Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli.

Abstract

Plasmid-encoded addiction genes augment the apparent stability of various low copy number bacterial plasmids by selectively killing plasmid-free (cured) segregants or their progeny. The addiction module of plasmid prophage P1 consists of a pair of genes called phd and doc. Phd serves to prevent host death when the prophage is retained and, should retention mechanisms fail, Doc causes death on curing. Doc acts as a cell toxin to which Phd is an antidote. In this study we show that host mutants with defects in either subunit of the ClpXP protease survive the loss of a plasmid that contains a P1 addiction module. The small antidote protein Phd is fully stable in these two mutant hosts, whereas it is labile in a wild-type host. We conclude that the role of ClpXP in the addiction mechanism of P1 is to degrade the Phd protein. This conclusion situates P1 among plasmids that elicit severe withdrawal symptoms and are able to do so because they encode both a cell toxin and an actively degraded macromolecule that blocks the synthesis or function of the toxin.Bacteriophage P1 lysogenizes Escherichia coli as a low copy number plasmid that is maintained with a loss frequency of about 10-5 per cell per generation (1). This remarkable stability is higher than can be accounted for by the mechanisms that ensure stringent control of plasmid replication and active partition of the replicas (2). The additional stabilization is provided by a "plasmid-addiction" module that selectively kills plasmid-free segregants or their progeny (3). The addiction module of Pl encodes two small proteins: Phd and Doc. Phd is responsible forprevention of host death in P1 lysogens; Doc causes death on curing. In cells that harbor a P1 prophage Phd must be maintained at a concentration sufficient to inhibit effectively the synthesis or the function of the toxin. We favor the view that Phd is an inhibitor of Doc function rather than an inhibitor of Doc synthesis. The latter hypothesis is difficult to reconcile with the apparent translational coupling of Doc synthesis to the synthesis of Phd (3) and with the delay of several generations after loss of the plasmid before withdrawal symptoms become manifest. On the other hand, if Phd is an antidote to Doc, then the death of cells that lose the plasmid could be most simply explained by the more rapid inactivation of the antidote compared to the toxin.Lability of the macromolecule that prevents toxin synthesis or function accounts for the selective killing of cells cured of addicting plasmids such as Rl or F. The products of the sok gene of Rl and of its homologs in other plasmids are labile antisense RNAs subject to rapid degradation by nucleases (4), whereas the protein products of ccdA of F (5) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.allows the toxins to be synthesized or released and to kill the cured cells.Lon is one of two well...

Energetics of Structural Transitions of the Addiction Antitoxin MazE

“…The prediction of structuring based on the mean hydrophobicity and mean net charge of the protein polypeptide chain is in good agreement with our observations and with recently reported results on addiction modules (11,47,89,90,102). Moreover, the large amount of the random-coil form found in free antitoxins in solutions (11,47,89,90,102) represents high vulnerability for their proteolytic cleavage (25,101,103).…”

A regulatory role for Staphylococcus aureus toxin–antitoxin system PemIKSa

“…Under favourable growth conditions genes of most TA systems are coexpressed and the deleterious activity of the toxin is prevented by the antitoxin 7 . However, various stress stimuli alter the toxin/antitoxin balance, usually by inducing increased proteolytic degradation of the labile antitoxin [8][9][10] . The activated toxin induces a dormant state or other adaptations that enable the bacteria to survive environmentally unfavourable conditions [11][12][13] .…”

Interference of mRNA Function by Sequence-specific Endoribonuclease PemK

“…In E. coli, some extrachromosomal elements are known to contain addiction modules causing the bacterial programmed cell death by the so-called postsegregational killing effect. The most studied extrachromosomal addiction modules are the phd-doc system on bacteriophage P1 (2)(3)(4)(5), the ccdA-ccdB system on factor F (6 -9), the kis-kid system on plasmid R1 (10 -13), and the pemIpemK system on plasmid R100 (14 -17). Interestingly, the E. coli chromosome also contains several addiction module systems, such as the relBE system (18 -21), the mazEF system (22)(23)(24)(25), and the chpB system (26 -28).…”