Regulation of gene expression in response to oxygen in Rhizobium etli: Role of FnrN in fixNOQP expression and in symbiotic nitrogen fixation

Previously, we reported finding duplicated fixNOQP operons in Rhizobium edi CFN42. One of these duplicated operons is located in the symbiotic plasmid (fixNOQPd), while the other is located in a cryptic plasmid (fixNOQPf). Al though a novel FixL-FixKf regulatory cascade participates in microaerobic e xpression of both fixNOQP duplicated operons, we found that a mutation infi xL eliminate fixNOQPf expression but has only a moderate effect on expressi on of fixNOQPd. This suggests that there are differential regulatory contro ls. Interestingly, only the fixNOQPd operon was essential for symbiotic nit rogen fixation (L. Girard, S. Brom, A. Davalos, O. Lopez, M. Soberon, and D . Romero, Mol. Plant-Microbe Interact. 13:1283-1292, 2000). Searching for p otential candidates responsible for the differential expression, we charact erized two fnrN homologs encoding transcriptional activators of the cyclic AMP receptor protein [CR-PI-Fnr family] in R. edi CFN42. One of these genes (fnrNd) is located on the symbiotic plasmid, while the other (fnrNchr) is located on the chromosome. Analysis of the expression of the fnrN genes usi ng transcriptional fusions with lacZ showed that the twofizrN genes are dif ferentially regulated, since only fnrNd is expressed in microaerobic cultur es of the wild-type strain while fnrNchr is negatively controlled by FixL. Mutagenesis of the two fnrN genes showed that both genes participate, in co njunction with FixL.FixKf, in the microaerobic induction of the fixNOQPd op eron. Participation of these genes is also seen during the symbiotic proces s, in which mutations in fnrNd and fnrNchr, either singly or in combination , lead to reductions in nitrogen fixation. Therefore, R. edi employs a regu latory circuit for induction of the fixNOQPd operon that involves at least three transcriptional regulators of the CRP-Fnr family. This regulatory cir cuit may be important for ensuring optimal production of the cbb(3), termin al oxidase during symbiosis.