Natacha Mine

Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM-yoeB toxin-antitoxin system

In Escherichia coli, the Lon ATP‐dependent protease is responsible for degradation of several regulatory proteins and for the elimination of abnormal proteins. Previous studies have shown that the overproduction of Lon is lethal. Here, we showed that Lon overproduction specifically inhibits translation through at least two different pathways. We have identified one of the pathways as being the chromosomal yefM‐yoeB toxin‐antitoxin system. The existence of a second pathway is demonstrated by the observation that the deletion of the yefM‐yoeB system did not completely suppress lethality and translation inhibition. We also showed that the YoeB toxin induces cleavage of translated mRNAs and that Lon overproduction specifically activates YoeB‐dependent mRNAs cleavage. Indeed, none of the other identified chromosomal toxin‐antitoxin systems (relBE, mazEF, chpB and dinJ‐yafQ) was involved in Lon‐dependent lethality, translation inhibition and mRNA cleavage even though the RelB and MazE antitoxins are known to be Lon substrates. Based on our results and other studies, translation inhibition appears to be the key element that triggers chromosomal toxin‐antitoxin systems. We propose that under Lon overproduction conditions, translation inhibition is mediated by Lon degradation of a component of the YoeB‐independent pathway, in turn activating the YoeB toxin by preventing synthesis of its unstable YefM antidote.

What Is the Benefit to Escherichia coli of Having Multiple Toxin-Antitoxin Systems in Its Genome?

The Escherichia coli K-12 chromosome encodes at least five proteic toxin-antitoxin (TA) systems. The mazEF and relBE systems have been extensively characterized and were proposed to be general stress response modules. On one hand, mazEF was proposed to act as a programmed cell death system that is triggered by a variety of stresses. On the other hand, relBE and mazEF were proposed to serve as growth modulators that induce a dormancy state during amino acid starvation. These conflicting hypotheses led us to test a possible synergetic effect of the five characterized E. coli TA systems on stress response. We compared the behavior of a wild-type strain and its derivative devoid of the five TA systems under various stress conditions. We were unable to detect TA-dependent programmed cell death under any of these conditions, even under conditions previously reported to induce it. Thus, our results rule out the programmed-cell-death hypothesis. Moreover, the presence of the five TA systems advantaged neither recovery from the different stresses nor cell growth under nutrient-limited conditions in competition experiments. This casts a doubt on whether TA systems significantly influence bacterial fitness and competitiveness during non-steady-state growth conditions.

Chromosomal Toxin-Antitoxin Systems May Act as Antiaddiction Modules

Toxin-antitoxin (TA) systems are widespread among bacterial chromosomes and mobile genetic elements. Although in plasmids TA systems have a clear role in their vertical inheritance by selectively killing plasmid-free daughter cells (postsegregational killing or addiction phenomenon), the physiological role of chromosomally encoded ones remains under debate. The assumption that chromosomally encoded TA systems are part of stress response networks and/or programmed cell death machinery has been called into question recently by the observation that none of the five canonical chromosomally encoded TA systems in the Escherichia coli chromosome seem to confer any selective advantage under stressful conditions (V. Tsilibaris, G. Maenhaut-Michel, N. Mine, and L. Van Melderen, J. Bacteriol. 189:6101-6108, 2007). Their prevalence in bacterial chromosomes indicates that they might have been acquired through horizontal gene transfer. Once integrated in chromosomes, they might in turn interfere with their homologues encoded by mobile genetic elements. In this work, we show that the chromosomally encoded Erwinia chrysanthemi ccd (control of cell death) (ccd(Ech)) system indeed protects the cell against postsegregational killing mediated by its F-plasmid ccd (ccd(F)) homologue. Moreover, competition experiments have shown that this system confers a fitness advantage under postsegregational conditions mediated by the ccd(F) system. We propose that ccd(Ech) acts as an antiaddiction module and, more generally, that the integration of TA systems in bacterial chromosomes could drive the evolution of plasmid-encoded ones and select toxins that are no longer recognized by the antiaddiction module.

New toxins homologous to ParE belonging to three-component toxin-antitoxin systems in Escherichia coli O157:H7.

