Antarctic Science 25(2), 219–228 (2013) & Antarctic Science Ltd 2013
doi:10.1017/S0954102012000831
Phylogeographic analysis of filterable bacteria with special
reference to Rhizobiales strains that occur in cryospheric habitats
RYOSUKE NAKAI1,2, ERI SHIBUYA1, ANA JUSTEL3, EUGENIO RICO4, ANTONIO QUESADA5,
FUMIHISA KOBAYASHI6, YASUNOBU IWASAKA6, GUANG-YU SHI7, YUKI AMANO8,9, TERUKI IWATSUKI8,9
and TAKESHI NAGANUMA1*
1
Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-hiroshima, Hiroshima 739-8528, Japan
2
Research Fellow of the Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo 102-8471, Japan
3
Departamento de Matemáticas, Universidad Autónoma de Madrid, 28049 Madrid, Spain
4
Departamento de Ecologı́a, Universidad Autónoma de Madrid, 28049 Madrid, Spain
5
Departamento de Biologı́a, Universidad Autónoma de Madrid, 28049 Madrid, Spain
6
Institute of Nature and Environmental Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
7
Institute of Atmospheric Physics, Chinese Academy of Science, Beijing 100029, China
8
Japan Atomic Energy Agency, Mizunami Underground Research Laboratory, 1-64 Yamanouchi, Akeyo-cho, Mizunami-shi,
Gifu 509-6132, Japan
9
Japan Atomic Energy Agency, Horonobe Underground Research Center, Hokushin 432-2, Horonobe-cho, Teshio-Gun,
Hokkaido 098-3224, Japan
*corresponding author: [email protected]
Abstract: Although the lower size limit of microorganisms was previously believed to be c. 0.2 mm, there
is evidence for the existence of microorganisms that can pass through 0.2 mm-pore-size filters called
ultramicrobacteria or nanobacteria. However, information on the phylogeny and biogeography of these
bacteria is limited. We obtained 53 isolates of 0.2 mm-passable bacteria from 31 samples collected at
26 locations worldwide, including the Arctic Svalbard Islands, deserts, and Maritime Antarctica.
Phylogenetic analysis of near full-length 16S rRNA gene sequences revealed that 18 of the 53 isolates were
, 97% homologous with previously cultured isolates, representing potentially novel species. Two isolates
(order Rhizobiales) (100% identical) collected from Byers Peninsula, Livingston Island in Maritime
Antarctica, were closely related (99.8% similarity) to an isolate collected from intertidal sediments in East
Antarctica. In addition, the sequence of this Antarctic isolate showed $ 97% similarity to 901 sequences
derived from known isolates and samples collected at geographically disparate locations under various
environmental conditions. Interestingly, among 13 sequences showing $ 99% similarity, ten were isolated
from cryospheric habitats such as Arctic, Antarctic, and alpine environments. This implies that such
Rhizobiales strains occur in the cryospheric regions, however, their abundance and biomass may be scarce
depending on the geographic location.
Received 7 November 2011, accepted 6 August 2012
Key words: 16S rRNA gene, Antarctica, Baas-Becking hypothesis, biodiversity, biogeography,
ultramicrobacteria
the 1970s. For example, it has been demonstrated that
bacteria smaller than 0.2 mm in diameter represent between
1% and 10% of all bacteria found in seawater (Hobbie et al.
1977, Zimmermann 1977, Watson et al. 1977). In addition,
in coastal waters, where bacterial populations are estimated
to range between 108 and 109 cells l-1, Fukuba et al. (2002)
reported that between 100 and 101 viable bacteria l-1 pass
through 0.2 mm-pore-size filters.
In 1981, two types of very small bacteria , 0.3 mm in
diameter were successfully cultured from seawater (Torrella
& Morita 1981). One displayed an increase in size following
incubation, whereas the other did not increase in size even
after incubation and was named ultramicrobacteria (UMB).
Since then, further isolations and phylogenetic identification
of 0.2 mm-passable microorganisms have been performed, and
Introduction
The occurrence of microorganisms smaller than 0.2 mm has
been questioned based on the minimum requisite size for
a complete set of genomic DNA, ribosomes, proteins,
cellular membranes, and other intracellular components
(Maniloff 1997). On the other hand, referring to the
genomes of Mycoplasma genitalium and other parasitic
bacteria, some argued that microbial cells can theoretically
be as small as 0.14 mm in diameter (Mushegian & Koonin
1996). The debate regarding the minimal cell size of
microorganisms remains unresolved.
Concurrent with this theoretical discussion, research on
microorganisms that pass through 0.2 mm-pore-size filters
(0.2 mm-passable microorganisms) has been ongoing since
219
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220
RYOSUKE NAKAI et al.
some of these isolates were UMB (MacDonell & Hood 1982,
Hood & MacDonell 1987, Haller et al. 2000, Elsaied et al.
