%0 Journal Article
%T Expression, Purification, and Functional Characterization of Atypical Xenocin, Its Immunity Protein, and Their Domains from Xenorhabdus nematophila
%A Jitendra Singh Rathore
%J International Journal of Bacteriology
%D 2013
%I Hindawi Publishing Corporation
%R 10.1155/2013/746862
%X Xenorhabdus nematophila, a gram-negative bacterium belonging to the family Enterobacteriaceae is a natural symbiont of a soil nematode from the family Steinernematidae. In this study cloning, expression, and purification of broad range iron regulated multidomain bacteriocin called xenocin from X. nematophila (66£¿kDa, encoded by xcinA gene) and its multidomain immunity protein (42£¿kDa, encoded by ximB gene) have been done. xcinA-ximB (N¡ä terminal 270£¿bp), translocation, and translocation-receptor domain of xcinA, ximB, and its hemolysin domain were cloned, expressed, and purified by single step Ni-NTA chromatography under native conditions. In the functional characterization, neutralization of xcinA toxicity by immunity domain of ximB gene was determined by endogenous assay. Exogenous toxic assays results showed that only the purified recombinant xenocin-immunity domain (10£¿kDa) protein complex had toxic activity. Atypical cognate immunity protein (42£¿kDa) of xenocin was fusion of immunity domain (10£¿kDa) and hemolysin domain (32£¿kDa). In silico analysis of immunity protein revealed its similarity with hemolysin and purine NTPase like proteins. Hemolytic activity was not observed in immunity protein or in its various domains; however, full-length immunity protein lacking Walker motif showed ATPase activity. Finally, using circular dichroism performed secondary structural analyses of all the recombinant proteins/protein complexes. 1. Introduction Bacteriocins are toxins produced by the bacteria to inhibit the growth of similar or closely related bacterial strain(s) during stress conditions [1]. They are structurally, functionally, and ecologically diverse, produced by almost all major lineages of Eubacteria and Archaebacteria [2]. Ribosomal encoded bacteriocins are generally secreted in the extracellular milieu by the producers where they recognize specific receptors on the surface of susceptible or target cells. They induce toxicity in the target cells by different mechanisms like enzymatic nuclease (DNase or RNase) or pore formation in cytoplasmic membrane [3]. Their structure comprises of three distinct domain organizations: (i) a domain involved in recognition of specific receptor R, (ii) a domain involved in translocation T, and (iii) a domain responsible for their toxic activity C. Molecular mass of ribosomal encoded bacteriocins vary from ~25 to 80£¿kDa and are broadly classified into two groups, group A and B, based on their cross-resistance [4]. These proteins have received increasing attention due to their potential use as preservatives in the
%U http://www.hindawi.com/journals/ijb/2013/746862/