Molecular microbial diversity of a soil sample and

ARTICLE IN PRESS
Microbiological Research 162 (2007) 15—25
www.elsevier.de/micres
Molecular microbial diversity of a soil sample and
detection of ammonia oxidizers from Cape Evans,
Mcmurdo Dry Valley, Antarctica
Bhupendra V. Shravage, Kannayakanahalli M. Dayananda,
Milind S. Patole, Yogesh S. Shouche
Molecular Biology Unit, National Centre for Cell Science, Ganeshkhind, Pune 411007, India
Accepted 3 January 2006
KEYWORDS
Antarctica;
SSU rDNA clone
library;
Soil diversity
Summary
The aim of our study was to estimate the uncultured eubacterial diversity of a soil
sample collected below a dead seal, Cape Evans, McMurdo, Antarctica by an SSU
rDNA gene library approach. Our study by sequencing of clones from SSU rDNA gene
library approach revealed high diversity in the soil sample from Antarctica. More
than 50% of clones showed homology to Cytophaga—Flavobacterium–Bacteroides
group; sequences also belonged to a, b, g proteobacteria, Thermus–Deinococcus and
high GC gram-positive group; Phylogenetic analysis of the SSU rDNA clones showed
the presence of species belonging to Cytophaga spp., Vitellibacter vladivostokensis,
Aequorivita lipolytica, Aequorivita crocea, Flavobacterium spp., Flexibacter sp.,
Subsaxibacter broadyi, Bacteroidetes, Roseobacter sp., Sphingomonas baekryungensis, Nitrosospira sp., Nitrosomonas cryotolerans, Psychrobacter spp., Chromohalobacter sp., Psychrobacter okhotskensis, Psychrobacter fozii, Psychrobacter
urativorans, Rubrobacter radiotolerans, Marinobacter sp., Rubrobacteridae, Desulfotomaculum aeronauticum and Deinococcus sp. The presence of ammonia oxidizing
bacteria in Antarctica soil was confirmed by the presence of the amoA gene.
Phylogenetic analysis revealed grouping of clones with their respective groups.
& 2006 Elsevier GmbH. All rights reserved.
Introduction
Corresponding author.
E-mail address: [email protected] (Y.S. Shouche).
The McMurdo Dry Valleys of Antarctica has a
unique climate. They are mostly ice-free throughout the year, with average annual temperatures
around 22 1C and relative humidity of 14–47%. The
0944-5013/$ - see front matter & 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.micres.2006.01.005
ARTICLE IN PRESS
16
strong katabatic winds from the polar ice plateau
create truly desert conditions (Friedmann et al.,
1987). The soils of dry valleys were assumed to be
abiotic (Horowitz et al., 1972). However, Friedmann
and Ocampo (1976) first described the presence of
microorganisms within the pore space of sandstones.
Since then a considerable number of different
microorganisms have been isolated from these
habitats (Baublis et al., 1991; Dawid et al., 1988;
Friedmann, 1980; Friedmann, 1982; Hirsch and
Gallikowski, 1985; Hirsch and Gallikowski, 1998;
Hirsch et al., 1988; Kappen and Friedmann, 1983).
However, these studies do not give a complete
picture about microbial community structure due
to inherent drawbacks of cultural methods; such
limitations are due to the reliance on selective
media and to the fact many bacterial populations
are refractory to cultivation (Head et al., 1998).
Consequently, they provide an incomplete assessment of the community structure. In recent years,
culture independent approaches, especially 16S
rDNA-based methods have been used extensively
for community analysis (Amann et al., 1995;
Hugenholtz et al., 1998; Paster et al., 2001). In
case of Antarctica most of the studies have
concentrated on the microbial diversity of sea
ice, lake sediments and cyanobacterial mat samples from lakes (Brambilla et al., 2001; Gordon et
al., 2000; Taton et al., 2003). The rRNA approach
has been used earlier to evaluate the bacterial
diversity of habitats such as sub-glacial lake ice and
perennial lake ice (Gordon et al., 2000), lake
sediments (Bowman et al., 2000a, b; Sjoling and
Cowan, 2003) and sediment core (Bowman et al.,
2000a, b; Torsvik et al., 1996) and sea ice (Bowman
et al., 1997) from Antarctica.
There is only one report of such study for the soil
samples from Schirmacher Oasis, Antarctica (Shivaji et al., 2003). The soil samples differ from the
lake sediments or mats because they provide
different ecological niches for microorganisms
(Shivaji et al., 2003). In this paper, we report a
study on a soil sample from McMurdo Dry Valley in
Antarctica using SSU ribosomal gene clone library
approach.
Materials and methods
Soil sampling
The soil sample was collected below a dead seal,
in Cape Evans, McMurdo Dry Valley, Antarctica,
771380 1000 S and 1661250 0400 E, in January 1995. No
vegetation was observed near the place of sample
B.V. Shravage et al.
collection. The sample was collected in sterilized
polyethylene bag, placed in ice and stored at
70 1C until further processing. The air temperature at the time of collection was +5 1C.
Isolation of DNA
Total community DNA was extracted from 1 g of
soil sample using protocol III as described earlier
with minor modifications (Yeates et al., 1997). In
brief, the soil sample was resuspended in extraction
buffer (100 mM Tris–Cl (pH 8.0), 100 mM Na2EDTA
(pH 8.0), 1.5 M NaCl) and incubated overnight at
37 1C by shaking at 150 rpm with lysozyme (200 mg/
ml) and Protenase K (100 mg/ml). The step that
includes the disruption of cells using glass beads was
omitted. The same sample was processed for the
DNA extraction again. The sample was processed for
third extraction and visualized for any DNA by
electrophoresis on 0.7% agarose gel and ethidium
bromide staining. The DNA was pooled from first and
second the extraction steps and used for PCR
amplification of 16S rDNA gene.
