Microbial Diversity in Inactive Chimney Structures from

Deep-Sea Hydrothermal Systems

Y. Suzuki, F. Inagaki, K. Takai, K.H. Nealson and K. Horikoshi
Subground Animalcule Retrieval (SUGAR) Project, Frontier Research System for Extremophiles, Japan Marine Science and Technology Center (JAMSTEC),
237-0061 Yokosuka, Japan
Received: 13 January 2003 / Accepted: 28 April 2003 / Online publication: 2 February 2004

Abstract

Massive chimney structures, which are characteristic of

many hydrothermally active zones, harbor diverse microbial communities containing both thermophilic and
hyperthermophilic microbes. However, vent chimneys
ultimately become hydrothermally inactive, and the
changes that occur in the microbial communities upon
becoming inactive have not been documented. We thus
collected inactive chimneys from two geologically and
geographically distinct hydrothermal fields, Iheya North
in the western Pacific Ocean and the Kairei field in the
Indian Ocean. The chimneys displayed easily distinguishable strata, which were analyzed with regard to both
mineralogical and microbiological properties. X-ray diffraction pattern and energy-dispersive spectroscopic
analyses revealed that the main mineral components of
the chimney substructures from Iheya North and the
Kairei field were barite (BaSO4) and chalcopyrite (CuFeS2), respectively. Microbial cell densities in the substructures determined by DAPI counting ranged from 1.7
107 cells g)1 to 3.0 108 cells g)1. The proportions of
archaeal rDNA in the whole microbial rDNA assemblages
in all substructures were, at most, a few percent as determined by quantitative fluorogenic PCR. The microbial
rDNA clone analysis and whole-cell fluorescence in situ
hybridization revealed a community that was decidedly
different from any communities previously reported in
active chimneys. Curiously, both samples revealed the
abundant presence of a group of Bacteria related to a
magnetosome-bearing bacterium, Magnetobacterium
bavaricum of the Nitrospirae division. These results
suggest that inactive chimneys provide a distinct microbial habitat.

Correspondence to: Y. Suzuki; E-mail: yohey@jamstec.go.jp

186

Introduction

Hydrothermal activity on the deep ocean floor is abundant and widespread, greatly affecting the geochemical
and isotopic mass balance of the oceans [5, 19, 20]. Deepsea hydrothermal vent environments exhibit complex
and dynamic features of microbial habitats where
reductive hot vent fluid interacts with oxidative, cold,
deep seawater. Microorganisms thriving in the extraordinary environments have been extensively studied by
cultivation-dependent and -independent approaches [10,
25 and references therein]. From these environments,
thermophilic members of the orders Thermococcales,
Methanococcales, Archaeoglobales, and Aquificales and
the mesophilic and thermophilic members of the e-subdivision of Proteobacteria have been detected. After
hydrothermal activity has ceased, chimney structures and
underlying mounds enriched with metal sulfides continue to interact with deep seawater. Despite the evidence
that inactive chimney structures are abundant, and that
metal sulfide oxidation can provide sufficient energy for
growth of chemolithotrophic microorganisms [7], microbial communities populating inactive chimneys have
drawn little attention. Thus, although chemoautotrophic
microorganisms on the surface of inactive sulfide structures have been characterized by cultivation-dependent
approaches [32], no culture-independent community
analyses have been reported.
In order to characterize microbial communities inhabiting inactive sulfide structures after deep-sea hydrothermal activity ceased, we collected inactive chimney
structures from two geographically distinct areas: the
Iheya North in the mid-Okinawa Trough, Western Pacific Ocean, and the Kairei field in the Central Indian
Ridge, Indian Ocean. Both the mineral compositions of
chimney structures and the tectonic settings of the areas
are known to be different [9, 12, 30]. In addition, the

DOI: 10.1007/s00248-003-1014-y

Volume 47, 186196 (2004)

 Springer-Verlag New York, LLC 2004

DEEP-SEA HYDROTHERMAL SYSTEMS

187

inactive chimneys used in this study had been oxidized to

different extents, resulting in formation of complex
substructures inside the chimneys that have distinct appearances. Microhabitats in chimney structures might
vary among substructures in which dissolution of minerals interacting with seawater provides chemically distinct microhabitats in terms of pH, redox potential, and
various chemicals. Thus, microbial communities and
mineral compositions in each substructure were determined using molecular biological and mineralogical
analyses.

freeze-dried sample was embedded into epoxy resin, and

the polished surface was analyzed by a JXA-8900L electron probe (JEOL, Tokyo, Japan) at 15 keV equipped
with energy-dispersive X-ray spectroscopy (EDS) to reveal chemical compositions of mineral phases. Backscattered electron imaging (BEI) that gives the contrast of
an image according to the chemical composition was
used to distinguish each of mineral phases present in the
subsamples. The rest of the subsample was thoroughly
ground using pestle and mortar, and then X-ray diffraction (XRD) pattern analysis was performed by a
Rint 2000 powder diffractometer (Rigaku Corporation,
Tokyo, Japan) at 40 kV and 30 mA.

