Post-segregational Killing Mechanisms Research Articles

Two New Plasmid Post-segregational Killing Mechanisms for the Implementation of Synthetic Gene Networks in Escherichia coli

SummaryPlasmids are the workhorse of both industrial biotechnology and synthetic biology, but ensuring they remain in bacterial cells is a challenge. Antibiotic selection cannot be used to stabilize plasmids in most real-world applications, and inserting dynamical gene networks into the genome remains challenging. Plasmids have evolved several mechanisms for stability, one of which, post-segregational killing (PSK), ensures that plasmid-free cells do not survive. Here we demonstrate the plasmid-stabilizing capabilities of the axe/txe toxin-antitoxin system and the microcin-V bacteriocin system in the probiotic bacteria Escherichia coli Nissle 1917 and show that they can outperform the commonly used hok/sok. Using plasmid stability assays, automated flow cytometry analysis, mathematical models, and Bayesian statistics we quantified plasmid stability in vitro. Furthermore, we used an in vivo mouse cancer model to demonstrate plasmid stability in a real-world therapeutic setting. These new PSK systems, plus the developed Bayesian methodology, will have wide applicability in clinical and industrial biotechnology.

MazEF homologous Modules System and A Post-segregational killing Mechanism (Bacteriostatic & Bactericidal Mechanism) of Novel Compound Isolated from Spondias monbinon Escherichia Coli and Bacillus Subtilis

MazEF homologous Modules System and A Post-segregational killing Mechanism (Bacteriostatic & Bactericidal Mechanism) of Novel Compound Isolated from Spondias monbinon Escherichia Coli and Bacillus Subtilis

A Post-segregational Killing Mechanism for Maintaining Plasmid PMF1 in Its Myxococcus fulvus Host.

Although plasmids provide additional functions for cellular adaptation to the environment, they also create a metabolic burden, which causes the host cells to be less competitive with their siblings. Low-copy-number plasmids have thus evolved several mechanisms for their long-term maintenance in host cells. pMF1, discovered in Myxococcus fulvus 124B02, is the only endogenous autonomously replicated plasmid yet found in myxobacteria. Here we report that a post-segregational killing system, encoded by a co-transcriptional gene pair of pMF1.19 and pMF1.20, is involved in maintaining the pMF1 plasmid in its host cells. We demonstrate that the protein encoded by pMF1.20 is a new kind of nuclease, which is able to cleave DNA in vitro. The nuclease activity can be neutralized by the protein encoded by pMF1.19 through protein–protein interaction, suggesting that the protein is an immune protein for nuclease cleavage. We propose that the post-segregational killing mechanism of the nuclease toxin and immune protein pair encoded by pMF1.20 and pMF1.19 is helpful for the stable maintenance of pMF1 in M. fulvus cells.

Type II Toxin-Antitoxin Systems in the Unicellular Cyanobacterium Synechocystis sp. PCC 6803.

Bacterial toxin–antitoxin (TA) systems are genetic elements, which are encoded by plasmid as well as chromosomal loci. They mediate plasmid and genomic island maintenance through post-segregational killing mechanisms but may also have milder effects, acting as mobile stress response systems that help certain cells of a population in persisting adverse growth conditions. Very few cyanobacterial TA system have been characterized thus far. In this work, we focus on the cyanobacterium Synechocystis 6803, a widely used model organism. We expand the number of putative Type II TA systems from 36 to 69 plus seven stand-alone components. Forty-seven TA pairs are located on the chromosome and 22 are plasmid-located. Different types of toxins are associated with various antitoxins in a mix and match principle. According to protein domains and experimental data, 81% of all toxins in Synechocystis 6803 likely exhibit RNase activity, suggesting extensive potential for toxicity-related RNA degradation and toxin-mediated transcriptome remodeling. Of particular interest is the Ssr8013–Slr8014 system encoded on plasmid pSYSG, which is part of a larger defense island or the pSYSX system Slr6056–Slr6057, which is linked to a bacterial ubiquitin-like system. Consequently, Synechocystis 6803 is one of the most prolific sources of new information about these genetic elements.

Toxin-Antitoxin Systems on the Large Defense Plasmid pSYSA of Synechocystis sp. PCC 6803

Bacterial toxin-antitoxin (TA) systems are genetic elements, which are encoded by plasmid as well as chromosomal loci and mediate plasmid and genomic island maintenance through post-segregational killing mechanisms. TA systems exist in surprisingly high numbers in all prokaryotes, but cyanobacterial TA systems have been only very poorly experimentally characterized so far. Cyanobacteria are the only prokaryotes that perform oxygenic photosynthesis. As such, cyanobacteria are of high ecological importance and are considered promising for the production of biofuels. Here, we present the molecular characterization of the sll7003/ssl7004 TA system encoded on plasmid pSYSA of the model cyanobacterium Synechocystis sp. PCC 6803 as involving a Mg(2+)-dependent RNA endonuclease activity targeting single-stranded RNA regions and demonstrate the functionality of four more TA systems encoded on this 100,749-bp plasmid. Furthermore, one additional type I, one additional type II, and three freestanding TA system components are predicted on pSYSA, all of which appear active judged by their expression. By harboring at least seven simultaneously active TA systems, pSYSA appears as the plasmid most strongly selected for among all plasmids studied in this respect thus far. These results point to a high biological relevance of pSYSA, whose coding capacity is 75% devoted to three distinct clustered regularly interspaced short palindromic repeats (CRISPR) systems mediating antiviral defense.

The hok mRNA family

The hok/sok toxin-antitoxin system of Escherichia coli plasmid R1 increases plasmid maintenance by killing plasmid-free daughter cells. The hok/sok locus specifies two RNAs: hok mRNA, which encodes a toxic transmembrane protein, and sok antisense RNA, which binds a complementary region in the hok mRNA and induces transcript degradation. During cell growth, the cis-encoded sok RNA inhibits expression of the Hok toxin. In plasmid-free segregants, the rapid decay of sok RNA relative to hok mRNA permits Hok translation, leading to cell death. This post-segregational killing mechanism relies upon the ability of the hok mRNA to adopt alternative structural configurations, which affect ease of translation and the susceptibility of the molecule to degradation. The full-length hok transcript is stable, highly structured and immune to ribosome and antisense RNA binding. Gradual 3′ end processing produces dramatic structural rearrangements in the mRNA, which render the molecule translationally active and expose the sok RNA binding site. During transcription, premature ribosome and sok binding are prevented through the formation of transient metastable hairpins in the 5′ end of the nascent transcript. Several hok mRNA paralogs have been identified in the genome of E. coli, and Hok protein orthologs found in the genomes of Enterobacteria. Using a combination of automated search and extensive manual editing, we compiled a multiple sequence alignment for the hok mRNA. All three experimentally validated hok mRNA structures are mapped onto this alignment, which has been submitted to the Rfam database for RNA families.

Stability of EcoRI restriction-modification enzymes in vivo differentiates the EcoRI restriction-modification system from other postsegregational cell killing systems.

Certain type II restriction modification gene systems can kill host cells when these gene systems are eliminated from the host cells. Such ability to cause postsegregational killing of host cells is the feature of bacterial addiction modules, each of which consists of toxin and antitoxin genes. With these addiction modules, the differential stability of toxin and antitoxin molecules in cells plays an essential role in the execution of postsegregational killing. We here examined in vivo stability of the EcoRI restriction enzyme (toxin) and modification enzyme (antitoxin), the gene system of which has previously been shown to cause postsegregational host killing in Escherichia coli. Using two different methods, namely, quantitative Western blot analysis and pulse-chase immunoprecipitation analysis, we demonstrated that both the EcoRI restriction enzyme and modification enzyme are as stable as bulk cellular proteins and that there is no marked difference in their stability. The numbers of EcoRI restriction and modification enzyme molecules present in a host cell during the steady-state growth were estimated. We monitored changes in cellular levels of the EcoRI restriction and modification enzymes during the postsegregational killing. Results from these analyses together suggest that the EcoRI gene system does not rely on differential stability between the toxin and the antitoxin molecules for execution of postsegregational cell killing. Our results provide insights into the mechanism of postsegregational killing by restriction-modification systems, which seems to be distinct from mechanisms of postsegregational killing by other bacterial addiction modules.

Proteic toxin-antitoxin, bacterial plasmid addiction systems and their evolution with special reference to the pas system of pTF-FC2

Genes encoding toxin-antitoxin proteins are frequently found on plasmids where they serve to stabilize the plasmid within a bacterial population. The toxin-antitoxin proteins do not increase the likelihood of a progeny cell receiving a plasmid but rather function as post-segregational killing mechanisms which decrease the proportion of cells that survive after losing the plasmid. These toxin-antitoxin couples therefore act as plasmid addiction systems. Several new proteic toxin-antitoxin systems have been identified and these systems appear to be ubiquitous on the chromosomes of bacteria and archaea. When placed on plasmids, these chromosomal systems also have the ability to stabilize plasmids and in at least one case, chromosomal- and plasmid-based toxin-antitoxin systems have been shown to interact. Recent findings regarding toxin-antitoxin systems and questions that have arisen as a result of these findings are reviewed.