Polymerase chain reaction (PCR) and
generation of SSU rDNA clone library
Eubacterial SSU rDNA gene universal primers
16F27N (50 -CCAGAGTTTGATCMTGGCTCAG-30 ) and
16R1525XP (50 -TTCTGCAGTCTAGAAGGAGGTGTWT
CCAGCC-30 ) (Brosius et al., 1978) were used to
amplify the 16S rDNA gene from total community
DNA. All PCR reactions were performed in a PE 9700
thermal cycler (Applied Biosystems, Inc., Foster
City, CA) under following conditions: Initial denaturation at 94 1C for 2 min followed by 35 cycles of
94 1C for 30 s, 64 1C for 1 min and 72 1C for 1 min,
with an additional extension step at 72 1C for 10
min. Reaction mixture of 50 ml contained 50 ng of
DNA template, 25 pm of each primer, 1 ml of 10 mM
deoxynucleoside triphosphates (Amersham biosciences) 5 ml of 10X ThermoPol reaction buffer
(20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM
(NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100) and 1U
Taq DNA polymerase (New England Biolabs). PCR
products were run on 0.8% agarose gel along with
standard molecular weight marker, stained with
ethidium bromide solution and visualized for
1.5 kbp product under UV transilluminator. The
amplified rDNA was cloned in pGEM-T vector
(Promega Corporation, USA) in accordance with
the manufacturer’s instructions. The presence of
ammonia oxidizing bacteria was determined by PCR
using the conserved primers for the gene encoding
the active site subunit of ammonia monoxygenase
ARTICLE IN PRESS
Molecular microbial diversity of a soil sample and detection of ammonia oxidizers from Antarctica
(amoA). The primer combination consisted of
amoA-1F and amoA-2R which has been proved most
reliable in earlier studies (Rotthauwe et al., 1997).
The forward primer used amoA-1F (50 -GGGGTTTC
TACTGGTGGT-30 ) targets a stretch corresponding to
positions 332–349 and the reverse primer used
(amoA-2R (50 -CCCCTCKGSAAAGCCTTCTTC-30 ) targets a stretch corresponding to positions 802–
822of the open reading frame for the amoA gene
sequence of Nitrosomonas europaea (McTavish
et al., 1993). PCR reactions were as described
above except that the annealing temperature was
60 1C for 1 min 30 s.
Nucleotide sequencing of cloned SSU rDNA
genes
Crude DNA lysates of clones were used for PCR
amplification of the clones using vector specific
primers. Resulting clones were screened by PCR
using vector specific primers. The nucleotide sequence of the cloned SSU rDNA gene was determined
using vector specific primers and sequenced directly
on ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Inc.) to obtain at least 500 bp from 50 end of
SSU rDNA gene. This stretch contains three hyper
variable regions, three variable regions and three
constant regions that help in the discrimination of
closely related species.
Sequence and phylogenetic analyses
All the sequences were checked for chimera
formation by using CHECK_CHIMERA software of the
ribosomal database project (Maidak et al., 1997).
Analysis of the cloned SSU rDNA gene sequences
was performed using basic local alignment search
tool (BLAST) (Altschul et al., 1997) at NCBI server.
Phylogenetic analyses were restricted to 482
nucleotide positions that were unambiguously
alignable in all sequences. A phylogenetic analysis
by neighbor joining method was done using the
PHYLIP version 3.63 (Felsenstein, 1989). The
number of clones representing a blast hit was
considered as clones representing a species and
species richness, which is the number of species in
the assemblage, was calculated using the default
parameters provided in EcoSim 7.0 software (Gotelli and Entsminger, 2001).
Results and discussion
The existing procedure (Yeates et al., 1997) was
modified slightly to get good quality high molecular
17
weight DNA from soil. Our extraction procedure
yielded 25–30 mg of good quality DNA from 1 g of
soil, which was used for PCR without further
purification. 1.5 kb region of almost complete SSU
rDNA gene was amplified using eubacteria specific
primers. To minimize the errors the PCR was done
in triplicates and the PCR products were pooled
before cloning into pGEMT-easy vector. A total of 86
clones were obtained. The insert DNA from clones
was PCR-amplified using vector specific primers and
sequenced. The sequences had at least 500
nucleotides from 50 -terminus of 16S rRNA gene.
This stretch contains three hyper variable regions,
three variable regions and three conserved regions.
Analysis of the cloned SSU rDNA gene sequences
was performed using the BLAST at NCBI server. None
of sequences was detected as potential CHIMERA.
Table 1 lists the clones along with percentage
similarity with nearest blast hits. The percentage
similarity with clone and its closest blast hits
ranged from 85% to 99% (Table 1). A phylogenetic
tree was drawn which clearly shows the affiliation
of different SSU rDNA clones with its closest BLAST
hits (Fig. 1). The clones were distributed in to three
main clusters, viz. proteobacter, Cytophaga/Flavobacterium/Bacteroides (CFB) and high GC/Deinococcus–Thermus group. The clone sequences
showed similarity to different subclasses of a, b, g
Proteobacteria, CFB group, High GC gram positive
and Thermus–Deinococcus groups. Nine of the
clones had more than 97% similarity to the closest
BLAST hits and the rest of the clones had less or
equal similarity to BLAST hits. This suggests the
presence of a large number of operational taxonomic units (OTUs) among the clones. In the soil
sample studied we found that some clones which
showed their closest relation to Nitrospira and
Nitorsomonas spp. As these bacteria may be
involved in ammonia oxidation process, their
functionality was checked by PCR for the presence
of ammonia monoxygenase gene (amoA). The PCR
was done with primers amoA-1F and amoA-2R
resulted in 491 bp PCR product (Fig. 5) that
confirmed the presence of amoA gene in Antarctic
soil sample.
