Abstract
Central to the genetic regulatory circuit that controls Bradyrhizobium japonicum nif and fix gene expression is the NifA protein. NifA activates transcription of several nif and fix genes and autoregulates its expression during symbiosis in soybean root nodules or in free-living microaerobic conditions. High O2 tensions result in the lack of nif expression, possibly by inactivation of NifA through oxidation of an essential metal cofactor. Several B. japonicum nif and fix promoters have upstream activator sequences (UAS) required for optimal activation. The UAS are located more than 100 bp from the -24/-12 promoter and have been proposed to be binding sites for NifA. We investigated the interaction of NifA with the nifD promoter region by using in vivo dimethyl sulfate footprinting. NifA-dependent protection from methylation of the two UAS of this promoter was detected. Footprinting experiments in the presence of rifampin showed that UAS-bound NifA led to the formation of an open nifD promoter-RNA polymerase sigma 54 complex. Shift to aerobic growth resulted in a rapid loss of protection of both the UAS and the promoter, indicating that the DNA-binding and the activation functions of NifA were controlled by the O2 status of the cell. After an almost complete inactivation by oxygen, the NifA protein began to degrade. Furthermore, metal deprivation also caused degradation of NifA. In this case, however, the rates of NifA inactivation and NifA degradation were not clearly distinguishable. The results are discussed in the light of a previously proposed model, according to which the oxidation state of a NifA-metal complex influences the conformation of NifA for both DNA-binding and positive control functions.