With many microbial model systems, molecular genetics provides powerful tools, complementary to high-throughput instrumental approaches, for defining gene function, regulation, and metabolism on a genome-wide scale. Reporter gene fusions can be used to learn how and when genes are turned on or off, which in turn can provide a clue(s) about their function. Systematic mutational analyses of genes in their normal chromosomal locations can generate insights into their roles. Conditional expression or hyperexpression of genes create novel phenotypes that are informative. Methods have also been developed for global analysis of cellular phenotypes in which hundreds or thousands of growth properties can be tested simultaneously, which also allow one to test and assay gene function. Here we will blend several newly developed and facile molecular genetic tools for genome manipulation of Escherichia coli K12 with physiology, phenotypic testing, and global gene expression profiling in order to study the E. co/i physiome with respect to inorganic orthophosphate (Pi) metabolism. We will carry out these experiments to test the following general hypothesis: Genetic regulatory mechanisms act in concert at the transcriptional and posttranscriptional levels in order to coordinate different steps of Pi metabolism with cell growth. These steps include pathways for the uptake of extracellular Pi (Pi (ext))into the cell and subsequent incorporation of intracellular Pi (Pi (int))into ATP and other organophosphates, which act as phosphoryl donors in cell metabolism.
Specific Aims are: (1) To identify new genes regulated by environmental Pi levels, to define their properties and functions, and to define how genes for Pi uptake that are unresponsive to Pi control are themselves regulated; (2) To identify regulatory factors that are important in coupling different steps in Pi metabolism and to describe regulatory interactions between the PhoR/PhoB two-component system and other two-component systems that appear to be connected to Pi metabolism; and (3) To identify the role(s) of acetyl phosphate in cell physiology as it relates to Pi metabolism by examining the regulatory consequences of perturbing acetyl phosphate metabolism.
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