RESEARCH LETTER

Members of the phylum Acidobacteria are dominant and

metabolically active in rhizosphere soil
Sang-Hoon Lee1, Jong-Ok Ka2 & Jae-Chang Cho1
1
Department of Environmental Sciences, Hankuk University of Foreign Studies, Yong-In, Korea; 2Department of Agricultural Biotechnology, Seoul
National University, Seoul, Korea

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Correspondence: Jae-Chang Cho,
Department of Environmental Sciences,
Hankuk University of Foreign Studies, Yong-In,
Korea 449-791. Tel.: 182 31 330 4350; fax:
182 31 330 4529; e-mail: chojc@hufs.ac.kr
Received 11 December 2007; accepted
20 May 2008.
First published online 16 June 2008.
DOI:10.1111/j.1574-6968.2008.01232.x
Editor: Juan Imperial
Abstract
A culture-independent survey was performed to search for 16S rRNA gene
sequences representing dominant and metabolically active bacteria in rhizosphere
soil. PCR- and reverse transcription-PCR-derived clone libraries were constructed
from DNA and RNA directly extracted from the soil sample. Acidobacteria-related
sequences occupied an unusually large proportion (450%) of both rDNA- and
rRNA-derived clone libraries. This study suggested that the bacteria belonging to
the phylum Acidobacteria might be numerically dominant as well as metabolically
active in the soil sample, implying that the phylum Acidobacteria might be highly
involved in the biogeochemical cycles of the rhizosphere soil.

Keywords
Acidobacteria ; rhizosphere; ecology.

four recently recognized species (Edaphobacter aggregans,

Introduction
Bacteria belonging to the phylum Acidobacteria have been
observed in a wide variety of environments. They have been
identified using molecular surveys based on 16S rRNA gene
sequences in community DNAs, which were directly
extracted from the environments, typically via PCR, cloning,
and sequencing (Ludwig et al., 1997; Hugenholtz et al., 1998;
Barns et al., 1999; Horn et al., 2003; Zimmermann et al.,
2005; Janssen, 2006; Penn et al., 2006). To date, more than
3000 sequences represent this phylum in public databases
and almost all Acidobacteria-related sequences have been
recovered from uncultivated microorganisms in soil samples
(Cole et al., 2005; Janssen, 2006). Considering their phylo-
genetic diversity and ecological distribution pattern, the
phylum Acidobacteria is a metabolically and genetically
diverse group compared with the well-understood phylum
Proteobacteria (Hugenholtz et al., 1998; Barns et al., 1999).
Despite the ubiquity and abundance of the bacteria
belonging to the phylum Acidobacteria, our knowledge of
this phylum is limited because of the relatively few species
that have been described. The phylum Acidobacteria con-
tains three species (Acidobacterium capsulatum, Geothrix
fermentans, and Holophaga foetida) described in ‘Bergey’s
Manual of Systematic Bacteriology’ (Garrity et al., 2004) and
Edaphobacter modestus, Chloracidobacterium thermophilum,
and Terriglobus roseus) (Bryant et al., 2007; Eichorst et al.,
2007; Koch et al., 2008). Although many laboratories are
constantly trying to obtain pure cultures and to elucidate
the ecological niche of the phylum Acidobacteria, only 99
isolates have been reported (Janssen et al., 2002; Sait et al.,
2002, 2006; Joseph et al., 2003; Stevenson et al., 2004; Davis
et al., 2005) and the ecological role of this phylum remains
unknown.
In this study, a culture-independent survey was per-
formed to search for 16S rRNA gene sequences representing
dominant and metabolically active bacteria in a soil sample
from the rhizosphere of a chestnut tree. The sampling site
was located in Gyeonggi, Korea, in an area rife with this
plant species, as chestnuts are the main agricultural product
of this area. We analyzed 16S rDNA- and rRNA-derived
clone libraries constructed from DNA and RNA directly
extracted from the rhizosphere soil. Because DNA obtained
from environmental samples might originate from cells in
low activity or dormancy stages (Felske et al., 1997), an
RNA-derived clone library was constructed to identify
metabolically active members of the microbiota in the soil
sample. We observed that members of the phylum Acido-
bacteria were dominant in both DNA- and RNA-derived

