RESUMO
The deep-sea denitrifier Pseudomonas sp. MT-1 has two gene clusters encoding dissimilatory nitrate reductases, periplasmic nitrate reductase (Nap) and membrane-bound nitrate reductase (Nar). In order to investigate the physiological role of these enzymes, we constructed the disrupted mutants of napA, narG, and narK (encoding the catalytic subunits of Nap and Nar, as well as the nitrate transporter, respectively). The napA mutant showed almost the same growth rate as the wild-type under both atmospheric and high pressure of 30 MPa. On the other hand, the narG and narK mutants showed growth deficiencies under atmospheric pressure which were more pronounced at a pressure of 30 MPa. Thus, Nar was shown to be the dominant dissimilatory nitrate reductase in MT-1, especially under high pressure, whereas Nap can support the growth with denitrification to some extent. Further, nitrate reductase activity of the soluble and membrane fractions of MT-1 was measured under high pressure. Both activities were highly piezotolerant even under a pressure of 150 MPa. Therefore, the stability of nitrate reductases under high pressure is not a limiting step for the growth of MT-1 under these conditions. Although the reason why Nar rather than Nap is dominant and the physiological role of Nap in MT-1 are still unclear, we have demonstrated the mechanisms of the denitrification system in the environment of the deep-sea.