Mechanism of radiosensitizing effect of chloride ion on E. coli

Cells of E. coli capable of repairing DNA damage are sensitized to radiation in the presence of NaCl. However, the enhanced radiolethality was suppressed by the addition of compounds such as an amino acid to the irradiation buffer. The protective efficiencies of these compounds depend on their reactivities with Cl-.2 or OH.. ATP synthesis in the cells irradiated in the presence of NaCl was severely inhibited depending on the dose of irradiation. This reduced rate of ATP synthesis can account for the inhibition of protein, RNA and DNA synthesis in the irradiated cells with NaCl.     

The influence of thiols on the pre-irradiation incubation effect of nitroimidazoles in E. coli cells

The increase in the degree of radiosensitization of Escherichia coli cells following prolonged pre-irradiation incubation with nitroimidazoles is not correlated with the loss of intracellular non-protein thiols (NPSH) alone. The rates of reduction of the nitro compounds and the NPSH removal do not show strong dependencies on the lipophilicities of the nitroimidazoles whereas the highly lipophilic compound RGW-609 effects an increase in radiosensitization in a much shorter incubation time than the other nitroimidazoles. Exogenous dithiothreitol (DTT) increased the rate of reduction of misonidazole in the cells but did not alter the fraction converted to the amine. Added DTT (0.15 mmol dm-3) completely protected against the pre-irradiation incubation effect of misonidazole (2.5 mmol dm-3) when added at the start of the incubation but only partially protected when added before irradiation. It is suggested that NPSH can intercept metabolite(s) (or their precursors) of nitroimidazoles which can potentiate cell killing by radiation.     

Radiation sensitization of E. coli B/r by nitrous oxide

E. coli B/r have been used to study radiation sensitization by nitrous oxide (N2O). Cells suspended in S?rensen's phosphate buffer show a large amount of sensitization by N2O (relative to the response in 100% N2). Cells in McIlvaine's phosphate-citric acid buffer, however, show no sensitization by N2O. Sensitization in S?rensen's buffer can be prevented by hydroxyl radical (.OH) removal or by catalase. Chemical assays for the amounts of H2O2 formed under various conditions provide the basis for the conclusion that the high concentration of the citrate ion in McIlvaine's buffer does not allow the build-up of H2O2. Sensitization by N2O requires that both H2O2 and OH radicals be present.     

Abnormal radiosensitizing and cytotoxic properties of ortho-substituted nitroimidazoles

Various 5-substituted 4-nitroimidazoles have been shown to be much more efficient radiosensitizers and much more toxic than would have been predicted from their electron affinities, as measured by values of one-electron reduction potential, E17. Using Chinese hamster V79 cells in vitro, a comparison has been made with some isomeric 4-substituted 5-nitroimidazoles. These compounds have E17 values some 64mV greater than the 4-nitroimidazoles, yet show much lower sensitizing efficiency and also lower toxicity. Neither series of compounds shows the greater toxicity towards hypoxic cells usually associated with nitroaromatic and nitroheterocyclic compounds. The second-order rate constants, k2, for reaction of these isomeric nitroimidazoles with glutathione and dithiothreitol were determined. Within each series the value of k2 increased with increasing electron affinity, however, the 4-nitroimidazoles were always more reactive than their corresponding 5-nitro isomers. The sensitizing and toxic properties of these compounds may involve depletion of intracellular thiols; this possibility is discussed.     

Prediction of effects of amino acid supplementation on growth of E. coli B/r

A mathematical model for the growth of a single cell of E. coli on medium containing amino acid is presented. A mixture of purified amino acids (glutamate, aspartate, serine, tyrosine, and leucine) combined in the ratios found in a natural digest (casein) were employed as the nitrogen source. Each of these amino acids is the representative of a different family of amino acids. The transport mechanisms and assimilation routes for each amino acid were inserted into the prototype model. The enzyme activities and saturation constants used in the model were based on literature data. The maximum velocities for uptake systems were calculated from experimental data. The formation and homeostasis of amino acid pools were regulated through cross-control of the activities of biosynthetic enzymes and of membrane transport of exogenous nutrients. The size of each amino acid pool was determined with mass balance equations that included terms for a transport system, a biosynthesis system, a transaminase enzyme system for interchange between the amino acid families, and a consumption system. The predictions of the extended model with regard to nutrient concentrations and growth rates compared well with the experimental data.     