2001, Miteva & Brenchley 2005, Dmitriev et al. 2007,
Geissinger et al. 2009). Hahn et al. (2003, 2009) isolated
Actinobacteria and Polynucleobacter Cluster (class
Betaproteobacteria) with ultramicro cell sizes (, 0.1 mm3),
even under eutrophic culture conditions, and called these
obligate UMB. It has also been reported that these obligate
UMB are cosmopolitan species found in freshwater regions
(Hahn 2003, Hahn et al. 2003).
Recent research has investigated community structure
and the functional potential of 0.2 mm-passable populations
in the environments using culture-independent methods
(Miyoshi et al. 2005, Naganuma et al. 2007, Nakai et al.
2011). Miyoshi et al. (2005) reported that c. 65% of all 16S
rRNA gene phylotypes from 0.1-mm-pore-size filtercaptured microorganisms in deep terrestrial aquifers were
affiliated with the candidate divisions OD1 and OP11,
potentially representing new bacterial phyla. Naganuma
et al. (2007) detected novel unaffiliated phylotypes only in
the 0.2 mm-passable fraction of deep-sea hydrothermal vent
samples. Nakai et al. (2011) indicated that the metagenome
of 0.2 mm-passable microorganisms in deep-sea hydrothermal
fluid contained novel genes pertinent to membrane functions.
These findings show that 0.2 mm-passable populations
include some phylogenetically and/or functionally important
species which need to be isolated and characterized.
In addition, our understanding of the biogeography for
0.2 mm-passable bacteria is at present limited to cosmopolitan
species found in freshwater (Hahn 2003, Hahn et al. 2003).
Here we tested samples taken from widely dispersed sites
for 0.2 mm-passable bacteria and investigated phylogenetic
relationships in order to preliminarily test the Baas-Becking
hypothesis that ‘‘everything is everywhere, but, the
environment selects’’ (Baas-Becking 1934), implying that
microscopic organisms do not have any large-scale spatial
pattern of distribution. Our results provide new information
on the phylogeography of 0.2 mm-passable bacteria.
Materials and methods
Sample collection
A total of 31 samples were collected from various
environments, including soil, gravel, seawater, and
freshwater, in 26 locations around the world, the majority
of which were in Japan. Arctic samples were collected in
August 2008 from Troll hot springs (79.48N, 13.48W) on
Spitzenberg Island in the Svalbard archipelago. This site is
the northernmost thermal springs documented on land
(Jamtveit et al. 2006). Antarctic samples were collected in
December 2008 from Byers Peninsula (6284'S, 6086'W),
Livingston Island, South Shetland Islands. Byers Peninsula
is one of the largest ice-free areas in the region and serves
as an important reference site (ASPA No. 126) within the
Antarctic ecosystem (Quesada et al. 2009). In addition,
gravel was collected from the Gobi Desert and eastern edge
of the Sahara Desert in November 2008 and December
2008, respectively. Although a significant number of taxa
of dust-borne microorganisms have been isolated and
characterized in both African and Asian desert systems,
only a few papers have reported a detailed identification of
culturable bacteria (Chen et al. 2011). Samples were
collected using sterile gloves and placed in sterile plastic
bottles or sample tubes and stored in the dark at room
temperature until processing.
Isolation and cultivation of 0.2 mm-passable bacteria
Bacteria were isolated using a method modified from
Elsaied et al. (2001). Liquid samples were filtered through
three-fold 0.2 mm filters (Advantec, Tokyo, Japan), and
concentrated R2A liquid medium (Massa et al. 1998) was
added to the filtrate to bring the final media concentration
to 13. Solid samples, such as soil or pebbles, were
suspended in phosphate buffer solution (8 g l-1 NaCl, 1.1 g l-1
Na2HPO4, 0.2 g l-1 KCl, and 0.2 g l-1 KH2PO4), and the
resulting suspension was processed in the same manner as the
liquid samples. The enrichment cultures were kept in the dark
at room temperature for at least three weeks. To confirm the
effectiveness on filtration through three-fold 0.2 mm filters,
Escherichia coli K12 (IFO 3301) cultures were used as
negative growth control. Subsequently, the culture liquids
were spread on 1.5% agar medium containing identical
ingredients. Single colonies were isolated and streaked on
fresh agar plates to purify colony-forming strains. This
procedure was repeated at least three times to ensure
reliable colony purification.
