Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum

Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two diff erent levels: transcriptional regulation of nif expression and posttranslat ional regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase- dinitrogenase reductase-activating glycohydrolase) system. We report here t he characterization of glnB, glnA, and nifA mutants and studies of their re lationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P-II) were constructed. While P-II-Y51F showed a lower nitrogenase activity than that of wild type, a P-II deletion mutant showed very little nif expression. This effect of P-II on nif expression is apparently the result of a requirement of P-II for NifA activation, whose activity is regulated by NH4+ in R. rubrum. The modification of glutamine s ynthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P-II might exist in R. rubrum and r egulate the modification of GS. P-II also appears to be involved in the reg ulation of DRAT activity, since an altered response to NH4+ was found in a mutant expressing P-II-Y51F. The adenylylation of GS plays no significant r ole in nif expression or the ADP-ribosylation of dinitrogenase reductase, s ince a mutant expressing GS-Y398F showed normal nitrogenase activity and no rmal modification of dinitrogenase reductase in response to NH4+ and darkne ss treatments.