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Materials and Methods

Sample Collection and Subsampling. An inactive
chimney structure was collected from an active hydrothermal vent field at the Iheya North in the midOkinawa Trough, Western Pacific Ocean (2747.48N,
12653.78E), at a water depth of 988m using the manned
submersible Shinkai 2000 in April of 2001 (JAMSTEC
Scientific Cruises NT02-06). The other inactive sulfide
chimney structures were obtained from the Kairei field
on Hakuho Knoll in the Central Indian Ridge, Indian
Ocean (2519.17S, 7002.43E), at a depth of 2443 m
utilizing the manned submersibles Shinkai 6500 in February of 2001 (JAMSTEC Scientific Cruises YK01-15),
respectively. The maps showing the locations of the sites
are available elsewhere [12, 30]. In both deep-sea
hydrothermal systems, active hydrothermal vents were
not distantly located (10 m away from >150C venting
and 5 m away from >250C venting in the Iheya North
and the Kairei fields respectively). The in situ temperatures of the deep seawaters around the inactive chimneys
were at 24C. During the retrieval of the inactive
chimneys using the submersibles, each of the chimneys
was exposed to, and reacted with, seawater from the
bottom to the surface of the oceans. The dead chimney
structures obtained were immediately placed onboard in
anaerobic bags filled with 100% N2 and stored at )80C
prior to subsampling in the laboratory.
Subsampling was aseptically conducted with the
frozen chimney structures in an anaerobic chamber in an
atmosphere of 10% H2 and 90% N2 in the laboratory.
Chimney substructures were separated from each other
using a sterile knife and spatula. Each of the substructures
was subjected to nucleic acid extraction, microscopic
observation, and characterization of mineral phases. We
subsampled four substructures from each of the Iheya
North and Kairei fields, and these subsamples were referred to as Ihe-1 to -4 and Ind-1 to -4, respectively.
Characterization of Mineral Composition. In order
to characterize mineral compositions in subamples, a
portion of the subsample was freeze-dried under anoxic
conditions to avoid oxidation during drying. Some of the

Microscopic Observation. Portions of the subsamples were fixed in sterilized synthetic seawater [28]
containing 3.7% (wt/vol) formaldehyde for 2 h, and the
mixture was vigorously agitated using a vortex mixer.
The suspension solution was briefly centrifuged (3000
g), and the supernatant was filtered with a 0.22-lmpore-size, 13-mm-diameter polycarbonate filter (Advantec, Tokyo, Japan). For staining, the filter was
incubated in deionized, distilled water (DDW) containing 4,6-diamidino-2-phenylindole (DAPI) (10 lg
mL)1) at 4C for 20 min. The filter was briefly rinsed
with DDW and examined under epifluorescence using
an Olympus BX51 microscope with the SPOT RT Slider
CCD camera system (Diagnostic Instruments Inc.,
Sterling Heights, MI, USA).
Extraction of DNA and Construction of Clone Librar-

Microbial DNA was directly extracted from each

subsample using a Soil DNA Kit (Mo Bio Lab., Solana
Beach, CA, USA), following the manufacturers instructions. Archaeal or bacterial rRNA genes (rDNA) were
amplified by polymerase chain reaction (PCR) using LA
Taq polymerase with GC buffer system (TaKaRa, Tokyo,
Japan). The oligonucleotide primers used were Arc21F
and Uni1492R for archaeal rDNA and Bac27F and
Uni1492R for bacterial rDNA [6, 14]. Reaction mixtures
were prepared in which the concentration of each
oligonucleotide primer was 0.1 lM and that of the DNA
template was about 0.1 ng/lL. Thermal cycling was
performed using a GeneAmp 9700 therma cycler with 30
cycles of denaturation at 96C for 20 s, annealing at 50C
(Archaea) or 53C (Bacteria) for 45 s, and extension at
72C for 120s. When no apparent product was recovered
after 30 cycles of reaction, the number of cycles was
extended to 40 cycles.
Amplified rDNA products with expected sizes were
excised from agarose gel after electrophoresis. The
products were purified using a Gel Spin DNA purification kit (Mo Bio Lab.). The DNA was precipitated with
ethanol, and the pellet was resuspended in DDW. The
purified rDNA was cloned in the vector pCR2.1 (Inviies.