FEMS Microbiol Lett 285 (2008) 263–269

c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
264
clone libraries. To the best of our knowledge, this is the first
report of metabolically active members of the phylum
Acidobacteria in rhizosphere soil.
Materials and methods
Soil sample and nucleic acid extraction
Soil samples (50 g) were taken from the rhizosphere of a
chestnut tree (Castanea crenata), stored at 4 1C for transport
to the laboratory (o30 min), and immediately subjected to
nucleic acid extraction upon arrival. Community DNA and
total RNA were directly extracted using a PowerSoil DNA
isolation kit (MoBio) and an RNA PowerSoil total RNA
isolation kit (MoBio), respectively. The physicochemical
properties of this soil were as follows: soil texture (USDA),
sandy loam; water content, 2.79%; water-holding capacity,
25.6%; total organic carbon, 0.08%; pH, 5.5; nitrate nitro-
gen, 7 mg g1; phosphorus, 4 mg g1; aluminum, 120 mg g1;
ammonia nitrogen, 5 mg g1; calcium, 1400 mg g1; chloride,
200 mg g1; ferric iron, 3 mg g1; magnesium, 13 mg g1;
manganese, 12 mg g1; nitrite nitrogen, 1 mg g1; and sulfate,
50 mg g1.
PCR, reverse transcription (RT)-PCR, cloning, and
sequencing
16S rRNA genes were directly amplified using universal
primers (27F: 5 0 -AGAGTTTGATCCTGGCTCAG-3 0 ,
1492R: 5 -AAGGAGGTGATCCAGCCGCA-3 0 ) designed to
0
anneal to a conserved position in the 3 0 and 5 0 regions of
bacterial 16S rRNA genes (Massol-Deya et al., 1995). The
reaction mixture included 25 mL of RED Taq ReadyMix PCR
Reaction mix with MgCl2 (Sigma), 1 mL each of the forward
and reverse primers (stock concentration, 20 mM), 200 ng of
template DNA extracted from the soil sample, and sterilized
distilled water to give a 50-mL final volume. The PCR
thermal profile was as follows: initial denaturation at 95 1C
for 5 min and 30 cycles consisting of denaturation at 95 1C
for 1 min, primer annealing at 55 1C for 1 min, and exten-
sion at 72 1C for 2 min. The final elongation step was
extended to 20 min. PCR amplification was performed with
a GeneAmp PCR system 9700 (Applied Biosystem).
For RT-PCR, segments of 16S rRNA gene (Escherichia coli
position 968–1401) were reverse transcribed and subse-
quently amplified using Tfl DNA polymerase (Access RT-
PCR system, Promega) and the primers U-968 (5 0 -AACGC
GAAGAACCTTAC-3 0 ) and L-1401 (5 0 -GCGTGTGTACAA
GACCC-3 0 ) (Nubel et al., 1996). The RT-PCR mixtures
(final volume, 50 mL) consisted of 10 mL of 5  avian myelo-
blastosis virus (AMV)-Tfl reaction buffer, 1 mL of 10 mM
each dNTP, 6 mL of 25 mM MgSO4, 1 mL (5 U) of AMV
reverse transcriptase, 1 mL (5 U) of Tfl DNA polymerase,
1 mL each of the forward and reverse primers, and 1 mL of


