MUTATIONS AFFECTING NITROGENASE SWITCH-OFF IN RHODOBACTER-CAPSULATUS

In vivo 'switch-off' and subsequent reactivation of nitrogenase activity in Rhodobacter capsulatus or Rhodospirillum rubrum in response to a variety of environmental stimuli, including the addition of fixed nitrogen, is thought to be due to the action of two nitrogenase Fe protein modifying activities; DRAT (dinitrogenase reductase ADP-ribosyl transferase) and DRAG (dinitrogenase reductase-activating glycohydrolase). Here it is demonstrated that strains, including one mutated in glnB, that constitutively express nif in the presence of fixed nitrogen are never-the-less capable of Fe protein modification. Thus the regulation of Fe protein modification is separate from that of its expression. The observations that Mn-deficient cultures are unable to fix nitrogen and that DRAG activity requires a divalent metal cation, most notably Mn2+ prompted the search for mutants (pseudo-prototrophs) capable of in vivo nitrogen fixation under Mn-deficient conditions. In the present study the isolation and partial characterization of several putative mutants is described. One, AF1, was shown to be altered in the in vivo regulation of N2ase activity in response to fixed nitrogen and to have an altered in vitro activity in glutamate grown cells. However, this strain was shown to possess in vitro DRAT activity and to have a modifiable Fe protein. Two-dimensional gel analysis indicates that this strain is altered in the synthesis of a 48 kDa protein of as yet unknown function. Thus, the mutation in this strain must affect, in an as yet undetermined manner, the response of the modifying system to fixed nitrogen.