Phylogenetic characterization
Genomic DNA extraction was performed as described
by Aljanabi & Martinez (1997). Bacteria-specific 27F (5'AGAGTTTGATCCTGGCTCAG-3') and universal 1492R
(5'-GGTTACCTTGTTACGACTT-3') primers were used to
amplify near full-length fragments of the 16S rRNA gene
(DeLong 1992). Reaction conditions for polymerase chain
reaction (PCR) amplification were the same as those used
in a previous report (Naganuma et al. 2007). After confirming
the presence of PCR products of the desired fragment size
(c. 1500 base pairs) by electrophoresis on 1.5% agarose gel,
PCR products were purified using the QIAquick PCR
Purification kit (Qiagen, Valencia, CA, USA). All purified
PCR products were sequenced using an ABI 3730XL
automatic DNA sequencer (Applied Biosystems, Foster
City, CA, USA). The 16S rRNA gene sequences obtained
were compared against known sequences using a BLASTN
search against the DDBJ/EMBL/GenBank nt-database and
the Greengenes prokaryotic multiple sequence alignment
(prokMSA) database (DeSantis et al. 2006). Greengenes is a
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Phylogenetic group
Isolate
Accession
number
Site
description
Isolation source
Sample
description
Closest organism
Isolation source
Accession
number
Sima
(%)
Culturability
of the filtrateb
Alphaproteobacteria
KNA-P
IZ3
GB9
AB539973
AB539970
AB539976
35.48N, 137.28E
35.48N, 132.78E
40.08N, 94.78E
groundwater
lake water
sand
Phaeospirillum fulvum isolate S3
Phaeospirillum sp. I08B-942
Phaeospirillum fulvum isolate S3
activated sludge from Korea AF508113
soil from western China
FJ529718
activated sludge from Korea AF508113
98.0
99.9
98.3
-
NP1
NP3
IZ6
AB539983
AB539984
AB539980
79.48N, 13.48E
79.48N, 13.48E
35.28N, 132.98E
Gifu, central Japan
Shimane, western Japan
Gobi desert, north-central
China
Svalbard, Arctic Norway
Svalbard, Arctic Norway
Shimane, western Japan
mud
mud
soil
activated sludge from Korea AF508113
activated sludge from Korea AF508113
unknown
EU697955
99.7
99.6
92.6
1
IZ12
AB539981
35.28N, 132.98E
Shimane, western Japan
sand
unknown
EU697955
92.5
1
IZ16
AB539982
35.28N, 132.98E
Shimane, western Japan
bark
unknown
EU697955
92.6
-
GB6
AB539977
40.08N, 94.88E
sand
92.7
1
AB540022
79.48N, 13.48E
root nodules from central
China
unknown
DQ100062
NP9
Gobi desert, north-central
China
Svalbard, Arctic Norway
Phaeospirillum fulvum isolate S3
Phaeospirillum fulvum isolate S3
Rhizobium huautlense strain
CCNWNX0087
Rhizobium huautlense strain
CCNWNX0087
Rhizobium huautlense strain
CCNWNX0087
Rhizobium sp. CCBAU 43060
EU697955
92.6
-
GB3
AB539978
36.08N, 104.08E
unknown
EU697955
92.3
-
GB10
AB539979
40.08N, 94.78E
unknown
EU697955
92.6
1
S-19
AB539971
62.78S, 61.08W
Gobi desert, north-central
China
Gobi desert, north-central
China
Maritime Antarctica
FJ889671
99.8
-
S-42
AB539972
62.78S, 61.08W
Maritime Antarctica
FJ889671
99.8
-
IZ17
IZ9-1
IZ10
IZ19-2
AB539985
AB539986
AB539987
AB539988
35.28N,
35.28N,
35.28N,
35.48N,
132.98E
132.98E
132.98E
132.78E
Shimane,
Shimane,
Shimane,
Shimane,
AF338177
AJ565423
AJ565423
DQ861288
99.8
99.2
99.1
99.9
1
1
1
IZ20-1
KNG
IZ7-1
Kha
AB539989
AB539993
AB539992
AB539994
35.48N,
34.38N,
35.28N,
34.28N,
132.78E
132.68E
132.98E
132.68E
AJ565423
AJ565423
AJ565430
DQ861290
99.6
99.2
99.9
99.9
1
1
1
Shr9
AB539995
DQ861290
99.7
-
IZ5-1
sandy sediment from East
Antarctica
sandy sediment from East
Antarctica
unknown
freshwater from Austria
freshwater from Austria
freshwater from
Switzerland
freshwater from Austria
freshwater from Austria
freshwater from Austria
freshwater from
Switzerland
freshwater from
Switzerland
freshwater from eastern
coastal China
freshwater from
Switzerland
river water from eastern
Japan
chromium contaminated
soil from India
lake water from East
Antarctica
lake water from East
Antarctica
freshwater from western
China
Betaproteobacteria
travertine
sand
sand
beach sand
Rhizobium huautlense strain
CCNWNX0087
Rhizobium huautlense strain
CCNWNX0087
Rhizobium huautlense strain
CCNWNX0087
Mycoplana sp. ZS1-12
Mycoplana sp. ZS1-12
Shimane, western Japan
Hiroshima, western Japan
Shimane, western Japan
Hiroshima, western Japan
lake water
river water
river water
river water
Hylemonella
Hylemonella
Hylemonella
Hylemonella
34.48N, 8.08E
Sahara Desert, Tunisia
salt lake water
Hylemonella gracilis isolate LL
AB539991
35.28N, 132.98E
Shimane, western Japan
river water
Hylemonella gracilis isolate WQT2
IZ8
AB539990
35.28N, 132.98E
Shimane, western Japan
river water
Hylemonella gracilis RR
IZ4
AB539996
35.48N, 132.78E
Shimane, western Japan
lake water
Beta proteobacterium KF034
KNA-A
AB539974
35.48N, 137.28E
Gifu, central Japan
groundwater
Acidovorax sp. G3DM-83
AB540008
34.28N, 132.68E
Hiroshima, western Japan
seawater
NOW
AB540009
29.58N, 124.28E
Okinawa, south-west Japan
seawater
IZ2
AB540010
35.48N, 132.78E
Shimane, western Japan
lake water
Saccharospirillum impatiens strain
EL-105
Saccharospirillum impatiens strain
EL-105
Pseudomonas pseudoalcaligenes
KS-1
Gammaproteobacteria kure
western
western
western
western
Japan
Japan
Japan
Japan
Afipia felis
Hylemonella gracilis, isolate WQP1
Hylemonella gracilis, isolate WQP1
Hylemonella gracilis, isolate ZL
gracilis, isolate WQP1
gracilis, isolate WQP1
sp. WQH1
gracilis isolate LL
AJ565424
100
1
DQ861289
99.2
1
AB376632
98.4
1
EU037287
99.4
-
AJ315983
96.7
1
AJ315983
96.0
1
EU815635
98.1
1
221
freshwater algal
mat
soil
river water
soil
lake water
PHYLOGEOGRAPHIC ANALYSIS OF NANO BACTERIA
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Table I. Phylogenetic affiliation of isolated strains and culturability of the filtrates.
