| This study addresses the molecular signals synthesized by the bacterium, Rhizobium meliloti, in order to establish a symbiotic association with its host plant, alfalfa. In my thesis work, I purified these soluble signal molecules by high-performance liquid chromatography based on their genetic correlation with the bacterial nodulation (nod) genes, nodABC. These genes are absolutely required for the formation of the nitrogen-fixing nodule on the host plant. I analyzed the purified compounds by fast-atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy, and determined that they belong to a class of acylated oligosaccharides, the Nod factors, which can cause nodule morphogenesis on the host plant. I developed a procedure which utilizes a polystyrene-divinylbenzene adsorption matrix to extract these lipo-oligosaccharides from large amounts of Rhizobium culture supernatants, and this material was subjected to further analysis to determine the exact structure of the purified oligosaccharides.; I also investigated the role that the nodABC genes play in Nod factor synthesis. I used a permeabilized cell assay with in vitro labeled ({dollar}sp{lcub}35{rcub}{dollar}S) -chitin oligosaccharide intermediates to define the biochemical roles of the NodA and NodB proteins in Nod factor synthesis. NodA and NodB were required for a chitin-N-acyltransferase activity, and NodB was required prior to NodA. Removal of the non-reducing N-acetyl group of the chitin substrate obviated the requirement for NodB in the assay, indicating that NodB is an N-deacetylase and NodA is likely an N-acyltransferase. I determined that the NodC protein has strong amino acid similarity to various {dollar}beta{dollar}-1,4-polysaccharide synthetic enzymes, indicating a likely role for NodC in polymerization of the {dollar}beta{dollar}-1,4-N-acetylglucosamine backbone of the Nod factors. |
