Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1

Journal of Japanese Society for Extremophiles (2006), Vol. 5, 27-33

ORIGINAL PAPER

Tamegai Ha, Furukawa Ta, Nitta Ja, Chikuma Sa, Miyazaki Mb,
Nogi Yb, Arakawa Sb, Kato Cb and Horikoshi Hb

Halomonas sp. strain DT-W, a halophile from the 11,000 m-depth of the Mariana
TrenchaDepartment of Chemistry, College of Humanities and Sciences, Nihon University, 3-25-40, Sakurajosui, Setagaya-ku,
Tokyo 156-8550, Japanb
Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology, 2-15, Natsushima-cho,
Yokosuka 237-0061, Japan
Corresponding author: Hideyuki Tamegai, htamegai@chs.nihon-u.ac.jp
TEL +81-3-3329-1151 ext. 5740, FAX +81-3-5317-9433
Received: April 26,2006 / Reviced: May 18,2006 / Accepted: May 23,2006
Agency for Marine-Earth Science and Technology(JAMSTEC) and many microorganisms have been
isolated from recovered deep-sea samples.
Theseexplorations have made significant contributions to thedevelopment of the studies of deep-sea microorganisms.
The mud of the Mariana Trench, one of the deepest
trenches in the world, was collected by a sterilized mudsampler using the unmanned submersible KAIKO
operated by JAMSTEC in 1996.
Genetic analysis
concerning biodiversity was carried out 6), and many
novel bacteria were isolated from the mud 4, 10, 16, 17, 19, 20).
Among the microorganisms isolated from the mudof the Mariana Trench, we observed some halophilicstrains.
Halophilic and halotolerant bacteria are
typically isolated from salt lakes, saline soils, salted
foods, and other saline environments 22).
Moderate
halophilles are also quite abundant in the water and mudof seas. Sometimes they compose up to 10% of thetotal marine microbial community 7).
Many halophiles
were isolated from the sea samples collected at the sea of
0-3000 m depths 7) and some of them were identified as
novel species of the genus Halomonas.
In the present study, we isolated one of the
halophilic strains from the mud of the Mariana Trenchand investigated the genetic and physiological propertiesof the strain.
This is the first report concerning
halophiles from the Mariana Trench. An examinationof such strains should provide novel insights regarding
life in the depths of deep-seas.

Abstract Moderately halophilic strain DT-W wasisolated from the mud of the Mariana Trench.
Cells ofthe organism were rod-shaped (1.5-2 µm x 0.5-0.8 µm)
with some flagella extruding from the cells. Growthoccurred in an NaCl concentration of about 0.1-15%(optimal: 3-5%), at pH of 6-9, and at temperatures
ranging from 4-51 °C (optimal: 30-37 °C). The resultsof 16S rDNA analysis and DNA-DNA hybridization
analyses showed that DT-W was closely related toHalomonas aquamarina, Halomonas axialensis andHalomonas meridiana.
Furthermore, additionalphysiological properties and the cytochrome contents ofDT-W were also analyzed.
Key words: Halomonas sp. strain DT-W, halophile,
Mariana Trench
Introduction
Microorganisms have been studied extensively becauseof their close relationship with humans. To date, many
microorganisms have been isolated from theenvironment and most of them were obtained from land.
However, land surfaces occupy only about 30% of theearth and another 70% of the earth is occupied by oceans.
Thus, the mud and water from oceans should be an
attractive source for the isolation of novelmicroorganisms. The deep-sea is especially of interest
because it is still a relatively unknown area because of
the difficulty in exploring these regions.
In Japan,many submersibles have been operated by the Japan27

Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1

Komagata 18).
DNA-DNA hybridization was carriedout by the method of Ezaki et al. 3) at 40 °C for 3h andthe results were assessed fluorometrically.
16S rDNA
sequences were determined as described previously 19).
The determined sequence was deposited in DDBJ,
EMBL and GenBank with the accession numberAB249883.

Materials and Methods
Isolation of the bacterium
Mud from the Mariana Trench (11°22.10' N, 142°25.85'
E, 10898 m depth) 19) was used as the source for isolationof organisms.
The mud was collected by a sterilized
mud sampler using the unmanned submersible KAIKO.
Approximately 5 g of mud were suspended in MT-1
medium 20) and the solids were removed by decantation.
A 0.5 ml aliquot of the supernatant was seeded on theplate of MT-1 agar medium (MT-1 medium containing
1.5% agar).
The plate was incubated at 30°C
overnight and the colonies that formed were isolated forfurther study.