CFB group
Cytophagales can be highly abundant in the
costal pelagic zone of Antarctica. They constitute
20–70% of the bacterial biomass in various seawater
samples. They have major roles to play in mineralization of macromolecules to micronutrients
(Glockner et al., 1999). 53.5% of the clones from
this soil sample showed similarity to CFB group. Out
ARTICLE IN PRESS
18
B.V. Shravage et al.
Table 1.
Sl.
No.
List of the SSU rDNA clones along with their accession numbers and BLAST hits
Name of the
clone
Acc. no. of
clones
Closest blast his
Cytophaga–Flavobacterium–Bacteroidetes
1
bh1.2
AF417864
Cytophaga sp. B12
2
bh1.19
AF417871
Cytophaga sp. B12
3
bh2.4
AF417881
Cytophaga sp. B12
4
bh2.54
AF417890
Cytophaga sp. B12
5
bh2.59
AF447178
Cytophaga sp. B12
6
bh2.65
AF417894
Cytophaga sp. B12
7
bh3.20
AF417899
Cytophaga sp. B12
8
bh3.37
AF417901
Cytophaga sp. B12
9
bh3.63
AF417903
Cytophaga sp. B12
10
bh5.7
AF417916
Cytophaga sp. B12
11
bh5.18
AF417919
Cytophaga sp. B12
12
bh5.13
AF447180
Cytophaga sp. B12
13
bh5.49
AF419200
Cytophaga sp. B12
14
bh6.11
AF419207
Cytophaga sp. B12
15
bh6.22
AF419208
Cytophaga sp. B12
16
bh7.22
AF420427
Cytophaga sp. B12
17
bh6.44
AF419211
Cytophaga sp. B12
18
bh6.45
AF419212
Cytophaga sp. B12
19
bh6.53
AF419214
Cytophaga sp. B12
20
bh6.61
AF419217
Cytophaga sp. B12
21
bh7.10
AF419280
Cytophaga sp. B12
22
bh4.59
AF417911
Cytophaga sp. 4301-10/1
23
bh2.35
AF417885
Vitellibacter vladivostokensis
24
bh3.26
AF417900
Vitellibacter vladivostokensis
25
bh1.33
AF417873
Aequorivita lipolytica Y10-2T
26
bh8.7
AF419285
Aequorivita crocea Y12-2T
27
bh8.55
AF419290
Flavobacterium sp. GWS-SE-H171
28
bh1.8
AF417865
Flavobacterium sp. V4.MS.12
29
bh1.44
AF417893
Flexibacter sp. OUB39
30
bh8.6
AF419284
Subsaxibacter broadyi strain P7
31
bh8.17
AF419286
Antarctic bacterium R-9217
32
bh4.63
AF419193
Antarctic bacterium R-7933
33
bh5.22
AF419195
Marine bacterium KMM 3937
34
bh1.62
AF417880
Bacterium Km4
35
bh8.26
AF419287
Unidentified bacterium
36
bh2.10
AF417882
Bacteroidetes bacterium GMDJE10E6
37
bh2.14
AF417883
Bacteroidetes bacterium GMDJE10E6
38
bh2.16
AF417884
Bacteroidetes bacterium GMDJE10E6
39
bh5.9
AF417917
Bacteroidetes bacterium GMDJE10E6
40
bh5.45
AF419199
Bacteroidetes bacterium GMDJE10E6
41
bh2.41
AF417886
Bacteroidetes bacterium GMDJE10E6
42
bh2.58
AF417892
Bacteroidetes bacterium GMDJE10E6
43
bh4.2
AF417902
Bacteroidetes bacterium MO48
44
bh4.20
AF417906
Bacteroidetes bacterium MO54
45
bh4.10
AF417904
Uncultured Bacteroidetes bacterium
46
bh6.8
AF419206
Uncultured Bacteroidetes bacterium clone
ML602J-37
Alpha proteobacter
1
bh3.3
AF417896
Roseobacter sp. ANT9270
2
bh7.13
AF419281
Roseobacter sp. ANT9270
3
bh7.15
AF419282
Roseobacter sp. ANT9270
4
bh4.32
AF417907
Sphingomonas baekryungensis
5
bh5.52
AF419202
Uncultured bacterium clone DC-5
Acc. no. of Similarity of SSU rDNA
sequence 50 end of the
closest
blast hit
clone (X500 bp) to its
closest match (%)
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF125327
AF536383
AF125327
AF125327
AF125327
AJ542652
AB071382
AB071382
AY027805
AY027806
AY332172
AJ244703
AB058913
AY693999
AJ441008
AJ440987
AF536386
AF367848
AJ786810
AY162091
AY162091
AY162091
AY162091
AY162091
AY162091
AY162091
AY553119
AY553122
AY701441
AF507870
95
95
93
96
97
93
96
94
96
91
94
97
97
94
95
94
96
90
96
97
97
87
92
92
92
96
93
90
93
94
97
97
89
93
94
88
87
88
87
90
85
86
88
93
92
93
AY167260
AY167260
AY167260
AY608604
AY529883
96
96
96
98
96
ARTICLE IN PRESS
Molecular microbial diversity of a soil sample and detection of ammonia oxidizers from Antarctica
19
Table 1. (continued )
Sl.
No.
