Abstract

The title of the research project is the ‘identification of bacteriocin produced by Klebsiella pneumoniae’.
In the experiment two different strains of K. pneumoniae were used; a bacteriocin producer strain K. pneumoniae 957 and a bacteriocin sensitive strain K. pneumoniae 757. The aim of this project was to isolate the bacteriocin so it could be identified by mass spectrometry.
The release of proteins in the cell lysate was confirmed by carrying out SDS poly acrylamide gel electrophoresis and the presence of bacteriocin was confirmed by bio assay test plate which showed the inhibition of growth of K. pneumoniae 757 by K. pneumoniae 957 cell lysate. To isolate the bacteriocin, native PAGE was used and for identification of the protein band on the gel bio assay test was performed.
The protein was isolated by ion exchange chromatography and identified by Mass/Spectrometry. BLAST search was used for finding the homologous peptide sequence. The search result directed towards new bacteriocin produced by K. pneumoniae. According to the BLAST search results the bacteriocin has similarities with cloacin and colicin, produced by Enterobacter cloacae and Escherichia coli respectively. Both bacteriocins are ribonuclease.
This new finding leads to many possible areas for exploring the similarities between cloacin and the new bacteriocin. To this stage new bacteriocin uses the same cell surface receptor for attachment and has a very similar cytotoxic domain and receptor binding domain that means the killing mechanism is the same as cloacin. But it is unknown if the victim is rRNA or tRNA, e.g., colicins E4 and E6 have sequence homology with colicin E3 but the E4 and E6 target amino acid incorporation and E3 directly cleaves 16s rRNA (Tomita et al, 2000). Therefore, if new bacteriocin has sequence homology with cloacin, it can have a different mechanism of action inside the sensitive cell for degrading RNA or it can have a different process of synthesis. All these facts can be used in future works to find out more about the new bacteriocin.