Rhizobium trifolii is a gram-negative bacterium which specifically infects clover root hairs and forms a root nodule symbiosis which fixes atmospheric nitrogen into ammonia. The overall aim of this research is to identify the biochemical events leading to successful infection of clover root hairs by R. trifolii as a model for cellular recognition between procaryotic and eucaryotic cells. The focus is on the structures of the cell surface molecules on the clover root and the bacterium required for specific molecular interaction during early stages of root hair infection. These are a glycoprotein lectin called trifoliin A and two bacterial receptors: the acidic capsular polysaccharide (CPS) and lipopolysaccharide (LPS). The lectin-binding CPS and LPS are biologically active molecules which modulate root hair infection when added to seedlings at very low concentration. During the previous 2.25 yrs of funding, we analyzed the CPS and LPS of two wild type R. trifolii and several non-infective mutant derivatives obtained by mutagenesis of the symbiotic plasmid (pSym) of the bacteria (strains provided by our collaborator, B. Rolfe). We found that mutation of certain essential nodulation genes residing within a 14 kb encoding region of pSym and required for invasion of the eucaryotic host cells alters the specific rhizobial attachment to root hairs, CPS & LPS chemistry (especially noncarbohydrate substitutions), and rhizobial interaction with trifoliin A in the root environment. We have elucidated the complete structure of the trifoliin A-binding repeating unit oligosaccharide of the CPS and have identified all of the glycosyl and fatty acid components of the LPS from wild type R. trifolii. For this competitive renewal, we propose to analyze the amino acid composition and sequence of trifoliin A, and the following properties of the isolated CPS & LPS of wild type and additional Tn5-induced non-infective mutants of R. trifolii grown in defined medium with and without apigenin (a plant flavone which increases expression of the nod genes): (i) glycosyl molar ratio; (ii) location of noncarbohydrate substitutions; (iii) trifoliin A-binding ability; and (iv) infection related biological activity. The analyses would include 1-D and 2-D NMR, GLC, GC/MS, FAB/MS, high resolution MS, and HPLC. The other measurements would be obtained by quantitative agglutination inhibition, solid-phase ELISA, and light microscopy of axenically grown seedlings. This information is relevant to studies which identify the underlying mechanisms of specific microbial invasive interactions with eucaryotic host cells as they relate to microbial pathogenesis and the establishment of the normal microbial flora in man.
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