FIX-regulated genes evolved through promoter duplication in Sinorhizobium meliloti

The FixLJ two-component system of Sinorhizobium meliloti is a global regulator, turning on nitrogen-fixation genes in microaerobiosis. Up to now, nifA and fixK were the only genes known to be directly regulated by FixJ. We used a genomic SELEX approach in order to isolate new FixJ targets in the genome. This led to the identification of 22 FixJ binding sites, including the known sites in the fixK1 and fixK2 promoters. FixJ binding sites are unevenly distributed among the three replicons constituting the S. meliloti genome: a majority are carried either by pSymA or by a short chromosomal region of non-chromosomal origin. Thus FixJ binding sites appear to be preferentially associated with the pSymA replicon, which carries the fixJ gene. Functional analysis of FixJ targets led to the discovery of two new FixJ-regulated genes, smc03253 and proB2. This FixJ-dependent regulation appears to be mediated by a duplication of the whole fixK promoter region, including the beginning of the fixK gene. Similar duplications were previously reported for the nifH promoter. By systematic comparison of all promoter regions we found 17 such duplications throughout the genome, indicating that promoter duplication is a common mechanism for the evolution of regulatory pathways in S. meliloti.