Regulation of Gene Expression in Response to Oxygen in Rhizobium etli: Role of FnrN in fixNOQPExpression and in Symbiotic Nitrogen Fixation

摘要

Previously, we reported finding duplicated fixNOQPoperons in Rhizobium etli CFN42. One of these duplicated operons is located in the symbiotic plasmid (fixNOQPd), while the other is located in a cryptic plasmid (fixNOQPf). Although a novel FixL-FixKf regulatory cascade participates in microaerobic expression of both fixNOQP duplicated operons, we found that a mutation in fixL eliminatesfixNOQPf expression but has only a moderate effect on expression of fixNOQPd. This suggests that there are differential regulatory controls. Interestingly, only thefixNOQPd operon was essential for symbiotic nitrogen fixation (L. Girard, S. Brom, A. Dávalos, O. Lopez, M. Soberón, and D. Romero, Mol. Plant-Microbe Interact. 13:1283–1292, 2000). Searching for potential candidates responsible for the differential expression, we characterized two fnrNhomologs (encoding transcriptional activators of the cyclic AMP receptor protein [CRP]-Fnr family) in R. etliCFN42. 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 fnrNgenes using transcriptional fusions with lacZ showed that the two fnrN genes are differentially regulated, since onlyfnrNd is expressed in microaerobic cultures of the wild-type strain while fnrNchr is negatively controlled by FixL. Mutagenesis of the two fnrN genes showed that both genes participate, in conjunction with FixL-FixKf, in the microaerobic induction of the fixNOQPd operon. Participation of these genes is also seen during the symbiotic process, in which mutations infnrNd and fnrNchr, either singly or in combination, lead to reductions in nitrogen fixation. Therefore,R. etli employs a regulatory circuit for induction of thefixNOQPd operon that involves at least three transcriptional regulators of the CRP-Fnr family. This regulatory circuit may be important for ensuring optimal production of thecbb ₃, terminal oxidase during symbiosis.