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Systematic and Applied Microbiology 33 (2010) 337–347
Contents lists available at ScienceDirect
Systematic and Applied Microbiology
journal homepage: www.elsevier.de/syapm
Isolation, genetic and functional characterization of novel soil
nirK-type denitrifiers
Silke Falk a , Binbin Liu b , Gesche Braker a,∗
a
b
Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse 10, D-35049 Marburg, Germany
Department of Chemistry, Biotechnology and Food Sciences, Norwegian University of Life Sciences, Ås N-1432, Norway
a r t i c l e
i n f o
Article history:
Received 16 February 2010
Keywords:
Denitrifiers
Soil
Uncultured denitrifiers
Cultivation
nirK
Phylogeny
a b s t r a c t
Denitrification, the reduction of nitrogen oxides (NO3 − and NO2 − ) to N2 via the intermediates NO and N2 O,
is crucial for nitrogen turnover in soils. Cultivation-independent approaches that applied nitrite reductase genes (nirK/nirS) as marker genes to detect denitrifiers showed a predominance of genes presumably
derived from as yet uncultured organisms. However, the phylogenetic affiliation of these organisms
remains unresolved since the ability to denitrify is widespread among phylogenetically unrelated organisms. In this study, denitrifiers were cultured using a strategy to generally enrich soil microorganisms. Of
490 colonies screened, eight nirK-containing isolates were phylogenetically identified (16S rRNA genes)
as members of the Rhizobiales. A nirK gene related to a large cluster of sequences from uncultured bacteria
mainly retrieved from soil was found in three isolates classified as Bradyrhizobium sp. Additional isolates
were classified as Bradyrhizobium japonicum and Bosea sp. that contained nirK genes also closely related
to the nirK from these strains. These isolates denitrified, albeit with different efficiencies. In Devosia sp.,
nirK was the only denitrification gene detected. Two Mesorhizobium sp. isolates contained a nirK gene also
related to nirK from cultured Mesorhizobia and uncultured soil bacteria but no gene encoding nitric oxide
or nitrous oxide reductase. These isolates accumulated NO under nitrate-reducing conditions without
growth, presumably due to the lethal effects of NO. This showed the presence of a functional nitrite reductase but lack of a nitric oxide reductase. In summary, similar nirK genotypes recurrently detected mainly
in soils likely originated from Rhizobia, and functional differences were presumably strain-dependent.
© 2010 Elsevier GmbH. All rights reserved.
Introduction
Denitrification is the dissimilatory reduction of oxidized nitrogen compounds (NO3 − and NO2 − ) to the gases nitric oxide (NO),
nitrous oxide (N2 O), and dinitrogen (N2 ) that are released concomitantly. NO and N2 O are important greenhouse gases, which
support global warming and the destruction of the stratospheric
ozone layer [20,24]. Denitrification processes in soils are a major
source of atmospheric N2 O and soils contribute to approximately
57% of the global emissions [55]. The process is mainly driven by
facultative anaerobic microorganisms, which use oxidized nitrogen
compounds as alternative electron acceptors for energy production [86]. The reduction of NO2 − to NO which is catalyzed by the
enzyme nitrite reductase is the key step of denitrification. This step
distinguishes denitrifiers sensu stricto from other nitrate respiring
microorganisms, since the gases produced cannot be assimilated
further by the organisms [86]. Two types of nitrite reductase exist, a
copper- and a cytochrome cd1 -containing form which are encoded
∗ Corresponding author. Tel.: +49 6421 178 733; fax: +49 6421 178 999.
E-mail address: [email protected] (G. Braker).
0723-2020/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.syapm.2010.06.004
by the genes nirK and nirS, respectively. Both types occur as single copy genes in the same genus but not in the same microbial
species. However, in one Thauera species two different functional
copies of nirS were found [25] and duplicate copies of nirS were
found in Thiobacillus denitrificans and Dechloromonas aromaticum,
whereas Magnetospirillum magneticum had three nirS genes [42].
Nitrite reductase genes have been frequently used as marker genes
to explore the functional group of denitrifiers in the environment
because a 16S rRNA gene-based approach is not suitable due to
the fact that the ability to denitrify is widespread among phylogenetically unrelated organisms [65]. Molecular studies of denitrifier
communities in, for example, soil, ground water, activated sludge
and marine habitats [14,26,31,33,37,38,72,81,82] have indicated
highly diverse natural denitrifier communities. For soils, they
have further shown a dominance of nitrite reductase genotypes
that were derived from unknown, presumably as yet uncultured,
organisms. For instance, phylogenetic analysis of cloned nirK gene
fragments from soils of different locations world-wide and with
distinct properties showed a preponderance of sequences that were
consistently grouped within a single cluster. This cluster currently
comprises more than 500 nirK sequences, mainly retrieved from
soil [4,14,60,81,85]. However, the only sequence within this cluster
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originating from a cultured organism is the nirK gene from Nitrosomonas sp. TA-921i-NH4, which is a bacterial ammonia-oxidizer
isolated from an estuary [15]. Other clusters within the nirK gene
tree entirely lack sequences from cultured representatives but
consist exclusively of environmental clone sequences. In contrast,
sequences of a number of nirK clones from activated sludge were
most closely related to nirK from cultured denitrifiers [32,59] suggesting that closely related functional genes might originate from
organisms that are phylogenetically related. However, to infer a
robust phylogenetic relationship of the organisms based only on
their functional genes for denitrification is currently not possible,
since horizontal gene transfer is likely to have occurred for denitrification genes [39,41]. Hence, cultivation studies are needed to
unravel the phylogenetic affiliation of the large group of phylogenetically unrelated denitrifiers that are relevant in the environment
and to study physiological properties of the organisms related to
denitrification. A common strategy is to selectively enrich nitrate
reducers, including denitrifiers from environmental samples, by
using nutrient rich media and anaerobic conditions with nitrogen
oxides as electron acceptors [73]. Although a variety of denitrifying
bacteria were isolated successfully from soil [19,21,29] these cultivation attempts generally resulted in denitrifying bacteria with
rather similar denitrification genotypes that showed little or no
overlap with the nirK genotypes dominating in soils. Although
results from molecular studies are also likely to be prone to bias, the
fraction of the microbial community that can be taken in culture is
known to be very small, thus leaving a large part of the community
diversity undetected. Recently, however, with the development of
advanced cultivation techniques, previously uncultured groups of
bacteria that are widespread and dominant in soil were isolated
successfully [23,41,43,61]. Hence, isolation approaches to overcome the ‘great plate count anomaly’ are promising for yielding
isolates that are supposedly relevant for the functioning of natural
communities.