Proteic toxin-antitoxin, bacterial plasmid addiction systems and their evolution with special reference to the pas system of pTF-FC2.

Genes encoding toxin-antitoxin proteins are frequently found on plasmids where they serve to stabilize the plasmid within a bacterial population. The toxin-antitoxin proteins do not increase the likelihood of a progeny cell receiving a plasmid but rather function as post-segregational killing mechanisms which decrease the proportion of cells that survive after losing the plasmid. These toxin-antitoxin couples therefore act as plasmid addiction systems. Several new proteic toxin-antitoxin systems have been identified and these systems appear to be ubiquitous on the chromosomes of bacteria and archaea. When placed on plasmids, these chromosomal systems also have the ability to stabilize plasmids and in at least one case, chromosomal- and plasmid-based toxin-antitoxin systems have been shown to interact. Recent findings regarding toxin-antitoxin systems and questions that have arisen as a result of these findings are reviewed.

Isolation of a Derivative ofEscherichia coli–Enterococcus faecalisShuttle Vector pAM401 Temperature Sensitive for Maintenance inE. faecalisand Its Use in Evaluating the Mechanism of pAD1par-Dependent Plasmid Stabilization

A derivative of theEscherichia coli–Enterococcus faecalisshuttle vector pAM401 was isolated by mutagenesis in anE. colimutator strain. This plasmid, designated pAM401ts, was more than an order of magnitude less stable at 38°C than at 30°C in theE. faecalishost strain JH2-2. TheE. faecalisplasmid pAD1-encodedparstability locus was cloned onto pAM401ts, and its effects on plasmid stability and host cell viability were assessed. It was found thatparstabilized pAM401ts at 38°C but also caused a substantial drop in cell viability three to four generations after a temperature shift from 30 to 38°C. After a maximum viability drop of 94%, culture growth recovered as plasmid-free cells began to accumulate. Provision of excess RNAII, the putativeparantidote,in transattenuated cell killing. These characteristics support a postsegregational killing mechanism forpar-mediated plasmid stabilization.

Contribution of different segments of the par region to stable maintenance of the broad-host-range plasmid RK2.

A 3.2-kb region of the broad-host-range plasmid RK2 has been shown to encode a highly efficient plasmid maintenance system that functions in a vector-independent manner. This region, designated par, consists of two divergently arranged operons: parCBA and parDE. The 0.7-kb parDE operon promotes plasmid stability by a postsegregational killing mechanism that ensures that plasmid-free daughter cells do not survive after cell division. The 2.3-kb parCBA operon encodes a site-specific resolvase protein (ParA) and its multimer resolution site (res) and two proteins (ParB and ParC) whose functions are as yet unknown. It has been proposed that the parCBA operon encodes a plasmid partitioning system (M. Gerlitz, O. Hrabak, and H. Schwabb, J. Bacteriol. 172:6194-6203, 1990; R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). To further define the role of this region in promoting the stable maintenance of plasmid RK2, the parCBA and parDE operons separately and the intact (parCBA/DE) par region (3.2 kb) were reintroduced into an RK2 plasmid deleted for par and assayed for plasmid stability in two Escherichia coli strains (MC1061K and MV10delta lac). The intact 3.2-kb region provided the highest degree of stability in the two strains tested. The ability of the parCBA or parDE region alone to promote stable maintenance in the E. coli strains was dependent on the particular strain and the growth temperature. Furthermore, the insertion of the ColE1 cer site into the RK2 plasmid deleted for the par region failed to stabilize the plasmid in the MC1061K strain, indicating that the multimer resolution activity encoded by parCBA is not by itself responsible for the stabilization activity observed for this operon. To examine the relative contributions of postsegregational cell killing and a possible partitioning function encoded by the intact 3.2-kb par region, stability assays were carried out with ParD provided in trans by a compatible (R6K) minireplicon to prevent postsegregational killing. In E. coli MV10delta lac, postsegregational killing appeared to be the predominant mechanism for stabilization since the presence of ParD substantially reduced the stability of plasmids carrying either the 3.2- or 0.7-kb region. However, in the case of E. coli MC1061K, the presence of ParD in trans did not result in a significant loss of stabilization by the 3.2-kb region, indicating that the putative partitioning function was largely responsible for RK2 maintenance. To examine the basis for the apparent differences in postsegregational killing between the two E. coli strains, transformation assays were carried out to determine the relative sensitivities of the strains to the ParE toxin protein. Consistent with the relatively small contribution of the postsegregational killing to plasmid stabilization in MC1061K, we found that this strain was substantially more resistant to killing by ParE in comparison to E. coli MV10delta lac. A transfer-deficient mutant of thepar-deleted plasmid was constructed for the stable maintenance studies. This plasmid was found to be lost from E. coli MV10delta lac at a rate three times greater than the rate for the transfer-proficient plasmid, suggesting that conjugation can also play a significant role in the maintenance of plasmid RK2.

