Constitutive expression of the nifA gene activates associative nitrogen fixation of Enterobacter gergoviae 57-7, an opportunistic endophytic diazotroph

Aims: This study was undertaken to investigate whether a nitrogen-fixing bacterium Enterobacter gergoviae 57-7, which was isolated from surface-sterilized maize (Zea nays L.) roots, can colonize in roots and whether constitutive expression of the nifA gene encoding the transcriptional activator of nitrogenase genes can activate nif gene expression in planta. Methods and Results: Maize seedlings grown in an agar medium were inoculated with Ent. gergoviae strains containing the green fluorescent protein reporter gene. Root colonization and expression of the dinitrogenase reductase gene (nifty) by Ent. gergoviae were observed by confocal laser scanning microscopy. gfp-tagged Ent. gergoviae was observed to colonize predominantly in cortical aerenchyma of primary and lateral roots and in stellar parenchyma cells and xylem vessels of primary roots. In planta nifH::gfp expression was not detected but after a constitutively expressed nifA gene was introduced into Ent. gergoviae. Conclusions: Enterobacter gergoviae 57-7 is an opportunistic endophyte because it can live in soil and colonize in maize roots in the gnotobiotic agar culture. In agreement with previous N-15-dilution evidence that Ent. gergoviae 57-7 did not fix N-2 in association with maize in pots whereas a derivative E7 containing a constitutively expressed nifA gene promoted plant growth partly through associative nitrogen fixation, constitutive expression of the nifA gene can activate bacterial nif gene expression in planta. Significance and Impact of the Study: This study and our previous studies suggest that manipulation of the promoter of the nifA gene in a nitrogen-fixing bacterium having a high colonization competence is a practical and promising approach to achieve a stable associative nitrogen fixation for cereals.