Dual control of the Bradyrhizobium japonicum symbiotic nitrogen fixation regulatory operon fixR nifA: analysis of cis- and trans-acting elements

Abstract

Aerobic expression of the fixR nifA operon in Bradyrhizobium japonicum was shown to depend on a cis-acting, promoter-upstream DNA sequence located between the -24/-12 promoter and position -86 relative to the transcription start site. An adenine at position -66 was essential for maximal expression. A chromosomal deletion of the upstream activator sequence (UAS) led to a symbiotically defective phenotype which was typical of nifA mutants. B. japonicum crude extracts contained a protein that bound to the UAS. By using chromosomally integrated fixR-lacZ fusions, the level of expression of the fixR nifA operon was found to be fivefold higher under reduced oxygen tension than under aerobiosis. This increase was due to autoactivation by the NifA protein and was partly independent of the UAS. Based on these data, we propose a model for the regulation of nitrogen fixation genes in B. japonicum that involves dual positive control of the fixR nifA operon. At high oxygen concentrations, the operon is expressed at a moderate level, subject to activation by the binding of a trans-acting factor to the UAS. Under such conditions, the nifA gene product is known to be inactive. At very low oxygen concentrations--a condition favorable to NifA activity--the NifA protein is the trans-acting factor which (i) enhances the level of fixR nifA expression (and hence its own synthesis) and (ii) activates other nif and fix genes.