Plasmid maintenance functions of the large virulence plasmid of Shigella flexneri.

The large virulence plasmid pMYSH6000 of Shigella flexneri contains a replicon and a plasmid maintenance stability determinant (Stb) on adjacent SalI fragments. The presence of a RepFIIA replicon on the SalI C fragment was confirmed, and the complete sequence of the adjacent SalI O fragment was determined. It shows homology to part of the transfer (tra) operon of the F plasmid. Stb stabilizes a partition-defective P1 miniplasmid in Escherichia coli. A 1.1-kb region containing a homolog of the F trbH gene was sufficient to confer stability. However, the trbH open reading frame could be interrupted without impairing stability. Deletion analysis implicated the involvement of two small open reading frames, STBORF1 and STBORF2, that fully overlap trbH in the opposite direction. These open reading frames are closely related to the vagC and vagD genes of the Salmonella dublin virulence plasmid and to open reading frame pairs in the F trbH region and in the chromosomes of Dichelobacter nodosus and Haemophilus influenzae. Stb appears to promote better-than-random distribution of plasmid copies and is a plasmid incompatibility determinant. The F homolog does not itself confer stability but exerts incompatibility against the activity of the Stb system. Stb is likely to encode either an active partition system or a postsegregational killing system. It shows little similarity to previously studied plasmid stability loci, but the genetic organization of STBORF1 and STBORF2 resembles that of postsegregational killing mechanisms.

Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein

The parDE operon, located within the 3.2-kb stabilization region of plasmid RK2, encodes antitoxin (ParD) and toxin (ParE) proteins that stabilize the maintenance of this broad-host-range plasmid via a postsegregational killing mechanism. A ParE protein derivative, designated ParE', was purified by construction of a fusion protein, GST-ParE, followed by glutathione-agarose binding and cleavage of the fusion protein. ParE' has three additional amino acids on the N terminus and a methionine residue in place of the native leucine residue. The results of glutathione-agarose affinity binding and glutaraldehyde cross-linking indicate that ParE' exists as a dimer in solution and that it binds to the dimeric form of ParD to form a tetrameric complex. The formation of this complex is presumably responsible for the ability of ParD to neutralize ParE toxin activity. Previous studies demonstrated that the parDE operon is autoregulated as a result of the binding of the ParD protein to the parDE promoter. ParE' also binds to the parDE promoter but only in the presence of the autoregulatory ParD protein. ParE', in the presence or absence of the ParD protein, does not bind to any other part of the 3.2-kb stabilization region. The binding of the ParE' protein to ParD did not alter the DNase I footprint pattern obtained as a result of ParD binding to the parDE promoter. The role of ParE in binding along with ParD to the promoter, if any, remains unclear.

Mechanism of Post-segregational Killing: Secondary Structure Analysis of the Entire Hok mRNA from Plasmid R1 Suggests a Fold-back Structure that Prevents Translation and Antisense RNA Binding

