The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA

The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA. Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling. Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling. Any deviations from these optimal levels decrease fis promoter activity. This strict dependence of fis expression on the superhelical density suggests that fis may be involved in 'fine-tuning' the homeostatic control mechanism of DNA supercoiling in E. coli.     

Escherichia coli DNA topoisomerase I mutants: Increased supercoiling is corrected by mutations near gyrase genes

Bacterial chromosomes and plasmid (pBR322) DNA from topoisomerase I-defective Escherichia coli strains have been characterized with respect to superhelical density. The topoisomerase I defect results in increased negative superhelical density of both the bacterial chromosome and pBR322. Thus topoisomerase I is involved in determining the level of supercoiling in bacteria. Three of the topoisomerase I-defective strains we studied carry secondary mutations that decrease superhelical density; these additional mutations are closely linked to the gyrB locus in two of the strains and to the gyrA locus in the third strain.     

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.     

Control of bacterial DNA supercoiling

Two DNA topoisomerases control the level of negative supercoiling in bacterial cells. DNA gyrase introduces supercoils, and DNA topoisomerase I prevents supercoiling from reaching unacceptably high levels. Perturbations of supercoiling are corrected by the substrate preferences of these topoisomerases with respect to DNA topology and by changes in expression of the genes encoding the enzymes. However, supercoiling changes when the growth environment is altered in ways that also affect cellular energetics. The ratio of [ATP] to [ADP], to which gyrase is sensitive, may be involved in the response of supercoiling to growth conditions. Inside cells, supercoiling is partitioned into two components, superhelical tension and restrained supercoils. Shifts in superhelical tension elicited by nicking or by salt shock do not rapidly change the level of restrained supercoiling. However, a steady-state change in supercoiling caused by mutation of topA does alter both tension and restrained supercoils. This communication between the two compartments may play a role in the control of supercoiling.     

Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein

Summary Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA ⁺ gene. When the dnaA gene was induced from either the plac or the pL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter pL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.     

Construction of an alkaline phosphatase fusion-generating transposon,mTn10phoA

We constructed a derivative of the mini-transposon mTn10 that generates translational fusions to the phoA gene from Escherichia coli and carries the Km^R determinant from Tn5. This new transposon, mTn10phoA, is carried on a mobilizable plasmid with both selectable and counterselectable markers. The plasmid carrying mTn10phoA was introduced into Legionella pneumophila. Southern hybridization analysis indicated that the mTn10phoA insertions were randomly distributed     

Torsional tension in intracellular bacteriophage T4 DNA. Evidence that a linear DNA duplex can be supercoiled in vivo

Torsional tension in intracellular bacteriophage T4 DNA and host cell DNA was measured in infected Escherichia coli cells using the trimethylpsoralen photobinding assay. Early in infection superhelical tension in the host E. coli DNA was gradually reduced until at 8 min post-infection there was no detectable tension. Negative torsional tension in the T4 DNA appeared transiently, reaching a maximum 4 to 6 min post-infection (at 32 °C) and declined to undetectable levels by 10 min. The maximum level of tension averaged over all infecting T4 DNA molecules was equivalent to superhelical density of about σ = ?0.03.Sedimentation studies of the psoralen-associated T4 DNA isolated from infected cells at 5 min post-infection indicated that this DNA was primarily in an intact linear form. This is the first evidence indicating that a linear DNA molecule can acquire torsional tension in vivo: the finding suggests that intracellular T4 DNA can be topologically restrained probably by interaction with other structures in the cell. Effects of inhibitors of DNA gyrase and effects of mutations in T4 gene 39 indicated that the observed torsional tension was introduced by E. coli DNA gyrase, not by the T4 topoisomerase. Studies of the number of nicks required to relax the tension suggest that the entire T4 genome is organized into one topological domain of supercoiling.The possible role of the negative superhelical tension in the initiation of T4 DNA replication was examined. Initiation of DNA replication occurred shortly after the accumulation of tension in T4 DNA. However, replication occurred at near-normal levels under conditions where acquisition of tension was blocked in T4 DNA. Results suggest that although a topoisomerase activity is required for the initiation of DNA replication, the observed torsional tension is not a prerequisite.     

Transcriptional regulation of nitrogen fixation genes by DNA supercoiling

DNA gyrase (ATP dependent topoisomerase type II, EC 5.99.1.3) was found to be essential for the expression of the Klebsiella pneumoniae nitrogen fixation gene cluster carried by plasmid pRD1 in Escherichia coli. In the absence of DNA gyrase activity, nitrogen fixation activity could be restored by providing a constitutively expressed nifA function in trans. Our results suggest that nif gene regulation by oxygen may be mediated through the alteration of the superhelical status of the promoter of the nifLA regulatory operon, in addition to the action of the nifL gene product.Communicated by J. Schell     

Chromosomal domains of supercoiling in Salmonella typhimurium

The chromosomes of enteric bacteria are divided into about 50 independently supercoiled domains. It is not known whether the net level of DNA supercoiling is similar in each domain, or whether the domains are differentially supercoiled. We have addressed this question genetically, using a supercoiling-sensitive promoter to probe the relative levels of supercoiling at defined points around the Salmonella typhimurium chromosome. We conclude that, within the limits of resolution of this approach, the level of supercoiling does not differ significantly between chromosomal domains, and that each domain responds in a similar fashion to factors that perturb supercoiling. These findings have implications for the organization of the bacterial genome.     

55.2, a Phage T4 ORFan Gene,Encodes an Inhibitor of Escherichia coli Topoisomerase I and Increases Phage Fitness

Topoisomerases are enzymes that alter the topological properties of DNA. Phage T4 encodes its own topoisomerase but it can also utilize host-encoded topoisomerases. Here we characterized 55.2, a phage T4 predicted ORF of unknown function. High levels of expression of the cloned 55.2 gene are toxic in E. coli. This toxicity is suppressed either by increased topoisomerase I expression or by partial inactivation of the ATPase subunit of the DNA gyrase. Interestingly, very low-level expression of 55.2, which is non-lethal to wild type E. coli, prevents the growth of a deletion mutant of the topoisomerase I (topA) gene. In vitro, gp55.2 binds DNA and blocks specifically the relaxation of negatively supercoiled DNA by topoisomerase I. In vivo, expression of gp55.2 at low non-toxic levels alters the steady state DNA supercoiling of a reporter plasmid. Although 55.2 is not an essential gene, competition experiments indicate that it is required for optimal phage growth. We propose that the role of gp55.2 is to subtly modulate host topoisomerase I activity during infection to insure optimal T4 phage yield.