Immunogold localization of the NodC and NodA proteins of Rhizobium meliloti

Abstract

Monospecific, polyclonal antibodies to the nodC and nodA gene products of Rhizobium meliloti were used in combination with immunogold labeling and transmission electron microscopy to localize the NodC and NodA proteins in cultures of R. meliloti. Both NodC and NodA were detected in the cytoplasm and cell envelope in thin sections of free-living rhizobia treated with luteolin, a known inducer of nod gene expression; however, only NodC was detected on cell surfaces when immunolabeling was performed with intact induced cells. In view of biochemical data characterizing NodC as an outer membrane protein with a large extracellular domain, the pattern of immunolabeling on thin sections suggests that NodC is produced on free cytoplasmic ribosomes prior to assembly in the membrane. The pattern of NodA labeling on thin sections is consistent with biochemical data detecting NodA in both soluble and membrane fractions of NodA-overexpressing strains of R. meliloti.

Biosynthesis of Rhizobium meliloti lipooligosaccharide Nod factors: NodA is required for an N-acyltransferase activity.

“…Our data indicate that the action of NodA follows that of NodC, and polymerized GlcNAc must therefore be available for the acylation reaction, either in the cytoplasm or in the membrane (28,29). Nod enzymes may possibly act as a complex in vivo, as has been suggested based on localization (28) and the overlap of NodA and NodB open reading frames in R. meliloti (30).…”

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).…”

Novel organization of the common nodulation genes in Rhizobium leguminosarum bv. phaseoli strains

“…Recent studies on the function of the Nod proteins suggest that the NodA and NodB proteins are involved in generating small, heat-stable compounds that stimulate the mitosis of various legume and nonlegume protoplasts (44). The NodA protein is found in the cytoplasm and cell envelope (21), and the NodB protein is also located in the cytosol (44). NodC is a cell surface protein with a eukaryotic receptor-like structure, which may serve as a transducer of an intracellular bacterial signal to root cells (20).…”