Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk) has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3). The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography. 1. Introduction Acinetobacter baumannii is an aerobic gram-negative bacterium, widely known for its multidrug resistance [1]. It is an opportunistic bacterial pathogen and possesses wide variety of resistance mechanisms [2–4]. It has high survival rate on abiotic surfaces and has the ability to persist in the hospital environment [3]. Hence, it is largely responsible for hospital-acquired infections as well as community-acquired infections [2, 3]. Previously, it has been reported that the infections caused by this pathogen were of moderate severity; however, the recently increased prevalence of this bacterial infection with serious consequences has been observed [3–5]. These include bacteremia, pneumonia, meningitis, urinary tract, and wound infections [4]. Nucleoside diphosphate kinase (Ndk) is involved in the catalysis of the transfer of -phosphate from nucleoside triphosphate to nucleoside diphosphate and thus maintains the nucleotide pool [6, 7]. The dNTPs that are formed are used as precursors for DNA and RNA synthesis. NTPs, specifically GTP, are important for cellular macromolecular synthesis and signalling mechanisms. Ndk has been found to play a pivotal role in crucial events like bacterial growth, signal transduction, and pathogenicity [8]. The role of Ndk in regulating growth, formation of NTP, and cell surface polysaccharides has been seen in pathogens like Pseudomonas aeruginosa and Mycobacterium tuberculosis [8–10]. Mycobacterial Ndk has been found to inhibit phagosome maturation and helps in the survival of the bacterium within the macrophage [11]. Ndk has also been shown to form different oligomeric structures in