K-129. Systematic Metabolic Perturbation Analysis of Transcriptional Regulation of Amino Acid and Nucleotide Metabolism in Escherichia coli

D. P. Sangurdekar;
Univ. of Minnesota, Saint Paul, MN.

Background: The genome of Escherichia coli encodes 4460 genes and over a 1000 reactions involving small metabolites. While the metabolic and transcriptional regulatory networks have been imputed from individual targeted experiments, the properties of these networks vis-à-vis their completeness can only be evaluated when activity of every gene, or at least of every relevant gene, in a genome has been perturbed. In this study, I focused on transcriptional regulation associated with metabolism of amino acids (aa) and nucleotides (nt) in E. coli by perturbing internal pools of end products. Method: Internal pools of aa and nt were perturbed by addition or starvation of individual metabolites in exponentially growing cell cultures, followed by survey of transcriptional responses using cDNA microarrays. The transcriptional data was analyzed in 3 stages: 1) responses of genes to individual perturbation were assessed; 2) responses were categorized according to the activity of annotated gene sets; 3) patterns of co-expression between genes and gene sets have been delineated. Promoter:GFP assays and gene-knockouts were used to validate the hypotheses generated in the transcriptional survey. Results: Genes encoding biosynthetic enzymes are repressed by almost all respective end product additions. Metabolic pathways that share common precursors (such as glutamate-arginine-purine-pyrimidines) exhibit significant degree of correlation in expression. Activity of the global regulator LRP, which modulates transcription of several aa metabolic pathways, is increased in response to addition of the known effectors alanine, leucine as well as cysteine, methionine, isoleucine and threonine. Nucleotide synthesis and arginine pathways were affected in more than 50% of individual conditions. . The analysis allowed functional classification of uncategorized genes. Conclusions: Excess or shortage of amino acids and nucleotides affects genome-wide transcription in a way that is consistent with the logic of metabolic regulation. Systematic surveys of transcriptional consequences of metabolic perturbations can be used to elucidate effector-mediated regulation of gene expression in bacteria.