). The sequences denoted OS FNIRK 1 and BS FNIRK
1 show higher homology with the sequences from the class II
copper-containing nitrite reductases from bacteria (69 to 79% sequence identities) and exhibited higher homology with the putative NirK sequences from fungi (48 to 50%) than with the multicopper oxidase sequence from M. infernorum V4 (30 and 31%, respectively).
PCR products from soil DNA were used to generate clone libraries
for each field using Perfect PCR Cloning Kits (5Prime, Gaithersburg, MD) following the manufacturer’s protocol. The clone libraries were sequenced with BigDye terminator (Life Technologies, Grand Island, NY) and an ABI 3130xl automated genetic analyzer (Life Technologies, Grand Island, NY).
Each sequence was checked for chimeric sequences using UCHIME in the FunGene Pipeline (Fish et al., 2013) and
searched against the NCBI non-redundant protein sequence
database using BLASTX. The sequences generated using the
PCR primers fnirK2F and fnirK1R were confirmed as nirK
genes through alignments with known nirK sequences utilizing clustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The
Boxshade program was then used to create an image using the clustalW2 alignment with conserved regions highlighted in black and semi-conserved regions highlighted in
gray (http://www.ch.embnet.org/software/BOX˙form.html). Phylogenetic analysis of the detected nirK genes was conducted with
reference sequences, which had high sequence similarities. The
sequences of archaeal nirK genes were analyzed separately due
to their smaller size (171 bp). The sequences of fungal and bacterial nirK genes were 468 bp. Amino acid sequences were deduced
from the nirK sequences and were aligned with the reference sequences using clustalW in the MEGA 5.1 software suite (Tamura
et al., 2011). MEGA was also used to construct phylogenetic trees
using a neighbor-joining Dayhoff algorithm from amino acid
sequences utilizing uniform rates among sites and completion–
deletion gap treatments with 1000 bootstrap iterations. Taxonomic identity was assigned to sequences based upon the sequence identity matches on NCBI BLAST (Table S1, Supporting
Information). Rarefaction as well as Shannon, Simpson, Ace and
Chao1 indices were calculated with an OTU cutoff of 97% using Mothur (Schloss et al., 2009). The sequences obtained from
agricultural soil were submitted to the NCBI database (accession
numbers KF481690-KF481912).
RESULTS AND DISCUSSION
PCR of fungal nirK genes from pure cultures and soil
samples
PCR with the primers fnirK2F and fnirK1R yielded 468 bp products from the DNA obtained from two fungal species (Fig. S1,
Supporting Information). None of the bacterial DNA showed
Confirmation of nirK sequence identity
Phylogenies of archaeal, bacterial and fungal nirK
genes
A phylogenic tree of NirK sequences in both bacteria and fungi
is presented in Fig. 2, while the phylogeny of archaeal NirK sequences is shown in Fig. 3. The NirK sequences in fungi and bacteria form four major clusters: two comprised of sequences similar to denitrifying bacteria (denitrifying bacteria clades 1 and
2), one similar to nitrifying bacteria (AOB clade) and one similar to fungi (fungal clade). Denitrifying bacteria clade 1 and the
AOB clade are closely related to class I copper-containing nitrite
reductases, while the fungal clade and denitrifying clade 2 are
closely related to class II copper-containing nitrite reductases as
defined by Boulanger and Murphy (2002). Denitrifying bacteria
clade 1 included bacterial NirK sequences identified using the
primer set developed by Braker et al., (1998), while the bacterial sequences detected with the primers fnirk2F and fnirk1R
clustered exclusively in the denitrifying bacteria clade 2. The
NirK sequences detected with qAnirk1F and qAnirk1R primers
clustered into one clade (soil archaeal clade) with previously described NirK sequences from soil archaea.
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FEMS Microbiology Ecology, 2015, Vol. 91, No. 2
Figure 1. Protein sequence alignment using clustalW and sequences obtained using the fnirk2F/fnirk1R primer set with the crystal structure of the class II coppercontaining nitrite reductase AniA from N. Gonorrhoeae, putative NirK sequences from the fungi P. chrysogenum and F. oxysporum, the bacteria R. solanacearum and W.
psychrotolerans RS3, and a multi-copper oxidase from M. infernorum V4. Conserved amino acid residues are highlighted in black and semi-conserved amino acid residues
are highlighted in gray. The symbol ∗ denotes putative type I copper (Cu) ligands.
