Fiona Gambanga and Monica Chander

Affiliation: Biology Department, Bryn Mawr College, Bryn Mawr, Pennsylvania


Identification of physiological activator of SoxR in Streptomyces

The medically important bacterium, Streptomyces coelicolor, naturally produces several antibiotics, including the redox-active polyketide antibiotic actinorhodin (Act).  Our lab is interested in determining how S. coelicolor avoids self-destruction by endogenous antibiotics.  We have previously produced evidence that one Act-detoxification mechanism may be regulated by the redox-sensing transcription factor, SoxR.  First, SoxR regulated genes are expressed in stationary phase when cells actively synthesize Act, but are not expressed in a mutant strain that does not produce Act (Dela Cruz et al., 2010).  Second, SoxR regulated genes include a membrane transporter, and several proteins that resemble antibiotic-metabolizing enzymes.  Finally, a soxR null mutant overproduces Act, suggesting that SoxR normally reduces the level of Act in S. coelicolor thereby preventing its accumulation to toxic levels.  If our hypothesis is correct and SoxR regulates an Act-detoxification mechanism in S. coelicolor, then one would expect that SoxR regulated genes would be expressed before the production of toxic molecules.  Given this reasoning, we propose that SoxR senses a redox-active precursor of Act.  The goal of this research project is to identify this molecule.  Towards this end we will block the Act biosynthetic pathway at specific steps. We will start by mutating the gene ACT VI-orf-2 for a mono-oxidoreductase in the S.coelicolor in the ACT biosynthetic pathway.  If the product of this reaction is the physiological signal for SoxR, then mutating the ACT VI-orf-2 gene will inactivate SoxR activity. If the physiological activator molecule for SoxR is downstream in the pathway, the ACT VI-orf-2 mutant will still maintain SoxR activity. The expression of SoxR-regulated gene will be monitored in each mutant background by quantitative real time PCR.


Work Cited

Dela Cruz, R., Y. Gao, S. Penumetcha, R. Sheplock, K. Weng, and M. Chander, 2010, Expression of the Streptomyces coelicolor SoxR regulon is intimately linked with actinorhodin production: Journal of bacteriology, v. 192, p. 6428-6438.