Doctoral Dissertations

Date of Award

8-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary Stacey

Committee Members

Beth C. Mullin, Neil Quigley. Peter M. Gresshoff

Abstract

This study explored the role of various chemical and physical factors in regulating nodulation gene expression in B. japonicum. In addition, experiments examined the expression of NodD1 and its physiological importance to Bradyrhizobium japonicum. Two xanthones - 1,3,7-trihydroxyxanthone and l,6-dihydroxy-2,8-dimethoxyxanthone - were found capable of inducing nodD1 and nodYABC gene expression. This is the first report that xanthone compounds can be nod gene inducers. When certain organic acids such as L-malate, succinate, fumarate, acetate, α-ketoglutarate, and oxaloacetate, were added to the cultures, nodD1 and nodYABC gene expression was significantly repressed. This inhibition was specific to nod gene expression and was not due to chelation of Ca2+ ions or pH change. This repression affected nod gene induction mediated by both NodD1 and NodV/NodW. L-malate and acetate added together to cultures exerted an additive inhibition exceeding 90%. A remarkable level of L-malate inhibition of nod gene expression occurred when cells were grown or resuspended in medium containing D-gluconate, L-glutamate, and yeast extract as the carbon and nitrogen sources. In contrast, lower or no inhibition occurred in cells grown or resuspended in medium containing glycerol as carbon source. It is believed that intracellular accumulation of L-malate, which occurred when cells did not readily utilize this organic acid, accounts for the different response of cells to L-malate. Other results showed that induction of nodYABC expression was suppressed when cells were grown under low oxygen tension. Furthermore, nod gene expression was tested at different pH values in RDY and MM. Nearly complete inhibition of nod gene expression in RDY occurred at pH 8.5 and pH 5.5 or lower in the presence or absence of &beta-malate. p-galactosidase activity in bacteroids containing a chromosomal nodC-lacZ fusion showed that nod gene expression was suppressed in bacteroids even though nod gene inducers were present. Since bacteroids in planta are exposed to an abundance of organic acids under microaerobic and acidic (pH 5.5 to 6.0) conditions, these results suggest that these nodule-specific conditions account for the suppression of nod gene expression in bacteroids.

The inducibility of nod gene expression was found higher in cells harvested from earlier growth phase or when induced at lower initial cell densities. Surprisingly, it was found that expression of NodD1 occurred in uninduced cultures at absorbance (A600mn) values greater than 0.4. These results suggest nodD1 expression may be regulated by a quorum sensing mechanism at higher cell densities. Elevated NodD1 expression may account for lower inducibility of cells at later growth phase and at higher cell densities.

Finally, it was found that NodDl is important for the normal growth of B. japonicum. Mutation of nodD1 resulted in a slower growth rate and lower cell viability. This effect was not due to defective production of Nod factors.

Download
Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS