May 16,2007
The Japan Agency for Marine-Earth Science and Technology

The First Genomic Analysis of Intercellular Chemoautotrophic Symbiont in Calyptogena Clam
A Step for Understanding of Deep Sea Chemosynthetic Ecosystem

1.Abstract

The Japan Agency for Marine-Earth Science and Technology (JAMSTEC; Mr. Yasuhiro Kato, President) conducted the complete genomic sequence of chemoautotrophic symbioses bacteria of Deep-sea clams (Pic1. Calyptogena okutanii), that were collected from the cold seep environment at the seafloor of Sagami bay, off Hatsushima, and clarified its genomic structure and gene´s characterization at the first time in the world.
By this study, it is discovered that bacterial symbionts of Calyptogena okutanii have genes that have a feature of autotrophic nutrition*1, for synthesizing all necessary energy and nutrition out of inorganic material (hydrogen sulfide and carbon dioxide) upwelling from sea bottom, and it is the smallest genome in autotrophic bacteria. Advancement has been made for elucidation of interaction between bacterial symbiont and its host in chemosynthetic ecosystem evolved in its own way at deep sea without sunlight.
Genome information analysis of Calyptogena okutanii have been perfomed by Marine Biology and Ecology Research Program (Dr. Tadashi Maruyama, Program Director), Extremobiosphere Research Center (XBR; Prof. Koki Horikoshi, Director-General) of JAMSTEC, and in cooperation with TAKARA BIO INC. (Ph.D. Ikunoshin Kato, President and CEO).
These results will be published on May 15 issue of American science journal "Current Biology".

2.Background

Chemosynthetic based animal community (Pic.1B) such as Calyptogena and Tubeworms had been discovered at hydrothermal vents andcold seeps in deep sea bottoms around the world, and it has already been known that they are chemosynthetic life*2 harboring bacterial symbionts in their cells including gill (Pic.1C) epithelial cells.
Bacterial symbiont produces energy by oxidizing sulfide and synthesize organic materials from carbon dioxide to provide their host. Chemosynthesis ecosystem depend on energy derived from inorganic chemicals upwelling from earth interior is one of the biggest discovery of the 20th century in biology history. Therefore, this discovery is attracting world wide scientists´ attention. However, chemosynthetic lives and bacterial symbionts are living at extreme pressure and low oxygen environment; it is difficult to reproduce such an environment in the laboratory, so that the understanding of this ecosystem has not been progressed much. Although its life had been formed since ancient earth, chemosynthetic ecosystem is known very little about its origin, symbioses and evolution mechanism; photosynthesis ecosystem is known very well on the other hand.
We conducted complete genomic analysis of bacterial symbiont of Calyptogena okutanii in cold seeps off Hatsushima in Sagami bay to analyze symbiosis mechanism of chemoautotrophic systems.

3.Study Method

Genomic analysis of Calyptogena okutanii symbiont was conducted after being collected alive from Calyptogena community around cold seep by the Remotely Operated Vehicle "HYPER DOLPHIN" at a depth of 1,157m off Hatsushima in Sagami Bay (35N 0.069´, 139E 13.444´), during the deep sea investigation cruise in June, 2004. Gill cell was fractured and host chromosomal DNA was degraded by DNase and only bacterial symbiont was separated. Then, genome libraries*3 were made with genome DNA extracted from isolated bacterial symbiont and genome sequence analyses*4 were performed. After the prediction of DNA´s performance analysis, metabolic map was constructed to show energy production and transport mechanism.

4.Result and discussion

Complete genome sequence of Calyptogena okutanii´s bacterial symbiont has been completed (Fig.1). It is 1,022,154 base pairs (bp) and its size is less than 1/4 of Escherichia coli´s. Performance analysis of 939 protein coding genes of this genome revealed metabolic system of the symbiont (Fig.2).

The results are as follows;

(1)

Synthesize energy material by sulfide oxidation metabolic pathways (indicated by yellow circle in fig.2), which substrate is hydrogen sulfide ingested into a host cell

(2)

Having carbon-fixation circuit (indicated by blue circle in fig.2, Calvin-Benson cycle) to produce organic materials from carbon dioxide in autotrophic system.