6
Beta
1
2
3
4
5
Name of the
clone
bh5.33
Proteobacter
bh1.16
bh1.36
bh8.1
bh8.2
bh5.50
Acc. no. of Similarity of SSU rDNA
sequence 50 end of the
closest
blast hit
clone (X500 bp) to its
closest match (%)
Acc. no. of
clones
Closest blast his
AF419197
Uncultured marine eubacterium HstpL100 AF159651
98
AF417869
AF417875
AF419283
AF420429
AF447181
Nitrosospira sp. Nsp65
Nitrosomonas cryotolerans
Uncultured bacterium clone KD4-7
Uncultured bacterium clone 234ds5
Uncultured earthworm cast bacterium
clone c286
AY123813
AF272423
AY218644
AY212681
AY154632
95
94
96
97
97
Uncultured Psychrobacter sp. clone
GAMMA4A
Uncultured Psychrobacter sp. Clone
GAMMA4A
Uncultured Psychrobacter sp. Clone
GAMMA4A
Uncultured Psychrobacter sp. Clone
GAMMA4A
Uncultured Psychrobacter sp. clone
GAMMA4A
Gamma proteobacterium KT0813
Gamma proteobacterium KT0813
Gamma proteobacterium KT0813
Gamma proteobacterium KT0813
Gamma proteobacterium KT0813
Gamma proteobacterium KT0813
Chromohalobacter sp. MAN K24
Psychrobacter okhotskensis
Psychrobacter okhotskensis
Psychrobacter okhotskensis
Psychrobacter fozii
Psychrobacter sp. HS5323
Psychrobacter sp. p3C
Psychrobacter sp. IC008
Psychrobacter urativorans
Rubrobacter radiotolerans
Marinobacter sp. MED106
Arctic sea ice bacterium ARK9987
Uncultured bacterium clone ARKDMS-9
AY494610
98
AY494610
96
AY494610
96
AY494610
97
AY494610
97
AF235130
AF235130
AF235130
AF235130
AF235130
AF235130
AB166934
AB094794
AB094794
AB094794
AY771717
AY443042
AJ495805
U85875
AJ609555
U65647
AY136121
AF468405
AF468252
93
92
96
93
96
96
93
99
95
98
99
95
99
98
97
93
94
99
93
Rubrobacteridae bacterium Ellin5415
Desulfotomaculum aeronauticum
AY673142
AY703033
96
95
Uncultured bacterium clone B-42
Uncultured bacterium clone B-42
Deinococcus sp. AA63
AY676482
AY676482
AJ585986
87
87
97
Gamma proteobacter
1
bh1.1
AF417862
2
bh1.11
AF417867
3
bh4.60
AF417912
4
bh4.66
AF417914
5
bh6.55
AF419215
6
bh1.37
AF417878
7
bh1.56
AF417879
8
bh2.44
AF417887
9
bh4.12
AF417905
10
bh4.40
AF417909
11
bh4.62
AF417913
12
bh4.45
AF417910
13
bh3.1
AF417895
14
bh5.19
AF419194
15
bh6.31
AF419209
16
bh3.5
AF417898
17
bh5.2
AF417915
18
bh2.51
AF417889
19
bh5.29
AF419196
20
bh5.53
AF419203
21
bh3.17
AF417897
22
bh3.31
AF447179
23
bh6.39
AF419210
24
bh8.50
AF419288
High GC gram +
1
bh7.44
AF420428
2
bh4.3
AF417908
Dienococcus–Thermus
1
bh6.6
AF419205
2
bh6.58
AF419216
3
bh8.52
AF419289
of 46 clones 22 belonged to Cytophaga sp. with
87–97% similarity in the BLAST. The intra clone
similarity was ranging between 59% and 91%, which
shows high diversity among Cytophaga sp. in the
soil sample. Seven clones showed 85–90% similarity
to Bacteroidetes bacterium isolate GMDJE10E6 and
had 82–99% intra clone similarity (data not shown);
two of the clones belonged to Bacteroidetes
bacterium clone MO48 and two of the Bacteroidetes were showing similarity to uncultured clones.
The clones representing Vitellibacter vladivostokensis, Aequorivita lipolytica, Flavobacterium sp.,
ARTICLE IN PRESS
20
B.V. Shravage et al.
