The ferredoxin (Fd) from Haloarcula japonica possesses a plant-type [2Fe-2S] cluster and is stable at high salt concentrations. Ha. japonica Fd (HjFd) includes an N-terminal additional domain rich in acidic amino acids, as well as a common core domain that contains the Fe-S cluster. The N-terminally HAT-tagged intact HjFd (HAT/HjFd) and spinach/Ha. japonica chimeric Fd (HAT/Sp/HjFd) were prepared and characterized. Escherichia coli-produced HAT/Sp/HjFd and Ha. japonica-produced HAT/HjFd were produced as holoproteins. On the other hand, E. coli-produced HAT/HjFd did not incorporate the Fe-S cluster. These results suggested that the N-terminal domain of HjFd contributed to the polypeptide folding and successive Fe-S cluster incorporation under high salt conditions. Both Ha. japonica-produced HAT/HjFd and E. coli-produced HAT/Sp/HjFd were stable at high salt concentrations (≥1.5 M NaCl), although a reduction in stability was observed at lower concentrations. Lack of the N-terminal domain did not affect the stability of HjFd, indicating that the core domain mainly contributed to the stability of HjFd at high salt concentrations. Solubility of E. coli-produced HAT/Sp/HjFd under high salt conditions was significantly lower than that of Ha. japonica-produced HAT/HjFd. It was revealed that substitution of the N-terminal domain of HjFd to that of spinach Fd injured the solubility of HjFd. Thus, it was concluded that the N-terminal domain of HjFd should perform the essential functions for halophilic adaptation from the folding process through the folded state.
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.