Conjugative plasmid-encoded toxin–antitoxin system PrpT/PrpA directly controls plasmid copy number

Toxin–antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin–antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

Type II Toxin-Antitoxin Systems: Evolution and Revolutions

ABSTRACT Type II toxin-antitoxin (TA) systems are small genetic elements composed of a toxic protein and its cognate antitoxin protein, the latter counteracting the toxicity of the former. While TA systems were initially discovered on plasmids, functioning as addiction modules through a phenomenon called postsegregational killing, they were later shown to be massively present in bacterial chromosomes, often in association with mobile genetic elements. Extensive research has been conducted in recent decades to better understand the physiological roles of these chromosomally encoded modules and to characterize the conditions leading to their activation. The diversity of their proposed roles, ranging from genomic stabilization and abortive phage infection to stress modulation and antibiotic persistence, in conjunction with the poor understanding of TA system regulation, resulted in the generation of simplistic models, often refuted by contradictory results. This review provides an epistemological and critical retrospective on TA modules and highlights fundamental questions concerning their roles and regulations that still remain unanswered.

The opportunistic marine pathogenVibrio parahaemolyticusbecomes virulent by acquiring a plasmid that expresses a deadly toxin

Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused byVibrio parahaemolyticusthat has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain ofV. parahaemolyticuscontains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of thePhotorhabdusinsect-related (Pir) toxins PirA and PirB. We determined the crystal structure of theV. parahaemolyticusPirA and PirB (PirAvpand PirBvp) proteins and found that the overall structural topology of PirAvp/PirBvpis very similar to that of theBacillusCry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirABvpheterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested thatpirABvpmay be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

Chromosomal Toxin-Antitoxin Systems May Act as Antiaddiction Modules

ABSTRACT 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) (ccdEch ) 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 ccdEch 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.

Maintenance Forced by a Restriction-Modification System Can Be Modulated by a Region in Its Modification Enzyme Not Essential for Methyltransferase Activity

ABSTRACT Several type II restriction-modification gene complexes can force their maintenance on their host bacteria by killing cells that have lost them in a process called postsegregational killing or genetic addiction. It is likely to proceed by dilution of the modification enzyme molecule during rounds of cell division following the gene loss, which exposes unmethylated recognition sites on the newly replicated chromosomes to lethal attack by the remaining restriction enzyme molecules. This process is in apparent contrast to the process of the classical types of postsegregational killing systems, in which built-in metabolic instability of the antitoxin allows release of the toxin for lethal action after the gene loss. In the present study, we characterize a mutant form of the EcoRII gene complex that shows stronger capacity in such maintenance. This phenotype is conferred by an L80P amino acid substitution (T239C nucleotide substitution) mutation in the modification enzyme. This mutant enzyme showed decreased DNA methyltransferase activity at a higher temperature in vivo and in vitro than the nonmutated enzyme, although a deletion mutant lacking the N-terminal 83 amino acids did not lose activity at either of the temperatures tested. Under a condition of inhibited protein synthesis, the activity of the L80P mutant was completely lost at a high temperature. In parallel, the L80P mutant protein disappeared more rapidly than the wild-type protein. These results demonstrate that the capability of a restriction-modification system in forcing maintenance on its host can be modulated by a region of its antitoxin, the modification enzyme, as in the classical postsegregational killing systems.

Mosaic Structure of p1658/97, a 125-Kilobase Plasmid Harboring an Active Amplicon with the Extended-Spectrum β-Lactamase Gene blaSHV-5

ABSTRACT Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum β-lactamase (ESBL) of the SHV type. The β-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the bla SHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.

Functional Interactions between Coexisting Toxin-Antitoxin Systems of the ccd Family in Escherichia coli O157:H7

ABSTRACT 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 CcdBO157 toxin targets the DNA gyrase, and the CcdAO157 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.

Comparative DNA Analysis of Two vanA Plasmids from Enterococcus faecium Strains Isolated from Poultry and a Poultry Farmer in Norway

ABSTRACT The DNA sequences of two plasmids carrying vanA, pVEF1 (39,626 bp) and pVEF2 (39,714 bp), were determined. Forty-three shared coding sequences were identified, and the only nucleotide difference was an 88-bp indel. A postsegregational killing system was identified. This system possibly explains the persistence of the vanA gene cluster in Norwegian poultry farms.