222
Phylogenetic group
Accession
number
Site
description
Isolation source
Sample
description
Closest organism
Isolation source
Accession
number
Sima
(%)
Culturability
of the filtrateb
AB540011
79.48N, 13.48E
Svalbard, Arctic Norway
hot spring water
Pseudomonas sp. PSA A4(4)
DQ628969
99.9
1
F9
KE
ShrSW
KJY
N2
AB540012
AB540013
AB540014
AB540000
AB540001
34.38N,
35.28N,
35.98N,
34.48N,
34.58N,
Hiroshima, western Japan
Yamaguchi, western Japan
Tunisia
Hiroshima, western Japan
Hiroshima, western Japan
seawater
seawater
seawater
river water
lake water
Reinekea blandensis MED297
Vibrio sp. PaD1.16b
Oceaniserpentilla haliotidis
Bacteroidetes bacterium 3B-2
Bacterium TG141
DQ403810
GQ406586
AM747817
GU117702
AB308367
99.0
99.6
98.5
94.4
93.0
no data
1
1
1
ArSB
AB539997
79.48N, 13.48E
Svalbard, Arctic Norway
hot spring water
Bacterium TG141
AB308367
93.4
1
GS
THS
AB539998
AB539999
78.28N, 15.58E
79.48N, 13.48E
Svalbard, Arctic Norway
Svalbard, Arctic Norway
glacial meltwater
hot spring water
Bacteroidetes bacterium 3B-2
Bacterium TG141
GU117702
AB308367
93.7
95.3
1
KIP
AB540002
31.98N, 130.08E
lake water
96.5
1
AB540003
31.98N, 130.08E
Gracilimonas tropica strain
CL-CB462
Flexibacter aggregans
EF988655
KIY
unknown
AB078038
96.6
1
YMK
AB540004
33.68N, 132.08E
Koshiki Island, western
Japan
Koshiki Island, western
Japan
Ohita, western Japan
beneath a high Arctic
glacier, Canada
Mediterranean Sea
unknown
Tasman Sea
marine sand from Korea
freshwater sediment from
eastern Japan
freshwater sediment from
eastern Japan
marine sand from Korea
freshwater sediment from
eastern Japan
unknown
estuary water
unknown
DQ486479
99.3
1
KOr
AB540005
33.98N, 130.98E
Yamaguchi, western Japan
seawater
South Pacific Ocean
AM990675
97.0
-
T1
AB540006
33.98N, 129.68E
Hiroshima, western Japan
seawater
96.4
1
AB540007
33.58N, 131.08E
Yamaguchi, western Japan
seawater
AB073588
98.9
1
GB1
AB540015
40.08N, 94.48E
sand
Blastococcus sp. YIM 65287
FJ214361
99.3
1
GB5
AB540016
40.08N, 94.78E
sand
98.6
1
AB539975
35.48N, 137.28E
groundwater
Blastococcus jejuensis strain
KST3-10
Micrococcus luteus strain INBI-1
DQ200983
KNA-M
Gobi desert, north-central
China
Gobi desert, north-central
China
Gifu, central Japan
EU438932
99.9
1
KNC
AB540017
34.38N, 132.68E
Hiroshima, western Japan
river water
AJ565413
99.7
1
NP3-12
AB540018
79.48N, 13.48E
Svalbard, Arctic Norway
mud
Microbacteriaceae bacterium
MWH-VicE1
Blastococcus sp. YIM 65287
offshore copepod from
western USA
red alga from southern
Japan
medicinal plant from
south-western China
marine sediment from
Korea
oil-polluted soil from
Russia
freshwater from Uganda
AB078044
Km
Flexibacteraceae bacterium
DG1232
Flexibacteraceae bacterium
MOLA 398
Flexibacter aurantiacus subsp.