Phylogenetic analysis
A phylogenetic tree was constructed based on the 16S
rDNA sequences. Database searches were carried out
by FASTA 13) on the Internet. Nucleotide substitution
rates 9) were determined and a distance-matrix tree wasconstructed by the neighbour-joining method 15) using
the CLUSTAL_W program 21) on the Internet.

Organisms and cultivation conditions
Strain DT-W was grown in MT-1 medium 20), normally at30 °C.
The reference strains used in this study,
Halomonas aquamarina DSM 30161T, Halomonas
axialensis DSM 15723T, and Halomonas venusta DSM
4743T were obtained from DSMZ, while Halomonas
meridiana NBRC 15608T was obtained from National
Institute of Technology and Evaluation BiologicalResource Center (NBRC).
These bacterial strains
were maintained on MT-1 agar medium at 30 °C.
Growth under various pressures for 20 h was monitored
as previously described 5).

Analysis of cytochrome content
The cells of DT-W (2g, wet weight) were suspended in
25 mM Na-phosphate buffer (pH 7.0) containing 0.1 M
EDTA and disrupted by a sonic oscillator (22.5 kHz, 100W). The suspension was centrifuged (10,000 x g, 10min) to remove unbroken cells and the cell-free extract
obtained was again centrifuged (100,000 x g, 1 h).
The supernatant was then used as the soluble fraction.
The pellet was resuspended in 25 mM Na-phosphatebuffer (pH 7.0), and the resulting suspension was used as
the membrane fraction.
Reduced minus oxidized
difference spectra of each fraction were measured
spectrophotometrically.
Protein concentrations ofeach fraction were determined by the method of Lowry
et al. 11) with slight modifications 2).

Physiological analyses
Physiological tests were performed with slight
modifications of the general procedures as described
previously 1).
Acid production from sugar wasassessed using MT-1 medium containing 1% of each
substrate and 0.03% of bromothymol blue.

Results and Discussion
Cells of the strain DT-W were rod-shaped with some
flagella apparent on the cell surface (Fig. 1).
The cells
were 1.5-2 µm long and 0.5-0.8 µm wide. Growthoccurred in an NaCl concentrations ranging from
0.1-15% (optimal: 3-5%), at pH of 6-9, and attemperatures from 4-51 °C (optimal: 30-37 °C).
These results indicated that DT-W was a moderate
halophile, and can grow under low temperature
conditions as found in the deep sea. Table 1 shows theeffects of pressure on the growth of DT-W. It appears
that strain DT-W is not a piezophilic bacterium, but istolerant to high pressures to some extent.

Physical and Chemical analyses
Morphology of the cells of DT-W was determined bytransmission electron microscopy as described
previously 12).
Cellular fatty acids and isoprenoid
quinones were analyzed according to the methods also
described previously 12).
Molecular biological studies
Chromosomal DNA was extracted from each strain by
the method of Saito and Miura 14). The G+C content
was determined by the method of Tamaoka and28

Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1

Fig. 1 Transmission electron micrograph of negatively stained cells of strain DT-W. Bar, 1.0 µm

Table 1

Effect of pressure on the growth of DT-W.

Pressure (MPa)
Optical density at 660 nm after 20h0.10.180

300.094

500.056700.018

to a subspecies of these Halomonads. This result is
not consistent with a recent study which showed that H.
axialensis is distinguishable from H. aquamarina and H.
meridiana 8).
However DNA-DNA relatedness
between H. venusta and the other strains was low enoughto show that there was no technical errors in ourprocedure. H. aquamarina was isolated from shallow
sea samples, H. axialensis was from a deep sea depth of
1,530 m depth and H. meridiana was from a lake. It isinteresting that a bacterium living in the Mariana Trench(11,000 m depth) is closely related with such bacteria
isolated from water depth quite distinct from its normal
habitat.

A phylogenetic tree was constructed on the basis of16S rDNA sequences (Fig. 2). The tree indicated that
DT-W could be classified in the genus Halomonas.
Specifically, it is closely related to H. axialensis, H.
aquamarina and H. meridiana. In order to analyze therelationshipbetweeneachstrain, DNA-DNA
hybridization was carried out (Table 2).
DT-W
showed comparatively high level of DNA-DNA
relatedness with H. axialensis, H. aquamarina and H.
meridiana (>70%).
These values were higher thanthose accepted for the phylogenetic definition of aspecies 23). Therefore, these strains might be classified
in the same species. Strain DT-W appeared to belong

Fig. 2. Phylogenetic tree showing
the relationships of strain DT-W
with related strains.
The tree was constructed by theneighbor-joining method andbased on 16S rDNA sequences.
The data for Zymobacter palmae
was used as the outgroup for thephylogenetic tree. Numbers
indicated bootstrap values
greater than 500. Bar, 0.1
nucleotide substitutions per site.

29Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1

Table 2 DNA-DNA hybridization using chromosomal DNA of DT-W and related strains.
Strain1234
5110085847720287100878622% Homology with DNA from:34838883
94
10090
81100
242352531
3126100

Strains: 1; Strain DT-W, 2; H. aquamarina DSM 30161T, 3; H. axialensis DSM 15723T, 4; H. meridiana NRBC 15608T,
5; H. venusta DSM 4743T.
Table 3

Phenotypic characteristics of strain DT-W and reference strains

Characteristic
Growth properties
Temperature (°C)
range
optimumpH
range
Salinityrange
optimum
Other properties
gelatin hydrolysis
casein hydrolysis
starch hydrolysis
Tween 80 hydrolysis
Catalase test
Oxidase test
GC content (%)
Acid production from:cellobiose
fructosegalactose
glucoselactosemannitolmannoseraffinose
rhamnosesucrosexylose12344 - 51
30 - 37

5 – 4020 – 25-1 - 3530-5 - 45
28 - 40

6-9

5 – 105 - 12

5 - 100.1 - 15
3-5

0 – 20
7.5 – 10

0.5 - 2440.01 - 25
1-3++
58.8+++
56.7++
54.7++++
56.9+++++-ND++++++
ND+++ND+ND
ND
ND-ND++++++
ND+++

Strains: 1; Strain DT-W, 2; H. aquamarina DSM 30161T, 3; H. axialensis DSM 15723T, 4; H. meridiana NRBC 15608T.
Data of substrate hydrolysis, catalase test, oxidase test and GC content for all strain were determined in this study.
Other data for 1 were determined in this study, for 2 and 4 were from reference and for 3 were from reference . ND;
no data available.
30

Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1Characteristics of strain DT-W and the reference
strains are shown in Table 3.
The results suggest thatstrain DT-W appears to be an aerobic chemoorganotroph.
Furthermore, neither nitrate nor nitrite supported growthof the organism with concomitant denitrification (datanot shown).
The G+C content of the DNA was
Table 4
Fatty acid
3-OH-12:0
14:0
16:0
16:1
18:1
19:1

calculated to be 58.8 mol%. In addition, the major
quinone isolated from strain DT-W was ubiquinone-9(Q-9).
The fatty acid compositions of DT-W and thereference strains are summarized in Table 4.
Themajor fatty acids in strain DT-W are C16:0(hexadecanoic acid) and C18:1 (octadecenoic acid).

Fatty acid composition of strain DT-W and reference strains
15322762

253211061

36421862

48524855
2

Values are percentages of total fatty acids. Strains: 1; Strain DT-W, 2; H. aquamarina DSM 30161T, 3; H. axialensis
DSM 15723T, 4; H. meridiana NRBC 15608T. Empty cells; not detected.

The cytochrome content of strain DT-W wasanalyzed spectrophotometrically as shown in Fig. 3.
These difference spectra showed that b-type (or/ando-type) and c-type cytochromes were expressed in themembrane fraction of the organism, and c-typecytochrome(s) in the soluble fraction.
There were noa-type or d-type cytochromes detected. This suggeststhat the terminal oxidase of DT-W is a b-type or o-typecytochrome.

.
Fig. 3 Reduced minus oxidized difference spectra of membrane fraction (6.1 mg/ml of protein, spectrum A) andsoluble fraction (2.2 mg/ml of protein, spectrum B) of strain DT-W.
Reduced forms were prepared by adding a small amount of Na2S2O4 and oxidized forms were prepared byadding a small amount of (NH4)2S2O8.

31

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In the present study, we isolated Halomonas sp. strain
DT-W from the mud of the Mariana Trench.
Furthermore, we characterized some of the physiologicalproperties on DT-W in detail.
These results represent
the first report concerning Halomonads from the MarianaTrench.
Acknowledgements
We wish to express our thanks to Professor M. Sugawara(Nihon University) for his technical assistance, and Mr.
K. Uematsu (JAMSTEC) for operating the electron
microscopic system. We also thank to Dr. H. K.
Kuramitsu for assistance in editing the manuscript.
This work was supported by a Moritani Foundation
Scholarship to H. T.
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