P1 is a bacteriophage of Escherichia coli and other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from 70 promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by the E. coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication.

P1 lysogens of Escherichia coli carry the prophage as a stable low copy number plasmid. The frequency with which viable cells cured of prophage are produced is about 10(-5) per cell per generation. Here we show that a significant part of this remarkable stability can be attributed to a plasmid-encoded mechanism that causes death of cells that have lost P1. In other words, the lysogenic cells appear to be addicted to the presence of the prophage. The plasmid withdrawal response depends on a gene named doc (death on curing), encoding a 126 amino acid protein. Expression of doc is not SOS-inducing and killing by Doc is recA-independent. In cells that retain P1 the killing is prevented by the product of a gene named phd (prevent host death), encoding a 73 amino acid protein. The genes phd and doc have been cloned and expressed from a 0.7 kb segment of P1 DNA. The two genes constitute an operon and the synthesis of Doc appears to be translationally coupled to that of Phd. Homologs of the P1 addiction genes are found elsewhere, but phd and doc are unrelated to previously described genes of other plasmids that also cause an apparent increase in plasmid stability by post-segregational killing.

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...

The P1 plasmid addiction operon increases the apparent stability of a plasmid that carries it by killing plasmid-free (cured) segregants. The operon consists of a gene encoding an endotoxin responsible for death on curing (doc), preceded by a gene encoding a relatively unstable antidote that can prevent host death (phd). When the copy number of the operon was increased, expression of a lacZ reporter fused to the promoter of the operon decreased, indicating that expression of the operon was stabilized by an autoregulatory circuit. Transcription of the lacZ reporter was repressed about 10-fold when phd, without doc, was expressed from an exogenous promoter. DNase I footprinting showed that Phd binds a perfect 10-base pair palindromic DNA sequence and, at higher concentrations, an adjacent, imperfect palindrome. The palindromic sites are located between the -10 region of the putative promoter and the start codon of phd. Electrophoretic mobility of DNA containing the promoter region was retarded in the presence of Phd and further retarded in the presence of Phd and Doc. When doc was co-expressed with phd, repression of the lacZ fusion was enhanced more than 100-fold. Thus, both products of the addiction operon participate in its autoregulation.