Abstract: Rebecca Sheplock
Mentor: Dr. Chander
This lab is studying the role and properties of the redox-sensing transcription factor SoxR in the antibiotic-producing bacterium Streptomyces coelicolor. SoxR has been well characterized in Escherichia coli where it senses oxidative stress via essential redox-active [2Fe-2S] clusters, and activates genes that protect against redox-cycling agents. While the E. coli and S. coelicolor SoxR proteins are highly homologous, SoxR does not play an oxidative-defense function in S. coelicolor. Preliminary evidence suggests that in S. coelicolor, SoxR senses the presence of endogenous antibiotics and regulates genes whose products export and modify these antibiotics. Existing evidence also indicates that S. coelicolor SoxR is regulated distinctly from its E. coli counterpart, since the former does not functionally complement an E. coli soxR mutant. My research will examine the potential regulatory roles of two features in S. coelicolor SoxR – [2Fe-2S] clusters and a 22-residue C-terminal region. The [2Fe-2S] clusters have been shown to be vital to the function of E. coli SoxR; thus the conservation of this feature in S. coelicolor SoxR suggests that these cofactors will also be important to the function of this protein. The 22-residue C-terminal region in S. coelicolor SoxR is absent in E. coli SoxR, thus this feature may contribute to the regulatory differences between the two homologs.
To determine if these two molecular features are essential to the function of SoxR, two mutants will be constructed by site-directed mutagenesis: one lacking the [2Fe-2S] clusters, and a second lacking the C-terminal region. The mutated soxR genes will be cloned into the vector pSET152 and introduced into a S. coelicolor soxR null mutant. The ability of the mutant genes to complement the soxR mutant will be assessed by monitoring (by quantitative real-time PCR) the transcriptional levels of two SoxR target genes, SCO2478 and SCO4266. If the mutants are able to complement the soxR mutant, then it can be concluded that these characteristics are not essential to SoxR function. Conversely, if complementation is not observed, then it can be inferred that the features are necessary for the function of SoxR.