(3)

Possess almost all amino acid biosynthesis system. Using energy material or organic compounds described in (1) (2), enable to synthesize nutrition material such as amino acid and coenzyme.

(4)

However, transport system of synthesized nutrition to a host cell lacks.

(5)

Its genome size is smaller than that of bacterial symbiont of other Chemosynthetic based animals. In the process of symbiosis and evolution with its host, it reduced its size drastically. Some essential genes (for cell division proteins, envelope proteins, etc.) that are important for growth of normal bacteria have also been lost.

Especially about (5), it is appeared to have the smallest genome among reported autotrophic systems in the world.
It is presumed that nutrients accumulated in bacterial symbiont are brought into host cell and digested by phagocytosis function. It is considered that bacterial symbiont are domesticated and controlled by host cell, some unknown mechanism is involved with proliferation of bacterial symbiont lacking cell division system. (Pic.2: cell division captured by electric microscope)

5.Prospect

Genome reduction of intracellular symbiont of Calyptogena okutanii evokes Mitochondria, since Mitochondria used to be bacteria and evolved in the process of symbiosis with a life form which is eukaryote cell´s ancestor. To this date, chemosynthetic organelle has not been found, but this chemosynthetic symbiont is possible to turn to be organelle in the future. From these reasons, chemosynthetic symbiont may be a good model for the cellular coevolution study.

Followings are our plans for future;

(1)

By comparing 1 million bp genome size Calyptogena okutanii symbiont and 2 or 3 million bp genome size related sulfur-oxidizing bacteria from same deep sea environment, clarify lost functions and genome reduction mechanism for about 1 or 2 million bp genes difference (1 or 2 thousands genes).

(2)

Promote the comparative study with other genome in other chemosynthetic symbiosis ecosystem (other kinds of Calyptogena, Bathymodiolus, Thyasiridae, Tubeworm, etc.), clarify coevolution mechanism.

(3)

Attempt expression and control analysis, artificially rearing of host cells and study its environmental response and adaptation under the circumstances.

We consider that these studies will contribute to elucidation of cellular coevolution mechanism and understanding of life evolution, and development of life science.

Glossary

*1: Autotrophic nutrition

By comparing 1 million bp genome size Calyptogena okutanii symbiont and 2 or 3 million bp genome size related sulfur-oxidizing bacteria from same deep sea environment, clarify lost functions and genome reduction mechanism for about 1 or 2 million bp genes difference (1 or 2 thousands genes).

*2: Chemosynthetic life form

PLife forms depend on organic materials produced out of reducing substances such as hydrogen sulfide or methane by chemosynthetic bacteria.

*3: Genome library

Library made by breaking down genome DNA in peaces and putting in plasmid vector at random. Require 5 to 10 times as much as general presumed genome size base sequences. In this study 20,000 analyses were done and 10 times as much as presumed genome size base sequences was determined.

*4: Genome sequence

Bind and edit and order each base sequence, fill the blank where the sequence is not identified. Complete genome sequence of circular bacterial symbiont with approximately 1 million bp has been succeeded.

*5: Phagocytosis

For example, myxameba takes mold spore or bacteria into its cell directly and absorbs nutrients in nature. This mechanism, a cell intakes solid body, called Phagocytosis.

*6: Organelle

It is an intracellular organ became a part of a eukaryotic cell by symbiosis with eukaryotic cell or its ancestor life form, such as Mitochondria and chloroplast.

Reference 1: Collaborative institute

Takara Bio Inc. http://www.takara-bio.com/

Headquarters: Seta 3-4-1, Otsu, Shiga 520-2193, Japan
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Contacts:

(For this study)

Dr. Tadashi Maruyama
Program Director, Marine Biology and Ecology Research Program
Extremobiosphere Research Center (XBR)
Mr. Noriyuki Murata, email; xbr@jamstec.go.jp
Manager, Research Promotion Office, XBR

(For publication)

Mr. Shinji Oshima, email; press@jamstec.go.jp
Manager, Planning Department, Press Office
JAMSTEC