copepodarum
Cytophaga sp. I-377
FJ214361
99.8
1
IZ19-1
AB540020
35.48N, 132.78E
Shimane, western Japan
lake water
Spirochaeta sp. MWH-HuW24
AJ565434
97.3
no data
IZ20-2
AB540019
35.48N, 132.78E
Shimane, western Japan
lake water
Spirochaeta sp. MWH-HuW24
AJ565434
97.4
no data
Shr3
AB540021
33.58N, 10.08E
Tunisia
sand
Desulfovibrio alkalitolerans strain
HSRB-E1
GQ863489
84.9
1
Isolate
Gammaproteobacteria ArSA
Bacterioroides
Actinobacteria
Spirochaetes
Unclassified bacteria
a
132.98E
129.08E
10.68E
124.78E
132.78E
lake water
medicinal plant from
south-western China
pond water from eastern
coastal China
pond water from eastern
coastal China
unknown
Similarity.
Liquid cultures of each isolate were filtered through three 0.2 mm-pore-size filters. The filtrates were added to R2A medium and observed after three weeks.15 growth was observed, - 5 no growth was
observed.
b
RYOSUKE NAKAI et al.
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Table I. Continued
PHYLOGEOGRAPHIC ANALYSIS OF NANO BACTERIA
223
Fig. 1. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences from Rhizobiales and Rhodospirillales
strains. The percentages of replicate trees in which the associated taxa clustered together in a bootstrap test (1000 replicates) are shown next
to the branches. There were a total of 1259 positions in the final dataset. Origins of the isolates and clones are in round brackets, and
nucleotide accession numbers are in square brackets. The scale bar indicates 0.02 substitutions per nucleotide site.
dedicated full-length 16S rRNA gene database with a curated
taxonomy based on de novo tree inference. The obtained
sequences and the highly homologous known sequences were
aligned using the CLUSTALX multiple alignment program
(Larkin et al. 2007). Phylogenetic trees were constructed
based on sequences after alignment using the neighbour
joining (NJ) method in MEGA 4.0 (Kumar et al. 2007).
Registration of the nucleic acid sequences
The 16S rRNA gene sequences obtained in this study have
been deposited in the DDBJ/EMBL/GenBank database
under the serial accession numbers AB539970–AB540022.
Evaluation of cell size under culture conditions using
electron microscopy and filtration
The cell morphology of isolates was examined with an
ultra-high-resolution field-emission scanning electron
microscope (FE-SEM) (S-5200, Hitachi High Technologies,
Tokyo, Japan). The cells were fixed in 1:20 dilution of 25%
glutaraldehyde solution, and an appropriate number of cells
were collected on a 0.1-mm-pore Anopore inorganic membrane
(Anodisc) filter (Whatman, Tokyo, Japan) for observation. The
membrane is composed of an alumina matrix that is
manufactured electrochemically. Filter-captured cells were
dehydrated with a series of ethanol washes (50%, 70%, 90%,
95%, 97.5%, 99.5%) and air-dried. In order to prevent
electrification and to generate clear images during electron
microscope observation, dehydrated cells were coated with a
c. 100-Å-thick (0.1 mm) layer of platinum via ion sputtering
(E-1030, Hitachi High Technologies, Tokyo, Japan).
We used E. coli K12 (IFO 3301) as control cells. However,
changes in cell morphology were unavoidable during sample
preparation, therefore cell size under culture conditions was
confirmed by passing culture liquids through three-fold 0.2 mm
filters (Advantec, Tokyo, Japan). Concentrated R2A liquid
medium (Massa et al. 1998) was added to the filtrate to bring
the final media concentration to 13. Because the pores of the
filter used for filtration are created by exposure to neutron
radiation, a portion of the pores overlap. As filters contain a
small number of pores that are larger than the nominal pore
size, we minimized the possibility of larger cells passing
through by filtering samples or culture suspensions through
three layers of filters. The resulting filtrate was incubated for at
least three weeks. To confirm the effectiveness on filtration
through three-fold 0.2 mm filters, E. coli K12 (IFO 3301)
cultures were used as negative growth control.
Results and discussion
Phylogeny of 0.2 mm-passable bacteria
We obtained 53 isolates of 0.2 mm-passable bacteria. The
phylogenetic analysis based on 16S rRNA gene sequences
classified them into five phyla of Proteobacteria
(34 isolates), Bacteroidetes (11), Actinobacteria (5), and
Spirochaetes (2), with one unclassified isolate showing less
than 85% similarity to known strains and isolates (Table I;
BLASTN search results against the Greengenes database
are shown in Table SI, which will be found at http://
dx.doi.org/10.1017/S0954102012000831).
Of the 34 protobacterial isolates, 15 were affiliated with the
class Alphaproteobacteria, 12 with the class Betaproteobacteria,
and seven with the class Gammaproteobacteria. No Delta,
Epsilon or other proteobacterial isolates, most of the known
species of which are microaerobic to anaerobic, were obtained
from the collected aerobic samples.
