Deletion of rifampicin-inactivating mono-ADP-ribosyl transferase gene of Mycobacterium smegmatis globally altered gene expression profile that favoured increase in ROS levels and thereby antibiotic resister generation

Sharmada Swaminath; Atul Pradhan; Rashmi Ravindran Nair; Parthasarathi Ajitkumar

doi:10.1016/j.crmicr.2022.100142

Deletion of rifampicin-inactivating mono-ADP-ribosyl transferase gene of Mycobacterium smegmatis globally altered gene expression profile that favoured increase in ROS levels and thereby antibiotic resister generation

Curr Res Microb Sci. 2022 May 31:3:100142. doi: 10.1016/j.crmicr.2022.100142. eCollection 2022.

Authors

Sharmada Swaminath^{1

2}, Atul Pradhan^{1

3}, Rashmi Ravindran Nair^{1

4}, Parthasarathi Ajitkumar¹

Affiliations

¹ Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, Karnataka, India.
² Department of Biology, University of San Diego, San Diego, CA, USA.
³ Department of Medicine, Renaissance School of Medicine, Stony Brook University, Stony Brook, New York, USA.
⁴ Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Abstract

The physiological role of mono-ADP-ribosyl transferase (Arr) of Mycobacterium smegmatis, which inactivates rifampicin, remains unclear. An earlier study reported increased expression of arr during oxidative stress and DNA damage. This suggested a role for Arr in the oxidative status of the cell and its associated effect on DNA damage. Since reactive oxygen species (ROS) influence oxidative status, we investigated whether Arr affected ROS levels in M. smegmatis. Significantly elevated levels of superoxide and hydroxyl radical were found in the mid-log phase (MLP) cultures of the arr knockout strain (arr-KO) as compared those in the wild-type strain (WT). Complementation of arr-KO with expression from genomically integrated arr under its native promoter restored the levels of ROS equivalent to that in WT. Due to the inherently high ROS levels in the actively growing arr-KO, rifampicin resisters with rpoB mutations could be selected at 0 hr of exposure itself against rifampicin, unlike in the WT where the resisters emerged at 12^th hr of rifampicin exposure. Microarray analysis of the actively growing cultures of arr-KO revealed significantly high levels of expression of genes from succinate dehydrogenase I and NADH dehydrogenase I operons, which would have contributed to the increased superoxide levels. In parallel, expression of specific DNA repair genes was significantly decreased, favouring retention of the mutations inflicted by the ROS. Expression of several metabolic pathway genes also was significantly altered. These observations revealed that Arr was required for maintaining a gene expression profile that would provide optimum levels of ROS and DNA repair system in the actively growing M. smegmatis.

Keywords: ABC transporter, ATP-Binding Cassette Transporter; AES, Allelic Exchange Substrates; CFU, Colony Forming Unit; DAVID database, Database for Annotation, Visualization and Integrated Discovery; DMPO, 5,5-Dimethyl-1-Pyrroline N-Oxide; DTPA, Diethylene Triamine Pentaacetic Acid; EPR, Electron Paramagnetic Resonance; ETC, Electron Transport Complex; GFP, Green Fluorescence Protein; Global gene expression; HPF, 3’-(p-Hydroxyphenyl) Fluorescein; LB, Luria-Bertani; MBC, Minimum Bactericidal Concentration; MLP, Mid-log phase; MSMEG, Mycobacterium smegmatis; Mono-ADP-ribosyl transferase; Mutation; Mycobacterium smegmatis; OD, Optical Density; PBS, Phosphate Buffered Saline; ROS, Reactive Oxygen Species; RRDR, Rifampicin Resistance Determining Region; Reactive oxygen species; Resister generation frequency; Rifampicin; Rifampicin resistance; VC, Vector Control; WT, wild type; arr-KO, arr knockout; cAMP, Cyclic Adenosine Mono Phosphate.