Type II toxin–antitoxin (TA) systems are considered as protein pairs in which a specific toxin is associated with a specific antitoxin. We have identified a novel antitoxin family (paaA) that is associated with parE toxins. The paaA–parE gene pairs form an operon with a third component (paaR) encoding a transcriptional regulator. Two paralogous paaR–paaA–parE systems are found in E. coli O157:H7. Deletions of the paaA–parE pairs in O157:H7 allowed us to show that these systems are expressed in their natural host and that PaaA antitoxins specifically counteract toxicity of their associated ParE toxin. For the paaR2–paaA2–parE2 system, PaaR2 and Paa2–ParE2 complex are able to regulate the operon expression and both are necessary to ensure complete repression. The paaR2–paaA2–parE2 system mediates ClpXP‐dependent post‐segregational killing. The PaaR2 regulator appears to be essential for this function, most likely by maintaining an appropriate antitoxin : toxin ratio in steady‐state conditions. Ectopic overexpression of ParE2 is bactericidal and is not resuscitated by PaaA2 expression. ParE2 colocalizes with the nucleoid, while it is diffusely distributed in the cytoplasm when PaaA2 is coexpressed. This indicates that ParE2 interacts with DNA‐gyrase cycling on DNA and that coexpression of PaaA2 antitoxin sequesters ParE2 away from its target by protein–protein complex formation.

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.

The decay of the chromosomally encoded ccdO157 toxin-antitoxin system in the Escherichia coli species.

The origin and the evolution of toxin–antitoxin (TA) systems remain to be uncovered. TA systems are abundant in bacterial chromosomes and are thought to be part of the flexible genome that originates from horizontal gene transfer. To gain insight into TA system evolution, we analyzed the distribution of the chromosomally encoded ccdO157 system in 395 natural isolates of Escherichia coli. It was discovered in the E. coli O157:H7 strain in which it constitutes a genomic islet between two core genes (folA and apaH). Our study revealed that the folA–apaH intergenic region is plastic and subject to insertion of foreign DNA. It could be composed (i) of a repetitive extragenic palindromic (REP) sequence, (ii) of the ccdO157 system or subtle variants of it, (iii) of a large DNA piece that contained a ccdAO157 antitoxin remnant in association with ORFs of unknown function, or (iv) of a variant of it containing an insertion sequence in the ccdAO157 remnant. Sequence analysis and functional tests of the ccdO157 variants revealed that 69% of the variants were composed of an active toxin and antitoxin, 29% were composed of an active antitoxin and an inactive toxin, and in 2% of the cases both ORFs were inactive. Molecular evolution analysis showed that ccdBO157 is under neutral evolution, suggesting that this system is devoid of any biological role in the E. coli species.

Replication functions of new broad host range plasmids isolated from polluted soils.

The nucleotide sequencing of replicons isolated from three new broad host range plasmids, pMOL98, pEMT8, and pEMT3, originating from polluted soils, showed a typical organization of iteron replicons replicating by the theta mode. In the pMOL98 replicon, the origin region and the rep gene were identified in complementation experiments. Sequence comparisons showed that the regions bearing these features are highly identical to regions in pIP02T and pSB102 and that the Rep proteins (but not the origin regions) of these three plasmids show some identity to the Rep proteins of the IncW group of plasmids. This suggests that pMOL98, pIPO2T, and pSB102 constitute a new Inc/Rep family, distantly related to the IncW group, but having an incompatibility phenotype different from the IncW phenotype. The pEMT8 replicon displayed an orf whose conceptually translated product is related to the Rep proteins of four plasmids, pSD20, pSW500, pMLb, and pALC1, not yet classified into any known incompatibility group. The vegetative origins of these plasmids were not similar, suggesting that the five plasmids could belong to a new family with similar Rep proteins but different incompatibility phenotypes. The pEMT3 replicon is clearly related to IncP replicons (sequence similarities and incompatibility phenotype), although sequence comparisons revealed some divergence with respect to the two well-documented subgroups IncPalpha and IncPbeta. This suggests that in these plasmids, despite the existence of a powerful system of centralized control over replication, maintenance, and transfer functions, plasticity and evolution of these functions are at work. Our analysis confirms the extreme genetic flexibility of plasmids and the absolute necessity of using multiple techniques (PCR, DNA sequencing, DNA chips, and databases) to analyze the role of broad host range plasmids in the capture, recombination and spread of genetic traits among bacteria.