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trogen, Carlsbad, CA, USA) using the Original TA

cloning kit, and rDNA clone libraries were constructed.
Sequencing of rDNA and Phylogenetic Analysis. The inserts of rDNA were amplified directly by PCR

from a randomly selected colony using M13 primers [16],

treated with exonuclease I and shrimp alkaline phosphatase (Amersham Pharmacia Biotech, Little Chalfont,
Buckinghamshire, UK), and sequenced by the dideoxynucleotide chain-termination method using a dRhodamine sequencing kit (PE Applied Biosystems, Foster
City, CA, USA) following the manufacturers recommendations. The Arc21F or Bac27F primer was used in
partial sequencing analysis. Single-strand sequences approximately 500 nucleotides in length were analyzed. The
sequence similarity among all of the single-strand sequences was analyzed using the FASTA program equipped with DNASIS software (Hitachi Software, Tokyo,
Japan). The rDNA sequences having 97% similarity as
determined by the FASTA program were tentatively assigned to the same phylogenetic clone type (phylotype),
and a representative rDNA sequence of each phylotype
was extended with various sequencing primers with bothstrand sequencing. Extended sequences were applied
to sequence similarity analysis with databases by the
gapped-BLAST search algorithms [1, 3]. After database
search, extended sequences were manually aligned according to the secondary structures using ARB (a software environment for sequence data) [23]. Chimeric
sequences were checked by comparing phylogenetic affiliations of the 5 and the 3 halves of each sequence.
Sequences were reduced to unambiguously alignable
positions using the Lane mask [14], and evolutionary
analysis of alignments was performed by distance methods, parsimony, and maximum likelihood using PAUP
[24].
Quantification
of
Archaeal
rDNA
Populations. Quantification of the archaeal rDNA in the whole

microbial DNA assemblages was performed by a quantitative fluorescent PCR method as previously described
[27]. An archaeal rDNA mixture containing equal
amounts of four species of archaeal rDNA (Haloarcula
japonica, Palaeococcus ferrophilus, Pyrobaculum sp., Sulfurisphaera sp.) was used as a standard [27].
Fluorescence in situ Hybridization (FISH). In order
to obtain an oligonucleotide probe specific to rDNA
clones closely related to candidate Magnetobacterium
bavaricum retrieved from dead chimneys, degeneracy
was introduced to an oligonucleotide probe previously
designed for M. bavaricum (5-GCCATCCCCTCGCTTACT-3 to 5-GCCMTCCCCTCRCTTACT-3) [22].
The degenerated probe (Mag655) sequence was analyzed
using the gapped-BLAST search algorithm to check if any

Figure 1. Photographs showing inactive chimney structures from

deep-sea hydrothermal fields. (A) The internal structure of a
chimney structure from Iheya North. An arrow points out a barite
grain disseminated in the core. (B) A whole chimney structure
collected from the Kairei field. (C) A section of the chimney
structure from the Kairei field. White lines indicates the boundaries between subsamples. Each number shown in the figures corresponds to subsample names.

other rDNA sequences have similarity to the Mag655

probe. Dot-blot hybridization analysis was conducted
with representative bacterial and archaeal rDNA clones
recovered in this study to confirm the specificity of
Mag655. As described above, rDNA was amplified from
the insert by direct PCR and purified products (100 ng)
were blotted onto a positively charged nylon membrane
(Boehringer Mannheim). The membrane was hybridized
with Mag655, the 5 end of which was labeled with digoxigenin (DIG), After hybridization, the membrane was
washed and the DIG-labeled probe hybridized with targeted rDNA was detected using the DIG luminescence
detection kit for nucleic acids (Boehringer Mannheim).

