Expression of the nodulation gene nod C of Rhizobium meliloti in Escherichia coli : role of the nod C gene product in nodulation

Abstract

The nod C gene of Rhizobium meliloti encodes a protein of mol. wt. 44 000 which is highly conserved in at least three Rhizobium species. In order to overproduce this protein, a gene fusion of λcI repressor sequences to a large fragment of nod C was constructed. The fusion was placed under control of the tac promoter on plasmid pEA305 to yield pJS1035. IPTG‐induced Escherichia coli cells harbouring pJS1035 accumulated the cI‐nod C hybrid protein up to 19% of total cellular protein. The synthesis of the hybrid protein drastically inhibits the growth rate of the bacterium. The fusion protein was purified by gel and hydroxyapatite chromatography in the presence of SDS. Antibodies raised against the purified fusion protein precipitated the mol. wt. 44 000 nod C proteins of R. meliloti and of the broad‐host range Rhizobium strain NGR234, which were both expressed in E. coli minicells. The hybrid protein is associated with the outer membrane of E. coli cells, and the cI‐nod C fusion protein appears to be an integral membrane protein. Nodulation of alfalfa by R. meliloti and of clover by R. trifolii was markedly inhibited (˜50%) by the addition of antibodies against the hybrid protein to plant growth medium and inoculum.

Immunogold localization of the NodC and NodA proteins of Rhizobium meliloti

“…3B). These results support genetic and biochemical data which have characterized NodC as a transmembrane protein with a large extracellular domain (12,13,25). Localization of NodC, but not NodA, as a surface antigen also correlates with published experiments in which only anti-NodC antibodies were capable of inhibiting nodulation when added to R. meliloti cells upon inoculation of alfalfa (12,25).…”

“…A combination of genetic, biochemical, and physiological methods has shown NodC to be a 46.8-kilodalton (kDa) dimeric, integral outer membrane protein with receptor like structure (12,13), while the 21.8-kDa NodA protein may be a soluble polypeptide (25) involved in synthesis of a diffusible plant growth factor (26). The use of immunocytochemistry to localize nodulation gene products has not previously been reported, although this approach has been employed effectively for the localization of other nodule constituents, including the oxygen-binding protein leghemoglobin (21), the nodule-specific enzyme uricase (18,28,29), and certain components of the peribacteroid and plasma membranes of infected cells (2,3,8 (12,25) and Western immunoblot analyses of nodule tissue and fractionated R. meliloti cells (13,25). For prolonged storage, antibodies were filtersterilized, frozen in liquid nitrogen, and kept at -70°C.…”

The NodC protein of Azorhizobium caulinodans is an N-acetylglucosaminyltransferase.

“…Only for NodB has a function been shown; it is an enzyme that removes the N-acetyl moiety from the nonreducing end of N-acetylglucosamine oligosaccharides (10). The role of NodC is controversial, because it was reported to share sequence homology with chitin synthases (11)(12)(13) and to be an outer membrane protein with receptor structure (14)(15)(16). Finally, NodA has been proposed to be an acyltransferase (17).…”

Immunodetection of grapevine fanleaf virus satellite RNA-encoded protein in infected Chenopodium quinoa

“…This vector overproduces the CI repressor under the control of the tac promoter (De Boer et al, 1983) upon induction by IPTG. Plasmid pEA305 has a single HindlII cloning site within the CI gene portion (John et al, 1985) which allowed insertion of the cDNA of the 3'-terminal part (nt 615 to nt 1114) of RNA 3 including the P3-coding sequence (Fuchs et al, 1989). The recombinant plasmid (pOM305) was used to transform E. coli strain W311OlaclqL8 as described by Maniatis et al (1982).…”