Applying antibiotics to susceptible bacterial cultures generates a minor population of persisters that remain susceptible to antibiotics but can endure them for extended periods. Recent reports suggest that antibiotic persisters (APs) of mycobacteria experience oxidative stress and develop resistance upon treatment with lethal doses of ciprofloxacin or rifampicin. However, the mechanisms driving the de novo emergence of resistance remained unclear. Here, we show that mycobacterial APs activate the SOS response resulting in up-regulation of the error-prone DNA polymerase DnaE2. The sustained expression of dnaE2 in APs led to mutagenesis across the genome and resulted in the rapid evolution of resistance to antibiotics. Inhibition of RecA by suramin, an anti- Trypanosoma drug, reduced the conversion rate of persisters to resistors in a diverse group of bacteria. Our study highlights suramin's novel application as a broad-spectrum agent in combating the development of drug resistance.

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The complex cellular architecture and the microenvironments within the biofilm give rise to a population that is both physiologically and genetically heterogenous. Transcriptome analysis of Mycobacterium smegmatis in biofilm culture and its transition phase into planktonic growth was performed to identify the genetic basis of heterogeneity in the biofilm. While there was an increase in the expression of mycobacterial mutasome consisting of dnaE2, imuA and imuB in the biofilm, the mutation burden was less compared to planktonic culture. Deletion of dnaE2 displays lower mutation frequency and bacterial fitness in comparison to the parental strain in biofilm culture. The expression of dnaE2 contributes to slower bacterial growth rate, potentially promoting persister formation. Our study uncovers the multiple benefits of dnaE2 expression in biofilm such as increasing genetic diversity and reducing growth rate; both of which are necessary for mycobacterial survival and adaptation.