Regulation and Characterization of Mutants of in .

Symbiosis between and requires tight control of redox balance in order to maintain respiration under the microaerobic conditions required for nitrogenase while still producing the eight electrons and sixteen molecules of ATP needed for nitrogen fixation. FixABCX, a cluster of electron transfer flavoproteins essential for nitrogen fixation, is encoded on the Sym plasmid (pRL10), immediately upstream of , which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically regulated NifA-dependent promoter upstream of (P), as well as an additional basal constitutive promoter driving background expression of (P). These were confirmed by 5'-end mapping of transcription start sites using differential RNA-seq. Complementation of polar and mutants (Fix strains) confirmed expression of from P in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant bacteroids were observed. One was larger than wild-type bacteroids and contained high levels of polyhydroxy-3-butyrate, a complex energy/reductant storage product. A second bacteroid phenotype was morphologically and compositionally different and resembled wild-type infection thread cells. From these two characteristic mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.