TY - JOUR
T1 - Regulation and characterization of mutants of fixABCX in Rhizobium leguminosarum
AU - Webb, Isabel U. C.
AU - Xu, Jiabao
AU - Sánchez-Cañizares, Carmen
AU - Karunakaran, Ramakrishnan
AU - Ramachandran, Vinoy K.
AU - Rutten, Paul J.
AU - East, Alison K.
AU - Huang, Wei E.
AU - Watmough, Nicholas J.
AU - Poole, Philip S.
N1 - Funding Information: Funding: This research was funded by Engineering and Physical Sciences Research Council grants EP/M002403/1 and EP/M02833X/1, Biotechnology and Biological Sciences Research Council grants BB/J014524/1, BB/M011224/1, and BB/N003608/1, and Natural Environment Research Council grant NE/M002934/1.
PY - 2021/10
Y1 - 2021/10
N2 - Symbiosis between Rhizobium leguminosarum and Pisum sativum 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 nifA, which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically regulated NifA-dependent promoter upstream of fixA (P
nifA1), as well as an additional basal constitutive promoter driving background expression of nifA (P
nifA2). These were confirmed by 59-end mapping of transcription start sites using differential RNA-seq. Complementation of polar fixAB and fixX mutants (Fix
- strains) confirmed expression of nifA from P
nifA1 in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of fixAB mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant fixAB 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 fixAB mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.
AB - Symbiosis between Rhizobium leguminosarum and Pisum sativum 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 nifA, which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically regulated NifA-dependent promoter upstream of fixA (P
nifA1), as well as an additional basal constitutive promoter driving background expression of nifA (P
nifA2). These were confirmed by 59-end mapping of transcription start sites using differential RNA-seq. Complementation of polar fixAB and fixX mutants (Fix
- strains) confirmed expression of nifA from P
nifA1 in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of fixAB mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant fixAB 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 fixAB mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.
KW - Bacteroid morphology
KW - DRNA-seq
KW - Electron transfer flavoproteins
KW - FixABCX
KW - NifA regulation
KW - Nitrogen fixation
KW - PHB
KW - Polyhydroxy-3-butyrate
KW - Redox balance
KW - Rhizobium leguminosarum bv. viciae
KW - Single-cell Raman microspectroscopy (SCRM)
UR - http://www.scopus.com/inward/record.url?scp=85118487310&partnerID=8YFLogxK
U2 - 10.1094/MPMI-02-21-0037-R
DO - 10.1094/MPMI-02-21-0037-R
M3 - Article
SN - 0894-0282
VL - 34
SP - 1167
EP - 1180
JO - Molecular Plant-Microbe Interactions
JF - Molecular Plant-Microbe Interactions
IS - 10
ER -