Degree Name

Master of Science (MS)

Semester of Degree Completion

1985

Thesis Director

Roger Darding

Abstract

Extracellular polysaccharides (EPS) are suspected to be involved in some aspect of the Rhizobium-legume symbiotic process. Extracellular polysaccharides produced by R. leguminosarum and R. trifolii mutants were isolated and partially characterized by gas chromatography, column chromatography, double diffusion, polyacrylamide gel electrophoresis, and immunoelectrophoresis.

The R. leguminosarum mutants, derived from strain 128C53, all are deficient in EPS production (exo-). Strain ANU54 is missing the symbiotic plasmid, while plasmid-containing strains ANU54 pJB5JI and ANU54 pBR1AN nodulate (nod+) peas and clover respectively, but do not fix nitrogen (fix-).

R. leguminosarum mutants produce an EPS that is similar to parental LPS in composition. Double diffusion studies indicate that all mutant LPS and EPS are antigenically identical. Both plasmid-containing mutants show an extra precipitin line in double diffusion analysis and unique antibody-binding bands when subjected to immunoelectrophoresis.

R. trifolii ANU794 produces normal amounts of EPS (exo+) and carries on normal symbiosis with clover. ANU437 (nod+ fix-exo-), is a Tn5 mutant of ANU794, but the insertion is not in the symbiotic plasmid. Strain ANU437RP4 (nod+ fix+,exo+) is strain ANU437 which has been complimented with a 4kb region from a wild-type R. trifolii.

ANU437 produces an EPS that has a sugar composition similar to parental LPS. The 4kb insertion contained in ANU437RP4 restores the production of normal amounts of EPS that is similar in composition to parental EPS.

Other R. trifolii mutants studied are affected in their ability to cause root hair curling. Strains ANU262 and ANU258 have a Tn5 insertion in the symbiotic plasmid, and cause severe root hair curling (hac++), but ANU258 is also affected in that it now has an increased host range.

Purification of LPS by Sepharose 4B column chromatography revealed that the void and included volumes of strains ANU258 and ANU262 contain KDO and heptose, while parental strain ANU843 did not. The pyruvate level is lower in the LPS from ANU258 than in the LPS from ANU262. Double diffusion studies indicate that LPS from ANU258 and ANU262 is antigenically identical to the parental LPS and to LPS from mutants that do not cause root hair curling. The EPS composition of ANU258 and ANU262 is similar to strain ANU843.

This study suggests that same small molecular weight molecules produced by plasmid-containing mutants of R. leguminosarum may have a role in the formation of nodules. Studies of R. trifolii EPS indicate that EPS may be important in the formation of effective nodules, but not in causing extreme root hair curling. Preliminary studies of LPS from mutants affected in root hair curling suggest that some differences exist in pyruvate levels and composition of Sepharose 4B peaks.

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