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Table 1. Mineralogic and preliminary microbiological characterizations of subsamples from inactive chimneys at the Iheya North and
Kairei field

Subsample
Iheya field
Ihe-1
Ihe-2
Ihe-3
Ihe-4
Kairei field
Ind-1
Ind-2
Ind-3
Ind-4

Mineral components

Population density
(cells g wet weight)1)

rDNA composition (%)b

Archaea/Bacteria

Distribution of M. bavaricumrelated cells (%)c

Barite
Barite
Barite + FeS + Chaa
Barite

1.7
9.2
1.2
1.8

107
107
108
107

0.3/99.7
0.8/99.2
2.2/97.8
3.7/96.3

NDd
ND
24.8
ND

Chalcopyrite + FeS
Chalcopyrite + FeS
Chalcopyrite
Chalcopyrite

3.0
2.4
9.8
5.0

108
108
107
107

3.1/96.9
1.9/98.1
4.2/95.8
1.8/98.2

ND
16.3
7.8
ND

All values are the means of, at least, duplicate measurements and range within less than 10% error, unless otherwise indicated.
a
Chalcopyrite.
b
rDNA composition was determined by using quantitative fluorogenic PCR [29].
c
Proportion of M. bavaricum-related cells in total bacterial cells in each subsample was determined by whole-cell hybridization.
d
Not detected.

For whole-cell hybridization, microbial cells in the

chimney subsample were fixed and separated from
chimney particles as described above, and then mounted
on a glass slide. The slide mounted with microbial cells
was dehydrated in an ethanol series (70, 80, and 90%,
v/v). Hybridization was conducted at 46C in a solution
containing 20 mM Tris-HCl (pH 7.4), 0.9 M NaCl, 0.1%
sodium dodecyl sulfate, 30% (v/v) formamide, and the
Mag655 and Eub338 probes (50 ng/lL), which were
labeled at the 5-end with Cy-3 and fluorescein, respectively. After hybridization, the slide was washed at 48C
in a solution lacking the probe and formamide at the
same stringency adjusted by NaCl concentration [15] and
stained with DAPI (33 lg/mL). Under epifluorescence,
the slides were examined using an Olympus BX51

microscope with a SPOT RT Slider CCD camera system

(Diagnostic Instruments Inc.).
Accession Numbers. Accession numbers for 16S
rDNA sequences from this study are available through
DNA Data Bank of Japan under accession numbers
AB099934AB099939 and AB099985AB100014.

Results
Description of Subsamples and Mineral Compositions. Hydrothermally inactive chimney structures were

collected from two geologically and geographically distinct deep-sea hydrothermal vent fields and subsampled
in the laboratory. Chimneys from both fields exhibited a

Figure 2. Powder X-ray

diffraction patterns of
subsamples that were obtained
from progressively deeper
sections of the inactive chimney
at Iheya North, Western Pacific
Ocean. Peaks from barite, FeS,
and chalcopyrite are labeled
with B, F, and C, respectively.

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Figure 3. (A, D, E) Backscattered electron images of

subsamples Ind-1, -2, and -4,
respectively. The brightness of
contrast is proportional to the
atomic weight of sample. (B,
C) EDS spectra typical of the
grains of chalcopyrite (Ch) and
the iron silicate (FS). Scale bars
are 1 lm.

thin reddish layer on the surface (Ihe-1 and Ind-1) that

overlaid a soft black layer (0.71.0 cm thick) referred to
as Ihe-2 and Ind-2, respectively (Fig. 1). Inside the black
layer in the chimney from the Iheya field, there was a
porous black core (Ihe-4) with patches of white precipitates (0.1 to 1 cm in diameter) (Fig. 1A) referred to as
(Ihe-3). In contrast, inside the black layer of the dead
chimney from the Kairei field (Ind-2), a metalliferous
brown layer (0.10.6 cm thick) containing fine grains was
found and referred to as Ind-3 (Fig. 1B, C). Underneath
this brown layer, there was a homogeneous and highly
metallic, yellowish green core (Ind-4).
Mineral components and their chemical compositions in each of the above strata were determined by XRD
pattern and EDS analyses (Table 1 and Figs. 2 and 3). In
all strata from the Iheya North, a major mineral component was barite (BaSO4, JCPDS# 76-0217) as shown in
Fig. 2. In Ihe-3, peaks from metal sulfides including FeS
(JCPDS# 80-1029) and chalcopyrite (CuFeS2, JCPDS#
71-0507) were detected by XRD pattern analysis (labeled
with F and C in Fig. 2, respectively), consistent with
chemical compositions obtained using EDS analysis (data
not shown). For Ihe-4, not enough material was obtained
for XRD analysis. However, EDS analysis revealed that

