Thomas M. Hill

Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis.

The 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 non‐toxic, 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, antibiotic‐insensitive state by potentiating a rapid transition to a dormant state upon application of stress.

Insertion of inverted Ter sites into the terminus region of the Escherichia coli chromosome delays completion of DNA replication and disrupts the cell cycle

To investigate the co‐ordination between DNA replication and cell division, we have disrupted the DNA replication cycle of Escherichia coli by inserting inverted Ter sites into the terminus region to delay completion of the chromosome. The inverted Ter sites (designated InvTer::spcr) were initially inserted into the chromosome of a Δtus strain to allow unrestrained chromosomal replication. We then introduced a functional tus gene by transforming the InvTer::spcr strain with a plasmid carrying the tus gene under control of an arabinose‐inducible promoter. In the presence of 0.2% arabinose, the cells formed long filaments, suggesting that activation of the inverted Ter sites by Tus arrested DNA replication and delayed the onset of cell division. Induction of sfiA, a gene in the SOS regulon, was observed following arrest of DNA replication; however, when a sfiB114 allele was introduced into InvTer::spcr strain, long filaments were still formed, suggesting that the sfi‐independent pathway also caused filamentation. Either recA::camr or lexA3 alleles suppressed filamentation when introduced in the InvTer strain. Interestingly, in both the recA::camr and lexA3 mutants, virtually all cells had a nucleoid, suggesting that cell division was proceeding even though DNA replication was not complete. These results suggest that DNA replication and cell division are uncoupled when recA is inactivated or when genes repressed by LexA cannot be induced.

Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus-Ter Complex

SUMMARY 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.

A molecular mousetrap determines polarity of termination of DNA replication in E. coli.

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 replication fork trap and termination of chromosome replication

Bacteria 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.

Cell division, guillotining of dimer chromosomes and SOS induction in resolution mutants (dif, xerC and xerD) of Escherichia coli

We have studied the growth and division of xerC, xerD and dif mutants of Escherichia coli, which are unable to resolve dimer chromosomes. These mutants express the Dif phenotype, which includes reduced viability, SOS induction and filamentation, and abnormal nucleoid morphology. Growth was studied in synchronous cultures and in microcolonies derived from single cells. SOS induction and filamentation commenced after an apparently normal cell division, which sheared unresolved dimer chromosomes. This has been called guillotining. Microcolony analysis demonstrated that cell division in the two daughter cells was inhibited after guillotining, and microcolonies formed that consisted of two filaments lying side by side. Growth of these filaments was severely reduced in hipA+ strains. We propose that guillotining at dif destroys the expression of the adjacent hipBA genes and, in the absence of continued formation of HipB, HipA inhibits growth. The length of the filaments was also affected by SfiA: sfiA dif hipA mutants initially formed filaments, but cell division at the ends of the filaments ultimately produced a number of DNA‐negative cells. If SOS induction was blocked by lexA3 (Ind−), filaments did not form, and cell division was not inhibited. However, pedigree analysis of cells in microcolonies demonstrated that lethal sectoring occurred as a result of limited growth and division of dead cells produced by guillotining.

Sequence-specific Interactions in the Tus-Ter Complex and the Effect of Base Pair Substitutions on Arrest of DNA Replication in Escherichia coli

Arrest of DNA replication in Escherichia coli is mediated by specific interactions between the Tus protein and terminator (Ter) sequences. Binding of Tus to aTer site forms a asymmetric protein-DNA complex that arrests DNA replication in an orientation-dependent fashion. In this study, mutant Ter sites carrying single base pair substitutions at 16 different positions were examined for their ability to bind purified Tus protein and arrest DNA replication.In vitro competition assays demonstrated that base pair substitutions at positions 8–19 had significant effects on the free energy of Tus binding (ΔΔG 0 of 1.5 to >4.0 kcal/mol). Concomitant with loss of binding affinity, mutations at these positions also showed significantly lower or undetectable replication arrest activities in vivo. Substitutions at positions 6, 20, and 21 had moderate effects on Tus-Terinteractions, suggesting that these base pairs contribute to, but are not absolutely critical for, Tus binding. Even though the effects on binding were minimal, these Ter mutants were not as efficient as wild type Tus-TerB complexes at arresting replication forks. Three new potential Ter sites, referred to as TerH, TerI, and TerJ, were identified by searching the E. coli genome for sequence similarity to a consensus Ter site sequence.

THE DESTRUCTION OF ESCHERICHIA COLI BIOFILMS USING HIGH-INTENSITY FOCUSED ULTRASOUND

High-intensity focused ultrasound (HIFU) has shown great potential for replacing surgery in many applications. In this work, HIFU was used to destroy Escherichia coli (E. coli) biofilms that had been grown on chambered microscope slides. Biofilms are central to the pathogenesis and persistence of nosocomial (hospital-acquired) infections associated with indwelling medical devices. The slides were exposed to 9.1 mus pulses at a pulse repetition frequency of 1000 Hz. The pulses were generated by a 1.1 MHz spherically focused source with a focal length of 6.3 cm and an active diameter of 7 cm. The peak rarefactional pressure for the pulses was varied as 3.1, 4.1, 5.2, 6.2 and 7.6 MPa in addition to a sham where the biofilms were not exposed. The effectiveness of the treatment was assessed by determining the number of colony forming units (CFU) remaining following exposure of the bacteria to HIFU. Most of the biofilms treated at the higher exposures of 6.2 and 7.6 MPa had no detectable CFU, indicating that bacteria in the biofilm were killed by the treatment or that treatment disrupted the biofilm and released bacteria from the slide. However, the ability of some bacteria to survive at the higher exposure settings needs to be resolved prior to implementing the treatment clinically.

Protein Microarray On-Demand: A Novel Protein Microarray System

We describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (∼3–7×10 −13 M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This “all DNA” microarray can be converted to a protein microarray on-demand without need for any additional capture reagent..

Tus-mediated arrest of DNA replication in Escherichia coli is modulated by DNA supercoiling.

In the absence of RecA, expression of the Tus protein of Escherichia coli is lethal when ectopic Ter sites are inserted into the chromosome in an orientation that blocks completion of chromosome replication. Using this observation as a basis for genetic selection, an extragenic suppressor of Tus‐mediated arrest of DNA replication was isolated with diminished ability of Tus to halt DNA replication. Resistance to tus expression mapped to a mutation in the stop codon of the topA gene (topA869), generating an elongated topoisomerase I protein with a marked reduction in activity. Other alleles of topA with mutations in the carboxyl‐terminal domain of topoisomerase I, topA10 and topA66, also rendered recA strains with blocking Ter sites insensitive to tus expression. Thus, increased negative supercoiling in the DNA of these mutants reduced the ability of Tus–Ter complexes to arrest DNA replication. The increase in superhelical density did not diminish replication arrest by disrupting Tus–Ter interactions, as Tus binding to Ter sites was essentially unaffected by the topA mutations. The topA869 mutation also relieved the requirement for recombination functions other than recA to restart replication, such as recC, ruvA and ruvC, indicating that the primary effect of the increased negative supercoiling was to interfere with Tus blockage of DNA replication. Introduction of gyrB mutations in combination with the topA869 mutation restored supercoiling density to normal values and also restored replication arrest at Ter sites, suggesting that supercoiling alone modulated Tus activity. We propose that increased negative supercoiling enhances DnaB unwinding activity, thereby reducing the duration of the Tus–DnaB interaction and leading to decreased Tus activity.