bh5.2
bh4.66
bh5.19
bh4.60
bh1.11
bh1.1
bh6.55
Uncultured Psychrobacter sp. clone G
Psychrobacter fozii
Psychrobacter sp. HS5323
Psychrobacter okhotskensis
Arctic sea ice bacterium ARK9987
bh3.1
bh6.31
bh5.29
Psychrobacter sp. IC008
bh2.51
Psychrobacter sp. p3C
Psychrobacter urativorans
bh3.5
bh5.53
Chromohalobacter sp. MAN K24
bh4.45
bh3.31
Marinobacter sp. MED106
bh8.50
Uncultured bacterium clone ARKDMS
bh1.16
bh1.36
Nitrosomonas cryotolerans
Nitrosospira sp. Nsp65
bh8.1
Uncultured bacterium clone KD4-7
Uncultured bacterium clone 234ds5
bh7.15
bh3.3
bh7.13
Roseobacter sp. ANT9270
Uncultured earthworm cast bacterium
Uncultured bacterium clone DC-5
Uncultured marine eubacterium HstpL
bh5.50
bh5.52
bh5.33
S. baekryungensis
bh4.32
bh6.39
bh1.56
bh4.62
bh4.40
bh2.44
Gamma proteobacterium KT0813
bh4.12
bh1.37
bh8.2
bh7.44
bh5.7
bh5.22
bh3.37
bh5.18
bh5.13
bh1.2
bh6.45
bh1.19
bh3.20
bh6.53
bh6.44
bh2.65
Cytophaga sp. B12
bh6.61
bh6.22
bh5.49
bh2.59
bh1.44
bh7.10
bh4.63
bh3.63
bh7.22
bh2.54
bh2.4
bh6.11
Marine bacterium KMM 3937
Antarctic bacterium R-7933
Flexibacter sp. OUB39
bh8.26
bh8.6
Subsaxibacter broadyi strain P7
Antarctic bacterium R-9217
bh8.17
Unidentified bacterium
bh8.7
Aequorivita crocea Y12-2T
Aequorivita lipolytica Y10-2T
bh8.55
Vitellibacter vladivostokensis
Bacterium Km4
Flavobacterium sp. GWS-SE-H171
bh1.62
bh2.35
bh3.26
bh1.33
bh1.8
bh4.59
Cytophaga sp. 4301-10/1
Flavobacterium sp. V4.MS.12
bh4.2
bh4.3
bh5.9
bh2.14
bh4.10
bh2.58
bh2.41
bh2.10
bh2.16
bh6.8
bh5.45
bh4.20
Uncultured Bacteroidetes bacterium
Bacteroidetes bacterium GMDJE10E6
Uncultured Bacteroidetes bacterium c
Bacteroidetes bacterium MO54
Bacteroidetes bacterium MO48
bh6.58
bh6.6
Uncultured bacterium clone B-42
Deinococcus sp. AA63
bh8.52
Desulfotomaculum aeronauticum
Rubrobacteridae bacterium Ellin5415
bh3.17
Rubrobacter radiotolerans
Halobacterium salinarum
Proteobacter
Group
CytophagaFlavobacteriumBacteroidetes
Group
High GC and
DeinococcusThermus Group
Figure 1. Neighbor joining tree representing the affiliation of the SSU rDNA clones to their closest related sequence.
Halobacterium salinarum is used as an out-group.
ARTICLE IN PRESS
Molecular microbial diversity of a soil sample and detection of ammonia oxidizers from Antarctica
Flexibacter sp., Subsaxibacter broadyi were also
found in the soil sample analyzed (Fig. 2). These
clones shared 92–97% similarity to their closest
relatives. Such observations with less than 95%
similarity to 16S rDNA gene sequences available in
the GenBank indicate the presence of novel and
uncharacterized bacterial lineage from Antarctic
soil. The soil sample from our study had large
number of members of CFB group and showed
similarity to the microbial flora of SIMCO (Brown
and Bowman, 2001) and differed completely from
the earlier study on soil sample form Antarctica
(Shivaji et al., 2004).
Proteobacter group
Nearly 40% of the clones from the library
belonged to Proteobacteria group (Fig. 3). Out of
35 clones six, five and 24 clones belonged to a, b
and g subgroups of proteobacteria, respectively. In
our study, gamma proteobacter subgroup constitutes 28% of the clones, which is similar to the earlier
observations of Shivaji et al. (2004), wherein 32% of
the clones belonged to gamma proteobacter subgroup. Six of the clones showed similarities to
gamma proteobacterium isolate KT0813, and
shared 73–97% intra clone similarities (data not
shown). Similarly five of the clones showed BLAST
hit to uncultured Psychrobacter sp. clone GAMMA4A
and shared 94–97% intra clone similarities. The
clones representing Roseobacter sp., Sphingomonas
baekryungensis, Nitrosospira sp., Nitrosomonas
cryotolerans, Chromohalobacter sp., Psychrobacter spp., Rubrobacter radiotolerans, Marinobacter
sp. have also been found in the library.
Ammonia oxidizers in Antarctica soil
Members of gamma proteobacteria group belonging to genera Psychrobacter and Marinobacter have
been isolated (Bowman et al., 2000a, b) and clones
representing their rDNA detected in earlier studies
(Brambilla et al., 2001). The gene encoding the
active site subunit of ammonia monoxygenase
(amoA) has been extensively used as a molecule
for cultivation-independent analysis of ammonia
oxidizer diversity (Mendum et al., 1999; Purkhold
et al., 2000; Purkhold et al., 2003; Rotthauwe et
al., 1997). Beta subgroup proteobacteria contained
sequences that showed 94–95% homologies to
Nitrospira and Nitrosomonas. These organisms are
known as ammonia oxidizers, which play a central
role in recycling nitrogen (Purkhold et al., 2003).
Their presence was further confirmed by the
presence of 491 bp PCR product for amoA gene
(Fig. 5) using the primers designed specific to the
ammonia oxidizers as described earlier (Rotthauwe
et al., 1997). The beta proteobacteria were
represented by three clone sequences with homologies to uncultured clones. This shows the complex
diversity of Antarctic bacteria.
Cytophaga-Flavobacterium-Bacteroidetes Group
25
20
No. of Clones
21
15
10
5
Vi
Cy
to
Cy
to
ph
ph
ag
te
a
a
g
lli
sp
ba a s
.B
p
ct
Ae
.
er
43 12
qu
01
vl
or
ad
-1
iv
iv
it
os 0/1
Ae a lip
to
Fl
ke
qu
ol
av
ns
yt
or
ob
is
ic
iv
ac
a
i
t
Y1
te
a
0Fl rium croc
av
ea 2T
ob sp.
