Physiological properties of Pseudomonas sp. strain MT-1, denitrifier from the 11,000 m-depth of Mariana Trench

極限環境微生物学会誌 Vol. 4, 2005

25

Journal of Japanese Society for Extremophiles (2005), Vol. 4, 25-31

Tamegai Ha, Nakamura Sa, Miyazaki Mb, Nogi Yb, Kasahara R a,b, Kato Cb and Horikoshi Kb

Physiological properties of Pseudomonas sp. strain MT-1, denitrifier from the 11,000 m-depth of Mariana Trench a Department of Chemistry, College of Humanities and Sciences, Nihon University

3-25-40, Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan b Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology

2-15, Natsushima-cho, Yokosuka 237-0061, Japan

Corresponding author: Tamegaia H, [email protected]

TEL +81-3-3329-1151 ext. 5740 FAX +81-3-5317-9433

Received: March 4, 2005, Accepted: March 24, 2005

Abstract In the present study, we investigated the

physiological properties of Pseudomonas sp. strain

MT-1 isolated from the mud of Mariana Trench.

Strain MT-1 was closely related with members of the

genus Pseudomonas, especially with Pseudomonas

chloritidismutans and Pseudomonas stutzeri on the basis

of 16S rDNA sequence. The DNA-DNA

hybridization values between strain MT-1 and

Pseudomonas reference strains were significantly lower

than those accepted as the phylogenetic definition of a

species. MT-1 had polar flagellum, and was

facultative anaerobe. The growth occurred in an

NaCl concentration of about 0-10% (optimum: 1-2%), in

pH of about 6-10 (optimum: 7-8), and in temperature of

about 4-45 °C (optimum: 32-35 °C). The G+C

content of the DNA was 60.5% mol%. The major

quinone was ubiquinone-9. The major fatty acid in

strain MT-1 was C16:0 (hexadecanoic acid), C16:1

(hexadecenoic acid) and C18:1 (octadecenoic acid).

The organism showed adaptational properties to

deep-sea environment compared with the reference

strains.

Key words: Pseudomonas sp. strain MT-1, denitrifier,

Mariana Trench

Introduction

Denitrification is one of the systems of anaerobic

respiration, and constitutes one of the main branches of

the environmental nitrogen cycle 26). Nitrogen is

incorporated into the biosphere with the biological

conversion from N2 to NH4+ (nitrogen fixation). It is

removed from there by oxidative conversion from NH4+

to NO3- (nitrification) and respiratory process which

reduce NO3- to NO2

-, NO, N2O and N2 subsequently

(denitrification). Many of the proteins are

participated in the system, and they have been studied

by many researchers 25, 26). In addition,

denitrification system is worthy to notice because it is

thought to be the ancient form of aerobic respiration

system 4, 13). Thus, the study of aerobic and

anaerobic respiratory system of the organism in the

isolated world (like deep sea) may provide a novel

knowledge for evolution of respiratory system.

However, little is known about respiratory system of

deep-sea organisms 9, 10, 17, 20, 24).

Denitrification is carried out by a number of

taxonomically diverse facultatively anaerobic

microorganisms. Pseudomonads contribute a large

number of denitrifying bacteria within a single genus 5,

7). Pseudomonas sp. strain MT-1 is one of the

denitrifying Pseudomonads isolated from the mud of

Mariana Trench (11°22.10'N, 142°25.85'E, 10898 m

dept), which was collected by the sterilized mud

sampler using the unmanned submersible KAIKO

operated by Japan Agency for Marine-Earth Science and

Technology (JAMSTEC) 16). This is the only

identified denitrifier isolated from deep sea, and some

genes for denitrification have been identified 18, 19).

Quite recently the organism have been classified as

原著論文


26

novel genomovar of Pseudomonas stutzeri by Sikorski

et al. (Sikorski, J., personal communication) They

performed the sequence analysis of 16s rDNA and

16S-23S rDNA internally transcribed spacer regions and

DNA-DNA hybridizations between many strains of P.

stutzeri. They also carried out the basic metabolic

tests on MT-1. However, detailed physiological

properties of the strain were still unclear. P. stutzeri

is known to show quite strong strain diversity, and the

members of the strain have been isolated world-wide

from various habitats including aquatic and terrestrial

ecosystems 14). Though, even each strain is closely

related with each other, they should adapt their own

environments. Thus, strain MT-1 is expected to

adapt its own environment, 11000-m depth of Mariana

Trench. However little is known about physiological

properties of this denitrifier from deep sea.

