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.     

Cellular responses to postsegregational killing by restriction-modification genes

Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA.     

The three vibrio cholerae chromosome II-encoded ParE toxins degrade chromosome I following loss of chromosome II

Three homologues of the plasmid RK2 ParDE toxin-antitoxin system are present in the Vibrio cholerae genome within the superintegron on chromosome II. Here we found that these three loci-two of which have identical open reading frames and regulatory sequences-encode functional toxin-antitoxin systems. The ParE toxins inhibit bacterial division and reduce viability, presumably due to their capacity to damage DNA. The in vivo effects of ParE1/3 mimic those of ParE2, which we have previously demonstrated to be a DNA gyrase inhibitor in vitro, suggesting that ParE1/3 is likewise a gyrase inhibitor, despite its relatively low degree of sequence identity. ParE-mediated DNA damage activates the V. cholerae SOS response, which in turn likely accounts for ParE's inhibition of cell division. Each toxin's effects can be prevented by the expression of its cognate ParD antitoxin, which acts in a toxin-specific fashion both to block toxicity and to repress the expression of its parDE operon. Derepression of ParE activity in ΔparAB2 mutant V. cholerae cells that have lost chromosome II contributes to the prominent DNA degradation that accompanies the death of these cells. Overall, our findings suggest that the ParE toxins lead to the postsegregational killing of cells missing chromosome II in a manner that closely mimics postsegregational killing mediated by plasmid-encoded homologs. Thus, the parDE loci aid in the maintenance of the integrity of the V. cholerae superintegron and in ensuring the inheritance of chromosome II.     

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.     

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

"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.     

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.     

Structural and functional analysis of the kid toxin protein from E. coli plasmid R1

We have determined the structure of Kid toxin protein from E. coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid-less daughter cells. Kid forms a two-component system with its antagonist, Kis antitoxin. Our 1.4 A crystal structure of Kid reveals a 2-fold symmetric dimer that closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli F plasmid despite the lack of any notable sequence similarity. Analysis of nontoxic mutants of Kid suggests a target interaction interface associated with toxicity that is in marked contrast to that proposed for CcdB. A possible region for interaction of Kid with the antitoxin is proposed that overlaps with the target binding site and may explain the mode of antitoxin action.     

Characterization of Bacillus subtilis clones surviving overproduction of Zeta, a pSM19035 plasmid-encoded toxin

The postsegregational killing system of pSM19035 plasmid consists of the proteins Zeta and Epsilon, a toxin and an antidote, respectively. Zeta mutants were isolated with the use of Bacillus subtilis strain with the zeta gene under control of an inducible promoter integrated into the chromosome. Results of mutant analysis point to the amino terminal part of the Zeta protein as being responsible for the toxicity.     

Functional interactions between coexisting toxin-antitoxin systems of the ccd family in Escherichia coli O157:H7

Toxin-antitoxin (TA) systems are widely represented on mobile genetic elements as well as in bacterial chromosomes. TA systems encode a toxin and an antitoxin neutralizing it. We have characterized a homolog of the ccd TA system of the F plasmid (ccd(F)) located in the chromosomal backbone of the pathogenic O157:H7 Escherichia coli strain (ccd(O157)). The ccd(F) and the ccd(O157) systems coexist in O157:H7 isolates, as these pathogenic strains contain an F-related virulence plasmid carrying the ccd(F) system. We have shown that the chromosomal ccd(O157) system encodes functional toxin and antitoxin proteins that share properties with their plasmidic homologs: the CcdB(O157) toxin targets the DNA gyrase, and the CcdA(O157) antitoxin is degraded by the Lon protease. The ccd(O157) chromosomal system is expressed in its natural context, although promoter activity analyses revealed that its expression is weaker than that of ccd(F). ccd(O157) is unable to mediate postsegregational killing when cloned in an unstable plasmid, supporting the idea that chromosomal TA systems play a role(s) other than stabilization in bacterial physiology. Our cross-interaction experiments revealed that the chromosomal toxin is neutralized by the plasmidic antitoxin while the plasmidic toxin is not neutralized by the chromosomal antitoxin, whether expressed ectopically or from its natural context. Moreover, the ccd(F) system is able to mediate postsegregational killing in an E. coli strain harboring the ccd(O157) system in its chromosome. This shows that the plasmidic ccd(F) system is functional in the presence of its chromosomal counterpart.     

