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

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] 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.