In the present study, we investigated the

physiological properties on strain MT-1, and found that

the organism displays adaptational properties to

deep-sea environment compared with the reference

strain. The organism can be a good object for the

study of physiological properties including

denitrification in the deep sea.

Materials and Methods

Organisms and cultivation conditions

Strain MT-1 was grown in MT-1 medium (0.5%

yeast extract, 1% tryptone, 3% NaCl, 0.01%

MgSO4·7H2O, 0.1% CaCl2·2H2O) 16), normally at 30 °C.

The reference strains used in this study, P. stutzeri IFO

14165T and Pseudomonas chloritidismutans DSM

13592T, was obtained from IFO and DSMZ, respectively.

These bacterial strains were maintained on MT-1 agar

medium (MT-1 medium containing 1.5% agar) at 30 °C.

Bacterial growth under various pressures was tested by

the methods described previously 16) with slight

modifications. Terminal electron acceptor (NaNO3

for MT-1 and P. stutzeri, and NaClO3 for P.

chloritidismutans) was added to each MT-1 medium for

maintaining the growth with anaerobic respiration.

Physiological analyses

Physiological tests were performed with a slight

modification of the general procedures as described

previously 1) Acid production from sugar was

assessed using MT-1 medium containing 1% of each

substrate and 0.03% of bromothymol blue.

Physical and Chemical analyses

Morphology of the cells of MT-1 was determined

by transmission electron microscopy as described

previously 6). Cellular fatty acids and isoprenoid

quinones were analyzed according to the methods

described previously 6).

Molecular biological studies

Chromosomal DNA was extracted from each

strain by the method of Saito and Miura 11). The G+C

content was determined by the method of Tamaoka and

Komagata 15). DNA-DNA hybridization was carried

out by the method of Ezaki et al.2) at 40 °C for 3h and

the results were measured fluorometrically.

Phylogenetic analysis

A phylogenetic tree was constructed based on the

16S rDNA sequences. Database search was carried

out by FASTA 8) on Internet. Nucleotide substitution

rates 3) were determined and a distance-matrix tree was

constructed by the neighbour-joining method 12) using

the CLUSTAL_W program 21) on internet.

Results and Discussion

In order to determine phylogenetic relationships

in detail, 16S rDNA sequence of strain MT-1 16) and

other known organisms were re-analyzed with

constructing phylogenetic tree. The results of

phylogenetic analyses (Fig. 1) clearly showed that the

strain MT-1 was classified into the genus Pseudomonas,

and were closely related to P. chloritidismutans and P.

stutzeri. On the results of DNA-DNA hybridization,

strain MT-1 showed quite low level of DNA-DNA

relatedness with P. chloritidismutans (<30%) and P.

stutzeri (<30%). This is significantly lower than that

accepted as the phylogenetic definition of a species 22).

These results were consistent with recent study

(Sikorski, J., personal communication).


27

Fig. 1 Phylogenetic tree showing the relationships of strain MT-1 within Pseudomonads.

The tree was constructed by the neighbor-joining method and based on 16S rDNA sequences.

Escherichia coli was used as the outgroup for the phylogenetic tree. Numbers indicated bootstrap values greater

than 500. Bar indicated 0.1 nucleotide substitutions per site.

Fig. 2 Transmission electron micrograph of negatively stained cells of strain

MT-1.

Bar indicated 1 µm.

Cells of the strain MT-1 were rod-shaped with

single polar flagellum (Fig. 2). Growth occurred in

an NaCl concentration of about 0-10% (optimal: 1-2%),

in pH of 6-10 (optimal: 7-8), and in temperature of

about 4-45 °C (optimal: 32-35 °C). MT-1 showed

optimal growth at comparatively higher temperature

than that at deep-sea. However, the organism showed

significant growth even at 4 °C although it was quite


28

slow 16). Further MT-1 showed optimal growth at

lower NaCl concentration than that of sea water.

This finding may be due to the fact that this organism

was isolated from mud of sea floor. Tolerance to

higher NaCl concentration may contribute to survival of

MT-1 in sea water. In the case of P.

chloritidismutans, growth occurs in an NaCl

concentration of about 0.1-4% (optimal: 2-4%), in pH of

7-9 (optimal: 7), and in temperature of about 10-37 °C

(optimal: 30 °C) 23). It is clear that MT-1 adapts to

wide-range environments compared with the reference

strain.