Phenylketonuria missense mutations in the Mediterranean.

Two missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of an Italian phenylketonuria (PKU) patient. Both mutations occurred in exon 7 of the PAH gene, resulting in the substitution of Trp for Arg at amino acid 252 (R252W) and of Leu for Pro (P281L) at amino acid 281 of the protein. Expression vectors containing either the normal human PAH cDNA or mutant cDNAs were constructed and transfected into cultured mammalian cells. Extracts from cells transfected with either mutant construct showed negligible enzyme activity and undetectable levels of immunoreactive PAH protein as compared to the normal construct. These results are compatible with the severe classical PKU phenotype observed in this patient. Population genetic studies in the Italian population revealed that both the R252W and the P281L mutations are in linkage disequilibrium with mutant restriction fragment length polymorphism (RFLP) haplotype 1, which is the most prevalent RFLP haplotype in this population. The R252W mutation is present in 10% and the P281L mutation is present in 20% of haplotype 1 mutant chromosomes. These mutations are both very rare among other European populations, suggesting a Mediterranean origin for these mutant chromosomes.     

Mutation in the glmS gene responsible for the cell wall synthesis increases resistance to the herbicide amitrole in the cyanobacterium Synechocystis sp. PCC6803

A DNA fragment transforming the cells of the cyanobacterium Synechocystis sp. PCC 6803 to amitrole (3-amino-1,2,4-triazole) resistance was cloned from the Atr2 mutant resistant to this herbicide. The transforming activity of the cloned fragment was shown to be related to the missence-mutation "Val250-->Leu250" in the glmS gene encoding glucosamine-6-phosphate synthase, a key enzyme of cell wall synthesis. The amino acid substitution is localized in the central nonconservative part of the GlmS protein, far from two reaction centers positioned at the ends of a polypeptide. It is suggested that the mutant protein has lost sensitivity to amitrole. In the wild type strain, this herbicide causes conditional glucosamine auxotrophy (exogenous glucosamine restores ability of the cells to row in the presence of the lethal amitrole concentrations). Val250 is proposed to be allosteric binding site of AM in the GlmS protein of cyanobacterium.     

Mechanism for oxidation of high-molecular-weight substrates by a fungal versatile peroxidase, MnP2

Unlike general peroxidases, Pleurotus ostreatus MnP2 was reported to have a unique property of direct oxidization of high-molecular-weight compounds, such as Poly R-478 and RNase A. To elucidate the mechanism for oxidation of polymeric substrates by MnP2, a series of mutant enzymes were produced by using a homologous gene expression system, and their reactivities were characterized. A mutant enzyme with an Ala substituting for an exposing Trp (W170A) drastically lost oxidation activity for veratryl alcohol (VA), Poly R-478, and RNase A, whereas the kinetic properties for Mn(2+) and H(2)O(2) were substantially unchanged. These results demonstrated that, in addition to VA, the high-molecular-weight substrates are directly oxidized by MnP2 at W170. Moreover, in the mutants Q266F and V166/168L, amino acid substitution(s) around W170 resulted in a decreased activity only for the high-molecular-weight substrates. These results, along with the three-dimensional modeling of the mutants, suggested that the mutations caused a steric hindrance to access of the polymeric substrates to W170. Another mutant, R263N, contained a newly generated N glycosylation site and showed a higher molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Interestingly, the R263N mutant exhibited an increased reactivity with VA and high-molecular-weight substrates. The existence of an additional carbohydrate modification and the catalytic properties in this mutant are discussed. This is the first study of a direct mechanism for oxidation of high-molecular-weight substrates by a fungal peroxidase using a homologous gene expression system.     

Substitution of Glu-59 by val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme

A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70–80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M _r of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.     