Mutations in the L-arabinose operon of Escherichia coli B/r with reduced initiator function.

Partial reversion mutants derived from a strain containing a strongly polar initiator-defective mutation (araI1036) in the L-arabinose operon were found to have several characteristics expected of mutants with reduced initiator function. These reversion mutations are cotransduced with the ara region and are probably within the araI region. Furthermore, they permit induction of the L-arabinose operon to a level only one-third of the normal wild-type level. These partially functional initiator regions reduce the expression of structural genes in the cis position only; they function quite independently of wild-type or defective initiator regions in the trans position. These mutants exhibit a two- to threefold increase in the rate of expression of ara operon genes within one-tenth of a generation after a shift of the growth temperature from 28 to 42 degrees C. This suggests that the temperature optimum for initiation of operon expression is higher for the partial revertant strains than it is for strains containing a wild-type initiator region.     

Indirect and intragenic suppression of the lexA102 mutation in E. coli B/r.

Summary In Escherichia coli B/r the expression of UV inducible (SOS) functions is under the control of the recA and lexA genes. In this study we have characterized mutants which are altered in their ability to express SOS functions. These mutants were isolated as UV resistant UV nonmutable (Rnm) derivatives of the lexA102 uvrA155 mutant strain WP51. The UV resistance of these Rnm strains is a result of the suppression of lexA102 mediated UV sensitivity. Genetic mapping of rnm mutations shows that the two predominant classes, rnmA and rnmB, map in or very near the lexA and recA genes respectively. rnmA mutations differ from rnmB with respectively recA protein synthesis. rnmA mutations do not restore the ability to express high levels of recA protein after UV treatment whereas rnmB mutations result in constitutive expression of high levels of recA protein. However, both rnmA and rnmB mutant strains inhibit postirradiation DNA degradation. This shows that in rnmA strains, high levels of recA protein are not needed to inhibit postirradiation DNA degradation.The genetic map location and constitutive expression of recA protein synthesis resulting from rnmB mutations suggests that they are operator constitutive mutations of the recA gene. The result that the lexA ⁺ gene is required for the expression of UV mutagenesis in rnmB mutants shows that high levels of recA protein do not circumvent the need for the lexA ⁺ gene product in this process. Thus, while the lexA gene product is required for the induction of recA protein synthesis, lexA must have an additional role in UV induced mutagenesis.     

Some mechanisms involved in the radiosensitization of E. coli B/r by paracetamol.

Paracetamol, a widely-used analgestic and antipyretic drug, sensitized E. coli B/r to 60Co gamma-rays under hypoxic conditions. Part of the sensitizing effect has been shown to be due to an electron adduct of the drug. Paracetamol inhibited both post-irradiation DNA and protein syntheses. The targets involved in the inhibition of post-irradiation DNA synthesis have been shown to be different in the presence of the sensitizer. Increased DNA degradation after irradiation was also observed when E. coli B/r were irradiated in the presence of the drug. The presence of paracetamol during hypoxic irradiation of E. coli B/r resulted in the enhancement of DNA single-strand scissions with no apparent effect on their rejoining.     

Radiation sensitization of E. coli B/r by mixtures of oxygen and nitrous oxide

Oxygen (O2) sensitizes bacterial cells in at least two mechanistically different ways, depending on the specific O2 concentration present during irradiation. Based on previous work from this laboratory, it has been proposed that nitrous oxide (N2O) and low concentrations of O2 share a common mechanism for damage. This mechanism, involving the production of superoxide anion radicals (O2-), is different from that which causes damage at high O2 concentrations. Others, however, have presented evidence that N2O and O2 (usually tested only at high concentrations) act in different ways to sensitize bacterial cells. We have now measured the radiation sensitivity in mixtures of N2O and O2 to observe additivity patterns and to determine if these two agents have any common processes for sensitization. We found that some low O2 concentrations do not increase the response in N2O, although they can have significant sensitizing effects in N2. This lack of additivity is taken as evidence for a common mechanism of damage from N2O and low concentrations of O2. In contrast, damage from high concentrations of O2 is additive to the damage from N2O. The greatest sensitivity, observed with a gas mixture of about 15 per cent O2/85 per cent N2O, is equivalent to the response in 100 per cent N2 plus the maximum amount of damage O2 can cause plus the maximum amount of damage N2O can cause. This additivity is taken as evidence that N2O and high concentrations of O2 sensitize in different ways. Thus, O2 is known to sensitize these bacteria in at least two different ways; one of these is apparently also the way N2O sensitizes.     

