Avijit Das

Expression of Phi11 Gp07 Causes Filamentation in Escherichia coli

Background: The Gp07 protein of aureophage Phi11 exhibits growth inhibitory effects when overexpressed in E. coli .The protein harbors two domains- an amino terminal Bro-like domain and a carboxy terminal Ant superfamily like KilA domain, of which the KilA domain retains the growth inhibitory effect of Gp07. Methods: We studied the effects exerted by the overexpression of Gp07 and its separate domains upon the growth rate as well as the morphology of the E. coli cells. Additionally, we generated a mutant of Gp07 (designated as ΔGp07) by deleting the first eleven amino acid residues from the amino-terminal region of Gp07, and studied its growth inhibitory effects upon E. coli. Results: Our results indicate that Gp07, ΔGp07 as well as the Carboxy-terminal region of Gp07 upon overexpression, retards the growth rate of the E. coli cells and also induces filamentation in the cells. Surprisingly, our data clearly suggests that the growth inhibition and filamentation induced by the the amino-terminal domain of Gp07 is temporal in nature. Conclusion: The carboxy-terminal of domain of gp07 is essential for its activity.

Cloning, overexpression and purification of a novel two-domain protein of Staphylococcus aureus phage Phi11

The genome of aureophage Phi11 reveals the presence of the gene gp07 which codes for the putative antirepressor protein (GenBank accession no. NC_004615.1). Antirepressor proteins are mainly involved in lytic cycle determination mechanisms of various bacteriophages. The Phi11 protein Gp07 consists of two domains-an amino terminal Bro domain and a carboxy terminal KilA domain. Despite the important role of antirepressor proteins in the developmental pathway of phages, there are no reports on the purification and characterization of aureophage antirepressor proteins. Here we describe a method to clone, overexpress and purify the full length Gp07 as carboxy terminal hexa histidine tag variant. The recombinant protein was expressed in Escherichia coli BL21(λDE3) cells and gradient of imidazole and NaCl were used for successful purification of the soluble recombinant protein to homogeneity. The protein exists as a dimer in solution as is evident from our gel filtration chromatography and glutaraldehyde cross-linking data. Further, we found that temperature has huge impact on the structural conformation of the protein. We expect that the purification of Gp07 will further our work in characterizing the role played by this protein during phage induction.

Changes in the Functional Activity of Phi11 Cro Protein is Mediated by Various Ions

AbstractPhi11, a temperate bacteriophage of Staphylococcus aureus, has been found to harbor a cro repressor gene which facilitates Phi11 to adopt the lytic mode of development. The Cro protein has been found to bind very specifically to a 15-bp operator DNA, located in the Phi11 cI–cro intergenic region [1]. To investigate the effects exerted by different ions upon the interaction between Cro and its cognate operator DNA, we have employed gel shift assays as well as circular dichroism spectral analysis. In this communication, we have shown that NH4+ and acetate− ions better facilitated the binding of Cro with its cognate operator as compared to Na+, K+ and Li+. Interestingly, Mg2+, carbonate2− and Citrate3− have an inhibitory effect upon the binding. The effect of the said ions upon the structure of Cro was also investigated by circular dichroism and it was found that other than Citrate3− ions, none of the other ions destabilised the protein. On the other hand, Mg2+ and carbonate2− ions maintained the structure of the protein but severely hampered its functional activity. Citrate3− ions severely unfolded Cro and also inhibited its function. Considering all the data, NH4+ and acetate− ions appeared to be more suitable in maintaining the biological activity of Cro.