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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034331-06
Application #
3285128
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-12-01
Project End
1991-12-31
Budget Start
1990-01-01
Budget End
1991-12-31
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Michigan State University
Department
Type
Schools of Medicine
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Lopez-Lara, I M; Orgambide, G; Dazzo, F B et al. (1995) Surface polysaccharide mutants of Rhizobium sp. (Acacia) strain GRH2: major requirement of lipopolysaccharide for successful invasion of Acacia nodules and host range determination. Microbiology 141 ( Pt 3):573-81
Lopez-Lara, I M; Orgambide, G; Dazzo, F B et al. (1993) Characterization and symbiotic importance of acidic extracellular polysaccharides of Rhizobium sp. strain GRH2 isolated from acacia nodules. J Bacteriol 175:2826-32
Salzwedel, J L; Dazzo, F B (1993) pSym nod gene influence on elicitation of peroxidase activity from white clover and pea roots by rhizobia and their cell-free supernatants. Mol Plant Microbe Interact 6:127-34
Orgambide, G; Philip-Hollingsworth, S; Cargill, L et al. (1992) Evaluation of acidic heteropolysaccharide structures in Rhizobium leguminosarum biovars altered in nodulation genes and host range. Mol Plant Microbe Interact 5:484-8
Dazzo, F B; Truchet, G L; Hollingsworth, R I et al. (1991) Rhizobium lipopolysaccharide modulates infection thread development in white clover root hairs. J Bacteriol 173:5371-84
Philip-Hollingsworth, S; Hollingsworth, R I; Dazzo, F B et al. (1989) The effect of interspecies transfer of Rhizobium host-specific nodulation genes on acidic polysaccharide structure and in situ binding by host lectin. J Biol Chem 264:5710-4
Philip-Hollingsworth, S; Hollingsworth, R I; Dazzo, F B (1989) Host-range related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum. J Biol Chem 264:1461-6
Gardiol, A E; Hollingsworth, R I; Dazzo, F B (1987) Alteration of surface properties in a Tn5 mutant strain of Rhizobium trifolii 0403. J Bacteriol 169:1161-7
Gardiol, A E; Truchet, G L; Dazzo, F B (1987) Requirement of succinate dehydrogenase activity for symbiotic bacteroid differentiation of Rhizobium meliloti in alfalfa nodules. Appl Environ Microbiol 53:1947-50
Hollingsworth, R I; Dazzo, F B; Mort, A J (1987) Reexamination of the presence and linkage of 3-hydroxybutyryl substituents in the acidic capsular polysaccharide of Rhizobium trifolii 0403. J Bacteriol 169:3369-71

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