Effect of ferricyanide onEscherichia coli

Ferricyanide, ferrocyanide and phosphates in concentrations greater than 0.07 M inhibit the respiration of NAD-linked substrates and succinate in washed cell suspension ofE. coli. Citrate accumulation in the medium under the inhibiting conditions indicates that these salts bring about a change in the permeability of the cell membrane of this bacterium.In general, ferricyanide does not resemble in action those compounds which uncouple respiratory-energy production. Energy production is prevented only when ferricyanide is the main electron acceptor. The inhibition studies and the addition of co-factors, indicate that cytochromes are not involved in the reduction of ferricyanide by cell-free extracts ofE. coli, either with NADH or succinate. An FAD-containing enzyme is probably involved in the reduction of ferricyanide by NADH, while succinic dehydrogenase seems to react directly with ferricyanide.We wish to thank Group Capt. Tom Gray-Young for technical advice and R. Filzroy for technical assistance.This work was supported by a grant from the Science Research Council.     

Effect of the essential oil fromAchillea fragrantissima onEscherichia coli cells

Escherichia coli cells treated with the essential oil from the plantAchillea fragrantissima released five polypeptides as well as K⁺ ions into the incubation medium. The oil also inhibited the respiration ofE. coli cells and reduced their ATP content. Electron micrographs showed that oil-treated cells were permeable to uranyl acetate. The effect of the essential oil on the cell membrane is discussed.     

Multiplicity of phosphorylation sites onEscherichia coli isocitrate dehydrogenase

Several lines of evidence indicate that the in vivo phosphorylation of isocitrate dehydrogenase (EC 1.1.1.42) inEscherichia coli occurs at multiple sites: first, the phosphorylated enzyme can be resolved by two-dimensional electrophoresis into three distinct spots differing in charge; second, the analysis of its phosphoamino acid content shows that it is modified at both serine and threonine residues; third, its extensive hydrolysis by proteolytic enzymes yields several different phosphopeptides.     

Enhancement of DNA polymerase II activity in E. coli after treatment with N-methyl-N'-nitro-N-nitrosoguanidine.

The G₁(G₀) arrest induced in NRK cells by picolinic acid was preceded by marked changes in iron metabolism. In contrast, picolinic acid did not significantly prevent zinc uptake and changes in intracellular zinc were small and clearly preceded by changes in iron. A kinetic study revealed that iron uptake by NRK cells was rapidly halted by picolinic acid. Experiments with radioiron-labeled cells indicated that picolinic acid, in a dose dependent manner, effectively removed iron from the cells. The dose of picolinic acid that exactly removed iron from the cells was also the concentration that induced the G₁(G₀) arrest. Picolinic acid, therefore, may induce the growth inhibition by selectively withholding iron from the cells. These data strongly suggest that iron availability may be a controlling factor in the initiation of DNA synthesis in NRK cells.     

The effect of free chlorine onEscherichia coli populations

The effect of free chlorine onEscherichia coli populations was studied by chlorination of a population of 10⁵ cells/ml. This cell density was low enough for the free-to-combined chlorine ratio to be 6.01 or greater. The predominance of free chlorine resulted in rapid and complete population death.Survivors obtained by dechlorination prior to complete population death were recovered equally well on nonselective and selective media. Although this suggests that survivors are not injured, evidence of survivor injury was observed.Colonies resulting from growth of these survivors had a smaller diameter than colonies from unchlornated controls. This suggests that the chlorinated cells have an increased lag and provides indirect evidence of survivor injury. Injury was indicated directly by an increase in the lag time of surviving cells in slide culture. Variability in the severity of free-chlorine-induced injury was indicated by a broadened range in the survivor lag times.     

DNA synthesis in nucleotide-permeable Escherichia coli cells. VII. Conversion of phi chi-174 DNA to its replicative form

On incubation with deoxynucleoside triphosphates and rATP, ether-treated (nucleotide-permeable) cells convert the single-stranded DNA of adsorbed bacteriophage φX174 particles to the double-stranded replicative forms. The main final product is the doubly-closed replicative form, RFI; a minor product is the relaxed form II. Interruptions in the nascent complementary strand of the viral DNA result in pieces corresponding to 5 to 10% of the unit length of the viral DNA. Pieces of similar size were previously seen in studies of the replication synthesis of Escherichia, coli DNA in ether-treated cells. Since the conversion of the single-stranded φX174 DNA to replicative form is known to be mediated entirely by host factors, it is argued that the viral single strands are replicated by macromolecular factors involed in the replication of E. coli DNA and that this is the reason why new φX174 DNA appears in short pieces. Possible consequences of this interpretation for an understanding of duplex replication are discussed. The joining of the short pieces of complementary φX174 DNA is inhibited at low deoxynucleoside triphosphate concentration (1 μM) but not by nicotinamide mononucleotide, which inhibits the NAD-dependent DNA ligase and blocks the conversion of RFII to RFI in ether-treated cells. The results are discussed with respect to previous studies on cell-DNA synthesis (Geider, 1972). It is argued that there are two polynucleotide joining mechanisms, of which only one requires NAD-dependent ligase action.     

