The rifampicin-inactivating mono-ADP-ribosyl transferase of Mycobacterium smegmatis significantly influences reactive oxygen species levels in the actively growing cells

Abstract

A classic example of antibiotic inactivating function in bacteria is the Mycobacterium smegmatis (Msm) encoded rifampicin-inactivating mono-ADP-ribosyl transferase (arr). Since its probable biological role has been proposed to be in DNA damage response, which is inflicted by reactive oxygen species (ROS), in the present study, we examined whether Msm Arr influences ROS levels. For this purpose, the levels of the ROS, hydroxyl radical and superoxide, were determined in the mid-log phase (MLP) cells of Msm arr knockout (arr-KO) strain, in comparison to those in the equivalently grown Msm arr+ wild-type (WT) strain. The MLP arr-KO cells generated significantly elevated levels of superoxide and hydroxyl radical, unlike the equivalently grown WT MLP cells. Complementation of arr-KO with arr, but not with empty vector, restored the ROS levels comparable to those in the WT strain. Elevated ROS levels in the arr-KO strain enabled selection of rifampicin-resistant mutants at 10-7 cfu/ml from the rifampicin-unexposed MLP cells of arr-KO, which is one-log10 higher than that for WT cells (10-8). Upon prolonged exposure to rifampicin, the susceptibility, persister formation, generation of elevated levels of hydroxyl radical by the persisters, rifampicin-resister generation frequency of the persisters and regrowth of the rifampicin-resistant mutants from the respective persisters were all comparable between the arr-KO and WT strains. These observations revealed that Arr influences ROS levels in the actively growing M. smegmatis cells but not in the rifampicin-exposed cells. We proposed the probable pathway through which Arr might be influencing ROS levels in the actively growing M. smegmatis cells. IMPORTANCEDiverse genera of bacteria consisting of pathogens, opportunistic pathogens and non-pathogens, possess Arr-type activities that confer equally efficient rifampicin resistance, thereby posing serious health hazard. Acquisition of this function by other bacteria through horizontal gene transfer enhances the hazard posed by the bacteria possessing it. M. smegmatis is an opportunistic human pathogen that causes infections of skin and soft tissues. Moreover, M. smegmatis is a genetically tractable model organism for M. tuberculosis with the potential to function even as tuberculosis vaccine. In view of these significant aspects of Arr and M. smegmatis, the study to find out the natural physiological role of Arr in M. smegmatis, gains importance for designing strategies to prevent antibiotic inactivation and to target the cellular function to contain the bacterium. Above all, the three-dimensional structure of M. smegmatis Arr reveals significant structural homology with eukaryotic ADP-ribosyltransferases and bacterial toxins, thereby giving the study broad significance.