Regulation of nitrogen fixation in rhizobia-legume symbioses

Rhizobia are soil-dwelling bacteria capable of infecting legume roots and forming a symbiotic association. Inside root nodules, low O2 levels trigger expression of the nitrogenase enzyme complex in rhizobia, allowing them to fix atmospheric N2 into NH3, and secrete this reduced nitrogen source to the host plant. Rhizobial N2 fixation is activated via the transcriptional regulator NifA, which is itself induced by low O2 concentration in legume root nodules, most commonly via the two-component sensor-regulator FixLJ or a modified version of this regulon. For rhizobia in the genus Mesorhizobium, it is currently unclear how low O2 tension is sensed and transduced into a signal to activate N2 fixation, with the FixLJ system shown to be dispensable for N2 fixation. Mesorhizobium ciceri CC1192, which forms a symbiosis with Cicer arietinum (chickpea) and is the commercial inoculant for this crop legume in Australia, harbours fixV, a putative LacI/GalR transcriptional regulator which may directly control the expression of nifA in this strain. To investigate the role of fixV and nifA within CC1192, inactivation vectors were constructed in the suicide vector pJQ200SK via Gibson Assembly and fixV and nifA deletion mutants generated, along with a nitrogenase null mutant (nifH) as a non-N2-fixing control. nifH mutants were symbiotically defective on C. arietinum, with nitrogenase activity completely abolished. Deletion of fixV resulted in reduced, but not abolished, nitrogenase activity on a per nodule basis and reduced foliage dry weights, while the ΔnifA mutant phenotype remains to be tested. FixV, therefore, is not essential for N2 fixation in CC1192 but likely plays a vital role alongside another as yet unidentified regulator