Research Projects

Recent findings show that the transposons encoded TnpB proteins are the ancestors of the CRISPR-Cas nucleases. Bioinformatics analysis uncovered that the TnpB shares a close relationship with the Cas12 family of nucleases. Originally, TnpB genes are present with TnpA, located within the insertion sequence (IS) elements, their role in transposition has always been denied. These TnpB genes are accompanied by a non-coding RNA that guides the TnpB endonuclease for site-specific cleavage. Although TnpB genes share this resemblance, they lack a CRISPR array, leading to speculation about their potential involvement in defense mechanisms or their role in other cellular functions. We aim to characterize the TnpB protein, which has not previously been studied in Brevibacillus laterosporus (Bla). BLAST analysis revealed that Bla exhibits 50% sequence similarity with Deinococcus radiodurans (ISDra2) TnpB. BlaTnpB consists of 361 amino acids, smaller than the well characterized ISDra2 TnpB (408 amino acids). Structural analysis using AlphaFold showed high similarity between the two proteins, particularly in the conservation of the active cleavage sites. Structural alignment of ISDra2 TnpB and BlaTnpB, via PyMol, showed high similarity with the preservation of active cleavage sites. The preliminary work has shown that the BlaTnpB has the potential to act as an endonuclease despite its small size. This nuclease activity can be used for the disruption of biofilm formed by the various bacterial species. These biofilms contain various components like polysaccharides, proteins, and extracellular DNA (eDNA). These eDNA provide cohesive support to the biofilm, which makes them a suitable target for novel therapeutics development. The BlaTnpB enzyme’s non-specific cleavage of DNA can be used in biofilm disruption making the bacteria more susceptible to antibiotics. The ability can significantly contribute to more effective treatments for infections caused by biofilm-forming pathogens. In contrast, the specific DNA cleavage capability of BlaTnpB, facilitated by RNA guidance, opens up exciting prospects for gene editing. Another objective includes the identification of antimicrobial peptides within the TnpB protein. Proteins having nuclease activity generally have a greater number of positively charged amino acids to interact with the negatively charged nucleic acids. Since TnpB protein is high positively charge, we analyzed the protein and found the cationic peptide regions. These regions might have the capability to interact with the negatively charged plasma membrane. This strategy has the potential to pave the way for creating novel peptides with antimicrobial capabilities against harmful bacteria. Currently, no other research has explored the use of TnpB protein as a source for disrupting biofilms and functioning as antimicrobial peptides. In our study, we aim to explore the applications of BlaTnpB protein in the fields of genome editing and antimicrobial peptides.