Of the sequences obtained with the primers targeting fungal
nirK genes, only 9 out of a total of 106 sequences shared more
than 75% sequence identities and clustered with known NirK
sequences of the fungal species, F. oxysporum (Kim et al., 2009),
P. chrysogenum (van den Berg et al., 2008), Coccidioides immitis RS
(EAS29309.2), Arthroderma gypseum CBS 118893 (XP˙003171471.1)
and F. lichenicola (ADB82651.1). Of these nine sequences, five were
from Jewel County, KS and four were from Currituck County, NC.
The remaining 97 sequences detected using fnirk2F and
fnirk1R primers were closely related to bacterial NirK sequences
in denitrifying bacteria clade 2. Members of denitrifying bacteria clade 2 are closely related to class II copper-containing nitrite
reductases (Boulanger and Murphy 2002). The known bacterial
NirK sequences, many of which are from known denitrifiers, in
denitrifying bacteria clade 2 (Fig. 2). AniA is a class II coppercontaining nitrite reductase with high homology to NirK and has
been shown to group with NirK sequences from other bacteria
(Mellies, Jose and Meyer 1997; Boulanger and Murphy 2002; Jones
et al., 2008). The bacterial NirK sequences in the denitrifying bacteria clade 2 were found in all of the soil communities.
The sequences from the amplicons generated using the
primers nirk1F and nirk5R formed two clades. Denitrifying
bacteria clade 1 consists of NirK sequences from previous
studies in agricultural fields (Henry et al., 2008; ABI19023.1,
ABI19222.1, ABI19249.1, ABI19558.1 and ADR10866.1), undisturbed soils (ABI19141.1) activated sludge (Hallin et al., 2006),
wetlands (AGI99571.1), lakes (Junier et al., 2009) and rice pad-
dies (Chen et al., 2010), as well as with known denitrifying bacteria, while the other clade has sequences that cluster with Nitrosomonas and Nitrosospira species, which are known AOB. Of
the 80 total sequences, 8 clustered with AOB and 72 clustered
with denitrifying bacteria. Bacterial NirK sequences in Beaufort
County, NC; Currituck County, NC; Boone County, IA and Jewell
County, KS clustered with denitrifying bacteria such as Sinorhizobium fredii (Schuldes et al., 2012), Mesorhizobium amorphae (Hao
et al., 2012), M. loti (WP˙020628950.1) Bradyrhizobium species (Mornico et al., 2012; Okubo et al., 2013), Rhizobium sp. 42MFCr.1
(WP˙018856207.1), Ochrobacterum sp. R-24467 (Heylen et al., 2006)
and Nitratireductor aquibiodomus (WP˙007010017.1) as well as uncultured bacteria isolated from other agricultural soils (Henry
et al., 2008; ABI19023.1, ABI19222.1, ABI19249.1, ABI19558.1),
undisturbed soils (ABI19141.1) activated sludge (Hanlin et al.,
2006), wetlands (AGI99571.1), lakes (Junier et al., 2008) and rice
paddies (Chen et al., 2010). A sequence from Jewell County, KS
also clustered with Pseudomonas chlororaphis (Loper et al., 2012).
Junier et al., (2008) reported a novel clade of nirK with only uncultured soil bacteria and uncultured bacteria from Lake Kinneret,
Isreal. Twelve sequences from Jewell County, KS clustered with
the clade from Lake Kinneret as well as with uncultured soil bacteria from agricultural soil (ADR10866.1).
All the NirK sequences in archaea form one major clade (soil
archaeal clade) with NirK sequences from uncultured grassland
and agricultural soil Thaumarchaeota (Bartossek et al., 2010)
as well as one NirK sequence from an enrichment culture of
Long et al.
5
Figure 2. Unrooted neighbor-joining phylogenetic tree of deduced NirK sequences from 468bp detected with primers targeting bacteria and fungi from agricultural
soils utilizing a Dayoff model. Abbreviations for sample sites are as follows: BS (Beaufort, NC sample site), CS (Currituck, NC sample site), KS (Jewell, KS sample site)
and OS (Boone, IA sample site). NIRK denotes that the sequences were obtained with primer set nirk1F/nirk5R, while FNIRK denotes that the sequences were obtained
with primer set fnirK2F/fnirK1R. Bootstrap numbers are a percentage of 1000 iterations.
