Postsegregational killing mediated by the P1 phage "addiction module" phd-doc requires the Escherichia coli programmed cell death system mazEF - PubMed (original) (raw)

Postsegregational killing mediated by the P1 phage "addiction module" phd-doc requires the Escherichia coli programmed cell death system mazEF

R Hazan et al. J Bacteriol. 2001 Mar.

Free PMC article

Abstract

"Addiction modules" consist of two genes; the product of the second is long lived and toxic, while the product of the first is short lived and antagonizes the lethal action of the toxin. The extrachromosomal addiction module phd-doc, located on the P1 prophage, is responsible for the postsegregational killing effect (death of plasmid-free cells). The Escherichia coli chromosomal addiction module analogue, mazEF, is responsible for the induction of programmed cell death. Here we show that the postsegregational killing mediated by the P1 phd-doc module depends on the presence of the E. coli mazEF system. In addition, we demonstrate that under conditions of postsegregational killing, mediated by phd-doc, protein synthesis of E. coli is inhibited. Based on our findings, we suggest the existence of a coupling between the phd-doc and mazEF systems.

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Figures

FIG. 1

The effect of the loss of the plasmids pGB2ts and pGB2ts::phd-doc on the growth of MC4100 and its Δ_mazEF_ derivative. (A) Growth on LB plates with spectinomycin. E. coli MC4100 (wild type) and its Δ_mazE_ derivative, both harboring either plasmid pGB2ts or pGB2ts::phd-doc, were plated on LB plates with 100 μg of spectinomycin/ml. The plates were incubated at 30 or at 42°C overnight. (B) Bacterial growth curves at 42°C in liquid LB medium without any added antibiotic. The bacteria were grown to an OD600 of 0.7 and then diluted to an OD600of 0.07. This step was repeated twice in order to cure the cells from the plasmids. The curves represent the values after the second dilution. Results for MC4100 are illustrated by black lines, and those for its Δ_mazEF_ derivative are illustrated by gray lines. Results with MC4100/pGB2ts (■), MC4100/pGB2ts::phd-doc (▴), Δ_mazEF_/pGB2ts ( formula image ), and Δ_mazEF_/pGB2ts::phd-doc (□) are shown.

FIG. 2

The effect of the E. coli mazEF system on the postsegregational killing mediated by the P1 phd-doc_addiction module. Plasmids pGB2ts and pGB2ts::phd-doc(Sptr) were used to transform E. coli MC4100 (wild type) and its Δ_mazEF derivative. The transformant MC4100Δ_mazEF_/pGB2ts::phd-doc was further transformed by plasmid pKK223_mazEF_(Ampr). Transformants were grown in LB liquid medium at 30°C to mid-logarithmic phase and were plated on LB plates without antibiotics at 30 and 42°C. The percentage of cell survivors was calculated by comparing the numbers of CFU at 42°C versus 30°C.

FIG. 3

The effect of the P1 phd-doc system on protein synthesis. Plasmid pGB2ts::phd-doc or pGB2ts (as a control) were used to transform E. coli MC4100 (wild type) and the MC4100 Δ_mazEF_ derivative. Plasmid curing was carried out in LB liquid medium as described in the legend to Fig. 1. The rate of protein synthesis was determined as described in Materials and Methods. Results for MC4100 are illustrated by black lines, and those for its Δ_mazE_ derivative are shown by gray lines. Results with MC4100/pGB2ts (■), MC4100/pGB2ts::phd-doc (▴), Δ_mazEF_/pGB2ts ( formula image ), and Δ_mazEF_/pGB2ts::phd-doc (□) are shown.

FIG. 4

A schematic representation for the coupling of the extrachromosomal P1 phd-doc system and the chromosomal_mazEF_ addiction module. (A) In the presence of plasmid-borne phd-doc. When the plasmid-borne_phd-doc_ system is expressed, there is a balance between the expression of the antitoxic Phd and its degradation by ClpX. This balance permits the neutralization of the toxic protein Doc by Phd. (B) When the plasmid is lost. Under conditions of plasmid loss, the P1_phd-doc_ system mediates postsegregational killing. Under these conditions, the level of the labile antitoxin Phd decreases below the threshold required for neutralizing Doc. Doc inhibits the translational machinery and thereby triggers programmed cell death mediated by the E. coli mazEF system. When protein translation is inhibited, the continuous expression of the labile antitoxic MazE protein is prevented. As a result, the stable toxin MazF causes cell death (for further details, see the text).

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Postsegregational killing mediated by the P1 phage "addiction module" phd-doc requires the Escherichia coli programmed cell death system mazEF - PubMed (original) (raw)