Thehok/soksystem of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The Hok mRNA is very stable and can be translated into Hok killer protein. Translation of the Hok mRNA is inhibited by the small unstable Sok antisense RNA. Translation ofhokis coupled to an overlapping reading frame termedmok. Translation ofmokis tightly regulated by Sok RNA, and Sok RNA thus regulateshoktranslation indirectly throughmok. The rapid decay of Sok RNA explains the onset of Hok synthesis in newborn plasmid-free segregants. However, a second control level is superimposed on this simple induction scheme, since the full-length Hok mRNA was found to be translationally inactive whereas a 3′-end truncated version of it was active. We have therefore previously suggested, that the 3′-terminal region of the full-length Hok mRNA encodes an element which prevents its translation. This element was termedfbi(fold-back inhibition). Here we describe thein vitrosecondary structure of the entire Hok mRNA. Our results suggest a closed structure in which the 3′-end of the full-length Hok mRNA folds back onto the translational initiation region ofmok. This structure explains why full-length Hok mRNA is translationally silent. The proposed structure was further supported by results obtained using mutations in the 3′-endfbielement. These “structure closing” mutations affected the structure much further upstream in themoktranslational initiation region and concomitantly prevented antisense RNA binding to the same region of the mRNA. These results lend further support to the induction model that explains onset of Hok mRNA translation in plasmid-free segregants. The most important regulatory element in this model is the FBI structure formed between the 3′-end and themoktranslational initiation region. This structure renders Hok mRNA translationally inactive and prevents antisense RNA binding, thus allowing the accumulation of a pool of mRNA which, by slow 3′-end processing, is activated in plasmid-free segregants, eventually leading to the elimination of these cells.

Mechanism of post-segregational killing by hok-homologue pnd of plasmid R483: Two translational control elements in the pnd mRNA

The pnd system of plasmid R483 mediates plasmid stabilization by killing of plasmid-free cells. The pnd mRNA is very stable and can be translated into PndA protein, a cell toxin which kills the cells from within by damaging the cell membrane. Translation of the pnd mRNA is inhibited by the PndB antisense, a small labile RNA of 63 nt. The rapid decay of the PndB antidote leads to onset of PndA synthesis in plasmid-free segregants or after addition of rifampicin. Surprisingly however, the full-length pnd mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was found to be active. We have therefore suggested previously, that the 3'-end of the full-length pnd mRNA encodes a fold-back inhibitory sequence (fbi), which prevents its translation. Here we present an analysis of the metabolism of the pnd mRNAs. A mutational analysis shows that single point mutations in the fbi motif results in more rapid truncation. The fbi mutations could not be complemented by second-site mutations in either of the pndA or pndC Shine-Dalgarno (SD) elements. Surprisingly, mutations in the pndC SD element also lead to a more rapid truncation. The effect of these latter mutations was, however, complemented by mutations in a proposed anti-SD element upstream of the pndC SD. Mutations in the anti-SD element were lethal. These results show, that the pnd mRNA contains two negative control elements, one located in its very 3'-end (fbi), and one located just upstream of the pndC SD region (the anti-SD element). These observations add to the complexity of the induction scheme previously proposed to explain activation of pndA expression in plasmid-free cells: In addition to its negative effect of translation, the fbi structure also maintains a reduced processing rate in the 3'-end of the mRNA. This permits the accumulation of a reservoir of pnd mRNA, which can be activated by 3'-end processing in plasmid-free cells. The anti-SD may prevent translation of the pnd mRNA during transcription, thus preventing detrimental synthesis of toxin.

Mechanism of post-segregational killing: translation of Hok, SrnB and Pnd mRNAs of plasmids R1, F and R483 is activated by 3'-end processing.

The gene systems hok/sok of R1, srnB of F and pnd of R483 mediate plasmid maintenance by killing of plasmid-free segregants. Translation of the very stable mRNAs encoding the killer proteins is regulated by small unstable antisense RNAs. The differential decay rates of the inhibitory antisense RNAs and the mRNAs encoding the killer proteins is the basis for the onset of killer mRNA translation in newborn plasmid-free segregants and the killing of these cells. We have suggested previously that this requires that the killer mRNAs occur in two forms. A translationally inactive form was proposed to be converted into a 3'-truncated, translationally active mRNA. In the presence of the antisense RNA, translation from this killer mRNA should be inhibited. In this communication we present in vivo and in vitro evidence that support this model. The requirement for 3'-processing for killer gene expression is demonstrated. By using in vitro techniques it is shown that full-length Hok mRNA is translationally inactive, whereas a 3'-end truncated version of the Hok mRNA is translationally active. In vitro secondary structure probing suggests that the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of the mok gene and thereby inhibits translation of the mRNA. By inference we conclude that the Pnd and SrnB mRNAs are regulated by a similar mechanism.

Mechanism of post-segregational killing: Sok antisense RNA interacts with Hok mRNA via its 5'-end single-stranded leader and competes with the 3'-end of Hok mRNA for binding to the mok translational initiation region.