In the present study, a molecular approach was first applied
to assess the structure of the nirK-type denitrifier community of
a typical temperate grassland soil. Then, cultivation of organisms
showing the preponderant nirK genotypes within the overall NirK
tree was attempted. Therefore, a strategy was used to generally
enrich organisms that dominate the soil microbial community by
mimicking the conditions prevalent in the environment (i.e. using
aerobic conditions and low nutrients, as well as a comparably low
isolation temperature). Resulting isolates were screened for the
presence of a nirK gene coding for the key enzyme of denitrification
and if they tested positive they were phylogenetically classified.
The isolates were further characterized for the occurrence of additional genes coding for nitrogen oxide reductases and for their
ability to denitrify.
medium, as described by Grosser et al. [30]. The medium was as
follows: 2.5 mM NH4 NO3 ; 1 mM NaNO3 ; 0.75 mM Na2 SO4 ; 4 mM
CaSO4 ; 0.25 mM K2 SO4 ; 2 mM MgCl2 ; 0.005 mM KH2 PO4 ; 0.02 mM
FeSO4 ; 15 mM MOPS; nutrient broth (1:10,000 diluted [w/v]), gelrite 8 g L−1 , pH 7.1. Plates were incubated aerobically at 15 ◦ C and
checked weekly for growth of colonies. After a total incubation
time of 4 weeks, colonies that had grown were screened for the
presence of a nirK gene by PCR using gene specific primers and
conditions described previously (Supplementary Table S1 [10,12]).
Therefore, cells from single colonies were suspended in 50 ␮L of
sterile double-distilled water and disrupted by freezing (−20 ◦ C,
3 min) and thawing (100 ◦ C, 3 min). Afterwards, cell debris was
removed by centrifugation (16,000 × g, 4 ◦ C, 10 min). The supernatant, containing the DNA, was then used as a template for the
screening procedure (see below).
Colonies containing a nirK gene were re-streaked onto fresh
plates and incubated again at 15 ◦ C for 4 weeks. To purify the isolates, this procedure was repeated seven times. Purity of the isolates
was proven by phase contrast microscopy and by sequence analysis
of the 16S rRNA genes from several colonies of a given isolate (see
below).
Since growth of the cultures in minimal medium was very
slow, different media were tested to optimize growth conditions
(nutrient broth [NB; 8 g L−1 ], yeast extract mannitol medium [YEM;
10 g L−1 mannitol, 0.5 g L−1 K2 HPO4 , 0.2 g L−1 MgSO4 , 0.1 g L−1 NaCl,
0.4 g L−1 yeast extract, 2 mM KNO3 , pH 7.0] and R2A medium
[0.5 g L−1 yeast extract; 0.5 g L−1 proteose–peptone; 0.5 g L−1
casamino acids; 0.5 g L−1 glucose; 0.5 g L−1 soluble starch; 0.3 g L−1
sodium pyruvate; 0.3 g L−1 K2 HPO4 ; 0.05 g L−1 MgSO4 ·7H2 O;
0.5 g L−1 potassium nitrate; adjusted to pH 7.0]).
DNA extraction from soil and pure cultures
Materials and methods
DNA extraction from 0.5 g field-fresh soil was carried out using
the Fast® DNA SPIN Kit for soil (BIO101, La Jolla, CA), according to
the manufacturer’s instructions.
Cells from 2 mL of a pure culture suspension were collected by
centrifugation at 16,000 × g at room temperature for 10 min. The
pellet was resuspended in 500 ␮L sterile double-distilled water and
the suspension was added to 0.5 g baked glass beads (diameter
0.17–0.18 mm, B. Braun Biotech International GmbH, Melsungen,
Germany). Cell lysis was undertaken with a bead beater (FastPrep® -24, MP Biomedicals, Heidelberg, Germany) for 45 s with
6.5 m s−1 , and samples were centrifuged to remove cell debris
(16,000 × g, 4 ◦ C, 5 min). DNA in the supernatant was purified from
proteins by phenol–chloroform extraction and subsequent ethanol
precipitation [3].
The purity and quantity of the extracted DNA were determined
by UV-spectrophotometry at 260 and 280 nm (NanoDrop® ND1000 UV/vis-spectrophotometer, Peqlab Biotechnologie GmbH,
Erlangen, Germany). The DNA was stored at −20 ◦ C.