Y1
ac
G
2W
te
2
riu SSE T
m
Su
Fl
s
H
bs
e
p.
ax xib
V4 171
a
ib
.M
ac cte
S.
r
t
12
An er b sp.
ro
O
ta
U
ad
rc
B3
An tic b yi s
tra 9
ta
ac
rc
i
t
n
tic eri
M
P7
ar
ba um
in
Rct
e
er
ba
iu 921
ct
m
7
er
Riu
79
m
Ba
33
K
ct
M
er
Ba M3
U
oi
ct
ni
de
9
e
d
te
riu 37
en
s
tif
m
Ba bac
ie
K
d
te
ct
U
riu
ba m4
er
nc
ct
oi
m
ul
er
B
d
G
tu
et
ac
iu
M
es
re
m
U
t
e
D
d
n
r
b
J
o
c
ac
Ba
E1
ul
id
te
tu
et
ct
0
r
E6
es
er
re
iu
oi
d
de
Ba bac m M
te
te
c
O
t
r
sb
er
48
oi ium
ac
de
te
M
te
riu
O
sb
5
m
ac 4
cl
te
on
r
iu
e
M
m
L6
02
J37
0
BLAST hits
Figure 2. Representation of the clones belonging to Cytophaga—Flavobacterium–Bacteroidetes group.
ARTICLE IN PRESS
22
B.V. Shravage et al.
Proteobacter Group
7
No. of Clones
6
5
4
3
2
1
U
U
Sp
Ro
s
hi eob
n
a
nc
g
n
o ct
ul
tu cult mo er s
re ur
na p.
d
m e d b s b a AN
ar
T
in act ekr 92
e e er
7
y
ub ium ung 0
en
ac
c
s
Ni teri lone is
u
t
U
U
nc Nit ros m H DC
nc
o
r
ul
s -5
s
os
ul
tu
o pir tpL
U tu
U red nc red mon a s 10
nc
p
0
u
b
ul ear ltur act as c . N
tu
s
re thw ed b eriu ryo p65
d
o
t
Ps rm act m c ole
lo ra
e
yc
c
hr ast rium ne ns
ob ba
c KD
G acte cte lon 4-7
am
r s riu e23
m
m
4d
Ch a p p. c
c
s
ro ro lon lone 5
t
eG c
m
oh eob
2
A
8
al ac
M 6
o
Ps ba teri MA
yc cte um
4
hr r s K A
o b p . T0
ac M 81
te
r AN 3
Ps Psy okh K2
yc
ch ots 4
hr
ob roba ken
s
Ps acte cte is
yc r s r f o
h
z
Ps ro p.
H ii
b
y
Ps c h r a c t S 5 3
yc ob er
23
s
a
h
Ru rob cter p. p
3C
br ac
s
p
ob ter
.IC
ac
u
M
A
0
r
U
0
nc rcti arin ter ativ 8
ul c s
ob rad or
tu
e
a
i
a
ot ns
re a i
ol
c cte
d
er
ba e b r s
a
ct ac p.
er
M ns
te
riu ED
iu
m
m
1
cl
on AR 06
e A K9
RK 98
D 7
M
S9
0
BLAST hits
Figure 3. Representation of the clones belonging to Proteobacter group.
100
Estimated species richness
Abundance
Average Diversity
10
Median Diversity
95% Conf. Low
95% Conf. High
1
1
5
9
13 17 21 25 29 33 37 41 45
Number of clones
Figure 4. Species richness estimate curves derived from SSU rDNA clone library data of Antarctica soil sample.
Thermophiles in Antarctica soil
Thermophillic microorganisms from Antarctica
have been reported from hot soils of Mount Erebus,
Ross Island (Boyd and Boyd, 1963). Antarctic soil is
known to harbor thermophillic strains in the northern Victoria Land but not in McMurdo Dry Valleys
(Logan et al., 2000). In the earlier studies on soil
sample analyzed (Shivaji et al., 2004), the presence of this group was not reported. Surprisingly,
from our study two clones bh8.53 and bh4.3 shared
their closest relation (97% and 95%, respectively)
with Deinococcus and Desufomaculum aeronauticum. However, their presence has to be supported
by culture-based studies to support the theory of
thermophillic microorganisms from Antarctic soils.
The analyses of species richness for the SSU rDNA
clones generated from Antarctica soil sample
revealed high diversity levels (Fig. 4). The average
diversity was falling between the 95% confident
values generated for the sample. In the SSU rDNA
clone library study, some genes were represented
by single clone while some genera were represented by large number of clones. Such observations have been reported earlier in studies that
were also based on SSU rDNA clone library approach
(Schmidt et al., 1991; Shivaji et al., 2004). These
observations may be attributed to several reasons
ARTICLE IN PRESS
Molecular microbial diversity of a soil sample and detection of ammonia oxidizers from Antarctica
and are guided by many factors (Amann et al.,
1995). Antarctica is a hotspot for those who are
interested in life at extreme environments. SSU
rDNA clone library analysis has shed light on those
aspects where once it was thought life could not
exist in extreme conditions. Our study revealed
high microbial diversity existing in such environments. We discovered SSU rDNA clones of bacteria
like Nitrosomonas and Nitrosospira spp. that are
known to play roles in ammonia oxidization, but
these clones had only 94–95% similarity to the
closest blast hits. As it was very difficult to assign a
functional role to these clones as similarities to it’s
nearest cultured neighbor were distant, we
checked for the presence of functional gene amoA
which is required for ammonia oxidation using
amoA specific primers which has been successfully
used earlier in similar studies. We confirmed the
presence of amoA functional gene in Antarctica soil
by the PCR amplication, which produced 491 bp
product (Fig. 5).
Also it is now a known fact that PCR primers
influence the sequence diversity within SSU rDNA
gene libraries (Marchesi et al., 1998). Also there
are many factors like PCR cycling parameters and
the amount of DNA template used in PCR reaction,
which may affect the coverage and hence overall
diversity in uncultured 16S rDNA library approach.