New data on the toxic and radioprotective effect of cysteine onEscherichia coli 15 T- cells

Summary Exponential phase cultures ofE. coli 15 T^- cells growing on glucosemineral medium were supplemented with 2 mM l-cysteine-HCl. The optical density, acid soluble SH (AS-SH) as well as the DNA, RNA, and protein synthesis of the cultures were examined during this treatment and after return to normal growth conditions. Similarly, the survival of cells irradiated by a standard X-ray dose in cysteine-free buffer was measured.The development of radioresistance during the cysteine treatment as well as the loss of this acquired radioresistance after return to normal growth conditions could be divided into two phases: a) an instantaneous and b) a slow change of radioresistance. Phase a seems to be related to the changes occurring in the AS-SH content of the bacteria, while phase b is apparently dependent on the alterations in the synthesis of macromolecules.This work was partly presented at the 6th Annual Meeting of the European Society for Radiation Biology, Interlaken 1968.     

DNA breakage due to metronidazole treatment

The mutagenicity of metronidazole [1-(hidroxyethyl)-2-methyl-5-nitroimidazole] (MTZ) has been shown in different prokaryotic systems. However, data on human cells are still contradictory. In this study DNA damage was determined by the single cell gel electrophoresis (SCGE) assay, in lymphocytes from 10 healthy subjects treated with therapeutic doses of this drug. Samples were obtained before treatment, as well as 1 and 15 days after ending treatment. Results showed a significant increase of DNA strand breaks 1 day after ending treatment, although, an inverse correlation between the amount of DNA damage and plasma concentrations of MTZ was obtained. Thus, the observed damage may be induced by some MTZ metabolite rather than by the parent drug. Interestingly, the amount of DNA damage returned to basal levels 15 days after ending treatment, except in two individuals. This persistent damage should be further investigated.     

DNA synthesis during bacterial conjugation. I. Effect of mating on DNA replication in Escherichia coli Hfr

Conjugation in Escherichia coli involves an oriented transfer of DNA from the Hfr to the F^?. We have examined the course of DNA replication in a donor cell while it is transferring its DNA. Using isotopic density shift for estimating replication, we have shown that mating is accompanied by initiation of a new round of DNA replication in Hfr cells. With the onset of F-mediated transfer replication, the normal vegetative replication in the Hfr appears to be suppressed. Experiments with F′ donors indicate that the transfer of the chromosome is necessary for switching off vegetative replication.     

Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli.

Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.     

Inductive effects of environmental concentration of atrazine onEscherichia coli andEnterococcus faecalis

Atrazine solutions (0.1, 1, 10 and 100 microg/L) inoculated with Escherichia coli and Enterococcus faecalis under natural conditions significantly increased (p < or = 0.05) the population levels of both test bacteria; it indicates the ability of bacterial cells to degrade atrazine and to use the original compound or its degradation products as nutrient(s). In some cases, alterations in the morphology of the colonies were also observed on selective solid media. Biochemical differentiation was also found and, on the other hand, a loss of culturability was recorded; this suggests that bacteria have entered in a viable but nonculturable state. A re-appearance of the colonies occurred after inoculation on tryptone-soy agar with atrazine.     

Effect of ionizing radiation on DNA synthesis in ataxia telangiectasia cells.

The effect of ionizing radiation on DNA synthesis in control and ataxia telangiectasia (AT) lymphoblastoid cell lines was determined. A dose dependent decrease in DNA synthesis was observed in control cells, and the rate and extent of thi decrease in synthesis increased with time after irradiation. No decrease in DNA synthesis was obtained in AT cells, immediately following irradiation, at doses up to 400 rads. At longer times postirradiation, inhibition of synthesis increased but the extent of inhibition was less in AT cell than controls at all doses used. An immediate depression of DNA synthesis was evident in control cells after a radiation dose of 200 rads reaching a maximum at 90 min postirradiation. Little or no decrease in DNA synthesis was evident in AT cells up to 60 min after the same radiation dose, but a decrease occurred between 60 and 90 min after irradiation. The rate of recovery of DNA synthesis to normal levels was more rapid in AT cells than in controls.