A continuous fluorometric assay for the assessment of MazF ribonuclease activity

Plasmids maintain themselves in their bacterial host through several different mechanisms, one of which involves the synthesis of plasmid-encoded toxin and antitoxin proteins. When the plasmid is present, the antitoxin binds to and neutralizes the toxin. If a plasmid-free daughter cell arises, however, the labile antitoxin is degraded (and not replenished) and the toxin kills the cell from within. These toxin-antitoxin (TA) systems thereby function as postsegregational killing systems, and the disruption of the TA interaction represents an intriguing antibacterial strategy. It was recently discovered that the genes for one particular TA system, MazEF, are ubiquitous on plasmids isolated from clinical vancomycin-resistant enterococci (VRE) strains. Thus, it appears that small molecule disruptors of the MazEF interaction have potential as antibacterial agents. The MazF toxin protein is known to be a ribonuclease. Unfortunately, traditional methods for the assessment of MazF activity rely on the use of radiolabeled substrates followed by analysis with polyacrylamide gel electrophoresis. This article describes a simple and convenient continuous assay for the assessment of MazF activity. The assay uses an oligonucleotide with a fluorophore on the 5' end and a quencher on the 3' end, and processing of this substrate by MazF results in a large increase in the fluorescence signal. Through this assay, we have for the first time determined K(M) and V(max) values for this enzyme and have also found that MazF is not inhibited by standard ribonuclease inhibitors. This assay will be useful to those interested in the biochemistry of the MazF family of toxins and the disruption of MazE/MazF.     

N-octyl-beta-valienamine up-regulates activity of F213I mutant beta-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease

Gaucher disease (GD) is the most common form of sphingolipidosis and is caused by a defect of beta-glucosidase (beta-Glu). A carbohydrate mimic N-octyl-beta-valienamine (NOV) is an inhibitor of beta-Glu. When applied to cultured GD fibroblasts with F213I beta-Glu mutation, NOV increased the protein level of the mutant enzyme and up-regulated cellular enzyme activity. The maximum effect of NOV was observed in F213I homozygous cells in which NOV treatment at 30 microM for 4 days caused a approximately 6-fold increase in the enzyme activity, up to approximately 80% of the activity in control cells. NOV was not effective in cells with other beta-Glu mutations, N370S, L444P, 84CG and RecNciI. Immunofluorescence and cell fractionation showed localization of the F213I mutant enzyme in the lysosomes of NOV-treated cells. Consistent with this, NOV restored clearance of 14C-labeled glucosylceramide in F213I homozygous cells. F213I mutant beta-Glu rapidly lost its activity at neutral pH in vitro and this pH-dependent loss of activity was attenuated by NOV. These results suggest that NOV works as a chemical chaperone to accelerate transport and maturation of F213I mutant beta-Glu and may suggest a therapeutic value of this compound for GD.     

Exploiting yoeB-yefM toxin-antitoxin system of Streptococcus pneumoniae on the selective killing of miR-21 overexpressing breast cancer cell line (MCF-7)

Toxin-antitoxin (TA) systems are two-component genetic modules widespread in bacterial and archaeal genomes, in which the toxin module is rendered inactive under resting conditions by its antitoxin counterpart. Under stress conditions, however, the antitoxin is degraded, freeing the toxin to exert its lethal effects. Although not evolved to function in eukaryotes, some studies have established the lethal activity of these bacterial toxins by inducing apoptosis in mammalian cells, an effect that can be neutralized by its cognate antitoxin. Inspired by the way the toxin can become active in eukaryotes cells, we produced an engrained yoeB-yefM TA system to selectively kill human breast cancer cells expressing a high level of miR-21. Accordingly, we generated an engineered yefM antitoxin gene with eight miR-21 target sites placed in its 3′untranslated region. The resulting TA system acts autonomously in human cells, distinguishing those that overexpress miR-21, killed by YoeB, from those that do not, remaining protected by YefM. Thus, we indicated that microRNA-control of the antitoxin protein of bacterial TA systems constitutes a novel strategy to enhance the selective killing of human cancer cells by the toxin module. The present study provides significant insights for developing novel anticancer strategies avoiding off-target effects, a challenge that has been pursued by many investigators over the years.     

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.