Analyzing the regulation of Streptomyces coelicolor SoxR by actinorhodin

Posted May 12th, 2010 at 1:29 pm.

Katie Weng

Mentor: Dr. Monica Chander

Oxidative stress damages cells and causes a plethora of diseases. Therefore, all organisms have mechanisms to protect against oxidative stress. In Escherichia coli, the transcription factor SoxR senses the presence of free radicals via redox-active iron-sulfur clusters and binds to the promoter of another transcription factor, soxS, to activate its expression. SoxS in turn activates the production of over forty genes that combat oxidative stress and repair cellular damage in the bacterium. SoxR is present in a variety of bacteria, such as the antibiotic producer, S. coelicolor. However, S. coelicolor does not have a soxS gene, which suggests that S. coelicolor SoxR plays a different physiological role than E. coli SoxR.

S. coelicolor produces four different antibodies, including actinorhodin. Any antibiotic-producing organism must have mechanisms to protect itself against self-toxicity. We hypothesize that SoxR is involved in actinorhodin resistance in S. coelicolor. This hypothesis stems from the observation that the promoters of two genes (SCO2478 and SCO4266), believed to modify and detoxify actinorhodin, contain highly conserved SoxR binding sites. Thus, as soon as actinorhodin is synthesized, it would activate SoxR, which would in turn activate the expression of the actinorhodin-detoxifying genes, SCO2478 and SCO4266.

The objective of this study is to examine two questions: i) is SoxR activated by actinorhodin? ii) does SoxR bind to the promoters of SCO2478 and SCO4266 and activate their transcription in the presence of actinorhodin? To address the first question, S. coelicolor soxR will be expressed in an E. coli strain that contains a soxS promoter-lacZ reporter fusion. The cells will be treated with actinorhodin. If SoxR is activated by actinorhodin, then we would expect to see reporter activity. Further, to determine if the iron-sulfur clusters are important in sensing actinorhodin, I will construct a SoxR mutant lacking iron-sulfur clusters and analyze its ability to respond to actinorhodin. The ability of SoxR to bind to the promoters of SCO2478 and SCO4266 will be tested by gel shift assays. In addition, to test if S. coelicolor SoxR activates the transcription of these two potential target genes, in vitro transcriptional assays will be performed in the presence and absence of actinorhodin.

Filed under: 2008,Chander, Dr. Monica,Weng, Katie by Ann Dixon

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