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FEMS Microbiology Ecology, 2015, Vol. 91, No. 2
Figure 3. Rooted neighbor-joining phylogenetic tree of deduced NirK sequences from 171bp detected with primers targeting archaea from agricultural soils utilizing a
Dayoff model with the hypothetical KNOR protein from Candidatus Nitrosopumilus koreensis AR1 serving as an outgroup. Abbreviations for sample sites are as follows: BS
(Beaufort, NC sample site), CS (Currituck, NC sample site), KS (Jewell, KS sample site) and OS (Boone, IA sample site). ANIRK denotes that the sequences were obtained
with primers targeting archaeal nirK. Bootstrap numbers are a percentage of 1000 iterations.
‘Candidatus Nitrososphaera gargensis’ (Spang et al., 2012) (Fig. 3).
Sequences from marine environmental samples did not cluster
with the sequences from any of the study sites. Archaeal sequences from this study exhibit similar clustering patterns to
the one previous study of soil archaeal NirK (Bartossek et al.,
2010).
Diversity and richness of nirK genes in archaea,
bacteria and fungi
Rarefaction curves for the different groupings of nirK genes are
presented in Fig. 4. Diversity and richness indices of the nirK
genes in different clusters and sites are presented in Table 2.
Overall, nirK sequences from denitrifying bacteria clade 1 exhibit
the highest diversity and richness, followed by nirK sequences
from denitrifying bacteria clade 2 which had the next highest
degree of diversity and richness. The archaeal nirK sequences
were the next most diverse and showed the next most richness,
followed by the AOB nirK sequences. The fungal nirK sequences
had the least diversity and richness. Beaufort soil community
had the highest diversity and richness for denitrifying bacteria
clade 2, while Currituck soil community showed the highest diversity and richness for denitrifying bacteria clade 1. Fungal nirK
genes had the same low diversity and richness in the two sites
where the genes were detected, and AOB had similarly low diversity and richness across all soil samples and was not detected
Figure 4. Rarefaction analysis of nirK genes in four agricultural fields using a 3%
distance cutoff calculated in the Mothur program.
in Beaufort soil community. AOA nirK genes were most diverse
and had the highest richness in Currituck soils.
Most of the literature on nirK gene diversity in soils is based
upon DNA fingerprinting techniques (e.g. DGGE, T-RFLP) and
thus could not be directly compared to the sequences from this
study (e.g. Throbak et al., 2004; Wertz et al., 2009; Smith, WagnerRiddle and Dunifield 2010; Bannert et al., 2011; Dandie et al., 2011;
Pastorelli et al., 2011; Zhou et al., 2011; Clark et al., 2012). Both
Long et al.
7
Table 2. Species diversity and richness of nirK in agricultural soils.
Site
Clade
Primer Set
Beaufort, NC
AOA
Denitrifying Bacteria Clade 1
Denitrifying Bacteria Clade 2
Denitrifying Fungi
AOB
AOA
Denitrifying Bacteria Clade 1
Denitrifying Bacteria Clade 2
Denitrifying Fungi
AOB
AOA
Denitrifying Bacteria Clade 1
Denitrifying Bacteria Clade 2
Denitrifying Fungi
AOB
AOA
Denitrifying Bacteria Clade 1
Denitrifying Bacteria Clade 2
Denitrifying Fungi
AOB
AOA
Denitrifying Bacteria Clade 1
Denitrifying Bacteria Clade 2
Denitrifying Fungi
AOB
qAnirK1F/qAnirK1R
nirk1F/nirk5R
fnirK2F/fnirK1R
fnirK2F/fnirK1R
nirk1F/nirk5R
qAnirK1F/qAnirK1R
nirk1F/nirk5R
fnirK2F/fnirK1R
fnirK2F/fnirK1R
nirk1F/nirk5R
qAnirK1F/qAnirK1R
nirk1F/nirk5R
fnirK2F/fnirK1R
fnirK2F/fnirK1R
nirk1F/nirk5R
qAnirK1F/qAnirK1R
nirk1F/nirk5R
fnirK2F/fnirK1R
fnirK2F/fnirK1R
nirk1F/nirk5R
qAnirK1F/qAnirK1R
nirk1F/nirk5R
fnirK2F/fnirK1R
fnirK2F/fnirK1R
nirk1F/nirk5R
Boone, IA
Currituck, NC
Jewell, KS
All Sites
1
Sequences
OTUs
Chao11
ACE1
Shannon1
Simpson1
9
20
34
10
12
23
3
11
20
34
4
3
8
20
6
5
2
38
72
97
9
8
7
13
19
4
6
2
1
8
17
14
1
2
4
10
2
1
1
17
45
41
2
5
9.0
35.5
49.3
4.5
7.5
2.0
1.0
10.5
69.5
16.5
1.0
2.0
4.5
46.1
2.0
1.0
1.0
23.1
171.1
99.5
2.0
6.5
12.2
71.3
153.9
7.1
8.8
0.0
1.0
14.7
98.9
16.9
1.0
3.0
5.8
40.1
2.0
1.0
1.0
48.3
356.3
174.9
2.0
8.7
1.677
2.359
2.603
1.089
1.633
0.179
0.000
2.020
2.762
2.484
0.000
0.637
1.277
1.677
0.637
0.000
0.000
2.490
3.481
3.159
0.687
1.494
0.111
0.074
0.084
0.356
0.152
0.913
1.000
0.055
0.021
0.066
1.000
0.333
0.191
0.290
0.467
1.000
1.000
0.093
0.035
0.070
0.444
0.143
Diversity indices were estimated at a 3% distance level.
