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. 2014 Feb 18;111(7):2734-9.
doi: 10.1073/pnas.1308241111. Epub 2014 Jan 21.

Toxin Kid Uncouples DNA Replication and Cell Division to Enforce Retention of Plasmid R1 in Escherichia Coli Cells

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Free PMC article

Toxin Kid Uncouples DNA Replication and Cell Division to Enforce Retention of Plasmid R1 in Escherichia Coli Cells

Belén Pimentel et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.

Keywords: PemK; RNase; mRNA interferase; parD; plasmid stability.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Kid does not kill E. coli. (A) Growth curves of DH10B cells carrying pBAD22copA plus either mR1KK, mR1hs, or mR1Ctrl and induced with arabinose to produce copA for the indicated times in minimal medium. (B) Relative (Rel.) number of dead (i.e., PI-permeable) cells in mR1KK (kiskid) and mR1hs (hoksok) samples from A, normalized against their control mR1Ctrl samples. (C) Proportion of cells carrying mR1KK that resume proliferation when sustained production of copA in A is ceased at the indicated times. Numbers are relative to those observed in control samples (i.e., cells carrying mR1Ctrl). n = 3; bars represent SEM.
Fig. 2.
Fig. 2.
Kid inhibits cell division and does not halt protein synthesis completely in E. coli. (A) Time-lapse images of DH10B cells carrying thermosensitive expression vector pPrTsLWCKid (Lower) or its empty control [Ctrl (pPrTsLWC); Upper] upon incubation at the inducing temperature (42 °C) for the times shown. (Scale bar: 2 μm.) (B) Incorporation of [35S]methionine (35S-Met) in DH10B cells hosting pPrTsLWCKid, relative to that in cells carrying empty pPrTsLWC, after being cultured at 42 °C for the indicated times. (C) 2D gel analysis of protein extracts prepared from the pPrTsLWC and pPrTsLWCKid samples at the 30-min time point in B. n = 3; bars represent SEM.
Fig. 3.
Fig. 3.
Kid restricts protein outputs in a UUACU-dependent manner. (A) Analysis of protein extracts from DH10B cells carrying expression vectors p177PraraKid and pTet-HS3FKis. Samples before induction (0 h), and at regular intervals after inducing Kid alone first, using arabinose (1–3 h), and then inducing Kis for another 3 h, using anhydrotetracycline (A-Tet) (3.25–6 h), were analyzed. Both 35S-labeled samples (Upper) and -unlabeled samples blotted with antibodies against Kid and Kis (Lower) are shown. WB, Western blot. (B) Analysis of 35S-labeled extracts from GCM2 cells, before (−) or at the indicated times after (with 0 h being immediately after) inducing them to produce Kid from pTet-Kid, using A-Tet. (C) Analysis of extracts from DH10B cells cotransformed with p177Prara-Kid18 (lanes 1–4) or p177Prara-Kid (lanes 5–8) and either pTet-H-EGFP-RepAr (lacking UUACU sites, lanes 1 and 2 and 5 and 6) or pTet-H-EGFP-RepAs (bearing UUACU sites; lanes 3 and 4 and 7 and 8). Odd lanes correspond to uninduced (−) samples. Even lanes correspond to samples induced (+) to produce Kid (or Kid18) for 1 h first and then His6-EGFP-RepAr (or His6-EGFP-RepAr) for another hour before labeling. Extracts from parallel unlabeled samples and blotted with an anti-EGFP antibody are also shown. (D) Analysis of 35S-labeled extracts from DH10B cells cotransformed with p177Prara-Kid plus either pTet-H-DnaBs (bearing UUACU sites, lanes 1 and 2) or pTet-H-DnaBr (lacking UUACU sites, lanes 3 and 4). Odd lanes correspond to uninduced (−) samples. Even lanes correspond to samples that were induced (+) to produce Kid alone for 1 h first, and then the corresponding DnaB variant, before labeling. Arrowheads (black for Kid and Kid18; white for Kis, RepA, and DnaB) mark the position of proteins in gels.
Fig. 4.
Fig. 4.
Kid uncouples DNA replication and cell division in E. coli. (A) Average numbers of oriC- (red) and oriR1- (green) foci in ILO1 cells carrying plasmids pWX6, mR1tetO240, and pPrTsLWCKid (Kid) and cultured at 42 °C for the indicated times. Values are normalized against those in parallel control samples carrying pWX6, mR1tetO240, and empty pPrTsLWC (Ctrl). Representative images of cells at different time points are shown. (B) Relative abundance of oriC-ter (OT) phenotypes observed in ILO6 cells carrying plasmids pWX6 and pPrTsLWC (Ctrl), after culturing them at 42 °C for the indicated times. Images of representative phenotypes in this sample are shown, and the average number of oriC (green) and ter (red) foci per cell, as well as oriC/ter ratios per cell and time point, are indicated. (C) Same as in B but in cells carrying pWX6 and pPrTsLWCKid. Average numbers of foci result from dividing the total number of each type of foci by the total number of cells (minimum of 600) analyzed per sample and time point.
Fig. 5.
Fig. 5.
Kid inhibits septum formation and divisome assembly in E. coli. (A) scanning EM (SEM) and transmission EM (TEM) images of 4–5 μm-long DH10B cells carrying either pPrTsHC and pPrTsLWC (Ctrl), pPrTsHC and pPrTsLWCKid (Kid), or pPrTsLWCKid and pPrTsHCKis (KidKis), and cultured at 42 °C for 2 h before analysis. The presence or absence of septum in cells is indicated with green and red arrows, respectively. (B) Primer extension of ftsZ-egfp and zapA-egfp mRNAs isolated from DH4FZGFP and DH4ZAGFP cells carrying plasmid pPrTsMCKid (Kid) or its parental control vector pPrTsMC (Ctrl), cultured at 42 °C for 30 min before analysis. Red arrows mark mRNA cleavage products, with the sequence cleaved shown on the axis. (C) Time-lapse bright-field (Top) and fluorescent (Middle) images of DH4FZGFP cells carrying pPrTsMCKid (Kid) or its parental control vector pPrTsMC (Ctrl). The images correspond to the same cells, imaged before (0′) and after (90′) placing them at 42 °C. (Bottom) Magnified views of cells stained with membrane dye FM4-64 (red) and demonstrating the distribution of FtsZ-EGFP (green) in Ctrl and Kid samples at the 90′ time point are also shown. (D) Same as in C but using DH4ZAGFP cells to examine ZapA-EGFP. (Scale bar: 2 μm.)
Fig. 6.
Fig. 6.
Kis-kid and hok-sok function sequentially and coordinately to facilitate survival of plasmid R1. When R1 CNs ensure plasmid transmission to both descendant cells, Kid remains neutralized and parental cells proliferate normally (1). However, Kid becomes active when R1 copy numbers are insufficient to warrant plasmid transmission to both daughter cells. This arrests parental cell division and stimulates RepA synthesis to rescue the plasmid. Once the latter is accomplished, the system resets, allowing host cells to proliferate again (2). If rescue fails, plasmid-free cells arise, and they are killed by Hok (3). White circles represent R1; light blue, red, yellow, and black spheres represent Kis, Kid, RepA, and Hok, respectively; and green rings and dark blue rods represent the divisome and the nucleoid, respectively.

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