The kilA operon of promiscuous plasmid RK2: the use of a transducing phage (lambda pklaA-1) to determine the effects of the lethal klaA gene on Escherichia coli cells.
The kil-kor regulon of promiscuous plasmid RK2 includes the replication initiator gene trfA and several potentially host-lethal kil loci (kilA, kilB, kilC, kilE), whose functions may be involved in plasmid maintenance or broad host range. The kilA locus consists of a single operon of three genes (klaA, klaB, klaC), each of which is lethal when expressed from the klaA promoter in the absence of repressors encoded by korA and korB. In this study, we examined the effects of the unregulated klaA gene on the host cell. Bacteriophage lambda was used to construct a transducing phage (lambda pklaA-1) that allows efficient introduction of the klaA gene into Escherichia coli. Cells lacking korA and korB (to allow uncontrolled expression of klaA) and expressing lambda repressor (to prevent phage lytic growth) are killed by lambda pklaA-1. Cell death is dependent on the klaA structural gene, independent of the SOS system of the host, and is prevented by the presence of korA and korB. lambda pklaA-1 was used to synchronously infect cells lacking korA and korB to determine the effects of klaA on the cells over time. The earliest effects, visible at two hours post-infection, are inhibition of growth of the culture, formation of elongated cells, and striking changes in the appearance of the outer membrane. After four to five hours, the viability of the culture declined sharply and macromolecular synthesis ceased. The distinct class of early events is consistent with the hypothesis that the KlaA polypeptide interacts with a specific target in the host cell.
Saltman
L H
LH
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Kim
K S
KS
Figurski
D H
DH
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005804:Genes, Lethal; D009876:Operon; D010641:Phenotype; D010957:Plasmids; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C067119:kilA protein, E coli