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.

ciation of SODD with the uncomplexed TNT-R1 then reestablishes the normal silent state for TNT-RI. This tight control of the duration of TNF signaling at the receptor level is somewhat analogous to the temporal regtllation of XF-KB activity by the YF-KB inhibitor IKB that occurs doansneam in the TPT signaling cascade. In addition to its role as a silencer of TNF-RI signaling, we ha\-e considered the possibility that SODD may also participate in transducing TNF signals once it is released from the activated receptor complex. However, at this time, we have no evidence for such a signaling role.It is likely that SODD also ft~nctions as an inhibitor of constitutive DR3 signaling because (i) SODD interacts with DR3 and TNF-RI equally \\ell in yeast hvo-hybrid and manmalian coprecipitation assays: (ii) DR3, like TNF-R1. signals independently of ligand when overexpressed (16): (iii) the death domains of DR3 and TSF-RI are highly related, sharing 45% sequence identity; and (iv) TNF-RI and DR3 both use TRADD, TRAF2. RIP, and FXDD for signal transduction (16). Finally, on the basis of these results. \\e predict that SODD-related proteins will be found that interact with and play a similar role in preventing spontaneous signaling by Fas. DR4. and DR5. In fact, a candidate protein having 61?h identity to the COOH-telminal 71 anlino acids of SODD is predicted to be encoded by expressed sequence tag cDNA clones (17) Kitson et a/., Nature 384, 372 (1996); S. Marsters et al., Curr. 9/01, 6, 1669. 17. GenBank accession numbers AA319013, AA362082, H10621, and T33545. 18. Rabbit polyclonal antisera were generated against a SODD peptide (amino acids 292 t o 313) and against purified His-tagged SODD produced in Escherichia coli. The upper band of the protein doublet in Fig. 1C is presumably phosphorylated SODD. This band predominates in cell lines that express low levels of SODD, whereas both bands are apparent in cell lines that express higher levels (12). 19, j. D. Woronicz, X. Gao, Z. Cao, M. Rothe, D. V.Coeddel, Science 278, 866 (1997). 20. We thank L. Huang for synthesizing the SODD peptide for antibody generation, V. Dixit for providing the DR3 cDNA, A. Ashkenazi for providing DR4 and DR5 cDNAs, D. Baltimore for supplying the parental mammalian cell expression vector for CST, pEBG, and L. Medin for help with the figures.9 October 1998; accepted 21 December 1998Silencing of Genes Flanking the P I Plasmid Centromere Oleg Rodionov, Malgorzata tobocka," Michael YarmolinskytPartition modules stabilize bacterial plasmids and chromosomes by actively promoting their segregation into daughter cells. The partition module of plasmid P I is typical and consists of a centromere site,parS, and genes that encode proteins ParA and ParB. We show that ParB can silence genes flankingpars (to which ParB binds), apparently by polymerizing along the DNAfrom a nucleation site at pars. Wild-type ParB contacts an extensive region of PI DNA; silencingdefective ParB proteins, which were found to be partition-defective, are less able to spread. Hence, ...

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.

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16 The rather low values for threonine and lysine obtained with both labeled aspartic acids in Lemna probably are significant and may indicate a "channeling" of aspartic acid, i.e., a preferential utilization of aspartic acid for incorporation into protein rather than for conversion to threonine or lysine. It will be noted, though, that the labeling of the lysine and the threonine are approximately equal, and that with acetate-2-C'4, all three protein amino acids exhibit nearly the same specific activity. 17 Probable mechanisms underlying the various labeling patterns observed will be discussed elsewhere.

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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...

Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation. This Phd domain is intrinsically disordered in solution and folds into an ␣-helix upon binding to Doc. The details of the interactions reveal the molecular basis for the inhibitory action of the antitoxin. The complex resembles the Fic (filamentation induced by cAMP) proteins and suggests a possible evolutionary origin for the phd/doc operon. Doc induces growth arrest of Escherichia coli cells in a reversible manner, by targeting the protein synthesis machinery. Moreover, Doc activates the endogenous E. coli RelE mRNA interferase but does not require this or any other known chromosomal toxin-antitoxin locus for its action in vivo.Small operons encoding a toxin and its antitoxin are common in the genomes of bacteria and archaea and are also found on certain plasmids and bacteriophages. These so-called toxinantitoxin (TA) 2 modules have been proposed to regulate the pace of metabolism and may induce a state of dormancy in case of nutritional stress (1-3). TA modules are highly abundant in opportunistic pathogens such as Mycobacterium tuberculosis (4), and their presence has been linked to persistence (5).On plasmids, TA modules act as addiction systems, aiding plasmid maintenance in the bacterial population by post-segregational killing (6), filling in a function related to apoptosis and programmed cell death in eukaryotes (7). Related effects have been observed for chromosome-located TA systems as some of them have been shown to diminish large scale genome reductions in the absence of selection (8). In the presence of the plasmid, both toxin and antitoxin are expressed, leading to a steady state equilibrium where the antitoxin counteracts the effect of the toxin. In its free state, the antitoxin, usually a modular protein that contains a functional intrinsically disordered region (9 -11), is under constant proteolytic attack. The toxinantitoxin complex acts as an autorepressor for the TA operon, ensuring that only small amounts of the proteins are present in the cell. Upon plasmid loss, the antitoxin is degraded by a specific intracellular protease, releasing the toxin. Without the possibility of replenishing the antitoxin population, the toxin action becomes irreversible, resulting in cell death.The phd/doc operon encodes a TA module aiding the maintenance of the plasmid-prophage P1 in Escherichia coli (12). Doc is an inhibitor of translation elongation through its association with the 30 S ribosomal subunit in a way similar to the antibiotic hygromycin (13). The action of Doc is suppressed by the antitoxin Phd, which consists of two domains. Its C-terminal domain (residues 52-73) harbors the interaction site with Doc and on its own prevents Doc-mediated growth arrest (14,15). The N-terminal region (residues 1-51) of Phd i...