Characteristics of strain MT-1 and the reference

strains were shown in Table 1. Strain MT-1 was

facultatively anaerobic chemoorganotroph, displaying

respiratory type of metabolism. Nitrate was available

for growth with denitrification. However, nitrite did

not support the denitrification growth. The organism

cannot grow with fermentation. The G+C content of

the DNA was 60.5% mol%. The major quinone was

ubiquinone-9 (Q-9)

Table 1 Phenotypic characteristics of strain MT-1 and reference strains

Characteristic 1 2 3

yellow pigment + - + flagellum polar polar polar denitrification with nitrate + + - denitrification with nitrite - ND - chlorate respiration - - + gelatin hydrolysis - - - casein hydrolysis - - - starch hydrolysis - + + Tween 80 hydrolysis + + + growth at 40 °C + + - growth at 4 °C + - - GC content (%) 60.5 60.6-66.3 63.9 Acid production from: glucose + + + xylose - - - mannitol + - + glycerol + - + fructose + - - L-arabinose + - - sucrose - - - sorbitol - - - raffinose - - - rhamnose - - - myo-inositol - - - lactose - - - trehalose - - - cellobiose - - - maltose - - + galactose + - - mannose + + +

Strains: 1; Strain MT-1, 2; P. stutzeri IFO 14165T, 3; P. chloritidismitans DSM 13592T. Data were from this study

and references 7, 16 and 23. ND; no data available.


29

Fatty acid compositions of MT-1 and reference

strains were summarized in Table 2. The major fatty

acid in strain MT-1 was C16:0 (hexadecanoic acid),

C16:1 (hexadecenoic acid) and C18:1 (octadecenoic

acid). The composition of fatty acid in strain MT-1

showed similarity with those in reference strain to some

extent. Significant difference was that percentage of

unsaturated fatty acids in total fatty acids was

significantly higher in strain MT-1 (66%) than in the

reference strains ( P. stutzeri: 22%, P. chloritidismutans:

56%).

Table 2 Fatty acid composition of strain MT-1 and reference strains

Fatty acid 1 2 3

3-OH-10:0 2 3 3

12:0 5 7 9

3-OH-12:0 1 2 2

14:0 1 1 2

14:1 1

16:0 21 23 26

16:1 29 21 24

17:0 2 3 1

18:0 2 34 1

18:1 35 32

19:1 1 1

Values are percentages of total fatty acids. Strains: 1; Strain MT-1, 2; P. stutzeri IFO 14165T, 3; P.

chloritidismitans DSM 13592T. Empty cells; not detected.

Table 3 Effect of pressure on the growth of MT-1 and reference strains.

Pressure (MPa) 1 2 3

0.1 100 100 100

30 33 10 26

Strains: 1; Strain MT-1, 2; P. stutzeri IFO 14165T, 3; P. chloritidismitans DSM 13592T. Growth under each

pressure was checked by OD at 660 nm after cultivation for 22 h. Values are percentages when OD under a

pressure of 0.1 MPa is defined as 100%. A pressure of 30 MPa corresponds to the pressure at about 3,000-m depth

of sea.

It has been already demonstrated that strain MT-1

shows optimal growth under atmospheric pressure.

However it shows adaptational property for high

hydrostatic pressure to some extent 16). Table 3

showed the effect of pressure on the growth of MT-1

and reference strains. MT-1 and P. chloritidismutans

seemed to adapt high hydrostatic pressure compared

with P. stutzeri to some extent. From the result of

phylogenetic analysis (Fig. 1), it appeared that the

phylogenetic relationship between MT-1 and P.

chloritidismutans is closer than that between MT-1 and

P. stutzeri. It is possible that adaptational property

for high hydrostatic pressure is specific to MT-1 and

closely related strains.

In the present study, we investigated about the

physiological properties on Pseudomonas sp. strain

MT-1 in detail. The results showed that the organism

can adapt to wide-range environments compared with


30

the reference strains. This fact may allow MT-1 for

life in deep-sea environment. The organism can be a

good object for the study of physiological properties

including denitrification in the deep sea.

Acknowledgements

We wish to express our thanks to Mr. Uematsu

(JAMSTEC) for operating electron microscopic system.

This work was supported by the Nihon University

Individual Research Grant and the Moritani Scholarship

Foundation for H. T.

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Physiological properties of Pseudomonas sp. strain MT-1, denitrifier from the 11,000 m-depth of Mariana Trench

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