Posttranslational modification of proteins is a prevalent method for the regulation of cells according to changes in the surrounding environment and diversifications. Pupylation, architecturally similar but not homologous to eukaryotic proteasomal degradation machinery, exists in a certain order of bacteria, especially actinobacteria. Pupylation supports the bacteria to survive under challenging environmental conditions like stress (physical or chemical) and nutrient starvation. Pupylation is also involved in iron homeostasis, which is necessary for cellular metabolism and the normal growth of bacteria. Pupylation is a posttranslational modification through which intrinsically disordered proteins are tagged for proteasomal degradation. Although this process is functionally reminiscent of ubiquitination in eukaryotes; it is carried out by a different set of enzymes in evolutionarily connected bacterial carboxylate-amine ligases. In this chapter, we will discuss the recent advances in the understanding of how proteins are tagged for proteasomal degradation in actinobacteria and its role in the survival of mycobacterium during pathogenesis in the host. Furthermore, we will examine the role of accessory factors associated with the proteasomal system in bacteria that function independently of proteolysis.