23
We strongly believe that combination of various
methods and larger sampling would help to resolve
the biases in studies involving molecular methods
to determine the diversity of microorganisms.
Further studies will also help in understanding the
survival and role of microorganisms in extreme
environments. The soil sample studied was collected below a dead seal with an interest to find
out the possibility of microbes, which may be
involved in recycling of macromolecules to microelements in harsh environments like Antarctica.
However the possibility of microflora originating
from the dead animal could not be ruled out. From
industrial and pharmaceutical point of view, such
extreme environments harbor microorganisms that
could be used as potential source of novel enzymes
and secondary metabolites/antibiotics. This was an
effort to study uncultured diversity of Antarctic soil
sample using SSU rDNA gene library based method.
Some of the clones, belonging to bacteroidetes
group, had similarity values less than 90% to their
closest blast hits, which indicate the presence of
new lineages in Antarctic soil. However, the
presence of ammonia oxidizers is significant from
the point of nitrogen recycling and the presence of
thermophiles needs further investigation.
References
Figure 5. PCR amplification of the specific 491 bp
fragment of the amoA gene from Antarctic soil. M,
1Kb+ DNA ladder (Invitrogen); lane1, amoA gene PCR
product.
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.,
Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped
BLAST and PSI-BLAST: a new generation of protein
database search programs. Nucleic Acids Res. 25 (17),
3389–3402.
Amann, R.I., Ludwig, W., Schleifer, K.H., 1995. Phylogenetic identification and in situ detection of individual
microbial cells without cultivation. Microbiol. Rev. 59
(1), 143–169.
Baublis, J.A., Wharton Jr., R.A., Volz, P.A., 1991.
Diversity of micro-fungi in an Antarctic dry valley. J.
Basic Microbiol. 31 (1), 3–12.
Bowman, J.P., McCammon, S.A., Brown, M.V., Nicholas,
D.S., McMeekin, T.A., 1997. Diversity and association
of psychrophillic bacteria in Antarctic sea ice. Appl.
Environ. Microbiol. 63, 3068–3078.
Bowman, J.P., McCammon, S.A., Rea, S.M., McMeekin,
T.A., 2000a. The microbial composition of three
limnologically disparate hypersaline Antarctic lakes.
FEMS Microbiol. Lett. 183, 81–88.
Bowman, J.P., Rea, S.M., McCammaon, S.A., McMeekin, T.A.,
2000b. Diversity and community structure within anoxic
sediment from marine salinity meromictic lakes and a
coastal meromictic marine basin, Vestfold Hilds, eastern
Antarctica. Environ. Microbiol. 2, 227–237.
Boyd, W.L., Boyd, J.W., 1963. Soil microorganisms of the
McMurdo Sound area, Antarctica. Appl. Microbiol. 11,
116–121.
ARTICLE IN PRESS
24
Brambilla, E., Hippe, H., Hagelstein, A., Tindall, B.,
Stackebrandt, E., 2001. 16S rDNA diversity of cultured
and uncultured prokaryotes of a mat sample from Lake
Fryxell, McMurdo Dry Valleys, Antarctica. Extremophiles 5, 23–33.
Brosius, J.M., Palmer, L., Kennedy, P., Noller, H.F., 1978.
Complete nucleotide sequence of the 16S ribosomal
DNA gene from Escherichia coli. Proc. Natl. Acad. Sci.
USA 75, 4801–4805.
Brown, M.V., Bowman, J.P., 2001. A molecular phylogenetic survey of sea-ice microbial communities (SIMCO). FEMS Microbiol. Ecol. 35 (3), 267–275.
Dawid, W., Galliowski, C.A., Hirsch, P., 1988. Psychrophilic myxobacteria from Antarctic soils. Polarforsch
58, 271–278.
Felsenstein, J., 1989. PHYLIP phylogeny inference package. Cladistics 5, 164–166.
Friedmann, E.I., 1980. Endolithic microbial life in hot and
cold deserts. Orig. Life 10 (3), 223–235.
Friedmann, E.I., 1982. Endolithic microorganisms in the
Antarctic cold desert. Science 15, 1045–1053.
Friedmann, E.I., Ocampo, R., 1976. Endolithic blue–
green algae in the dry valleys: primary producers and
the Antarctic desert ecosystem. Science 193,
1247–1249.
Friedmann, E.I., McKay, C.P., Nienow, J.A., 1987. The
cryptoendolithic microbial environment in the Ross
Desert of Antarctica: satellite-transmitted continuous
nanoclimate data, 1984 to 1986. Polar Biol. 7,
273–287.
Gordon, D.A., Priscu, J., Govannoni, S., 2000. Origin and
phylogeny of microbes in living in permanent Antarctic
lake ice. Microb. Ecol. 39, 197–202.
Gotelli, N.J., Entsminger, G.L., 2001. EcoSim: Null
Models Software for Ecology, version 7.0. Acquired
Intelligence Inc. & Kesey-Bear. http://homepages.together.net/gentsmin/ecosim.htm.
Glockner, F.O., Fuchs, B.M., Amann, R., 1999. Bacterioplankton compositions of lakes and oceans: a
first comparison based on fluorescence in situ
hybridization. Appl. Environ. Microbiol. 65 (8),
3721–3726.
Head, I.M., Saunders, J.R., Pickup, R.W., 1998. Microbial
evolution, diversity, and ecology: a decade of ribosomal DNA analysis of uncultivated microorganisms.
Microb. Ecol. 35, 1–21.
Hirsch, P., Gallikowski, C.A., 1985. Microorganims in soil
samples from Linneaus Terrace southern Victoria
Land: preliminary observations. Antarctic J. US 19,
183–186.