Characterization of the phd-doc and ccd Toxin-Antitoxin Cassettes from Vibrio Superintegrons

Toxin-antitoxin (TA) systems have been reported in the genomes of most bacterial species, and their role when located on the chromosome is still debated. TA systems are particularly abundant in the massive cassette arrays associated with chromosomal superintegrons (SI). Here, we describe the characterization of two superintegron cassettes encoding putative TA systems. The first is the phd-doc(SI) system identified in Vibrio cholerae N16961. We determined its distribution in 36 V. cholerae strains and among five V. metschnikovii strains. We show that this cassette, which is in position 72 of the V. cholerae N16961 cassette array, is functional, carries its own promoter, and is expressed from this location. Interestingly, the phd-doc(SI) system is unable to control its own expression, most likely due to the absence of any DNA-binding domain on the antitoxin. In addition, this SI system is able to cross talk with the canonical P1 phage system. The second cassette that we characterized is the ccd(Vfi) cassette found in the V. fischeri superintegron. We demonstrate that CcdB(Vfi) targets DNA-gyrase, as the canonical CcB(F) toxin, and that ccd(Vfi) regulates its expression in a fashion similar to the ccd(F) operon of the conjugative plasmid F. We also establish that this cassette is functional and expressed in its chromosomal context in V. fischeri CIP 103206T. We tested its functional interactions with the ccdAB(F) system and found that CcdA(Vfi) is specific for its associated CcdB(Vfi) and cannot prevent CcdB(F) toxicity. Based on these results, we discuss the possible biological functions of these TA systems in superintegrons.

Method for concentrating and purifying recombinant autonomous parvovirus vectors designed for tumour-cell-targeted gene therapy

Recent work has highlighted the use of parvoviruses as potential vectors for tumour-cell-targeted gene therapy. The oncotropic properties of the prototype strain of minute virus of mice (MVMp) suggest that this virus might be a useful vehicle for introducing selectively therapeutic genes, e.g. lymphokine or suicide genes, into tumour cells and preferentially expressing them. But the low titre of recombinant virus stocks (10(5)-10(6) infectious units per ml) and their high level of contamination by cell proteins make it practically impossible to evaluate their efficacy in in vivo systems. A technique is described for producing cellular contaminant-free stocks of recombinant virus particles, with titres up to 5 x 10(8) IU/ml.

A novel MVMp-based vector system specifically designed to reduce the risk of replication-competent virus generation by homologous recombination.

Recent work highlights the potential usefulness of MVM-based vectors as selective vehicles for cancer gene therapy (Dupont et al, Gene Therapy, 2000; 7: 790–796). To implement this strategy, however, it is necessary to develop optimized methods for producing high-titer, helper-free parvovirus stocks. Recombinants of MVMp (rMVMp) are currently generated by transiently co-transfecting permissive cell lines with a plasmid carrying the vector genome and a helper plasmid expressing the capsid genes (replaced with a foreign gene in the vector genome). The resulting stocks, however, are always heavily contaminated with replication-competent viruses (RCV), which precludes their use in vivo and particularly in gene therapy. In the present work we have developed a second-generation MVMp-based vector system specifically designed to reduce the probability of RCV generation by homologous recombination. We have constructed a new MVMp-based vector and a new helper genome with minimal sequence overlap and have used the degeneracy of the genetic code to further decrease vector–helper homology. In this system, the left homologous region was almost completely eliminated and the right sequence overlap was reduced to 74 nt with only 61% homology. We were thus able to substantially reduce (∼200 ×), but not completely eliminate, generation of contaminating viruses in medium-scale rMVMp preparations. Since the remaining sequence homology between the new vector and helper genomes is weak, our results suggest that contaminating viruses in this system are generated by nonhomologous recombination. It is important to note, unlike the autonomously replicating helper viruses produced from the first-generation vector/helper genomes, the contaminating viruses arising from the new packaging system cannot initiate secondary infection rounds (so they are not ‘replication-competent viruses’). Our findings have important implications for the design of new MVMp-based vectors and for the construction of trans-complementing packaging cell lines.