Abstract
The Bradyrhizobium sp. DOA9 strain displays the unusual properties to have a symbiotic plasmid and to fix nitrogen during both free-living and symbiotic growth. Sequence genome analysis shows that this strain contains the structural genes of dinitrogenase (nifDK) and the nifA regulatory gene on both the plasmid and chromosome. It was previously shown that both nifDK clusters are differentially expressed depending on growth conditions, suggesting different mechanisms of regulation. In this study, we examined the functional regulatory role of the two nifA genes found on the plasmid (nifAp) and chromosome (nifAc) that encode proteins with a moderate level of identity (55%) and different structural architectures. Using gusA (β-glucuronidase) reporter strains, we showed that both nifA genes were expressed during both the free-living and symbiotic growth stages. During symbiosis with Aeschynomene americana, mutants in only one nifA gene were not altered in their symbiotic properties, while a double nifA mutant was drastically impaired in nitrogen fixation, indicating that the two NifA proteins are functionally redundant during this culture condition. In contrast, under in vitro conditions, the nifAc mutant was unable to fix nitrogen, and no effect of the nifAp mutation was detected, indicating that NifAc is essential to activate nif genes during free-living growth. In accordance, the nitrogenase fixation deficiency of this mutant could be restored by the introduction of nifAc but not by nifAp or by two chimeric nifA genes encoding hybrid proteins with the N-terminus part of NifAc and the C-terminus of NifAp. Furthermore, transcriptional analysis by RT-qPCR of the WT and two nifA mutant backgrounds showed that NifAc and NifAp activated the expression of both chromosome and plasmid structural nifDK genes during symbiosis, while only NifAc activated the expression of nifDKc during free-living conditions. In summary, this study provides a better overview of the complex mechanisms of regulation of the nitrogenase genes in the DOA9 strain that involve two distinct NifA proteins, which are exchangeable during symbiosis for the activation of nif genes but not during free-living growth where NifAc is essential for the activation of nifDKc.
Highlights
Rhizobium-legume symbiosis is considered as the major contributor of biologically fixed nitrogen to terrestrial ecosystems
It has been shown that the two genes are part of the same transcript (Thöny et al, 1987), suggesting that fixR nifA found on the chromosome (nifAc) forms an operon in the DOA9 strain
The divergence of NifAp is not limited to the absence of the N-terminal GAF domain, since phylogenetic analysis showed that this protein formed an outgroup that was well separated from the NifA proteins identified in Bradyrhizobium strains (Figure 1D)
Summary
Rhizobium-legume symbiosis is considered as the major contributor of biologically fixed nitrogen to terrestrial ecosystems. The reduction of atmospheric N2 is catalyzed by the nitrogenase enzyme complex, which requires high-energy input in the form of ATP and electrons to break the triple bond. This enzymatic complex is highly sensitive to molecular oxygen, which irreversibly inactivates the enzyme. A master regulator of nitrogen fixation is the NifA protein, which acts in association with the RNA polymerase sigma factor RpoN (σ54) to activate the expression of nif genes by binding to an upstream activating sequence (UAS; 5 TGT-N10-ACA-3 ). The central domain interacts with the σ54-RNA polymerase and possesses ATPase activity, while the C-terminal domain shows a helixturn-helix (HTH) motif involved in DNA-binding. In Sinorhizobium meliloti, nifA expression is activated by the FixLJ two-component regulatory system in response to low oxygen tension, while in Bradyrhizobium japonicum, the fixR-nifA operon is controlled by the redox-responsive two-component system RegSR (Bauer et al, 1998)
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