Cultivation and screening procedure
PCR amplification of 16S rRNA genes and functional genes
Grassland soil (alluvial soil) was sampled next to a river (Lahn)
in Marburg, Germany (50◦ 50 27, 37 N und 8◦ 45 39, 81 E) in June
2006. A grab sample of the upper 10 cm of the soil was taken, transported to the laboratory and immediately homogenized and sieved
at <2 mm. It had the following characteristics: pH (CaCl2 ) 5.19,
total nitrogen 0.31%, total carbon 3.19%, NO3 –N(2 M KCl) 2.90 ␮g g−1
dry weight and NO2 –N(2 M KCl) 3.43 ␮g g−1 dry weight. An aliquot
(10 g) of the soil was suspended in 90 mL Ringer solution. In two
parallel stepwise dilution series, aliquots (10 mL) of the soil suspensions were further tenfold diluted in 90 mL Ringer solution
down to a dilution step of 10−10 . Aliquots (100 ␮L) of each of
the dilutions 10−6 to 10−10 were spread plated on solid minimal
From the eight isolates that tested positive for nirK, 16S rRNA
genes, as well as the functional genes narG, napA, nirK, nirS, cnorB,
qnorB and nosZ, were PCR-amplified using gene specific primer
pairs and conditions published previously (Supplementary Table
S1). A typical amplification reaction mixture contained 25 pmol of
each primer, 200 ␮M of each deoxynucleoside triphosphate (Roche
Molecular Diagnostics GmbH, Mannheim, Germany), 400 ng ␮L−1
bovine serum albumin (Roche Molecular Diagnostics), and 1.25 U
of REDAccuTaq LA DNA polymerase (Sigma–Aldrich Chemie GmbH,
Steinheim, Germany) in 1× reaction buffer provided by the manufacturer. Template DNA (1 ␮L) and sterile water were added to a
final volume of 25 ␮L reaction solution.
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339
Table 1
Isolates containing a nirK gene retrieved from grassland soil. Preferred growth medium, phylogenetic affiliation of the isolates as determined by BLAST search of 16S rRNA
genes, results of PCR-based detection of denitrification genes, fraction of nitrate converted to N2 O and cell density. N2 O and OD measurements are means ± standard deviation
of n = 3.
Isolate
Medium
GSM-406
GSM-467
GSM-471
GSM-469
YEM
R2A
R2A
YEM
GSM-187
GSM-205
GSM-373
R2A
R2A
YEM
GSM-484
YEM
a
b
c
d
N2 O from NO3 − (%)b
OD580 a
+
+
−
+
6.6 ± 11.2
123.2 ± 9.6
21.4 ± 1.6
59.5 ± 41.4
0.013 ± 0.000
0.128 ± 0.059
0.075 ± 0.028
0.331 ± 0.002
+/−
−/−
−/−
+
−
−
108.0 ± 67.2
n.d.c
−d
0.239 ± 0.010
−
−
−/−
−
−
−
16S rRNA gene
Denitrification gene
Nearest cultured relative (BLAST identity,
length of query)
narG/napA
nirK (cluster)
cnorB/qnorB
nosZ
Bradyrhizobium sp. 2RO3 (99.1%, 1270 bp)
Bradyrhizobium group (97.9%, 1260 bp)
Bradyrhizobium group (98.5%, 1402 bp)
Bradyrhizobium sp. and B. canariense
(99.4%, 1268 bp)
Bosea sp. PD 19 (99.6%, 1403 bp)
Devosia limi (96.9%, 1371 bp)
Mesorhizobium sp. CCBAU11299 (99.8%,
1373 bp)
Mesorhizobium sp. CCBAU11217 (98.2%,
1264 bp)
−/+
−/+
−/+
−/+
+ (I)
+ (I)
+ (I)
+ (II)
+/−
+/−
+/−
+/−
−/+
−/−
+/−
+ (IV)
+ (IV)
+ (V)
+/−
+ (V)
Determined after 15 days incubation.
N2 O-production after acetylene inhibition.
Not determined.
N2 O production from NO3 − –N <0.001%.
Thermocycling was carried out as described previously (narG
[58]; napA [28]; cnorB/qnorB [9]; nosZ [45]). Amplification of the
16S rRNA gene was carried out using the primers described by
Amann et al. [1], as modified by Moyer (personal communication)
and using the conditions described by Braker et al. [11]. The quality
and quantity of PCR products were determined by electrophoresis
of an aliquot of each reaction on a 1.5% (w/v) agarose gel (Biozym
Scientific GmbH, Oldendorf, Germany) and by visualization of the
bands by UV excitation after staining the gel with ethidium bromide
(0.5 mg L−1 ).
Cloning, sequencing, and phylogenetic analysis
Amplicons of nirK from the soil DNA extract were cloned using
the pGEM-T cloning kit (Promega, Mannheim, Germany), according to the manufacturer’s instructions. White colonies picked at
random were screened for inserts of the correct size by PCR amplification with vector-specific primers, as described elsewhere [4].
Inserts of the proper length (514 bp) from 54 colonies were chosen
for sequencing.
PCR amplicons of the 16S rRNA genes and of functional genes
of the isolates, as well as clones obtained from soil DNA, were
purified with the Wizard SV Gel and PCR Clean-Up System
(Promega). Subsequently, they were sequenced in both directions
using dye terminator chemistry and gene specific primers (isolates) or M13/T7 primers (clones), and analyzed on an ABI 377 DNA
sequencer (Applied Biosystems, Foster City, CA). The coverage C of
the clone library was calculated to describe the extent to which
the sequences in the library represented the total population (i.e.
the clone library). The equation for calculating the coverage (CX )
for a single sample (X) is CX = 1 − (Nx /n), where Nx is the number
of unique sequences in the sample and n is the total number of
sequences.
