Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Speaker Abstracts

Visualizing the Mycobacterial Mutasome Michael A. Reiche 1 , Dirk Lang 2 , Valerie Mizrahi 1,3 , Digby F. Warner 1,3 .

1 University of Cape Town, Cape Town, Western Cape, South Africa, 2 University of Cape Town, Cape Town, Western Cape, South Africa, 3 University of Cape Town, Cape Town, Western Cape, South Africa. Previous work in our laboratory identified a DNA damage-inducible mutagenic DNA repair system that is required for adaptive mutagenesis, including the development of drug resistance, in Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis (TB). Moreover, genetic studies established that this so-called “mycobacterial mutasome” comprises a minimum of three essential components: a dnaE2 -encoded PolIIIα subunit, and imuB - and imuA ’- encoded accessory factors. Current work aims to investigate the recruitment dynamics and sub-cellular localization of the mutasome components following exposure of bacilli to genotoxic stress. To this end, we constructed a panel of M . smegmatis ( Msm ) reporter mutants encoding fluorescently tagged mutasome proteins. The DNA damage survival and induced mutagenesis functions of the recombinant proteins were assessed, and fluorescence visualized and quantified in a series of DNA damage assays. In addition, population-wide expression characteristics were assessed by flow cytometry. Results indicate that expression of enhanced green fluorescent protein (EGFP) from the DNA damage-responsive imuA’ promoter initiates 60 minutes post exposure to mitomycin C (MMC), a known genotoxin, with maximal EGFP expression achieved 360 minutes post treatment. These results indicate that Msm responds rapidly to DNA damage, with dynamics and level of expression correlating with the amount of damage incurred. Moreover, cellular localization of mutasome components indicates differential recruitment and localization of ImuA’ and ImuB in MMC-exposed cells. In combination, our results establish the utility of combining fluorescence imaging with functional genetics to elucidate the mechanisms regulating expression and activity of a major error-prone damage tolerance pathway in Mtb .

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