c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
S.-H. Lee et al.
appropriately diluted RNA (1 mg). The RT-PCR thermal
profile was as follows: cDNA synthesis at 48 1C for 45 min,
initial denaturation at 95 1C for 2 min, and 30 cycles
consisting of denaturation at 94 1C for 30 s, primer anneal-
ing at 56 1C for 30 s, and extension at 68 1C for 40 s. The final
elongation step was extended to 7 min. PCR without the
reverse transcription step was performed to verify the
absence of DNA.
The PCR amplicons prepared from community DNA and
total RNA were purified using a QIAquick PCR purification
kit (Qiagen) and cloned into PCR 2.1-TOPO cloning vector
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with a TOPO TA cloning kit (Invitrogen) to construct
rDNA- and rRNA-derived clone libraries respectively.
Clones were randomly selected from the two libraries and
recombinant plasmids were purified with a Plasmid Mini-
prep kit (Takara). Inserts were sequenced multiple times on
each strand using BigDye terminator chemistry with an
automated capillary sequencer (Applied Biosystems).
Phylogenetic analyses
Cloned sequences were checked for possible chimeric ori-
gins using MALLARD software based on the Pintail algorithm
(Ashelford et al., 2005). A few potentially suspicious
sequences were excluded in subsequent analyses. Phylum-
level phylogenetic positions of the cloned sequences were
determined using the naı̈ve Bayesian classifier (Wang et al.,
2007). RDP’s Sequence Match (RDP database) and NCBI-
BLAST (GenBank database) were used to find closely related
sequences. Cloned sequences, along with reference
sequences, were edited, aligned using CLUSTALW (Thompson
et al., 1994), and subjected to phylogenetic reconstruction
using MEGA software (Kumar et al., 2004). The evolutionary
distances were calculated according to Kimura’s 2-parameter
model (Kimura, 1980). Phylogenetic trees were inferred
using the neighbor-joining algorithm and the tree topology
was statistically evaluated by 1000 bootstrap resamplings.
Nucleotide sequence accession numbers
The 16S rRNA gene sequences obtained in this study have
been deposited in the GenBank database under accession
numbers EF588327–EF588427.
Results and discussion
PCR-amplified 16S rRNA genes from community DNA,
extracted directly from the rhizosphere soil, were cloned,
sequenced, and phylogenetically analyzed (Fig. 1). Sixty-five
percent of the 49 analyzed sequences belonged to the
phylum Acidobacteria, while 22% were members of the
phylum Proteobacteria (Fig. 2). In the Acidobacteria clade,
three bootstrap-supported monophyletic groups, which
corresponded to acidobacterial subdivisions 1, 2, and 3
FEMS Microbiol Lett 285 (2008) 263–269
Acidobacteria in rhizosphere soil 265

100 WSD-014
WSD-048
Environmental clone 614 (Y11632)
WSD-045
Acidobacterium capsulatum (D26171)
100 77 WSD-038
92 WSD-039
WSD-006 1
100 WSD-008
52 WSD-036
86 WSD-046
52 WSD-015
61 WSD-029
74 Environmental clone SJA 149 (AJ009495)
Environmental clone 002 (AF013515)
100 WSD-049 3
94 Environmental clone 613 (Y11631)
75

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55 WSD-024
92 WSD-031
100 WSD-009
100 WSD-037 Acidobacteria
WSD-027
100 WSD-023
72 WSD-030
100 73 WSD-011
WSD-040
100 WSD-005
82 WSD-013
98
WSD-028 2
100 WSD-004
82 WSD-032
Environmental clone DA052 (Y07646)
86 88 WSD-021
52 WSD-044
65 WSD-007
WSD-042
100 WSD-019
67 WSD-025
54 Methylococcus capsulatus (X72771)
99 WSD-018
Burkholderia sp. SAP II (AF052387)
100 WSD-026
100 WSD-003
69 WSD-017
90 83 WSD-002
100 WSD-022
61 WSD-041 Proteobacteria
Fig. 1. Phylogenetic relationships of PCR-ampli- Environmental clone DhA-73 (Y12596)
fied 16S rRNA gene sequences from the rhizo- 100 Environmental isolate (AJ001345)
73
WSD-043
sphere soil. The cloned sequences were 95 Methylopila capsulata (AF004844)
compared with the most closely related 90 WSD-047
sequences obtained from the database (RDP-II), 86 Environmental clone JTB131 (AB015245)
100 WSD-001
as well as other representatives of major bacterial 100 WSD-010
groups. The phylogenetic distances of each 100 WSD-016
100 WSD-020
sequence were calculated using the Kimura 2- Thermomicrobium roseum (M34115)
Clostridia
parameter model and the tree was constructed Thermoanaerobacter ethanolicus (L09162)
WSD-033
using the neighbor-joining algorithm. The num-
100 WSD-034
bers at the nodes indicate the bootstrap score (as Environmental clone C019 (AF013522) Verrucomicrobia
a percentage) and are shown for frequencies at or 100 WSD-035
Environmental clone OPS3 (AF018188)
above the threshold of 50%. The scale bar Planctomycetes
100 WSD-012
represents the expected number of changes per
nucleotide position. 0.05