Hirsch, P., Gallikowski, C.A., 1998. Preliminary characterization and identification of1984/85 continental
Antarctic soil micro-organisms of Linnaeus Terrace
(altitude 1600 m, McMurdo Dry Valleys). Polarforsch
58, 93–101.
Hirsch, P., Hoffamnn, B., Gallikowski, C.A., Mevs, U.,
Siebert, J., Sittig, M., 1988. Diversity and identification of heterotrophs from Antarctic rocks of the
McMurdo Dry Valleys (Ross Desert). Polarforsch 58,
261–269.
B.V. Shravage et al.
Horowitz, N.H., Cameron, R.E., Hubbard, J.S., 1972.
Microbiology of the dry valleys of Antarctica. Science
176, 242–245.
Hugenholtz, P., Goebel, B.M., Pace, N.R., 1998. Impact of
culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180
(18), 4765–4774.
Kappen, L., Friedmann, E.I., 1983. Ecophysiology of
lichens in the dry valley of southern Victoria Land,
Antarctica. CO2 gas exchange in cryptoendolithic
lichens. Polar Biol. 1, 227–232.
Logan, N.A., Lebbe, L., Hoste, B., Goris, J., Forsyth, G.,
Heyndrickx, M., Murray, B.L., Syme, N., WynnWilliams, D.D., De Vos, P., 2000. Aerobic endosporeforming bacteria from geothermal environments in
northern Victoria Land, Antarctica, and Candlemas
Island, South Sandwich archipelago, with the proposal
of Bacillus fumarioli sp. nov. Int. J. Syst. Evol.
Microbiol. 50 (Pt 5), 1741–1753.
Maidak, B.L., Olsen, G.J., Larsen, N., Overbeek, R.,
McCaughey, M.J., Woese, C.R., 1997. The RDP
(ribosomal database project). Nucleic Acids Res. 25
(1), 109–111.
Marchesi, J.R., Sato, T., Weightman, A.J., Martin, T.A.,
Fry, J.C., Hiom, S.J., Wade, W.G., 1998. Design and
evaluation of useful of useful bacterium-specific PCR
primers that amplify genes coding for bacterial 16S
rRNA. Appl. Environ. Microbiol. 64, 795–799.
Mendum, T.A., Sockett, R.E., Hirsch, P.R., 1999. Use of
molecular and isotopic techniques to monitor the
response of autotrophic ammonia-oxidizing populations of the beta subdivision of the class proteobacteria in arable soils to nitrogen fertilizer. Appl.
Environ. Microbiol. 65, 4155–4162.
Paster, B.J., Boches, S.K., Galvin, J.L., Ericson, E.R.,
Lau, C.N., Levanos, V.A., Sahasrabuddhe, Dewhrist,
F.E., 2001. Bacterial diversity in subgigival plaque. J.
Bacteriol. 183, 3770–3783.
Purkhold, U., Andreas, A.P., Stefan, J., Markus, C.S.,
Hans-Peter, K., Michael, W., 2000. Phylogeny of all
recognized species of ammonia oxidizers based on
comparative 16S rDNA and amoA sequence analysis:
implications for molecular diversity surveys. Appl.
Environ. Microbiol. 66, 5368–5382.
Purkhold, U., Wagner, M., Timmermann, G., Pommerening-Roser, A., Koops, H.P., 2003. 16S rRNA and amoAbased phylogeny of 12 novel betaproteobacterial
ammonia-oxidizing isolates: extension of the dataset
and proposal of a new lineage within the nitrosomonads. Int. J. Syst. Evol. Microbiol. 53 (Pt 5), 1485–1494.
Rotthauwe, J.H., Witzel, K.P., Liesack, W., 1997. The
ammonia monooxygenase structural gene amoA as a
functional marker: molecular fine-scale analysis of
natural ammonia-oxidizing populations. Appl. Environ.
Microbiol. 63 (12), 4704–4712.
Schmidt, T.M., Delong, E.G., Pace, N.R., 1991. Analysis of
marine picoplankton community by 16S rRNA gene
cloning and sequencing. J. Bacteriol. 173, 4371–4378.
Shivaji, S., Reddy, G.S., Aduri, R.P., Kutty, R., Ravenschlag, K., 2004. Bacterial diversity of a soil sample
ARTICLE IN PRESS
Molecular microbial diversity of a soil sample and detection of ammonia oxidizers from Antarctica
from Schirmacher Oasis, Antarctica. Cell. Mol. Biol.
(Noisy-le-grand). 50 (5), 525–536.
Sjoling, S., Cowan, D.A., 2003. High 16S rDNA bacterial
diversity in glacial meltwater lake sediment, Bartina
Island, Antarctica. Extremophiles 7, 275–282.
Taton, A., Grubisic, S., Brambilla, E., De Wit, R.,
Wilmotte, A., 2003. Cyanobacterial diversity in
natural and artificial microbial mats of Lake Fryxell
(McMurdo Dry Valleys, Antarctica): a morphological
and molecular approach. Appl. Environ. Microbiol. 69
(9), 5157–5169.
25
McTavish, H., Fuchs, J.A., Hooper, A.B., 1993. Sequence
of the gene coding for ammonia monooxygenase
in Nitrosomonas europaea. J. Bacteriol. 175,
2436–2444.
Torsvik, V., Sroheim, R., Goksoyr, J., 1996. Total bacterial
diversity in soil and sediment communities—a review.
J. Ind. Microbiol. 17, 170–178.
Yeates, C., Gillings, M.R., Davison, A.D., Altavilla, N.,
Veal, D.A., 1997. PCR amplification of crude microbial
DNA extracted from soil. Lett. Appl. Microbiol. 25 (4),
303–307.