Mutagenesis and Functional Characterization of the glnB , glnA , and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

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Abstract
Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational 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 the characterization of glnB , glnA , and nifA mutants and studies of their relationship 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 NH 4 + in R. rubrum . The modification of glutamine synthetase (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 regulate the modification of GS. P II also appears to be involved in the regulation of DRAT activity, since an altered response to NH 4 + was found in a mutant expressing P II -Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH 4 + and darkness treatments.