The hok/sok system of plasmid R1, which mediates plasmid stabilization by killing of plasmid-free segregants, codes for two RNA species, Hok mRNA and Sok antisense RNA. The lethal expression of hok is inhibited post-transcriptionally by the 67 nt Sok-RNA. In this paper, we analyse the secondary structure of Sok-RNA and the binding of Sok-RNA to Hok mRNA in vitro. The reaction between the two RNAs leads to the formation of a complete duplex in which Sok-RNA is hybridized over its entire length to Hok mRNA. The second-order rate constant of duplex formation was determined to be approximately 1 x 10(5) M-1s-1. Mutations in the 5'-end single-stranded leader of Sok-RNA severely reduced the binding rate to wt Hok mRNA, whereas loop mutations in Sok-RNA had no such effect. The reduced binding rates were paralleled by abolished in vivo regulatory properties. These results suggest that, unlike in other well-characterized antisense/target RNA systems, the initial recognition reaction between Sok-RNA and Hok mRNA takes place between the single-stranded 5'-end of Sok-RNA and the complementary region in Hok mRNA, without the involvement of an antisense loop in the initial binding step. Furthermore, the finding that Sok-RNA competes with the 3'-end of full-length Hok mRNA for binding to the mok translational initiation region adds to the complexity of killer gene regulation.

Mechanism of post-segregational killing by the hok/sok system of plasmid R1 : Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene

The hok/sok locus of plasmid R1, which mediates plasmid stabilization by killing of plasmid-free segregants, codes for two RNAs, Hok mRNA and Sok antisense RNA. Hok mRNA encodes the Hok killer protein of 52 amino acid residues. Expression of hok is regulated post-transcriptionally by Sok antisense RNA. Killing of plasmid-free daughter-cells by the hok/sok system is accomplished through differential decay of the Hok and Sok-RNAs: Hok mRNA is very stable while Sok-RNA decays rapidly, thus leading to derepression of Hok mRNA translation in plasmid-free segregants, ensuring a rapid and selective killing of these cells. Sok antisense RNA is complementary to the leader region of the Hok mRNA. However, the region of complementarity does not overlap with the hok Shine-Dalgarno sequence. Thus, Sok-RNA regulates hok translation indirectly by an as yet unknown mechanism. We show here that Sok antisense RNA regulates the translation of another reading frame located in the hok/sok locus. This new reading frame, which overlaps with almost the entire hok gene, was denoted mok ( mediation of killing). Point-mutations that prevent mok translation through the hok translational initiation region abolish efficient expression of hok. Furthermore, these mutations abolish the Sok-RNA-mediated control of hok gene expression. Hence, the antisense-RNA-mediated regulation of the hok gene seems to occur via translational coupling between the hok and mok reading-frames.

The parB (hok/sok) Locus of Plasmid R1: A General Purpose Plasmid Stabilization System

The parB locus of plasmid R1, comprising at most 580 base pairs of DNA, mediates efficient plasmid stabilization via postsegregational killing of plasmid free cells. The locus encodes two small genes, hok and sok. The hok gene product is a potent cell killing protein, the expression of which is regulated by the sok product, an anti-sense RNA complementary to the hok mRNA. The hok mRNA is extraordinarily stable, while the sok-RNA is rapidly degraded. The mechanism of postsegregational killing is explained by the differential decay of the hok and sok-RNA's: In newborn plasmid free cells the prolonged persistence of the hok mRNA leads to synthesis of the Hok protein, thus ensuring a rapid and selective killing of these cells. As predicted from this simple model, any unstably inherited plasmid should become stabilized by carrying the parB locus. This important prediction was confirmed by testing a variety of different replicons. Even plasmids replicating in as distantly related organisms as Eschericia coli, Serratia marsescense, and Pseudomonas putida were efficiently stabilized. The parB locus therefore constitutes a convenient and efficient plasmid stabilization casette, useful in many gram negative species.

Translational control and differential RNA decay are key elements regulating postsegregational expression of the killer protein encoded by the parB locus of plasmid R1

The parB locus of plasmid R1, which mediates plasmid stability via postsegregational killing of plasmid-free cells, encodes two genes, hok and sok. The hok gene product is a potent cell-killing protein. The hok gene is regulated at the translational level by the sok gene-encoded repressor, a small anti-sense RNA complementary to the hok mRNA. The hok mRNA is extraordinarily stable, while the sok RNA decays rapidly. The mechanism of postsegregational killing is explained by the following model; the sok RNA molecule rapidly disappears in cells that have lost a parB-carrying plasmid, leading to translation of the stable hok mRNA. Consequently, the Hok protein is synthesized and killing of the plasmid-free cell follows.