Characterization of the growth inhibition phenotype of the kilAtelAB operon from IncPα plasmid RK2Te^r

Abstract

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.

Characterization of a region of the IncHI2 plasmid R478 which protects Escherichia coli from toxic effects specified by components of the tellurite, phage, and colicin resistance cluster

“…The klaA gene product has been shown to inhibit bacteriophage development and to interfere with cell division in E. coli (24,25,37), resulting in cells that are 10 to 30 times longer than normal (37). Filamentous cells were found to be nonseptated with surface blebs (37), similar to the phenotypes induced by pKFW4A in the present study (Fig. 6).…”

“…A second operon on RK2, parDE, also specifies cell division inhibition and is lethal to cells grown on minimal media (22). An amplified klaA filamentation phenotype was observed with cells grown on minimal media compared to cells grown on richer media (37). In our study, cells were extremely long and size was difficult to measure.…”

Identification and molecular genetic analysis of multiple loci contributing to high-level tellurite resistance in Rhodobacter sphaeroides 2.4.1

“…This operon encodes latent high-level tellurite resistance in E. coli, which becomes apparent after a point mutation in klaC. The mutant form of klaC has been designated telB (33,35,38). To determine if other homologs of the components of the kilA operon were also present, we sequenced downstream and upstream of R. sphaeroides telA.…”

Expression of Escherichia coli TehA gives resistance to antiseptics and disinfectants similar to that conferred by multidrug resistance efflux pumps

“…Construction of pEMR11 was performed with PCR to move the emrE gene from pASP505 behind the tac promoter in pMS119EH. The 5Ј primer was designed to provide an improved ribosome binding site and intervening sequence which worked well for protein expression with other proteins in our laboratory (2,38,39).…”