Phylogenetic analyses were undertaken with ARB [46].
Sequences of the 16S rRNA genes and the functional genes
nirK, cnorB, and nosZ were aligned to sequences from public
databases using the ARB Fast Aligner tool. Trees were reconstructed using the distance matrix-based neighbor joining method
(ARB) and the overall topology was confirmed by trees calculated with the parsimony and maximum likelihood methods
(PHYLIP [27]) based on nucleotides and transduced amino acids
for 16S rRNA and functional genes, respectively. Filters were calculated to exclude insertions and deletions due to ambiguous
positional homology. The 16S rRNA gene-based tree included
1211 nucleotide positions; for nirK, 72 amino acid positions
were included in the tree calculations, 100 positions for norB
and 117 positions for nosZ. Nucleotide sequences retrieved from
the isolates and environmental clones have been deposited
in the EMBL database under accession numbers FN600559FN600636.
Gas emission measurements
N2 O production of the isolates was measured in triplicate in
120 mL serum bottles that were filled with 30 mL of the preferred media (Table 1) supplemented with 5 mM KNO3 . The bottles
were capped with butyl rubber septa, oxygen was removed by
flushing the bottles with N2 for 3 min, and the pressure was
adjusted to normal pressure. The reduction of nitrous oxide to
dinitrogen was inhibited by replacing 10% of the headspace with
acetylene. The anaerobic cultures were shaken at 150 rpm and
incubated at 25 ◦ C for 2 weeks. Before each measurement, cultures were shaken manually to equilibrate between the gas and
the liquid phase. Samples were taken with 0.5 mL PressureLock-Syringes® (VICI, Baton Rouge, LA, USA) that were flushed
with N2 before gas samples were taken. N2 O was measured by
a gas chromatograph (Carlo Erba Instruments GC 8000) connected to a 63 Ni-electron capture detector (ECD). Data were
evaluated with the software Peak Simple (version 2.66, SRI
Instruments, Torrance, CA). The percentage conversion of nitrate
to N2 O was determined as the molar fraction of N2 O produced in relation to the 5 mM potassium nitrate added to the
medium.
The gas emission of two cultures (GSM-373 and GSM-484)
was monitored in a robotized incubation system, as described in
detail by Molstad et al. [53]. For each culture, three parallel flasks
were set up. The serum flasks (120 mL) containing 50 mL liquid
medium (YEM) were capped with butyl rubber septa and gastight aluminum crimp seals with a small amount of laboratory
grease. The flasks with medium were made anaerobic by repeated
evacuation/filling with helium (He), as described previously [53].
Then, the flasks were inoculated with 2 mL aerobic preculture
(OD580 ≈ 0.02) and 1% oxygen was added to activate cell growth.
Culturing was carried out at 28 ◦ C in a tempered water bath by
constantly stirring the medium with sterile Teflon magnetic bars
in the flasks.
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Results and discussion
Molecular diversity of nirK genes in grassland soil
The aim of this study was to isolate the as yet unknown nirK-type
denitrifiers that were shown by culture-independent approaches to
dominate the natural denitrifier communities in soils. However, no
attempt was made in this study to assess the nirS-type denitrifier
community present in the soil. At first, the diversity and structure
of the nirK-type denitrifier community in a typical temperate grassland soil was assessed by PCR amplification of the nirK gene that
codes for copper-containing nitrite reductase as a marker gene. The
PCR product was cloned and 54 clones were sequenced, which was
sufficient to achieve coverage of 85% of the clone library based on
deduced amino acid sequences. Phylogenetic analysis of deduced
nirK amino acid sequences included sequences from uncultured
grassland soil bacteria, which were obtained in this study, and nirK
sequences from cultured as well as from uncultured denitrifiers,
which were available from the public databases. First, a neighbor
joining tree was calculated based on all NirK sequences available
(Supplementary Fig. S1a and S1b). Subsequently, to avoid crowding
of the tree, environmental sequences retrieved by other cultureindependent studies and redundant NirK sequences from isolates
of identical phylogenetic affiliation (except for those obtained by
Hashimoto et al. [34]) were removed manually without changing
the overall tree topology. Finally, based on this reduced dataset,
trees were calculated using the neighbor joining, maximum parsimony and maximum likelihood methods (Fig. 1; Supplementary
Fig. S2 and S3). Clusters within the NirK tree were defined if
sequences were consistently grouped together by all three methods. Based on the sequences included in the analysis five clusters
were found, and clones from the grassland soil were grouped into
three of them (clusters I, III, and V). The most dominant group of
sequences (34) from this soil was grouped in cluster III that only
consisted of sequences from as yet uncultured organisms. Within
this cluster, the NirK sequences that were most closely related to
our clones were retrieved from soil and rhizosphere [60,66,81].
Sequences of the second most abundant group of clones from this
soil (18) were grouped within the largest cluster (cluster I; ∼600
sequences) of NirK sequences in the overall NirK tree, which consisted almost exclusively of sequences from soils. Within cluster I,
our sequences were most closely affiliated with NirK from uncultured organisms (85–100% amino acid sequence identity) and to a
lesser extent to NirK from Nitrosomonas sp. TA-921li-NH4 (69–87%
amino acid sequence identity), the only cultured organism containing a related nirK gene. To the best of our knowledge, sequences
placed in this cluster have been found in each culture-independent
study on soil nirK-type denitrifier communities published to date.