the Ihe-4 mineral phase contained only Ba, S, and O and

was therefore most likely barite (data not shown). In all
samples from the Kairei field dead chimney, chalcopyrite
was a major component (data not shown). Observations
using BEI coupled EDS analysis revealed that iron silicate
grains were abundant (Fig. 2, A and B). In Ind-2 and
Ind-3, iron silicate was found to form on the rim of
chalcopyrite grains (Fig. 2, C and D). In Ind-4, chalcopyrite grains without the rim were observed (Fig. 2, E).
These results indicate that the iron silicate is the product
of oxidative weathering of chalcopyrite and that, in Ind2, chalcopyrite was being oxidized to form the iron-silicate rim. As no XRD peaks were observed, the iron
silicate was inferred to be amorphous.
Microbial Population Density and Quantification of
Archaeal rDNA Populations. Microbial population den-

sity was evaluated by direct count of DAPI-stained microbial cells (Table 1). Among subsamples from the Iheya
North, population densities ranged from 1.7 107 to 1.2
108 cells g)1 (wet weight), with the highest cell density
observed in Ihe-3, the secondary black layer underneath
the surface black layer. For the Kairei field, the highest
population density was observed in the surface layers (3.0

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Table 2. Distribution of representative clone types of archaeal and bacterial rDNA in inactive chimney from the Iheya North, Western

Pacific Ocean
No. of rDNA clones occurred per subsample
Clone type

Archea (kingdom, division)

Crenarchaeota, Marine Group I
Ihe-A2-1
Euryarchaeota, Benthic Marine Group E
IheA4-4
Ihe-A4-21
Total archaeal rDNA clones analyzed Bacteria (division, subdivision)
Proteobacieria, Alphaproteobacteria
IheB2-3
IheB1-27
Proteobacteria, Gammaproteobacteria
IheB2-13
IheB2-23
IheB2-31
Proteobacteria, Deltaproteobacteria
IheB4-26
Acidobacteria
IheB3-42
Actinobacteria
IheB3-34
Chlorobi
IheB3-7
Nitrospirae
IheB3-8
IheB3-31
Total bacterial rDNA clones analyzed

20

15

11
31

15

18

14
12
31

20

2
2
9
22

8
7
2
12
2
6
3
1

42

36

36
27
76

2
20

Representative rDNA clones grouped by partial rDNA sequence analysis having more than 97% similarity and represented more than twice in the whole
libraries.

108 cells g)1). The lowest density was found in the core
part of the chimney structure (5.0 10)7 cells g)1).
The relative amounts of archaeal and bacterial rDNA
in the whole microbial DNA assemblages extracted from
the subsamples were estimated by quantitative fluorogenic PCR (Table 1). Throughout the subsamples, the
proportion of bacterial rDNA was much higher than that
of archaeal rDNA; the proportion of the archaeal rDNA
was at most a few percent (Table 1).
Molecular Phylogenetic Analysis. Archaeal and
bacterial rDNA clones were obtained from the subsamples and then characterized by partial sequencing (ca. 500
nucleotides) and sequence similarity analysis. The number of archaeal or bacterial rDNA clones partially sequenced varied from 15 to 85 per subsample (Tables 2
and 3). Partial sequences with similarity more than 97%
were grouped as the same clone type. Representative
rDNA clone sequences, prefixed as IheA- and IndA- for
archaea and IheB- and IndB- for bacterial rDNA clones,
respectively, were extended to approximately 1.2 kb in
length for phylogenetic analysis of their affiliations.
Archaeal rDNA phylotypes obtained from the Iheya
North and Kairei field were mainly associated with two
phylogenetic groups: marine crenarchaeotic group I

(MGI) and marine benthic euryarchaeotic group E

(MBGE) [3] (Tables 2 and 3). Proportions of MBGE in
the dead chimneys from both fields increased from the
surface to the inner parts.
Phylogenetically diverse bacterial rDNA phylotypes
were obtained from dead chimney structures by cultureindependent molecular analysis (Tables 2 and 3). An evolutionary distance dendogram of bacterial rDNA phylotypes from inactive chimney structures and available
databases is shown in Fig. 2. Ihe-1 and -2, rDNA clones
clustered with the c-subdivision of Proteobacteria, were
predominant (31 of 42 IheB1 clones and 17 of 36 IheB2
clones). The rDNA clones (IheB2-13, -23, and -31) positioned within c-Proteobacteria were related to thioautotrophic gill symbionts of deep-sea clams and mussels (e.g.,
Calyptogena, Bathymodiolus) or methanotrophic bacteria.
In Ihe-3, rDNA clones belonging to the Nitrospirae
division were especially closely related to candidate
M. bavaricum, representing 38 of 43 IheB3 clones. In
white precipitates (Ihe-4), rDNA clones closely related to
Geobacter spp. were abundant (12 out of 20 IheB4 clones).
In the reddish layer formed at the surface of the
Kairei field chimney (Ind-1), the dominant phylotypes
were clustered within the a-subdivision of Proteobacteria
(22 of 85 clones) and the Bacteroidetes division (13 of 85