(Hugenholtz et al., 1998; Janssen, 2006), were observed, and
accounted for 22.5%, 36.7%, and 6.1%, respectively, of the
clones in our library. The minor groups included the phyla
Clostridia (8%), Verrucomicrobia (2%), and Planctomycetes
(2%). These results were in contrast to those from pre-
liminary studies using cultures. Cultures on both oligo-
trophic (R2A agar, Difco) and copiotrophic (Nutrient agar,
Difco) media showed that the majority (99%) of isolates
belonged to the phylum Firmicutes likely due to culturing
biases (data not shown).
Unlike most other soil bacterial community structures
determined by 16S rRNA gene clone libraries, the phylum
Acidobacteria occupied an unusually large proportion (65%)
of our clone library and outnumbered the phylum Proteo-
bacteria by approximately three to one. Although our
observations should not be generalized to the soil microbial
community because of the limited size of our clone library
and an additional sampling of bacterial clones required to
determine the full extent of bacterial community diversity,
our results do nevertheless suggest that the phylum

FEMS Microbiol Lett 285 (2008) 263–269

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
266
70
60
50
Contribution (%)
40
30
20
10
0
DNA RNA
Fig. 2. Contribution of 16S rRNA gene sequences belonging to phylum
Acidobacteria (black bar) and phylum Proteobacteria (gray bar) in DNA-
and RNA-derived libraries. Striped, slashed, and checked bars indicate
acidobacterial subdivisions 1, 2, and 3, respectively.
Acidobacteria might be the predominant bacterial group in
our rhizosphere soil sample.
In a comprehensive review of the dominant soil bacterial
taxa in 16S rRNA gene libraries (Janssen, 2006), members of
the phylum Proteobacteria represented an average of 39%
(range, 10–77%) of libraries constructed from soil bacterial
communities, and the members of the phylum Acidobacteria
made up an average of 20% (range, 5–46%). As reviewed by
Janssen(2006), the phylum Proteobacteria, which is metabo-
lically versatile and genetically diverse, comprises the largest
fraction of the bacterial community in soil ecosystems,
including the rhizosphere (Filion et al., 2004; Sanguin et al.,
2006). Even in other natural and human-made ecosystems
(e.g. marine, freshwater, wastewater, hot spring microbial
mats, and the oral cavity), the phylum Proteobacteria is more
dominant than the phylum Acidobacteria (LaPara et al.,
2000; Layton et al., 2000; Sievert et al., 2000; Smit et al.,
2001; Paster et al., 2002; Martiny et al., 2003; Polymenakou
et al., 2005; Penn et al., 2006). To our knowledge, there are
only two other reports that show that the phylum Acidobac-
teria contributes substantially (450%) to the total bacterial
community. In the study conducted by Kuske et al.(1997),
the phylum Acidobacteria was the most dominant bacterial
group in 16S rRNA gene clone libraries, which were con-
structed from soil samples taken from the Sunset Crater
National Monument (53.6%) and Coconino National Forest
(57.1%) in Arizona, United States. Later, Dunbar et al.
(1999) observed that Acidobacteria-related sequences were
most abundant in clone libraries constructed from soil
samples collected from the same geographic location. Based
on the distribution of 16S rRNA gene sequences among the
bacterial phyla found in the literature, Smit et al. (2001)
suggested that the ratio between the number of Proteobac-
teria and Acidobacteria might be determined by the trophic
level of the soil. The ratio of Proteobacteria to Acidobacteria


c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
S.-H. Lee et al.
was o 0.34 for oligotrophic soil and 40.46 for copio-
trophic soil, indicating that oligotrophic environments have
a selective effect for Acidobacteria. They considered the two
Arizona soils (Kuske et al., 1997; Dunbar et al., 1999) to be
oligotrophic (ratio, 0.16). Based on their hypothesis, the
trophic level of our rhizosphere soil sample could be
marginally oligotrophic (ratio, 0.34). However, Marilley
et al. (1999) found that the rhizosphere, which is a relatively
copiotrophic niche for bacteria, has a selective effect toward
the phylum Proteobacteria and is detrimental to the phylum
Acidobacteria.
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To determine the metabolically active members of bacter-
ial community in our soil sample, we extracted total RNA
from the soil sample and constructed a crDNA (comple-
mentary ribosomal RNA gene) library. Because the ribo-
some per cell ratio is approximately proportional to the
growth rate of bacteria, and a higher number of ribosomes
suggests metabolically active cells (Wagner, 1994; Pace,
1997), such 16S crDNA libraries are assumed to better
reflect metabolically active bacteria. Indeed, the rrn operon
copy number, which is largely unknown for uncultured
bacteria, did not affect the level of rRNA (Fogel et al., 1999).
RT-PCR amplicons were cloned, sequenced, and phylo-
genetically analyzed (Figs 2 and 3). Fifty-six percent of 52
analyzed clones fell into the phylum Acidobacteria, while
31% was related to the phylum Proteobacteria. Similar to the
result from the clone library constructed with the commu-
nity DNA, three subdivisions 1, 2, and 3 were observed in
the phylum Acidobacteria, representing 17.3%, 9.6%, and
28.8%, respectively, of the total crDNA library. To compare
the sequences in crDNA library and rDNA library, we used a
local alignment approach using NCBI-BLAST (http://
www.ncbi.nlm.nih.gov/blast/Blast.cgi), because the
RT-PCR-amplified region of 16S rRNA gene was different
from the PCR-amplified region of 16S rRNA gene (to avoid
inhibition of reverse transcription caused by the secondary
structure of the 16S rRNA gene molecules). The NCBI-BLAST
showed that the closest relatives of Acidobacteria-related
sequences in DNA- and RNA-derived clone libraries were
identical. This suggested that the cloned 16S rRNA genes
could originate from the highly similar phylotypes that
dominated the DNA-derived clone library. Hence, we con-
cluded that the phylum Acidobacteria might be numerically
dominant as well as metabolically active in our rhizosphere
soil. However, because we performed the nucleic acid
extractions after the soil samples were transported to the
laboratory, changes in population structure as well as the
rRNA composition might occur during the sample trans-
portation even at 4 1C. We considered our conclusions to be
valid under the assumption that no significant changes
occurred during the sample storage period.
The phylum Acidobacteria occupied a large proportion of
DNA- and RNA-derived clone libraries, indicating that the
FEMS Microbiol Lett 285 (2008) 263–269
Acidobacteria in rhizosphere soil 267

67 WSR-049
69 WSR-035
WSR-040
55
72 WSR-026
83 WSR-022
WSR-001 1
80 Acidobacterium capsulatum (D26171)
81 Environmental clone 614 (Y11632)
82 WSR-034
WSR-003
72 WSR-019
WSR-028
WSR-024
92 WSR-043
94 WSR-029 2
86 51 WSR-009
91 Environmental clone DA052 (Y07646) Acidobacteria
59 WSR-016