Finally, a single NirK clone sequence was most closely related to
NirK sequences from Mesorhizobium sp. in cluster V, in particular
to NirK from strain 4FB11, a halobenzoate-degrader isolated from
marine sediment [68]. However, none of the sequences from our
clones were placed in cluster IV which, in the majority of cases,
contained sequences from cultured species.
Cultivated denitrifiers from grassland soil
To isolate nirK-type denitrifiers from soil, an unspecific
approach was applied to generally enrich microorganisms that
dominate the soil microbial community. Therefore, dilute media
(NB diluted 100-fold), a small inoculum size (100 ␮L of dilutions
10−6 to 10−10 ) and incubation times of up to 4 weeks were used.
Compared to conventional enrichment techniques these modifications were shown previously to result in the growth of rarely
isolated microbial groups [23]. Moreover, Hashimoto et al. [34] also
recently used diluted (100-fold) nutrient broth, but with anaer-
obic conditions in the presence of nitrate, to successfully culture
mostly oligotrophic denitrifiers from soil. Of all our colonies (687)
that grew on plates within 4 weeks of incubation that were transferred onto fresh media, only 490 showed growth after the first
transfer. Among them, the screening procedure, using the same
PCR conditions as for the environmental sample, detected eight
nirK-containing colonies which were then purified to single isolates (GSM-187, -205, -373, -406, -467, -469, -471, and -484). While
colonies 187 and 205 had already been visible after 16 days, the
majority (GSM-373, -406, -467, -469, -471 and -484) grew only after
29 days incubation, suggesting that they belonged to slow-growing
bacterial species that frequently remained undetected by cultivation approaches. From these isolates, the nirK gene fragments were
sequenced and the deduced amino acid sequences were included
into the NirK tree. These NirK sequences were grouped in cluster I (GSM-471, -406, and -467), cluster II (GSM-469), cluster IV
(GSM-187 and -205), and cluster V (GSM-373 and -484). Within
cluster I, the NirK sequence of isolate GSM-471 was identical to
that of a clone from the same habitat, while isolates GSM-406 and
-467 showed only 83–86% sequence identity to the most closely
related clone from the same soil. None of these isolates contained
a nirK gene that was closely related to that of Nitrosomonas sp. TA921i-NH4. The NirK sequence of isolate GSM-469 (cluster II) was
highly related to the NirK sequence of Bradyrhizobium japonicum
USDA 110, a symbiotic nitrogen-fixing bacterium isolated from soybean nodules. The NirK sequences of isolates GSM-373 and -484
grouped with those of several Mesorhizobium strains [34,67] within
cluster V, and with an environmental NirK sequence from the grassland soil. Cluster IV contained NirK sequences of several isolated
strains belonging to a variety of different species, for instance,
Acidovorax sp., Agrobacterium tumefaciens, Bacillus sp., Bosea sp.,
Diaphorobacter sp., Enterococcus sp., Pseudomonas sp., Rhizobium
sp., and Staphylococcus sp., as well as those of isolates GSM-187
and -205. These organisms were reported to be readily cultivated
in other studies by applying the conventional approach to use an
oxidized nitrogen compound as an electron acceptor under anaerobic conditions [21,39,68]. In our study, however, no isolate could
be cultured containing a nirK gene that was related to the environmental clones of cluster III, which numerically dominated the clone
library.
Phylogenetic affiliation of denitrifiers from grassland soil
Based on their 16S rRNA gene sequences, all isolates were
classified as Alpha proteobacteria and members of the order Rhizobiales (Table 1, Fig. 2). More specifically, isolates GSM-406, -467,
-469 and -471 were assigned to Bradyrhizobium species. Isolate
GSM-406 showed 99% sequence identity to the 16S rRNA gene
sequence of bacterium 2RO3, which is affiliated to Bradyrhizobium
sp. [36], and isolates GSM-467 and -471 were affiliated to organisms belonging to Bradyrhizobium spp. [64] with a sequence identity
of 97.9 and 98.5%, respectively. Isolate GSM-469 was most closely
related to Bradyrhizobium canariense [71] and Bradyrhizobium sp.
[57]. Isolates (GSM-373 and -484) affiliated to Mesorhizobium sp.
with a sequence identity of 99.8 and 98.2%, respectively, were also
cultured from the grassland soil. A large fraction (85%) of the oligotrophic denitrifiers that were isolated by Hashimoto et al. [34]
from upland soil, was also classified as Alphaproteobacteria (the
Bradyrhizobium, Agromonas, Nitrobacter and Afipia [BANA] cluster
and Rhizobium/Mesorhizobium cluster). In addition, most of them
contained nirK genes that were related to nirK from Bradyrhizobium
sp. and Mesorhizobium sp. Furthermore, when we reanalyzed their
nirK sequence data, the NirK sequences from their strains were also
related to those of our isolates and environmental clones in clusters I, II, IV, and V. Moreover, it became obvious that a number of
their Bradyrhizobium sp. NirK sequences also grouped in cluster I
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341
Fig. 1. Neighbor joining tree of deduced amino acids of partial nirK gene fragments (72 amino acid positions) from cultured microorganisms and clones retrieved from
grassland soil. Clusters were defined if sequences clustered consistently together when trees were calculated with different calculation methods. Numbers in front of clone
or species designations indicate number of sequences included. Sequences obtained within this study are shown in bold, and accession numbers are given in parentheses.
