Characterization of the growth inhibition phenotype of the kilAtelAB operon from IncP alpha plasmid RK2Ter

The cryptic tellurite resistance (Ter) determinant from RK2Ter has been previously cloned into a pUC8 plasmid (pDT1558). The Ter.determinant was identified as the kilA locus and comprises an operon of three genes: kilA, telA, and telB (also referred to as klaA, klaB, and klaC on RK2(Tes)). Cultures harboring the plasmid pDT1558 displayed an extended lag phase in rpoS- hosts, but grew normally in rpoS+ strains. Each of the genes from RK2Ter was subcloned into the expression vector pJF118EH behind an inducible tac promoter using polymerase chain reaction (PCR). The PCR primers were used to engineer an efficient ribosome-binding site and an adjacent sequence to improve protein expression. Expression plasmids were modified by inclusion of different resistance markers for selection during complementation. The killing phenotype of the kilAtelAB operon was studied with the overexpressing plasmids. Each individual gene specified growth inhibition of Escherichia coli cells, but with different combinations of the genes giving rise to differing degrees of the inhibition. Additionally, the bacteriostatic and bacteriocidal effects of the genes were found to be different depending on whether or not the cultures were grown in minimal salts medium with glucose or in Luria-Bertani medium. Cells harboring the kilA gene alone or the three genes of the kilAtelAB operon expressed either in trans or cis behind the tac promoter were found to form nonseptated filaments up to 10-30 times the length of control cells. The effect of filamentation was greater for cells grown in minimal salts medium with glucose as the carbon source. This study demonstrates that each of the gene products from kilA, telA, and telB, when expressed at high levels either alone or together with another gene in the operon, exhibit some degree of growth inhibition of host cells. This growth inhibition is considered paramount in the stable maintenance of the plasmid within its host.