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Table 3. Distribution of representative clone types of archaeal and bacterial rDNA in inactive chimney from the Kairei field, Indian

Ocean
No. of rDNA clones occurred per subsample
Clone type

Archea (kingdom, division)

Crenarchaeota, Marine Group I
IndA1-21
IndA2-6
Euryarchaeota, Benthic Marine Group E
IndA3-37
Total archaeal rDNA clones analyzed Bacteria (division, subdivision)
Proteobacteria, Alphaproteobacteria
IndB1-2
IndB1-38
IndB1-49
IndB1-58
IndB2-5
IndB3-43
Proteobacteria, Gammaproteobacteria
IndB3-17
IndB1-2
Proteobacteria, Deltaproteobacteria
IndB3-24
IndB4-15
IndB2-42
IndB4-24
Proteobacteria, Epsilonproteobacteria
IndB1-30
IndB2-33
Actinobacteria
IndB1-23
Nitrospirae
IndB4-27
Bacteroidetes
IndB1-22
IndB4-4
IndB4-9
IndB4-25
Planctomycetes
IndB2-72
Verrucomicrobia
IndB1-5
Total bacterial rDNA clones analyzed

7
3

28
45

19
28

40
47

33
41

6
7
3
4
1
1

3
1
1

1
1

1
1

3
8
2

1
1
2

5
2

19

18

1
1

2
7
5
2

42

1
75

13

1
85

40

Representative rDNA clones grouped by partial rDNA sequence analysis having more than 97% similarity and represented more than twice in the whole
libraries.

clones) (Table 3). In Ind-2, rDNA clones closely related

to candidate M. bavaricum made up 19 of 42 IndB2
clones, whereas in Ind-3, clones positioned within the dand e-subdivisions of Proteobacteria, as well as clones
closely related to M. bavaricum and Actinobacteria,
were numerous (Table 3). The bacterial rDNA clone type
(IndB4-27) within the Nitrospirae division found in the
intermediate structures of the Kairei field dead chimney
was most closely related to the Nitrospirae rDNA phylotypes (IheB3-8 and IheB3-31) recovered from the Iheya
North dead chimney (Fig. 4). The rDNA sequences
within d-Proteobacteria in IndB3 rDNA clone library
were similar to Geobacter and Desulfobulbus spp. In
contrast, the core part of the Kairei field chimney (Ind-4)

contained many rDNA sequences clustered within Bacteroidetes (16 of 40 IndB4 clones).
Whole-Cell Hybridization. In order to confirm the
predominance and localization of bacteria related to M.
bavaricum in dead chimneys, whole-cell hybridization
using a ribosomal RNA-targeted oligonucleotide probe
was conducted. The specificity of the designed probe
(Mag655) was confirmed by a sequence analysis using the
gapped-BLAST search algorithm as described above. It
was also confirmed that the designed probe hybridized
only with the targeted rDNA sequence in the clone libraries obtained from the inactive chimneys by using
dot-hybridization analysis.

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Figure 4. Evolutionary distance

dendogram of bacterial clones from
inactive chimneys and other environmental
clones and culture organisms based on
1131 nucleotides of rDNA. Accession
numbers for GenBank/EMBL/DDBJ
databases are provided if the clone or
organism name is not unique. Divisions or
subdivisions are bracketed at the right of
the figure. Branch points supported by
distance, maximum-likelihood, and
parsimony estimations (percentage of 100
bootstrap resamplings 75%) are indicated
by solid circles. Marginally supported
branch points (supported by most
phylogenetic analyses with bootstrap values
of 50 to 74%) are indicated by open circles.
Branch points without circles are not
supported by the majority of analyses.
Evolutionary distances are indicated by the
sum of horizontal branch lengths. The scale
bar represents changes per nucleotide.

