Effect of oxygen level on simultaneous nitrogenase and sMMO expression and activity in Methylosinus trichosporium OB3b and its sMMO(C) mutant, PP319: aerotolerant N2 fixation in PP319.

ABSTRACT

Soluble methane monooxygenase (sMMO) expression and activity were monitored under conditions that either promoted or suppressed the expression of nitrogenase in Methylosinus trichosporium OB3b wild-type (WT) and in its sMMO-constitutive mutant, PP319. Both WT and mutant cultures had reduced sMMO activity and protein levels under elevated O2 conditions (188 microM) compared with low O2 conditions (24 microM). Simultaneous N2 fixation also reduced sMMO activity in both cultures when O2 was low. However, when O2 levels were increased, nitrogenase expression ceased and sMMO activity was reduced by approximately 77% in the WT, whereas sMMO and nitrogenase expression and activity in PP319 were relatively unaffected by the higher O2 levels. Western immunoblot analysis showed that the nitrogenase Fe protein resolved as two components (apparent molecular mass of 30.5 and 32 kDa) in both the WT and PP319 when O2 levels were low. When O2 levels were high, only the 32-kDa form of the Fe protein was present in PP319, whereas neither form was detectable in the WT. Aerotolerant N2 fixation appears to be associated with the 32-kDa Fe protein in M. trichosporium OB3b.