Investigation of a Streptococcal Phosphatase, an Unexpected Potential Iron Sensory Mechanism

Iron serves an important role in the catalytic production of cytotoxic reactive oxygen species. Because of the harmful nature of these intermediates, bacteria have evolved mechanisms for minimizing iron-based toxicity. An example of this mechanism is the Regulator of Iron Transport (RitR) in Streptococcus pneumonia. RitR has been identified as a strong repressor of pneumococcal iron uptake (piu) transporter synthesis, though its regulatory network has yet to be fully characterized. RitR interacts with a eukaryote-like Serine-Threonine kinase phosphatase pair, Stkp and PhpP, respectively. These three proteins have been implicated in roles of iron uptake, oxidative stress relief, and DNA repair. We hypothesized that, in the event that there is an abundance of internal iron and further extracellular signaling, a sensory mechanism preventing the further intake of iron existed in a PhpP-RitR interaction. We also hypothesized that internal iron ions bind to PhpP, thus altering the nature of the PhpP-RitR interaction such that RitR remains bound, actively repressing the piu operon and preventing unnecessary sequestration of iron. To date, we have successfully cloned PhpP, expressed it in Escherichia coli cells, and purified it via a series of selective precipitation methods and chromatographic procedures. Once purified, experimentation involving PhpP, pNPP (a phosphatase substrate used to mimic the natural substrate of PhpP, phosphorylated RitR), and iron solutions was conducted via spectrophotometric methods to characterize the effect of iron on the enzymatic activity of PhpP. We are currently conducting further spectrophotometric experiments to provide further characterization of the iron regulatory network of RitR in Streptococcus pneumoniae