SummaryThe ability of a high frequency (10 ----2 ) of Escherichia coli to survive prolonged exposure to penicillin antibiotics, called high persistence, is associated with mutations in the hipA gene. The hip operon is located in the chromosomal terminus near dif and consists of two genes, hipA and hipB . The wild-type hipA gene encodes a toxin, whereas hipB encodes a DNA-binding protein that autoregulates expression of the hip operon and binds to HipA to nullify its toxic effects. We have characterized the hipA7 allele, which confers high persistence, and established that HipA7 is nontoxic, contains two mutations (G22S and D291A) and that both mutations are required for the full range of phenotypes associated with hip mutants. Furthermore, expression of hipA7 in the absence of hipB is sufficient to establish the high persistent phenotype, indicating that hipB is not required. There is a strong correlation between the frequency of persister cells generated by hipA7 strains and cell density, with hipA7 strains generating a 20-fold higher frequency of persisters as cultures approach stationary phase. It is also demonstrated that relA knock-outs diminish the high persistent phenotype in hipA7 mutants and that relA spoT knock-outs eliminate high persistence altogether, suggesting that hipA7 facilitates the establishment of the persister state by inducing (p)ppGpp synthesis. Consistent with this proposal, ectopic expression of relA ¢ ¢ ¢ ¢ from a plasmid was shown to increase the number of persistent cells produced by hipA7 relA double mutants by 100-fold or more. A model is presented that postulates that hipA7 increases the basal level of (p)ppGpp synthesis, allowing a significantly greater percentage of cells in a population to assume a persistent, antibioticinsensitive state by potentiating a rapid transition to a dormant state upon application of stress.

Persistence is an epigenetic trait that allows a small fraction of bacteria, approximately one in a million, to survive prolonged exposure to antibiotics. In Escherichia coli an increased frequency of persisters, called "high persistence," is conferred by mutations in the hipA gene, which encodes the toxin entity of the toxin-antitoxin module hipBA. The high-persistence allele hipA7 was originally identified because of its ability to confer high persistence, but little is known about the physiological role of the wild-type hipA gene. We report here that the expression of wild-type hipA in excess of hipB inhibits protein, RNA, and DNA synthesis in vivo. However, unlike the RelE and MazF toxins, HipA had no effect on protein synthesis in an in vitro translation system. Moreover, the expression of wild-type hipA conferred a transient dormant state (persistence) to a sizable fraction of cells, whereas the rest of the cells remained in a prolonged dormant state that, under appropriate conditions, could be fully reversed by expression of the cognate antitoxin gene hipB. In contrast, expression of the mutant hipA7 gene in excess of hipB did not markedly inhibit protein synthesis as did wild-type hipA and yet still conferred persistence to ca. 10% of cells. We propose that wild-type HipA, upon release from HipB, is able to inhibit macromolecular synthesis and induces a bacteriostatic state that can be reversed by expression of the hipB gene. However, the ability of the wild-type hipA gene to generate a high frequency of persisters, equal to that conferred by the hipA7 allele, may be distinct from the ability to block macromolecular synthesis.

During chromosome synthesis in Escherichia coli, replication forks are blocked by Tus bound Ter sites on approach from one direction but not the other. To study the basis of this polarity, we measured the rates of dissociation of Tus from forked TerB oligonucleotides, such as would be produced by the replicative DnaB helicase at both the fork-blocking (nonpermissive) and permissive ends of the Ter site. Strand separation of a few nucleotides at the permissive end was sufficient to force rapid dissociation of Tus to allow fork progression. In contrast, strand separation extending to and including the strictly conserved G-C(6) base pair at the nonpermissive end led to formation of a stable locked complex. Lock formation specifically requires the cytosine residue, C(6). The crystal structure of the locked complex showed that C(6) moves 14 A from its normal position to bind in a cytosine-specific pocket on the surface of Tus.