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RYOSUKE NAKAI et al.
Fig. 2. Distribution of the sample collection sites corresponding to the sequences derived from known isolates and environmental
samples related to the Antarctic strain S-19. The x- and y-axes represent 08 latitude and longitude respectively. Sequences related to
the S-19 sequence were identified from the DDBJ/EMBL/GenBank database, and grouped by level of similarity to it (97%, 98%,
99%, or 100% similarity: E-value 5 0). The number of sequences similar to the S-19 sequence increased from one to 901 when the
similarity threshold was changed from 100% to 97%. Sequences similar to the S-19 sequence on the database but lacking
information on country of isolation source were not included in the graphs.
Of the 15 Alphaproteobacterial isolates, seven similar
isolates were only remotely related (at 92–93% similarity)
to the genus Rhizobium, five were closely related (. 98%)
to a Phaeospirillum species/strain, two Antarctic isolates
(of which the 16S rRNA gene sequences are 100%
identical) were almost identical (99.8%) to an Antarctic
Mycoplana strain, and one was almost identical (99.8%)
to Afipia felis. In contrast, the Betaproteobacterial isolate
pool was dominated by a Hylemonella species/strain (10/12
isolates). Gammaproteobacterial isolates were scattered
in five genera of Saccharospirillum, Pseudomonas,
Oceaniserpentilla, Reinekea, and Vibrio.
About half of the Bacteroidetes isolates were
affiliated with the genera of Flexibacter, Cytophaga, and
Gracilimonas, while the other half was loosely related
(at , 96% similarities) to two unidentified Bacteroidetes.
Isolates of Actinobacteria and Spirochaetes were closely
related (. 97%) to previously reported species/strains.
The last isolate ‘‘Shr3’’ from the Sahara is totally unrelated
(, 85%) to known cultured organisms and thus is of
great taxonomic interest, but will be described and
discussed elsewhere.
Relative dominance of Proteobacteria and Bacteroidetes
is affected by the recovery of isolates belonging to an
unnamed group of Rhizobiales and the genus Hylemonella
(Proteobacteria) as well as Flexibacter and Cytophaga
(Bacteroidetes). To the best of our knowledge this is the
first report of 0.2 mm-passable Rhizobiales, although certain
Rhizobium species such as R. leguminosarum have shown a
negative relationship between cell size and low nutrient
conditions (Postma et al. 1988). Our results suggest that
this relationship may hold for other groups in the
cryosphere. The Hylemonella strains are well-studied
0.2 mm-passable fast-growing freshwater bacteria that
have slender spirillum-shaped cell morphology (Wang
et al. 2007). Flexibacter and Cytophaga species also
exhibit 0.2 mm-passable slender filamentous forms (Hood
& MacDonell 1987, Hahn 2004, Hahn et al. 2004) as well
as 0.2 mm-passable dwarf cells during their life cycles
(Elsaied et al. 2001).
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PHYLOGEOGRAPHIC ANALYSIS OF NANO BACTERIA
Phylogeography of 0.2 mm-passable bacteria
Generally, microorganisms have large population sizes and
very short generation times resulting in high dispersal rates.
Studies have shown some protist, fungal, and bacterial
taxa with cosmopolitan distribution, rather than restricted
geographic distribution (reviewed by Green & Bohannan
2006). The Baas-Becking (or ubiquity) hypothesis is
supported by findings that identical bacterial 16S rRNA
gene sequences have been detected from lakes in both the
Arctic and Antarctic (Pearce et al. 2007). Conversely, there
are reports that some microbial taxa have restricted
geographic distribution from dispersal limitations, which
implies that not all microorganisms can disperse globally
(reviewed by Martiny et al. 2006).
The seven isolates belonging to the order Rhizobiales
were mutually closely related (97.2–100% similarity), and
formed a separate cluster within the phylogenetic tree
(Fig. 1; phylogenies of all isolates and their closest known
sequences are presented in Fig. S1, which will be found at
http://dx.doi.org/10.1017/S0954102012000831). This cluster
of seven isolates included a clone (GQ264001) isolated
from a waste site in North America. In addition, the five
isolates belonging to the genus Phaeospirillum (class
Alphaproteobacteria) collected in the Arctic, Japan, and
China were also mutually closely related (97.2–100%
similarity). Given that similar isolates were collected from
geographically disparate locations, it would appear that
these 0.2 mm-passable bacteria are more ubiquitous in
terrestrial environments than aquatic environments. To test
this conjecture, it will be necessary to isolate and culture a
larger sample of 0.2 mm-passable bacteria from a wider
range of terrestrial samples. In contrast, the eight novel
strains belonging to the phylum Bacteroidetes isolated from
the Arctic and Japan were not mutually closely related
(75.5–93.0% similarity).
The KNC strain isolated from Japanese river water was
very closely related (99.7% similarity) to Microbacteriaceae
bacterium MWH-VicE1 belonging to the Luna cluster
(phylum Actinobacteria) isolated by Hahn et al. (2004).
