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

摘要

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 PII) were constructed. While PII-Y51F showed a lower nitrogenase activity than that of wild type, a PII deletion mutant showed very little nif expression. This effect of PII on nif expression is apparently the result of a requirement of PII for NifA activation, whose activity is regulated by NH4⁺ 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 PII might exist in R. rubrum and regulate the modification of GS. PII also appears to be involved in the regulation of DRAT activity, since an altered response to NH4⁺ was found in a mutant expressing PII-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 NH4⁺ and darkness treatments.