Date of Award

1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Randall C. Gayda

Abstract

Chapter 1. Production of exopolysaccharides by Rhizobium has been linked with efficient invasion and nodulation of leguminous plant roots by the bacteria. Exopolysaccharide-deficient (exo) mutants of Rhizobium fredii USDA191 were isolated by Tn5-insertion mutagenesis. Five phenotypically unique exo mutants were investigated for exopolysaccharide synthesis and their ability to nodulate soybeans. The exopolysaccharides produced by these mutants were analyzed for polysaccharide composition by column chromatography and thin layer chromatography. Two mutants designated exo3 and exo5 were deficient in both the neutral glucan and exopolysaccharide synthesis, but still made effective nodules on Glycine max cv. Peking, although with decreased efficiency. The remaining three mutants (exo1, exo2, and exo4) synthesized neutral glucans at various levels compared with wild type and exhibited partial exopolysaccharide-deficiencies. One of these exo mutants, exo4, induced nodules on soybean, Peking. These data imply that neither exopolysaccharides nor neutral glucans are necessary for the formation of spherical nodules by R. fredii. Chapter 2. Rhizobium fredii USDA191 is a fast-growing symbiont that nodulates soybeans as well as slow-growing Bradyrhizobium species. R. meliloti exo DNA clones (obtained from Graham Walker) were introduced by triparental plasmid matings into five exo mutants of R. fredii USDA191. These exo mutants of R. fredii each exhibited a unique Tn5 insertion pattern and a depressed exopolysaccharide synthesis. In two R. fredii exo mutants, exopolysaccharide expression was restored by introduction of R. meliloti exo DNA. R. fredii YKL288 (exo4) was complemented for exopolysaccharide synthesis when plasmid pD56 (exoF/B) but not plasmid pD2 (exoB) was introduced into it, and R. fredii YKL293 (exo5) was complemented for exopolysaccharide production when it contained plasmid pD15 (exoC). Significantly, plasmid pLYK5293 containing DNA sequences flanking the Tn5 of YKL293 hybridized with DNAs of plasmid clones pD56 (exoF/B), pD2 (exoB), and pD5 (exoD) of R. meliloti. Putative wild type exo5 genes also were cloned into a phage lambda NM1149 using plasmid pLYK5293 as a probe DNA. The data suggest the existence of common pathways for exopolysaccharide synthesis in R. fredii as well as in R. meliloti. The data also suggest a possible linkage in R. fredii USDA191 of exoC and exoD gene homologues of R. meliloti.

Pages

119

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