This cluster consists of strains isolated from lakes and ponds
in Europe, East Africa, and China, and is known to be an
obligate ultramicrobacterial cluster.
The two Antarctic isolates (S-19 and S-42) belonging to
the order Rhizobiales isolated from two samples (beach
sand and freshwater algal mat) collected from Maritime
Antarctica were found to be closely related (99.8%
similarity) to Mycoplana sp. ZS1-12 isolated from sandy
intertidal sediments of the Larsemann Hills coast (Princess
Elizabeth Land, East Antarctica) (Yu et al. 2010). In
addition, BLASTN search results showed that the sequence
of Antarctic isolate S-19 showed $ 97%, $ 98%, $ 99%,
and 100% similarity to 901, 226, 13, and one 16S rRNA gene
sequences of known isolates and/or from environmental
samples, respectively. The latitudes and longitudes of the
225
sample collection sites corresponding to these sequences
are shown in Fig. 2 (accession numbers of all sequences
showing $ 97% similarity to each of 0.2 mm-passable
isolates, together with their sample collection sites and
sources, are listed in Table SII (which will be found
at http://dx.doi.org/10.1017/S0954102012000831). Sample
collection sites corresponding to the 901 sequences with
$ 97% similarity were geographically disparate and from
diverse environmental conditions. On the other hand,
samples corresponding to 13 sequences with $ 99%
similarity included those from cryospheric habitats, such
as Ellesmere Island in the Canadian Arctic (one sequence)
and Antarctica (five sequences). Sequences closely related
($ 99%) to the S-19 sequence were also isolated from
samples collected at the mid-range latitudes: a biofilm
found in the Central Alps in Switzerland (one sequence)
and glaciers and snow recovered from China (three
sequences). These four sequences were also from
cryospheric habitats. One sequence 100% identical to
S-19 was the other Antarctic isolate (S-42) obtained in this
study. In summary, 10 of the 13 sequences with $ 99%
similarity to the S-19 sequence (c. 77%) were isolated from
samples from cryospheric habitats, such as Arctic,
Antarctic, and alpine environments. This implies that
such Rhizobiales strains occur in the cryospheric regions,
but their abundance and biomass may be scarce depending
on the geographic location.
In clear contrast to the Antarctic isolate described earlier,
Spirochaeta strain IZ-19-1 isolated from lake water
collected in western Japan was related to only one
Chinese coastal strain. No known isolates or environmental
sequences were found to be similar to IZ-19-1 as of June 2011
even with a 97% similarity threshold (Fig. S2 & Table SII,
which will be found at http://dx.doi.org/10.1017/
S0954102012000831). This implies that Spirochaeta
strain IZ-19-1 belongs to the Asian endemic group that
inhabits a restricted region.
Candidates of new taxa obtained from
0.2 mm-passable fractions
The results of BLASTN searches on the obtained 16S
rRNA gene sequences indicate that a total of 18 isolates
(seven belonging to the order Rhizobiales (class
Alphaproteobacteria), two to the genus Saccharosprillium
(class Gammaproteobacteria), eight to the phylum
Bacteoidetes, and the isolate ‘‘Shr3’’) were less than 97%
homologous with known strains and isolates, suggesting novel
taxa at species, genera, class, or even higher levels (Table I).
This result may arise as faster-growing bacteria were
effectively excluded by filtration (Hahn 2004), allowing
isolation of slower-growing bacteria that had been
previously undetectable. Even a minor species could be
recovered by a culture method designed to select for
multiplication of minorities (Pedrós-Alió 2006). Our design
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226
RYOSUKE NAKAI et al.
Fig. 3. Scanning electron micrographs of
strain KNC (phylum Actinobacteria)
that exhibit typical solenoid or
crescent-shaped cell morphology.
Scale bars 5 1 mm, pore
sizes 5 0.1 mm in diameter.
was to exclude a majority of fast-growing heterotrophic
bacteria by filtration with triple-serial 0.2 mm-pore filters.
More than 99.9% of bacterial cells in natural environments
are excluded, leaving only a fraction as small as , 0.1%
that would pass the filters (Fukuba et al. 2002). As a result,
the presence of novel microbial lineages that had
previously been obscured by faster-growers was revealed.
Cell size of 0.2 mm-passable bacteria under
culture conditions
Field-emission SEM images of the cell morphology of NP1
and NP3 (genus Phaeospirillum, class Alphaproteobacteria)
and IZ6, GB3 and S-42 (order Rhizobiales, class
Alphaproteobacteria) showed that the cells were fat
and rod-shaped, with minimum diameters between 0.3
and 0.4 mm. In contrast, cells of GB9 (Phaeospirillum) and
GB10 (Rhizobiales) were larger, with diameters between
0.5 and 1.0 mm. Larger cells may occur in culture
suspensions despite filtration through 0.2 mm filters by
cell polymorphism. In the present study, liquid cultures
were filtered twice, and although growth activity was
observed in the filtrate of GB10 cultures (Table I), no
diminutive cells were detected. It is possible that such
bacteria that grow larger than 0.2 mm under culture
conditions may also generate cells smaller than 0.2 mm
temporarily, which passed through the 0.2 mm filters. For
example, Cytophaga species have cyclic morphological
change, namely young filament and old coccus, and the
coccid cells are known to release sub-0.2 mm cells (Elsaied
et al. 2001). In the future, detailed observation of cell
morphology over the course of all lifecycle stages is needed.
