The affects of genistein on the Bradyrhizobium japonicum bacterial transcriptome [abstract]

Abstract only availableFaculty Mentor: Gary Stacey, Plant Microbiology and PathologyBradyrhizobium japonicum forms a symbiotic relationship with the roots of the soybean plant (Glycine max). This bacterium is of great importance because of its ability to provide the soybean with a source of nitrogen by the conversion of atmospheric dinitrogen to ammonia. In order to establish this symbiosis, the bacteria must attach to the root hair surface and initiate development of a root nodule. The bacterium responds to plant flavonoids through production of nod factor, a product of the nod genes, which facilitates bacterial root entry and initiates nodule organogenesis. Therefore, we have investigated the expression of nod genes after treatment with a plant derived flavonoid inducer, genistein. Free-living cultures of Bradyrhizobium japonicum were treated and cells were harvested six hours after treatment and total RNA was extracted. Semi-quantitative and quantitative RT-PCR was performed to confirm induction of the nodY and nodC genes. The RT-PCR results confirmed that nodY and nodC are upregulated in the presence of genistein compared to an ethanol-treated control. To further define genistein regulation on the nod genes, a DNA microarray experiment was performed on the above extracted RNA to define the bacterial transcriptional response to genistein, the results of which will be presented.Bradyrhizobium japonicum forms a symbiotic relationship with the roots of the soybean plant (Glycine max).  This bacterium is of great importance because of its ability to provide the soybean with a source of nitrogen by the conversion of atmospheric dinitrogen to ammonia.   In order to establish this symbiosis, the bacteria must attach to the root hair surface and initiate development of a root nodule.  The bacterium responds to plant flavonoids through production of nod factor, a product of the nod genes, which facilitates bacterial root entry and initiates nodule organogenesis. Therefore, we have investigated the expression of nod genes after treatment with a plant derived flavonoid inducer, genistein. Free-living cultures of Bradyrhizobium japonicum were treated and cells were harvested six hours after treatment and total RNA was extracted.  Semi-quantitative and quantitative RT-PCR was performed to confirm induction of the nodY and nodC genes. The RT-PCR results confirmed that nodY and nodC are upregulated in the presence of genistein compared to an ethanol-treated control.  To further define genistein regulation on the nod genes, a DNA microarray experiment was performed on the above extracted RNA to define the bacterial transcriptional response to genistein, the results of which will be presented

Development and validation of a DNA microarray for analysis of the Bradyrhizobium japonicum transcriptome

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 24, 2010).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. Gary Stacey.Vita.Ph. D University of Missouri--Columbia 2009.[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Bradyrhizobium japonicum, forms a nitrogen fixing symbiosis with soybean. The ability to survive in soil environments, colonize the rhizosphere and establish effective plant symbioses requires that B. japonicum modify gene expression in response to the prevailing environmental conditions. To better understand the adaptation of B. japonicum to its environment, a whole genome oligonucleotide DNA microarray was constructed to monitor gene expression. Validation of the DNA microarray was performed by profiling gene expression in a variety of free-living conditions requiring known modifications of the B. japonicum transcriptome for which the appropriate changes in gene expression were documented. In addition, analysis of the B. japonicum NtrC regulon was performed. NtrC is a response regulator controlling gene expression in relation to cellular nitrogen status. Analysis of the B. japonicum NtrC regulon during nitrogen limitation revealed modification of the central nitrogen assimilation pathways and induction of genes with transport functions, presumably for acquisition of nitrogenous compounds from the environment. Finally, to better utilize the emerging transcriptomics data for B. japonicum, a new mutagenesis strategy was developed based upon the recombinational cloning strategies developed by the E. coli research community. This technology allows simple, targeted and efficient construction of B. japonicum mutants and can generate multiple, unmarked and nonpolar mutations in a single background, thereby, greatly enhancing the ability to study gene function.Includes bibliographical references