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WSR-004
WSR-033
WSR-030
WSR-018
96 WSR-039
WSR-038
WSR-032
WSR-012
Environmental clone 613 (Y11631) 3
Environmental clone 002 (AF013515)
Environmental clone SJA-149 (AJ009495)
WSR-047
86 WSR-042
WSR-023
WSR-045
84 WSR-020
67 WSR-007
98 Polyangium sp. (M94280)
95 Polyangium cellulosum (M94282)
94 WSR-044
WSR-002
91 Desulforhabdus amnigenus (X83274)
WSR-011
74 Environmental clone GKS98 (AJ224990)
WSR-048
Pseudomonas saccharophila (AB021407)
98 Environmental isolate DhA-73 (AF125877)
WSR-036
71 WSR-005
Frateuria aurantia (AJ010481)
100 WSR-013
Environmental clone DA111 (Y12596)
WSR-006
Azospirillum brasilense (Z29617) Proteobacteria
WSR-017
88 Acidosphaera rubrifaciens (D86512)
59 WSR-027
97 Mesorhizobium loti (Y14159)
100 93 Mesorhizobium plurifarium (Y14161)
96 WSR-021
WSR-025
100 WSR-008
98 WSR-010
Caulobacter sp. (AJ227767)
Fig. 3. Phylogenetic relationships of RT-PCR- WSR-050
amplified 16S rRNA gene sequences from the 94 Methylocystis sp. WI14 (AF153281)
Environmental clone DA067 (Y07582)
rhizosphere soil. The cloned sequences were Bradyrhizobium genosp. G (Z94804)
compared with the most closely related 100 WSR-037
100 Environmental clone WCHD3-88 (AF05 0561)
sequences obtained from the database (RDP-II), WSR-014
as well as other representatives of major bacterial Environmental isolate (AF083615) Verrucomicrobia
groups. The phylogenetic distances of each 100 WSR-015
52 Environmental clone OPB9 (AF027043)
sequence were calculated using the Kimura 2- 68 WSR-046
83 Environmental clone SAR307 (U20798) Clostridia
parameter model and the tree was constructed WSR-041
using the neighbor-joining algorithm. The num- 72 Actinomyces sp. TM220 (X92707)
WSR-051
bers at the nodes indicate the bootstrap score 61 Frankia sp. (M55343)
75 WSR-052
(as a percentage) and are shown for frequencies Actinobacteria
96 Environmental clone DA016 (Y07606)
at or above the threshold of 50%. The scale bar 94 WSR-031
represents the expected number of changes per
nucleotide position. 0.02

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c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
268
members of the phylum Acidobacteria might be highly
involved in biogeochemical cycles in the soil. Although
Acidobacteria-related sequences have been retrieved from
diverse environments, this ubiquitous bacterial taxon is
known as an uncultured bacterial group (at least it is
difficult to culture from environmental samples), and its
ecological function remains largely unknown. The develop-
ment of effective cultivation methods, followed by genetic
and physiological characterization of the bacteria belonging
to the phylum Acidobacteria, as well as the metagenome
analysis suggested by Quaiser et al. (2003), is a relevant and
ecologically significant task that would elucidate their eco-
logical roles.
Acknowledgements
This work was supported by the GRRC program (PR07011)
of Gyeonggi province and by the KOSEF grant R01-2006-
000-11061-0.
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Supplementary material
The following supplementary material for this article is
available online:
Table S1. Summary of PCR-amplified 16S rRNA gene
sequences from rhizosphere soil.
Table S2. Summary of reverse transcription-PCR-amplified
16S rRNA sequences from rhizosphere soil.
This material is available as part of the online article
from: http://www.blackwell-synergy.com/doi/abs/10.1111/
j.1574-6968.2008.01232.x (This link will take you to the
article abstract.)
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