IZ16 and NP9 isolates (Rhizobiales), which were seen
on FE-SEM to be relatively large, did not grow after
secondary filtration, and it is possible that the cells are
small in the environment from which the samples were
collected. This effect may result from cells entering
dormancy and/or entering a viable but non-culturable
(VBNC) state in the environment. Cells in these states
are typically of reduced size (e.g. Hood & MacDonell
1987), presumably to adapt to low organic concentrations
by increasing the surface-to-volume ratio or a starvationsurvival strategy. Alternatively, even though these isolates
may form sub-0.2 mm cells at some point of their life cycle,
the number of diminutive cells might have been negligible,
and the cells were not identified in the filtrate. In genus
Afipia (class Alphaproteobacteria), the isolated IZ17 cells
were small with minimum diameters between 0.2 and
0.3 mm.
Cells of the IZ19-2 isolate (genus Hylemonella, class
Betaproteobacteria), the IZ4 isolate (related to an unidentified
species belonging to the class Betaproteobacteria), and
the ShrSW isolate (genus Oceaniserpentilla, class
Gammaproteobacteria) were relatively small and rod-like,
with minimum diameters between 0.2 and 0.3 mm. Cells of
the KE isolate (genus Vibrio, class Gammaproteobacteria)
were extremely small, with a minimum diameter of
c. 0.15 mm.
Cells of the KIP, KIY, and KOr isolates belonging to the
CFB group (phylum Bacteroidetes) were relatively long
and rod-like, with maximum diameters between 1.5 and
3.0 mm. No obvious sub-0.2 mm cells were observed among
these isolates. Although it is possible that cell morphology
is altered by fixation and dehydration, the minimum
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PHYLOGEOGRAPHIC ANALYSIS OF NANO BACTERIA
diameters of the cells were between 0.2 and 0.3 mm. These
cells are likely to pass through the 0.2 mm pores, not
because of their diminutive cell size, but because of their
long rod shape. This will need to be validated by other
experiments such as detailed observation of the filtrates.
Eight of the 11 isolates belonging to the phylum
Bacteroidetes grew in the twice-filtered culture.
The isolated KNC cells were selenoid and closely related
to the obligate UMB belonging to the Luna cluster (phylum
Actinobacteria) (Fig. 3). Other members of the Luna cluster
have also been reported to form selenoid or crescent-shaped
cells (Hahn et al. 2003). The name Luna cluster is derived
from this crescent-shaped cell morphology, which may be
associated with flexibility and thus 0.2 mm-passability of
the cells.
Conclusions
Phylogeographic analysis of 0.2 mm-passable bacteria in
this study demonstrated that some isolates were related
closely to geographically very distant strains, while others
were found only in a restricted region. Thus, the isolates
identified in this study showed diverse distribution patterns.
In future, we will consider 0.2 mm-passable bacteria showing
characteristic distributions as reference microorganisms to test
the Baas-Becking hypothesis. However, the possibility exists
that the presence of bacteria in the environment occurred from
inadvertent selectivity and/or sampling effort. To validate our
results, we would use isolation and phylogenetic identification
techniques on the bacteria of the 0.2 mm-filtrable fraction in
the cryosphere and other environments. Further studies would
require additional techniques such as multi-locus sequence
typing and phenotypic characterization.
Acknowledgements
We are grateful to Drs Synnøve Elvevold and Kim Holmén
at the Norwegian Polar Institute and Dr Yoshihide Ohta
at University of Oslo, who helped us immensely in
conducting the investigation at Svalbard in the Arctic.
We are grateful to the crew of the RV Toyoshio-maru,
Hiroshima University, and the cruise participants for their
on-board assistance. We also acknowledge the Natural
Science Centre for Basic Research and Development of
Hiroshima University for FE-SEM analysis. This work was
supported by a Grant-in-Aid for Scientific Research
(No. 18255005) and Research Fellowships for Young
Scientists (No. 11J30005) from the Japan Society for the
Promotion of Science and a grant from the Institute for
Fermentation, Osaka, Japan. Antarctic samples were collected
during the Spanish program LIMNOPOLAR International Polar
Year 2007–08 campaign with the support of the Spanish
Polar Programme, Ministerio de Ciencia e Innovación
(POL2006-06635/ANT). This article was published thanks
to the financial support given by the Ministerio de Ciencia e
227
Innovación (Spain) with the grant ref. CTM2011-12973-E.
The constructive comments of the reviewers are also
gratefully acknowledged.
Supplemental material
Two supplemental figures and two supplemental tables will
be found at http://dx.doi.org/10.1017/S0954102012000831
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