The rifampicin-inactivating mono-ADP-ribosyl transferase ofMycobacterium smegmatissignificantly influences reactive oxygen species levels in the actively growing cells

Abstract

ABSTRACTA classic example of antibiotic inactivating function in bacteria is theMycobacterium 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 whetherMsmArr influences ROS levels. For this purpose, the levels of the ROS, hydroxyl radical and superoxide, were determined in the mid-log phase (MLP) cells ofMsm arrknockout (arr-KO) strain, in comparison to those in the equivalently grownMsm arr+wild-type (WT) strain. The MLParr-KO cells generated significantly elevated levels of superoxide and hydroxyl radical, unlike the equivalently grown WT MLP cells. Complementation ofarr-KO witharr, but not with empty vector, restored the ROS levels comparable to those in the WT strain. Elevated ROS levels in thearr-KO strain enabled selection of rifampicin-resistant mutants at 10-7cfu/ml from the rifampicin-unexposed MLP cells ofarr-KO, which is one-log10higher 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 thearr-KO and WT strains. These observations revealed that Arr influences ROS levels in the actively growingM. smegmatiscells but not in the rifampicin-exposed cells. We proposed the probable pathway through which Arr might be influencing ROS levels in the actively growingM. smegmatiscells.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. smegmatisis an opportunistic human pathogen that causes infections of skin and soft tissues. Moreover,M. smegmatisis a genetically tractable model organism forM. tuberculosiswith the potential to function even as tuberculosis vaccine. In view of these significant aspects of Arr andM. smegmatis, the study to find out the natural physiological role of Arr inM. 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 ofM. smegmatisArr reveals significant structural homology with eukaryotic ADP-ribosyltransferases and bacterial toxins, thereby giving the study broad significance.