Using Mag655 and a bacteria-specific probe

(Eub338), whole-cell hybridization was carried out with
all subsamples from the Iheya North and Kairei fields to
quantify the proportion of M. bavaricum cells in total
bacterial cells. Microbial cells that hybridized both with
Mag655 and Eub338 were observed in Ihe-3 and Ind-2
and -3 (Table 1). As shown in Fig. 5, the cells are rods,
approximately 45 lm long and 0.71.0 lm wide, and
the high intensity of probe-conferred fluorescence indicated that there was a high rRNA content potentially
associated with high physiological activity. Over the total
cells hybridized with Eub338, Mag655-hybridized cells
accounted for 24.8, 16.3, and 7.8% in Ihe3 and Ind-2 and
-3, respectively, although much higher proportions of

rDNA sequences related to M. bavaricum were obtained from the PCR-amplified bacterial rDNA clone
libraries.
Discussion

Deep-sea hydrothermal activities occur mainly on midocean ridges or back-arc basins. The Okinawa Trough is a
back-arc basin formed by extension within continental
crust behind the Ryukyu island arc, while the Kairei
hydrothermal field is a ridge-type deep-sea hydrothermal
system located close to the Rodrigez triple junction in the
Indian Ocean. In both fields, there are active black
smoker chimneys indicating the active formation of

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Figure 5. Whole-cell hybridization of fixed cells in the inactive

chimneys with the fluorescein-labeled Eub338 probe and the
Cy-3-labeled Mag655 probe viewed by epifluorescence microscopy
in which fluorescein-specific filters (A) and Cy-3-specific filters
(B) were used.

metal (primarily iron) sulfides. The inactive chimneys in

these fields, however, are quite different from each other
in their mineral compositions. Chimneys formed in Iheya
North were mainly made up of a sulfate mineral, barite,
with a relatively minor iron sulfide mineral content. In
contrast, chimneys formed in the Kairei field contained a
sulfide mineral, chalcopyrite. The simplest interpretation
is that the Iheya North inactive chimneys are more highly
oxidized by their microbial populations, with sulfate
being produced and deposited as insoluble BaSO4, while
those of the Kairei field are still in the nascent stages of
iron sulfide oxidation.
In keeping with this notion, the microbial diversity
seen in the Iheya communities is decidedly less than that
seen in the Kairei samples (Fig. 6). Each stratum of the
Iheya chimney is dominated (6094%) by one major
bacterial group, and only three or fewer divisions are seen

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in each stratum. In contrast, each layer of the Kairei

chimney samples contains at least five divisions, and the
dominant divisions tend to be 50% or less. Furthermore,
the dominant bacterial divisions between the two are
substantially different between the two samples. Whether
this is an inherent difference between the geologically and
geographically different samples or whether it is a function of the extent of chimney oxidation cannot be distinguished at this point.
One group of Bacteria that appeared in this study
was the lineage represented by M. bavaricum; these
bacteria were found as dominant members in layers Ihe3, Ind-2, and Ind-3. M. bavaricum was originally found
in freshwater sediment samples collected from Lake
Chiemsee, Germany, and through a combination of PCRbased rRNA sequence retrieval and fluorescent oligonuleotide probing [22], it was characterized as a Bacterial
type that contains high numbers of magnetosomes. It is
as yet uncultivated and phylogenetically distinct from the
other characterized magnetotactic bacteria [4]. Measurements with an oxygen microelectrode have revealed
that sediment layers enriched with M. bavaricum are
microaerobic [22]. As enrichment cultures of M. bavaricum contain sulfur inclusions as well as magnetite, it
has been suggested that this organism may have an ironand/or sulfur-dependent energy metabolism under
microaerobic conditions. Because of the unusual nature
of this organism, and because its presence in marine
systems was previously unknown, we attempted to localize and quantify the number of M. bavaricumlike cells
in the various strata. In the surface of the dead chimney
structure from the Iheya North (the subsamples Ihe-1
and -2) containing no sulfide minerals but barite, neither
bacterial rDNA clones nor cells related to M. bavaricum were found. In sharp contrast, M. bavaricum
related bacteria were predominant in the inside part of
the chimney (Ihe-3) containing metal sulfldes besides
barite. In Ihe-4, which was collected from the same location in the chimney structure as Ihe-3 as shown in
Fig. 1A and contained no metal sulfldes, the rDNA signal
from M. bavaricum was absent. These findings clearly
demonstrated that the presence of metal sulfides is
strongly associated with the occurrence of M. bavaricumrelated bacteria.
Among subsamples from the Kairei field, all of which
contained chalcopyrite as a major mineral constitute, M.
bavaricumrelated bacteria were present in all strata,
although substantial variations in the numbers were
noted (Table 1). In the intermediate two layers between
the most outer and inner parts of the Kairei field chimney
where partial oxidation of chalcopyrite grains was evident, M. bavaricumrelated bacteria were highly enriched, indicating that the M. bavaricumrelated
organisms might thrive in certain interface zones of dead
chimneys between the infiltrated oxidative seawater and

