Regulatory Role of Rhizobium etli CNPAF512 fnrN during Symbiosis

Abstract

The Rhizobium etli CNPAF512 fnrN gene was identified in the fixABCX rpoN 2 region. The corresponding protein contains the hallmark residues characteristic of proteins belonging to the class IB group of Fnr-related proteins. The expression of R. etli fnrN is highly induced under free-living microaerobic conditions and during symbiosis. This microaerobic and symbiotic induction of fnrN is not controlled by the sigma factor RpoN and the symbiotic regulator nifA or fixLJ, but it is due to positive autoregulation. Inoculation of Phaseolus vulgaris with an R. etli fnrN mutant strain resulted in a severe reduction in the bacteroid nitrogen fixation capacity compared to the wild-type capacity, confirming the importance of FnrN during symbiosis. The expression of the R. etli fixN, fixG, and arcA genes is strictly controlled by fnrN under free-living microaerobic conditions and in bacteroids during symbiosis with the host. However, there is an additional level of regulation of fixN and fixG under symbiotic conditions. A phylogenetic analysis of the available rhizobial FnrN and FixK proteins grouped the proteins in three different clusters.Soil bacteria belonging to the genera Rhizobium, Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, and Sinorhizobium (collectively referred to as rhizobia) elicit the formation of nodules on the roots of their leguminous hosts. In these specialized organs, the bacteria are released into the plant cells and differentiate into bacteroids that fix atmospheric nitrogen into ammonia that can be assimilated by the host plant. The nodules provide the microoxic conditions required for functioning of the oxygen-sensitive nitrogenase enzyme complex.In rhizobia, NifA activates transcription of several nitrogen fixation genes in conjunction with 54 RNA polymerase (12). In Rhizobium etli CNPAF512, NifA is strictly required for nitrogen fixation activity in nodules of Phaseolus vulgaris and controls the expression of several genes involved in nitrogen fixation, including nifH, iscN, and orf180-rpoN 2 (13,33,34

Characterization of the NifA-RpoN Regulon in Rhizobium etli in Free Life and in Symbiosis with Phaseolus vulgaris

“…Moreover, we confirmed the presence of the genes previously reported to be NifA-RpoN dependent in R. etli, including bacS, prxS, rpoN2, and iscN (16,31,43), and the presence of operons predicted in silico, including the iscN-nifUSW (16), nifHDKE1-yhd00103-yhd00102-fdxB, nifHDK3E2NX, and nifHD2-hemNd2-yhd00090 operons. For the last three operons, RNA transcripts that were the correct length were detected in Northern blots (data not shown).…”

“…However, NifAdependent gene activation occurs only at low oxygen concentrations (43,55). To date, only a few targets, including nifHDK, iscN nifUS, fixABC, prxS-rpoN2, melA, and bacS, have been reported to be members of the R. etli NifA-RpoN2 regulon during symbiosis with P. vulgaris (15,17,27,31,43,55).…”

Effective Symbiosis between Rhizobium etli and Phaseolus vulgaris Requires the Alarmone ppGpp

“…In several control experiments, the expression of gusA fusions was either unaffected or reduced in nifA or rpoN mutant backgrounds, making it unlikely that the observed increase in relA expression is indirectly caused by a defect in nitrogen fixation (data not shown). Also, the inactivation of the second regulatory protein, FnrN, controlling the oxygen-dependent expression of bacteroid genes involved in respiration and leading to a decrease in nitrogen fixation of approximately 80% (29), had no effect on relA expression. An interaction between the quorum-sensing and (p)ppGpp regulatory circuits was previously described for A. tumefaciens (48) and Pseudomonas aeruginosa (39).…”

Experimental Evolution of Legume Symbionts: What Have We Learnt?

“…However, depending on rhizobium species, the expression of these genes is controlled by either a single NodD regulatory protein [78] or multiple, both positive and negative, regulators organized in intricate networks [99][100][101][102][103]. Likewise, expression of nitrogen fixation (nif -fix) genes is under the control of the conserved NifA protein, but upstream from NifA the regulatory cascade is highly variable among rhizobia, involving different two-component systems (FixLJ, RegSR, NtrBC, ActRS, FixL-FxkR) responding to various signals [104][105][106][107] and specific downstream transcription regulators (FixK1, FixK2, FnrN) [108][109][110]. Surface polysaccharide biosynthesis genes are other striking examples of essential symbiotic genes subjected to very complex regulations involving a multitude of interconnected regulators that largely differ between rhizobium species [111,112].…”