Doctoral Dissertations

Jae-Seong So

Date of Award

8-1988

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary Stacey

Committee Members

Beth Mullin, Raymond Beck, Stuart Riggsby

Abstract

Nodulation of soybean by Bradyrhizobium japonicum is a complex developmental process involving intimate interactions between both plant and bacterial genes. To study this process at the molecular level, 25 symbiotically defective mutants of B. japonicum strains USDA 110 and 61A101C were isolated by random transposon Tn5 mutagenesis. Research was focused on four nodulation-defective mutants of strain USDA 110 and one Nod- mutant of strain 61A101C. The mutated DNA from each of these mutants was cloned and shown to be responsible for the observed phenotype by homogenotization of the cloned fragments into wild-type strains. The four USDA 110 mutations were found to map to two distinct regions; one of which encoded essential functions for histidine biosynthesis. Hybridization of these cloned regions, as well as the cloned 61A101C region, to the previously cloned nod regions of Rhizobium meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, indicating that these regions define new nodulation loci in B. japonicum. Using the cloned mutated DNA as probes, the wild type clones were successfully obtained from gene libraries by colony hybridization.

Additional attention was placed on the cloned region from 61A101C because of the interesting phenotype of mutation in this locus. The region was found to be well conserved among various Bradyrhizobium species whereas no homology was detected with Rhizobium species examined. To determine the structure, organization and possible function of the region, it was characterized by physical and genetic means. A detailed restriction map of the region III was generated. The essential nod gene region was narrowed to a 2.9 kb XhoI fragment, which was the smallest fragment responsible for the conserved hybridization pattern. DNA sequences of parts of the 2.9 kb XhoI fragment were determined. Search for homology of the sequences against the DNA sequence data bank failed to find any significant homology to the previously known gene sequences.

In addition, the phenotype of a mutation in the region was characterized. Although the mutant was originally shown to be Nod-, in further nodulation experiments with various cultivars of soybean it formed anomalous nodule-like structures at varying frequencies (0 to 54% of plants inoculated) depending on the cultivar used. Ultrastructural analysis of these structures revealed no evidence for bacterial invasion as the plant cells were completely devoid of bacteria. The mutant could induce root hair curling but failed to induce infection thread formation.

The mutant was found to be different from wild type with respect to its cell surface. A large portion of cells in liqiud medium formed aggregates resulting in sizable clumps. Moreover, when the cells were collected by centrifugation, the mutant cells formed a "rocket"-shaped pellet unlike the compact round pellet formed by wild type cells. Chemical analysis of the exopolysaccharide (EPS) samples from the mutant and wild type cells showed that the mutant's EPS contained a considerably higher glucose content, which is presumably due to overproduced beta-2- glucan. Further, the mutant strain JS314 lacked a membrane polypeptide of 14 kd in size, that was present in the wild-type strain.

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