The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process

SummaryBacteria that have a circular chromosome with a bidirectional DNA replication origin are thought to utilize a 'replication fork trap' to control termination of replication. The fork trap is an arrangement of replication pause sites that ensures that the two replication forks fuse within the terminus region of the chromosome, approximately opposite the origin on the circular map. However, the biological significance of the replication fork trap has been mysterious, as its inactivation has no obvious consequence. Here we review the research that led to the replication fork trap theory, and we aim to integrate several recent findings that contribute towards an understanding of the physiological roles of the replication fork trap. Likely roles include the prevention of over-replication, and the optimization of post-replicative mechanisms of chromosome segregation, such as that involving FtsK in Escherichia coli.

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A polar DNA replication barrier is formed when the DNA-binding protein Tus forms a complex with any of the four 23-base-pair terminator (ter) sites found in the terminus region of the Escherichia coli chromosome. We have used a plasmid DNA replication system reconstituted with purified proteins in vitro to investigate the interaction ofthe Tus protein with the replication fork. Purified Tus protein alone is necessary and sufficient to arrest DNA replication on CoIEltype plasmid templates containing ter sites. Tus proteincatalyzed termination depends upon the orientation of the ter site in the plasmid DNA. Nucleotide resolution mapping of the terminated nascent DNA shows that leading-strand DNA synthesis arrests at the point of contact with the Tus protein, while the rial lagging-strand primer sites are 50-70 nucleotides upstream. In addition, the distribution of leading-strand arrest sites changes when the composition of the proteins on the lagging-strand side of the replication fork is altered.

Arrest of DNA replication inReplication forks approaching from the non-permissive side of the Tus-Ter complex are arrested, but replisomes approaching from the permissive side can pass through the complex. The Ter sites are oriented in the chromosome to permit DNA replication in the origin-to-terminus direction, but restrict replication forks traveling in the terminus-to-origin direction. Thus, the Tus-Ter complexes form a replication fork trap and prevent DNA replication forks from meeting in regions other than in the chromosomal terminus.The functional polarity demonstrated by Tus is reflected in the asymmetry of the protein-DNA complex, whose crystal structure has been recently solved (2). Tus binds as a monomer (3, 4) and contacts both strands of the Ter site on the nonpermissive of the complex, but only a single strand on the permissive side (2, 5). The Ter site DNA is nestled into a cleft formed by the two primary domains of Tus (amino and carboxyl domains) and the interstrand ␤-sheets that connect the two domains (2). The primary determinants of base pair recognition and binding are mediated by the main two interstrand ␤-sheets, which penetrate deeply into the major groove of the Ter site, making both polar and hydrophobic contacts with the bases. Binding is also enhanced by extensive contacts between Tus and the phosphates in the DNA backbone. A total of 42 amino acid residues stretched along the length of the protein make contacts with the DNA.Tus binds to the chromosomal Ter sites with a very high affinity. The K obs for Tus binding to the TerB site ranges between 3.4 ϫ 10 Ϫ13 M and 7.5 ϫ 10 Ϫ13 M, depending on the buffer conditions used (5, 6). Half-lives (t1 ⁄2 ) of the protein-DNA complex were determined to be 550 to 149 min, respectively, in these studies. The high affinity of the Tus-Ter interaction in conjunction with the distribution of protein-DNA contacts has been used to suggest that Tus can arrest DNA replication by functioning as a clamp on the DNA and preventing the unwinding activity of the DnaB helicase (2,5,7,8). Alternately, protein-protein interactions between Tus and the DnaB helicase have been postulated to mediate replication arrest. This latter model is based upon the specificity of Tus function (9, 10), differential ability of Tus to halt helicase unwinding when presented with different templates (11), and mutational studies on Tus (6,12).Ter sequences were originally identified as 22-23 base pairs in length, based on sequence identity between TerA, TerB, and TerC (13,14). As additional sites were identified both in the chromosome and in plasmid replicons, it became apparent that the essential conserved elements of the Ter site were an 11-base pair "core" sequence (positions 9 -19) and an upstream G-C base pair at position 6 (Fig. 1). Nucleoside analogs have been used to partially map the determinants of Tus binding (15,16) and it was shown that (i) the G residues at positions 10, 13, and 17 within the core sequence contributed both major and minor groove interactions, (ii) the c...