The identification of phosphate starvation-inducible genes and the role of the phosphate-specific transport (Pst) system andphoU genes in phosphate regulation in Escherichia coli

Paul Michael Steed, Purdue University

Abstract

All living cells require phosphorus for growth. The means by which the enteric bacterium Escherichia coli satisfies this requirement and the coordinate regulation of the expression of genes involved in the utilization of various phosphorus sources is a model for cellular response to nutrient deprivation. When cells are limited for inorganic phosphate (Pi), the preferred phosphorus source, the transcription of the PHO regulon genes is markedly induced. This expression is dependent upon the PhoB and PhoR proteins which comprise a two-component regulatory system. The products of the pstSCAB-phoU operon are also involved in this regulation because mutations in these genes render the PHO regulon constitutive. The pst genes encode the high-affinity phosphate-specific transport system (Pst) while PhoU is thought to be a regulatory protein because a missense allele of phoU, phoU35, has no effect on transport by Pst. There are two interesting problems regarding the PHO regulon and its control that have been addressed in this work. First, previous studies have indicated that more proteins have higher levels of synthesis during Pi starvation than have been thus far identified. Through the analysis of a set of random lacZ fusions to unknown promoters (generated by Mu d1) isolated for phosphate starvation-inducible (psi) Lac expression, unknown Pi-regulated genes were studied. Several psi genes were identified by DNA sequence analysis of the psi::lacZ(Mu d1) fusion junctions and novel regulatory interactions have been explored by the study of the regulation of psi::lacZ fusions. The mechanisms of control of the PHO regulon have also been investigated. Most of the work implicating the genes of the pstSCAB-phoU operon in Pi repression of the PHO regulon involves the analysis of mutations, including phoU35, isolated under conditions that should not have permitted Pst$\sp-$ cells to grow. Defined deletions of the pstSCAB-phoU operon have been constructed, verified, and studied. These analyses also indicate that cells which lack PhoU but retain a functional Pst system have a severe growth defect. Additionally, when these phoU null mutants are starved for Pi they become sensitive to Pi and can only recover from these starvation conditions when provided with an alternate phosphorus source in the absence of Pi. Thus, PhoU is a bifunctional protein involved in Pi utilization and in PHO regulon control.

Degree

Ph.D.

Advisors

Wanner, Purdue University.

Subject Area

Molecular biology|Genetics

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