Serine/Threonine Protein Kinases (STPKs) phosphorylates target proteins thereby regulates various important cellular signal transduction pathways such as cell division and cell wall synthesis. It has been demonstrated that the STPKs regulate peptidoglycan biosynthesis by phosphorylating penicillin binding proteins (PBPs). We extensively characterized both PknI (STPK) and DacB2 (PBP) roles individually as well as combining by genetic knockout and phenotypic characterization studies. In the present study, we analyzed the role of PknI and DacB2 in cell division and virulence. The double knockout (DKO) strain growth was reduced under stress conditions like acidic pH, nutrient depletion media and low oxygen availability conditions. We also found that the DKO growth was significantly reduced in macrophage cell line and it was hypersensitive to oxidative and nitrosative stress condition. The DKO strain significantly attenuated in guinea pig model which was measured by reduced bacillary load, gross pathological and histopathological damages. Overall, these results clearly demonstrated that both PknI and DacB2 together play an important role in cell division under stress conditions, the DKO strain significantly attenuated both in vitro and in vivo models.