The scale bar indicates the number of changes per sequence position.
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S. Falk et al. / Systematic and Applied Microbiology 33 (2010) 337–347
Fig. 2. Neighbor joining tree of 16S rRNA genes (1211 positions) from cultured microorganisms retrieved from grassland soil and genes from closely related isolates retrieved
from the database. Sequences obtained within this study are shown in bold, and accession numbers are given in parentheses. The scale bar indicates the number of changes
per sequence position.
of the NirK tree, especially when we included the NirK sequence
of Nitrosomonas sp. TA-921li-NH4. Organisms of the BANA group
are phylogenetically very close to B. japonicum [62] and their common feature is an oligotrophic life style [35,52]. Hence, both data
sets supported the conclusion that the NirK sequences derived
from as yet unknown organisms of cluster I likely originated from
organisms within the Bradyrhizobia. B. japonicum was shown previously to be present at high densities of 104 to 105 cells g−1 dry
soil in fields under paddy-upland rotation, and all isolates (103)
that were isolated from nodules were denitrifiers [2]. Another
study had reported that a high fraction (270/321) of B. japonicum culture collection strains screened were able to denitrify
[75].
The 16S rRNA genes of two isolates, GSM-187 and -205, in cluster
IV were 99.6% identical to the 16S rRNA gene of Bosea PD19 [21] and
96.9% identical to the 16S rRNA gene of Devosia limi, respectively.
These organisms belong to the families of the Bradyrhizobiaceae and
Hyphomicrobiaceae within the Rhizobiales, respectively, and, while
Bosea spp. were shown to denitrify [21,22], no report exists as yet
on the ability of Devosia spp. to denitrify.
Detection of additional denitrification genes
Furthermore, our isolates were screened for the presence of
additional reductase genes involved in the denitrification process
by PCR using gene specific primers (for sequences and references see supplementary Table S1) to detect narG and napA
(membrane-bound and periplasmic nitrate reductase, respectively), nirS (cytochrome cd1 -containing nitrite reductase), norB
(nitric oxide reductase) and nosZ (nitrous oxide reductase). All isolates affiliated with Bradyrhizobium spp. (GSM-406, -467, -469 and
-471) and Bosea spp. (GSM-187) contained a napA-gene, while isolates GSM-373 and -484 (Mesorhizobium sp.) contained narG-genes
(Table 1). B. japonicum was shown previously to contain a NapABtype periplasmic dissimilatory nitrate reductase [44] but no narG
gene has been detected as yet for Mesorhizobium spp. None of the
isolates contained a nirS-gene, which agrees with the fact that
co-occurrence of both types of nitrite reductase within the same
organism has never been described. A norB gene, as well as a nosZ
gene, were amplified from all Bradyrhizobium isolates and the Bosea
sp. except for isolate GSM-471, where no nosZ gene was found. Neither norB nor nosZ were detected for isolates GSM-373 and -484
(Mesorhizobium spp.) and the only denitrification gene that could
be amplified from Devosia isolate GSM-205 was nirK. However, it
is well known that most Rhizobiales do not contain the full suite
of denitrification genes [54]. For instance, while B. japonicum and
Sinorhizobium meliloti contain the nap, nir, nor and nos genes, Rhizobium galegae (formerly Pseudomonas G-179 [7]) harbors nap, nirK
and norB genes but lacks a nosZ gene [83]; Azospirillum brasilense
Sp245 contains napABC genes [70] but organisms belonging to the
genera Azospirillum and Herbaspirillum were variable with regard
to the denitrification genes they contained [45]. Interestingly, only
a nirK gene seems to be present in Rhizobium sullae (formerly R.
hedysari [74]).
Heylen et al. [39,40] found an incongruence of nirK and norB phylogenies and suggested an independent evolution of these genes
among the Rhizobia. However, comparative phylogenetic analysis
of nirK, norB and nosZ genes (if applicable) underlined that they
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S. Falk et al. / Systematic and Applied Microbiology 33 (2010) 337–347
343
Fig. 3. Neighbor joining tree of deduced amino acids of partial norB gene fragments (100 amino acid positions) from cultured microorganisms. Sequences obtained within
this study are shown in bold, and accession numbers are given in parentheses. The scale bar indicates the number of changes per sequence position.
Fig. 4. Neighbor joining tree of deduced amino acids of partial nosZ gene fragments (117 amino acid positions) from cultured microorganisms. Sequences obtained within
this study are shown in bold, and accession numbers are given in parentheses. The scale bar indicates the number of changes per sequence position.
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S. Falk et al. / Systematic and Applied Microbiology 33 (2010) 337–347
Fig. 5. Oxygen consumption and NO production by Mesorhizobium sp. isolates GSM-373 (a) and GSM-484 (b). Only one of the three replicate flasks is shown for each strain.
The initial NO3 − concentration was 2 mM. Oxygen concentrations in the liquid are shown as a shaded area (␮M). Oxygen consumption is the cumulative consumption
of O2 (closed circles, ␮mole/flask); headspace NO production is also cumulative production of NO corrected for losses by dilution (open squares, ␮mole/flask); actual NO
concentration in the liquid not corrected for dilution is shown by closed squares (␮M).
were closely related in isolates GSM-406, -467, -471, and -469,
while the respective genes of Bosea sp. GSM-187 grouped more
distantly (Figs. 1, 3 and 4). Generally, the phylogenetic relationships observed within the functional genes also agreed with the
phylogeny of the organisms based on their 16S rRNA genes (Fig. 2)
which likely excludes horizontal gene transfer as the evolutionary mechanism for individual denitrification genes in our isolates.
