Doctoral Dissertations

Date of Award

8-1988

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary Stacey

Committee Members

Jeffrey Becker, Beth Mullin, Stuart Riggsby

Abstract

The Rhizobium - legume symbiosis is a complex association between two symbiotic partners that results in the formation of a root nodule. This process depends on the expression of a number of plant and bacterial genes that are considered critical for the maintenance of the symbiotic state. In order to identify some of the bacterial genes that are involved in nodulation, the DNA in and around the coninon nodulation genes of B. japonicum USDA 110 was analyzed. A number of novel symbiotic loci were identified by site-directed Tn5 mutagenesis. Specifically, four loci were identified that appear to play a role in host specific nodulation by Bradyrhizobium japonicum.

A DNA locus essential for the nodulation of Macroptilium atropurpureum (siratro) was identified. A locus essential for the nodulation of Glycine max (soybean) was also identified and encoded new nodulation gene(s) since none of the previously identified genes hybridized to it. A detailed restriction map of this DNA region was generated to study this new nodulation region at a molecular level. Analysis of Tn5 mutants in this region led to the definition of three loci: nodT, nodU and nodV. These loci were very well conserved among various Bradyrhizobiumspecies, however, no homology was detected to the DNAs of any Rhizobium species examined indicating that these loci were unique to the genus. Bradyrhizobium. The regulation of this region was studied with the aid of mini-Mu-lac insertion fusions. The nodT:lacZ and nodU:lacZ fusions were found to be induced by soybean seed exudate and isoflavones. The role of nodP in the induction of nodT was examined and it was found that induction of nodT required the nodD gene product.

A mutation in the nodT locus resulted in the loss of ability of the mutant to nodulate soybean. This mutant, NAD165, was also unable to nodulate several cultivars of soybean and other hosts of B. japonicum but could nodulate G. soja, suggesting that the nodT locus could possibly encode host specificity functions. Light microscopy revealed that the mutant could induce root hair curling but failed to induce cortical cell division.

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