Escherichia coli cells with increased levels of DnaA and deficient in recombinational repair have decreased viability

The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, beta clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, beta clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the beta clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 micro m) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional approximately 100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.     

The marine viromes of four oceanic regions

Viruses are the most common biological entities in the marine environment. There has not been a global survey of these viruses, and consequently, it is not known what types of viruses are in Earth's oceans or how they are distributed. Metagenomic analyses of 184 viral assemblages collected over a decade and representing 68 sites in four major oceanic regions showed that most of the viral sequences were not similar to those in the current databases. There was a distinct “marine-ness” quality to the viral assemblages. Global diversity was very high, presumably several hundred thousand of species, and regional richness varied on a North-South latitudinal gradient. The marine regions had different assemblages of viruses. Cyanophages and a newly discovered clade of single-stranded DNA phages dominated the Sargasso Sea sample, whereas prophage-like sequences were most common in the Arctic. However most viral species were found to be widespread. With a majority of shared species between oceanic regions, most of the differences between viral assemblages seemed to be explained by variation in the occurrence of the most common viral species and not by exclusion of different viral genomes. These results support the idea that viruses are widely dispersed and that local environmental conditions enrich for certain viral types through selective pressure.     

Kinetics of the formation and dissociation of actin filament branches mediated by Arp2/3 complex

The actin filament network at the leading edge of motile cells relies on localized branching by Arp2/3 complex from "mother" filaments growing near the plasma membrane. The nucleotide bound to the mother filaments (ATP, ADP and phosphate, or ADP) may influence the branch dynamics. To determine the effect of the nucleotide bound to the subunits of the mother filament on the formation and stability of branches, we compared the time courses of actin polymerization in bulk samples measured using the fluorescence of pyrene actin with observations of single filaments by total internal reflection fluorescence microscopy. Although the branch nucleation rate in bulk samples was nearly the same regardless of the nucleotide on the mother filaments, we observed fewer branches by microscopy on ADP-bound filaments than on ADP-P(i)-bound filaments. Observation of branches in the microscope depends on their binding to the slide. Since the probability that a branch binds to the slide is directly related to its lifetime, we used counts of branches to infer their rates of dissociation from mother filaments. We conclude that the nucleotide on the mother filament does not affect the initial branching event but that branches are an order of magnitude more stable on the sides of new ATP- or ADP-P(i) filaments than on ADP-actin filaments.     

A model for the initiation of replication in Escherichia coli

The role of the protein DnaA as the principal control of replication initiation is investigated by a mathematical model. Data showing that DnaA is growth rate regulated suggest that its concentration alone is not the only factor determining the timing of initiation. A mathematical model with stochastic and deterministic components is constructed from known experimental evidence and subdivides the total pool of DnaA protein into four forms. The active form, DnaA.ATP, can be bound to the origin of replication, oriC, where it is assumed that a critical level of these bound molecules is needed to initiate replication. The active form can also exist in a reserve pool bound to the chromosome or a free pool in the cytoplasm. Finally, a large inactive pool of DnaA protein completes the state variables and provides an explanation for how the DnaA.ATP form could be the principal controlling element in the timing of initiation. The fact that DnaA protein is an autorepressor is used to derive its synthesis rate. The model studies a single exponentially growing cell through a series of cell divisions. Computer simulations are performed, and the results compare favorably to data for different cell cycle times. The model shows synchrony of initiation events in agreement with experimental results.     

Diagnostic Accuracy in Venous Thrombosis

A group of 111 surgical patients at high risk of venous thrombosis were studied after operation by independent clinical assessment and with ¹²⁵I-fibrinogen to detect venous thrombosis. Almost half of the patients developed venous thrombosis. Of these, two-thirds were not suspected clinically despite careful scrutiny. In the patients in whom a clinical diagnosis of venous thrombosis was made this diagnosis was falsely positive in a quarter. More than half of all thrombotic episodes were detectable on the day after operation.The prevalence of venous thrombosis, together with the difficulty in diagnosing it, strongly supports the argument that a reduction in the incidence of pulmonary embolism must depend on widespread adoption of effective prophylaxis, especially in the large number of patients at high risk of venous thrombosis. Prophylactic trials must be objectively assessed, and it is in this field that the ¹²⁵I-fibrinogen technique probably has the most to offer.     

Metagenomic analyses of an uncultured viral community from human feces

Here we present the first metagenomic analyses of an uncultured viral community from human feces, using partial shotgun sequencing. Most of the sequences were unrelated to anything previously reported. The recognizable viruses were mostly siphophages, and the community contained an estimated 1,200 viral genotypes.     

Oscillations in a model of repression with external control

A mathematical model for control by repression by an extracellular substance is developed, including diffusion and time delays. The model examines how active transport of a nutrient can produce either oscillatory or stable responses depending on a variety of parameters, such as diffusivity, cell size, or nutrient concentration. The system of equations for the mathematical model is reduced to a system of delay differential equations and linear Volterra equations. After linearizing these equations and forming the limiting Volterra equations, the resulting linear system no longer has any spatial dependence. Local stability analysis of the radially symmetric model shows that the system of equations can undergo Hopf bifurcations for certain parameter values, while other ranges of the parameters guarantee asymptotic stability. One numerical study shows that the model can exhibit intracellular biochemical oscillations with increasing extracellular concentrations of the nutrient, which suggests a possible trigger mechanism for morphogenesis.The work of this author was supported in part by NSF grants DMS-8603787 and DMS-8807360The work of this author was supported under the REU program of NSF by grant DMS-8807360The work of this author was supported under the REU program of NSF by grant DMS-8807360