Minor variations that were observed in the positioning of single
genes could be due to the fact that, within the Rhizobia, denitrification genes were found to be dispersed over the chromosome in
B. japonicum [44,50] or located together on a plasmid in S. meliloti
[5,17,18].
Denitrification activity
Since it is not good, better: misleading, inadequate, insufficient
to conclude that denitrification activity exists for organisms based
only on the genetic potential for denitrification, potential denitrification activity was assessed after optimization of growth of
the isolates in liquid medium at 25 ◦ C. None of the isolates grew
best on the minimal medium used for isolation, therefore, yeast
extract mannitol (YEM) medium or R2A were preferred (Table 1).
Isolate GSM-205 (Devosia sp.) did not grow on solid medium in
the presence of a nitrogen oxide as an electron acceptor. Also, it
did not grow in liquid medium independent of whether oxygen or
nitrate were provided as terminal electron acceptors, which prohibited testing its ability to denitrify using the assay applied. The
remaining cultures were grown anaerobically in liquid medium in
the presence of nitrate as an electron acceptor and acetylene to
inhibit N2 O reduction, as described by Tiedje [73]. Growth of the
Bradyrhizobium isolates GSM-406 and -471 was very slow and after
15 days incubation they reached only very low cell densities of
OD580 of 0.013 and 0.075, which correlated with a low molar fraction of 6.6 and 21.4% nitrate converted to N2 O, respectively. Among
the remaining Bradyrhizobium isolates, GSM-469 grew comparably
well (OD580 of 0.331) but produced only approximately 60% N2 O
from the nitrate added. B. japonicum ATCC 10324 was also described
as a very inefficient NO3 − converter on a mole per g cell protein
basis, although it converted NO3 − stoichiometrically [47]. On the
other hand, a high conversion of nitrate was observed from isolate
GSM-467 (123.2%) despite moderate growth (OD580 of 0.128). A
high production of N2 O from nitrate at comparably high cell densities was also observed for Bosea sp. isolate GSM-187. Hence, all
isolates that contained the full suite of denitrification genes (except
for lack of nosZ in GSM-471) were able to denitrify. However, only
isolates GSM-467 (Bradyrhizobium sp.) and -187 (Bosea sp.) met
all the criteria of Mahne and Tiedje [47] and Tiedje [73] for defining true denitrifiers which: (i) convert a nitrogen oxide (NO3 − or
NO2 − ) stoichiometrically to N2 O and N2 , (ii) produce gas rapidly,
and (iii) contain a dissimilatory nitrite, nitric oxide, or nitrous oxide
reductase.
The two isolates affiliated with Mesorhizobium spp. (GSM-373
and -484), for which only narG and nirK were detected via PCR,
showed no measurable growth if cultured anaerobically in the presence of nitrate. Therefore, our Mesorhizobium spp. isolates were
tested for the presence of a functional nitrite and nitric oxide reductase. They were grown at an initial concentration of 1% O2 in the
headspace of gas-tight vessels, and gas consumption (O2 ) and production (NO, N2 O, and N2 ) upon the transition from aerobic to
anaerobic growth was measured using a robotized incubation system, as described by Molstad et al. [53]. Both isolates accumulated
NO in the headspace, but the gas kinetics were different (Fig. 5).
For isolate GSM-373, the NO concentration increased when oxygen had been practically depleted and then NO quickly reached
a plateau at 4.2 ␮mol in the headspace and 2.7 ␮M in the liquid.
Thus, a clear shift from aerobic respiration to anaerobic denitrification occurred. After reaching the plateau no net production
of NO occurred and the decrease of NO in the liquid was due to
the dilution of sampling. This suggests that at this point the culture stopped all metabolic activity (hence presumably growth).
For isolate GSM-484, NO production started before oxygen had
been depleted and quickly accumulated at a concentration of about
1.5 ␮mole in the headspace and 0.9 ␮M in the liquid. Then, this
culture continued both denitrification (i.e. net NO production) and
respiration, albeit at a lower speed than before, thus keeping the
NO concentration in the liquid constant. The growth of this culture may theoretically have proceeded throughout, as indicated by
a continued oxygen consumption rate and continued (but slower)
NO production rate. However, whether growth was arrested after
the upshot of NO or not remains unknown, since OD was not measured throughout the incubation for both cultures, but no growth
was detectable in the initial test for denitrification. In all flasks, only
marginal amounts (<0.001% of NO3 − –N) of N2 O accumulated after
NO reached the plateau, and N2 production was not detectable (data
not shown). Thus, isolates GSM-373 and -484 behaved according to
our expectations based on the apparent lack of norB. The marginal
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S. Falk et al. / Systematic and Applied Microbiology 33 (2010) 337–347
accumulation of N2 O was probably due to a chemical conversion
of iron by trace amounts in the medium, since similar conversions have been demonstrated previously. For instance, the purified
cytochrome cd1 -containing nitrite reductase from Pseudomonas
stutzeri produced NO as the main product from 1 mM nitrite, as well
as a small amount of N2 O [79] which could be eliminated by adding
chelators such as EDTA [84]. Moreover, the nitric oxide-reducing
enzymatic process was perturbed by a non-enzymatic reduction
through iron(II) ascorbate in neutral to alkaline aqueous solution
[87].
Although the NO production accounted for only a small portion (<0.1%) of the NO3 − –N present, both isolates produced large
amounts of NO. The high concentrations of NO seemed to be lethal
to the cells since no detectable anaerobic growth occurred. Nitric
oxide has cytotoxic effects and inhibits a number of metalloproteins found in bacterial respiratory chains [78]. Braun and Zumft
[13] found that a NO reductase defective mutant strain (Nor− ) of
P. stutzeri discontinued anaerobic growth on nitrate after NO accumulated. On the other hand, a strain containing a Nir− Nor− double
mutation, that inactivated both nitrite reductase and NO reductase,
was viable under anaerobic conditions. Monza et al. [54] isolated
fast-growing Mesorhizobium spp. which also contained a nirK gene
but not norB or nosZ. These strains were not able to grow under
nitrate respiring conditions and did not accumulate nitrous oxide
in the growth medium. Rhizobium sullae HCNT1, which also cannot grow anaerobically with nitrate or N2 O [16] and which was
unique to date in harboring and expressing only a nirK-type nitrite
reductase, produced high amounts of 15 ␮M NO within 5 min after
addition of 100 ␮M nitrite [74]. In contrast, denitrifiers possessing a NO reductase generally keep the free concentrations of NO in
the nanomolar range to avoid cytotoxic effects [76], for example,
200 nM NO by Rhodobacter sphaeroides 2.4.3 [48]. Toffanin et al. [74]
speculated that nitrite concentrations in the environment that are
typically in the nanomolar range [8] are sufficiently low to prevent
an accumulation of NO to cytotoxic levels even under anaerobic
conditions. An alternative hypothesis would be that organisms
such as Mesorhizobium spp. isolates GSM-373 and -484 require
partners in the environment to scavenge free NO. Organisms containing a cnorB gene but lacking a nitrite reductase gene have been
reported previously [40] and two Thioalkalivibrio stains that were
co-cultured from a hypersaline alkaline lake acted as tandem partners to perform complete denitrification [69]. However, the steps
catalyzed in these two organisms were NO3 − -reduction to NO2 −
and NO2 − -reduction to N2 . A third explanation to the truncation of
the denitrification pathway genes to only nirK would be a distinct
function of NirK, such as its recently discovered ability to reduce
toxic selenite to elemental selenium in R. sullae HCNT1 [6]. This
strain was originally isolated from soils with high selenite content
but we are not aware of similar environmental constraints with
regard to the soil from which the isolates in this study originated.
Thus, the role of NirK in these and previously cultured Mesorhizobium spp. remains to be resolved.
Environmental implications
The majority of sequences in the overall nirK gene tree originated from uncultured bacteria; however, a number of strains with
closely related nirK genotypes could be cultured. Although similar
denitrification genes may have been distributed also to different
phylogenetic groups within the prokaryotes via horizontal gene
transfer, a major part of the genes from as yet uncultured organisms likely originated from organisms affiliated with the Rhizobia.
Closely related genotypes were recurrently detected by cultivationindependent studies exploring denitrifier communities in soils and
most of the strains isolated that contained similar nirK genes were
proven to denitrify in batch cultures. That organisms affiliated
345
with the Rhizobia actively denitrify in soils is supported by the
detection of nirK transcripts related to nirK gene sequences that
presumably originated from members of the Rhizobiales [66,80].
Rhizobiales are nitrogen-fixing bacteria that usually live in symbiosis with legumes, but can also be found free-living in soil when
leguminous plants are absent. As free-living cells, Rhizobia grow
preferentially as aerobic heterotrophic microorganisms that utilize O2 as the final electron acceptor of the respiratory chain for the
generation of ATP. However, nirK, as well as norB, were expressed in
free-living cells under low oxygen conditions and in the presence of
a nitrogen oxide [51,77]. Symbiotic expression of B. japonicum denitrification genes (nir, nor and nos) was also shown in soybean root
nodules [49]. Although the nirK, norB and nosZ genotypes of our isolates, which were affiliated with Bradyrhizobium sp., were closely
related, their ability to grow under nitrate-reducing conditions and
to denitrify was variable. Hence, denitrification in individual strains
seems to depend on additional factors apart from the nitrogen oxide
reductase genotype which remain to be elucidated.
Another interesting finding is the presence of a functional nitrite
reductase and the concurrent lack of a norB gene, as well as of
a NO-reducing enzyme, in strains of Mesorhizobium spp. Organisms belonging to this genus are generally quite versatile with
regard to their ability to denitrify. Besides the nirK gene in the
strains described above, Mesorhizobium spp. have been found to
additionally contain narG as well as norB (data not shown) and
nosZ [54] homologs, while no denitrification gene is present in the
complete genome sequence or in the symbiotic island of two M.
loti strains (see http://genome.kazusa.or.jp/rhizobase). Moreover,
many of them were described as diazotrophs [63], and some are
aerobic denitrifiers [56].
Overall, our results suggest that other properties of the organisms apart from denitrification, such as their ability to fix nitrogen,
are factors that probably select for these microorganisms in the soil.
Acknowledgments
Lars R. Bakken, Frederic Gich, and Jörg Overmann are acknowledged for fruitful scientific discussions. We are grateful to Eulogio
J. Bedmar, Catherine Dandie and Bongkeun Song for providing us
with Bradyrhizobium japonicum USDA 110, Bosea sp. PD 19 and
Mesorhizobium sp. 4FB11, respectively. This work was supported
by grants from the Max Planck Society (Munich, Germany) and the
German Federal Ministry for Education and Research within the
BIOLOG Biodiversity Program (01LC0021).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.syapm.2010.06.004.
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