AUTHOR=Wongdee Jenjira , Boonkerd Nantakorn , Teaumroong Neung , Tittabutr Panlada , Giraud Eric 

TITLE=Regulation of Nitrogen Fixation in Bradyrhizobium sp. Strain DOA9 Involves Two Distinct NifA Regulatory Proteins That Are Functionally Redundant During Symbiosis but Not During Free-Living Growth

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01644

DOI=10.3389/fmicb.2018.01644

ISSN=1664-302X

ABSTRACT=<p>The <italic>Bradyrhizobium</italic> 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 (<italic>nifDK</italic>) and the <italic>nifA</italic> regulatory gene on both the plasmid and chromosome. It was previously shown that both <italic>nifDK</italic> 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 <italic>nifA</italic> genes found on the plasmid (<italic>nifAp</italic>) and chromosome (<italic>nifAc</italic>) that encode proteins with a moderate level of identity (55%) and different structural architectures. Using <italic>gusA</italic> (β-glucuronidase) reporter strains, we showed that both <italic>nifA</italic> genes were expressed during both the free-living and symbiotic growth stages. During symbiosis with <italic>Aeschynomene americana</italic>, mutants in only one <italic>nifA</italic> gene were not altered in their symbiotic properties, while a double <italic>nifA</italic> mutant was drastically impaired in nitrogen fixation, indicating that the two NifA proteins are functionally redundant during this culture condition. In contrast, under <italic>in vitro</italic> conditions, the <italic>nifAc</italic> mutant was unable to fix nitrogen, and no effect of the <italic>nifAp</italic> mutation was detected, indicating that NifAc is essential to activate <italic>nif</italic> genes during free-living growth. In accordance, the nitrogenase fixation deficiency of this mutant could be restored by the introduction of <italic>nifAc</italic> but not by <italic>nifAp</italic> or by two chimeric <italic>nifA</italic> 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 <italic>nifA</italic> mutant backgrounds showed that NifAc and NifAp activated the expression of both chromosome and plasmid structural <italic>nifDK</italic> genes during symbiosis, while only NifAc activated the expression of <italic>nifDKc</italic> 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 <italic>nif</italic> genes but not during free-living growth where NifAc is essential for the activation of <italic>nifDKc</italic>.</p>