ABSTRACTBacteria respond to stressful growth conditions through a conserved phenomenon of stringent response mediated by synthesis of stress alarmones ppGpp and pppGpp [referred to as (p)ppGpp]. (p)ppGpp synthesis is known to occur by ribosome-associated RelA. In addition, a dual-function protein, SpoT (with both synthetase and hydrolase activities), maintains (p)ppGpp homeostasis. The presence of (p)ppGpp is also known to contribute to antibiotic resistance in bacteria.Mycobacterium smegmatispossesses Arr, which inactivates rifampin by its ADP ribosylation. Arr has been shown to be upregulated in response to stress. However, the roles Arr might play during growth have remained unclear. We show that Arr confers growth fitness advantage toM. smegmatiseven in the absence of rifampin. Arr deficiency inM. smegmatisresulted in deficiency of biofilm formation. Further, we show that while Arr does not interact with the wild-typeEscherichia coliribosomes, it interacts with them when theE. coliribosomal protein L11 (a stringent response regulator) is replaced with its homolog fromM. smegmatis. The Arr interaction withE. coliribosomes occurs even when the N-terminal 33 amino acids of its L11 protein were replaced with the corresponding sequence ofM. smegmatisL11 (Msm-EcoL11 chimeric protein). Interestingly, Arr interaction with theE. coliribosomes harboringM. smegmatisL11 orMsm-EcoL11 results in the synthesis of ppGppin vivo. Our study shows a novel role of antibiotic resistance genearrin stress response.IMPORTANCEMycobacterium smegmatis, like many other bacteria, possesses an ADP-ribosyltransferase, Arr, which confers resistance to the first-line antituberculosis drug, rifampin, by its ADP ribosylation. In this report, we show that in addition to its known property of conferring resistance to rifampin, Arr confers growth fitness advantage toM. smegmatiseven when there is no rifampin in the growth medium. We then show that Arr establishes species-specific interactions with ribosomes through the N-terminal sequence of ribosomal protein L11 (a stringent response regulator) and results in ppGpp (stress alarmone) synthesis. Deficiency of Arr inM. smegmatisresults in deficiency of biofilm formation. Arr protein is physiologically important both in conferring antibiotic resistance as well as in mediating stringent response.