the diversity and richness of nirK genes in denitrifying bacteria
clades 1 and 2 from all four sites fell into the mid-range of diversity and richness reported from alpine soil (Kelly et al., 2013),
turf grass soil (Dell et al., 2010), and palsa and acidic peat (Palmer
and Horn 2012; Palmer, Biasi and Horn 2012), while exhibiting
higher richness and diversity than from rice paddy soils (Yoshida
et al., 2012), temperate mixed forest soils (Katsuyama et al., 2008)
and some agricultural soils (Braker, Dorsch and Bakken 2012). If
comparing site by site, this study presents diversity and richness in the lower ranges of the previous studies. This may be
due to the separation of nirK genes into different taxonomical
and functional groups before estimating diversity and richness.
The diversity and richness of archaeal nirK genes found in this
study fell into the mid-range of diversity and richness reported
from Monterey Bay, the California current and San Francisco Bay
(Lund et al., 2012).
CONCLUSIONS
This study presents the first report comparing the diversity and
richness of nirK genes in archaea, bacteria and fungi, of which
fungal nirK genes were not previously detected. Based on the current primer sets available, denitrifying bacteria have the highest degree of nirK gene diversity and richness. Fungal nirK genes
were detected in only two sites, while bacterial and archaeal nirK
genes were obtained from all four of the sample sites. The low
detection of fungal nirK genes may reflect low specificity of the
primers to detect the targeted genes. However, the new primers
allow the detection of class II bacterial nirK genes, which cannot be observed with the primers of Braker et al., (1998) and
Throback et al., (2004). Thus, the primers from this study can
greatly enhance our understanding of the diversity of microor-
ganisms that reduce NO2 − to NO, which is a source of N2 O production in denitrification and nitrification. Bacteria that carry
out anaerobic ammonium oxidation and denitrifying anaerobic
methane oxidation are also capable of dissimilatory reduction of
NO2 − to NO, which is converted to N2 in both pathways without
the production of N2 O Ettwig et al., 2010; Kartal et al., 2011). The
ultimate fate of the intracellularly produced NO (conversion to
N2 or N2 O) is dependent on the N cycling pathways present in the
microorganism in which the NO was produced. The bacteria or
fungi carrying class II nirK genes might be more relevant in agricultural soils and may have a higher contribution towards N2 O
production than those carrying class I nirK genes. Future studies
should be followed to evaluate the importance of different NO2 −
respiring microorganisms in soil N2 O production.
ACKNOWLEDGEMENTS
We acknowledge Dr Joshua Heitman for providing soil samples from four agricultural fields and Dr Stein for kindly providing cell pellets of a methane-oxidizing bacterium, Methylocystis sp. Rockwell and four AOB species, including N. eutropha, N.
ureae, N. europaea and N. multifromis. We would also like to thank
the anonymous reviewers who helped shape and improve this
article.
FUNDING
This study was supported by the US National Institute of Food
and Agriculture grant (NCR-2008-02832) and a general fund of
Virginia Institute of Marine Science, College of Williams & Mary.
The paper is Contribution No. 3414 of the Virginia Institute of
Marine Science, College of William & Mary.
8
FEMS Microbiology Ecology, 2015, Vol. 91, No. 2
SUPPLEMENTARY DATA
Supplementary data is available at FEMSEC online.
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