Doctoral Dissertations

Date of Award

5-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Gary Stacey

Committee Members

Beth Mullin, Peter Gresshoff, Evans Roth

Abstract

Two new symbiotic genes, nfeC,/u> and hsfA, essential for nodulation competitiveness and host specific nitrogen fixation, respectively, were identified and sequenced from the soybean root nodule bacterium, Bradyrhizobium japonicum. A Tn5 insertion (NAD14) in nfeC did not affect nitrogen fixation but caused a significant delay in soybean (Glycine max) nodulation. In addition, this mutant exhibited a reduction in its competitive ability to nodulate soybean when co-inoculated with the wild type. DNA sequence analysis of the mutated region revealed that the NAD14 Tn5 insertion mapped within an open reading frame of 825 bp. Primer extension using B. japonicum mRNA from three different growth conditions, aerobic, anaerobic, and bacteroids (i.e. symbiotic form), indicated that the upstream region of the gene contained two promoters, which were differentially regulated in response to the growth conditions. One promoter was active in bacteroids, but not under aerobic or anaerobic free-living conditions. The other promoter was functional only under aerobic conditions. Therefore, the regulation of the nfeC gene appears to be unique. The bacteroid-specific promoter has a 5' consensus sequence suggesting a requirement for RpoN (i.e., σ54). A bacteroid- specific activator of nfeC is postulated based on comparison to other RpoN-dependent promoters. A mutation in hsfA exhibited a host specific nitrogen fixation phenotype. This mutant induced nitrogen-fixing nodules on Glycine max, G. soia, and siratro (Macroptilium atropurpureum), but ineffective nodules on cowpea (Viona unquiculata). Microscopic analysis of a deletion mutant, lacking hsfA, revealed that, while some bacteroids were found in the infected cowpea nodule cell cytoplasm, they appeared to be readily degraded and usually failed to develop into mature bacteroids. On soybean, nodule cells infected by this strain differentiate bacteroids in a manner similar to wild-type infected cells. These results suggest that hsfA may play a role maintaining the integrity of B. japonicum in cowpea but not soybean nodules. DNA sequence analysis of the hsfA region showed an open reading frame of 96 amino acids. Primer extension results indicated that hsfA also has a bacteroid-specific RpoN-dependent promoter. This work extends the genetic characterization of the nodulation region of B. japonicum by identifying two new genetic loci involved in nodule formation and function.

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