Purification of Soluble c-type Cytochromes from a Barotolerant Bacterium Shewanella sp. Strain DSS 12
Mitsunori YAMADA*a, Hideyuki TAMEGAIa, Ron USAMIa, Chiaki KATOb, and Koki HORIKOSHIa,b
a Department of Applied Chemistry, Faculty of Engineering, Toyo University, 2100 Kujirai Kawagoe-shi Saitama 350, Japan
b The DEEPSTAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima- cho, Yokosuka 237, Japan
Deep-sea environment is known as a place of very low temperature with very high hydrostatic pressure. Microorganisms living at the sea-bottom must adapt special mechanisms for living under extreme conditions. Recently, large variety of bacteria isolated from the samples of deep-sea sediments. A barotolerant bacterium strain DSS 12 was isolated from a deep-sea mud of the Ryukyu Trench (1). This bacterium showed optimal growth at the pressure of 30 MPa and at the temperature of 8℃. The results of the study about cydD gene of strain DSS 12 indicated that expression of the respiratory system in this bacterium is regulated by pressure (2), and the quantitative expression of c-type cytochrome(s) in the cells grew under high pressure was lesser than in the cells grew at atmospheric pressure by spectrophotometric analysis. This (or these) cytochrome(s) may be considered to play some roll in baroadaptibility of this bacterium (3). In the present study, we purified two soluble c-type cytochromes to electorophoretically homogeneous state from the barotolerant bacterium Shewanella Sp. strain DSS12 grown at atmospheric pressure (0.1 MPa).
Both c-type cytochromes (named cA and cB) were purified by an ion exchange chromatography and a gel filtration. The spectra of cytochrome cA showed a peak at 410 nm in the oxidized form, and a peaks at 419, 525 and 553 nm in the reduced form. The pyridine ferrohemochrome spectrum showed peaks at 521,550 nm. These spectral features represent the presence of heme c in the molecule. The result of SDS- PAGE revealed that cytochrome cA consists of one polypeptide of 8.5 kDa. The absorption spectra of cytochrome cB showed a peak at 416 nm in oxidized form and 410, 522, 553 nm in the reduced form. The pyridine ferrohemochrome spectrum showed peaks at 521,550 nm. These spectral features also represent the presence of heme c in the molecule. The result of SDS-PAGE revealed that this cytochrome also consists of one polypeptide with molecular mass of 23 kDa.
The results in the present study will be one of the step to exhibit the relationship between baroadaptibility and respiratory system.
1. Kato, C., Sato, T., and Horikoshi, K. (1995) Biodiv. Conserv., 4, 1-9.
2. Kato, C., Tamegai, H., Ikegami, A., Usami, R., and Horikoshi, K. (1996) J. Biochem., 120, 301- 305.
3. Tamegai, H., Kato, C., and Horikoshi, K. (1997) J. Biochem. Mol. Biol. Biophys., in press.