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Figure 6. Graphic representations of the

distributions of bacterial 16S rDNA clones in
each subsample from the Iheya North (A) and
Kairei fields (B) shown in Tables 2 and 3.
Gamma, Alpha, Delta and Epsilon refer
to the c-, a-, d-, and e-subdivisions of
Proteobacteria, respectively. Nitrosp, Chlorob,
Acido, Actino, Plancto, Verruco, and Bacterio
refer to the divisions of Nitrospirae,
Chlorobi, Acidobacteria, Actinobacteria,
Planctomycetes, Verrucomicrobia, and
Bacteroidetes, respectively.

the pristine metal sulfides. Detailed geochemical analysis

of the dead chimneys will be required to elucidate the in
situ physicochemical conditions of microhabitats inside
the inactive chimneys, in which M. bavaricumrelated
bacteria predominate. In the absence of such data, it is
not possible to infer the physiological traits of M. bavaricum. In addition, although magnetite and sulfur
inclusions have been observed in M. bavaricum [22],
we were not able to find either magnetite or any inclusion
bodies in the M. bavaricumrelated cells, making any
physiological analogies to the freshwater bacteria impossible. However, localization of the M. bavaricum
related bacteria with metal sulfides and the expected
redox interface suggests that these organisms may utilize
sulfur or iron for their metabolism. Enrichment of the
M. bavaricumrelated bacteria is currently under way.
Although the number of Archaea (based on quantitative rDNA analysis) was small, the distribution was
notable. In the more oxidized Iheya samples, Crenarchaeota (MG1) dominated the outer strata, and
Euryarchaeota (MBGE) dominated the inner two. In
contrast, in the Kairei samples, all strata were dominated
by MBGE-like cells. This is consistent with the Iheya
sample being a more mature (i.e., more completely oxidized) inactive chimney, and with the MBGE group being
early colonizers (along with the Bacteroidetes and Nitrospirae types), and MG1 group being later successors
(along with the c-Proteobacteria).
To this end, the only environmental rDNA sequences
belonging to MBGE have been detected from deep-sea
sediments in the Atlantic Ocean and an active sulfide
chimney structure at Myojin Knoll in the Western Pacific
Ocean [26, 31] as a minor component in the archaeal
rDNA clone libraries. Until this study, no rDNA signals
related to either MBGE or M. bavaricum had been
detected as a major component in our complementary
microbiological investigations of active chimney structures, plumes, and deep-sea waters in either of these
hydrothermal systems (unpublished data). In addition,
no predominance of either of these phylotypes has been
reported in previous microbiological investigations of
microbial habitats in active deep-sea hydrothermal

environments [2, 10, 13, 17, 18, 25, 26, 29]. Thus, it is
inferred that both archaeal and bacterial community
structures in deep-sea hydrothermal vent chimneys dramatically change after hydrothermal emission ceases.
A central and important question with regard to
these findings is whether the differences we observe are
due to differences in aging of the inactive chimneys, or
perhaps are simply geographically and geologically influenced. We believe that the primary differences are due
to the oxidative metabolism of the sulfides, and that these
chimneys simply represent different points in time.
Clearly, this can be distinguished by more samples of
chimneys of different ages, and perhaps some laboratory
studies of artificially aged chimneys under different
conditions.
Massive sulfide deposits (millions of tons) that
consist of chimneys and underlying mounds are formed
by deep-sea hydrothermal activity in tectonic settings [8,
11, 21, 33]. Since most of the chimneys are in fact inactive, the microbial communities found there may be
relevant to the energetics of the deep-sea floor in ways
previously not appreciated.
Acknowledgments

We thank the captains and crews of the R/Vs Natsushima

and Yokosuka and the DSVs Shinkai 2000 and 6500 operation groups for their technical expertise. We also
thank Katsuyuki Uematsu and Tadashi Yokoyama for
their help with the electron microprobe and X-ray
powder diffractometer, respectively. We are grateful to
Hisako Hirayama, Takuro Nunoura, Hanako Oida, and
Masae Suzuki for their laboratory assistance. Mineralogical characterization was partly performed at the facility of the Mineralogical Institute of University of
Tokyo.
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