Mutational and dark-repair specificities in U.V.-irradiated di-auxotrophs of E. coli B/r

Summary U.V. irradiation experiments have been performed with three tyr^-leu^- diauxotrophs of E. coli B/r; 36-10, 36-18 and 36-32. These have in common the tyr^- marker WU-36. Three types of mutations were scored; tyr⁺leu^-, tyr^-leu⁺, and tyr⁺leu⁺. The percentages of total mutants scored which are of the tyr⁺leu⁺ type depend, firstly, on the strain. The tyr⁺leu⁺ revertants are rare in 36-10 and common in 36-18 and 36-32. Secondly, the percentages of tyr⁺leu⁺ among total revertants are determined by the nature of the plating medium, being altered by supplementation with a low level of the required amino acids, a low level of nutrient broth, or by caffeine. These differential effects upon tyr⁺leu⁺ and tyr⁺leu^- or tyr^-leu⁺ reversions can be interpreted at the levels of dark-repair and the later stages in the process leading to phenotypic expression. It is suggested that states of repression and derepression may be implicated in determining susceptibility of lesions in suppressor loci to repair processes. Phenotypically different classes of mutations have different pathways leading to final expression.This work was initiated in Dr. Ruth F. Hill's laboratory, Department of Radiology, Columbia University, New York.     

Studies on the biosynthesis of acylphosphatidylglycerol in Escherichia coli B and B/r.

The present study has demonstrated that one molecule of acylphosphatidylglycerol was synthesized from two molecules of phosphatidylgycerol by the transacylation reaction in which phosphatidylglycerol acted both as an acyl donor and an acceptor. Phosphatidylethanolamine was identified as an another acyl donor, participating in acylphosphatidylglycerol formation. These results are discussed in terms of a new pathway for the turnover of phosphatidylglycerol in Escherichia coli.     

Aggregation of Escherichia coli B/r A during agar filtration: effect on morphometric measurements.

We have investigated the phenomenon of particle aggregation in a sample of 71,038 Escherichia coli B/r A cells in balanced exponential growth, during preparation for electron microscopy by agar filtration. The bacteria were photographed in a transmission electron microscope and the dimensions and spatial relationships among all the members of each aggregate were recorded using an interactive image processing system. The proportion of aggregated cells, 22%, is much greater than that found by direct count in a light microscope (7%), implying that most aggregation takes place during the preparation stages. The aggregated cells are about 1% narrower than the free cells, because of mutual compression, and 1.5% longer, because of a selection bias in favor of longer cells. From a statistical analysis of the data, we conclude that the clustering of cells into aggregates in the course of sample preparation is the result of random encounters during the settling on the collodion membrane and of the changing surface tension during the drying process. A method is proposed to correct morphometric measurements for the distortion caused by cellular aggregation of this kind.     

Evolution of the D-ribose operon on Escherichia coli B/r.

The D-ribose operon (rbs) of Escherichia coli K-12 maps at 83 min and is inducible. The rbs operon of E. coli B/r maps at 2 min and is constitutive. Evidence is presented showing that a second inducible copy of the rbs operons is present in E. coli B/r mapping at 83 min. The data indicated that the duplication of the rbs operon represented a transposition of the 83-min region to 2 min. The identification of a second copy of the rbs operon in B/r and the determination of its inducibility were based on the reactivation, through mutagenesis, of inducible rbs expression, mapping by P1 transduction of the mutation site to 83 min, and merodiploid complementation analysis of the D-ribokinase expression in E. coli B/r. We also show that the rbs transposition to 2-min continued to generate transposable elements coding for the 1- to 2-min region of the chromosome and transposing onto extrachromosomal DNA target molecules such as pBR322.     

The araIc mutation in Escherichia coli B/r.

The araIc allele is a cis-acting mutation which has been used to define the araBAD promoter in Escherichia coli B/r. Nineteen araIc mutants were originally isolated by Englesberg and co-workers as Ara+ "revertants" of an araC deletion mutant (Englesberg et al. J. Mol. Biol. 43:281-298, 1969). The mutants constitutively expressed araBAD gene products in the absence of functional araC activator protein. Eight of the araIc mutations have been cloned by in vivo recombination onto pBR322-ara hybrid plasmids. Restriction and DNA sequence analysis of these araIc mutations showed that they result from a single base-pair change located at -35 in the araBAD promoter.