Post-irradiation Plating Medium Research Articles

Decreased survival of UV-irradiated Staphylococcus aureus in the presence of 8-methoxypsoralen in the post-irradiation plating medium

For Staphylococcus aureus, the presence of 8-methoxypsoralen (8-MOP) in the post-irradiation plating medium increased the lethal effect of far-UV light (FUV; ∼254 nm) and of 8-MOP plus near-UV light (8-MOP+NUV; ∼365 nm), an effect similar to that caused by acriflavine which inhibits DNA repair. In the repair-proficient strain, the presence of 8-MOP in the plating medium was almost as effective in inhibiting the repair of damage caused by FUV as that caused by 8-MOP photoadditions. Survival data obtained with Rec −-like and Uvr −-like strains suggest that 8-MOP in the plating medium, although possibly inhibiting recombination repair, was much more effective in inhibiting excision repair of FUV damage. Regarding 8-MOP+NUV treatment, 8-MOP in the plating medium had a lesser effect in the repair-deficient strains, differing from that observed after FUV treatment, which is consistent with the notion that different types of damage are caused by the two treatments.

Glucose and related catabolite repressors are powerful inhibitors of pKM101-enhanced UV mutagenesis in Escherichia coli

When stationary phase Escherichia coli K12 trp (amber) cells were exposed to UV doses ranging from 180–540 J m −2, we found that we could not recover any induced Trp + revertants unless the irradiated cultures were first supplied with the Muc + mutation-enhancing IncP plasmid pKM101 (by conjugation). We also found that the numbers of UV-induced Trp + revertants recovered from pKM101 + cultures varied quite dramatically depending upon which of several commonly-used carbon sources were present in the post-irradiation plating medium, e.g., there were always significantly fewer revertants on minimal glucose plates than on minimal glycerol plates. More importantly, there were also fewer UV-induced revertants on glycerol+glucose plates than on `glycerol-only' plates. We then tested two glucose-related compounds which are known to depress intracellular cyclic AMP (cAMP) levels even more effectively than glucose (glucose-6-phosphate and the non-utilisable methyl-α- d-glucopyranoside) and found that they too were able to exert powerfully antimutagenic effects in UV-treated pKM101-containing bacteria. Taken together, these results provide strong additional support for our working hypothesis that at least one component of the mutational pathway which operates in UV-irradiated pKM101-containing cells is extremely sensitive to classical cAMP-mediated catabolite repression.

The sensitivity of an actinic reticuloid cell strain to near-ultraviolet radiation and its modification by trolox-C, a vitamin E analogue.

Near-UV radiation (365 nm)-induced lethality, as measured by colony-forming ability, showed an actinic reticuloid cell strain to be sensitive relative to normal human fibroblasts, when irradiated at 25 degrees C. This effect was not seen after far-UV (254 nm) irradiation. Trolox-C, a water-soluble analogue of vitamin E, incorporated in the pre-irradiation growth medium or in the post-irradiation plating medium, protected the actinic reticuloid cells to the extent that they were as resistant as normal cells. Plating medium containing Trolox-C did not provide differential protection against inactivation of the two cell strains by wavelengths in the far-UV region. The protection provided by Trolox-C, an analogue of the natural antioxidant vitamin E, suggests some free radical involvement in the aetiology of the disease.

Effects of peroxide and catalase on near ultraviolet radiation sensitivity in Escherichia coli strains.

The role of peroxide and catalase on NUV radiation sensitivity was examined in two repair competent E. coli strains, AB1157 and B/r. Exponential phase B/r is considerably more sensitive to NUV radiation than exponential phase AB1157. However, resistance to 5 mmol dm-3 H2O2 was induced in both AB1157 and B/r by pretreating growing cells with 30 mumol dm-3 H2O2. Pretreatment also induced resistance to broad-band NUV radiation in these strains. The addition of catalase to the post-irradiation plating medium increased survival to the same extent as that provided by pretreatment with 30 mumol dm-3 H2O2, in both strains. The NUV radiation sensitivity seen in B/r does not appear to be due to a deficiency in enzymes that scavenge H2O2, as a catalase deficient mutant, E. coli UM1, is more resistant to NUV radiation than B/r. Also, assays for H2O2 scavenging ability show little difference between AB1157 and B/r in this respect. Two hypotheses are put forward to account for the sensitivity of exponential phase B/r. Whilst it is apparent that peroxides and catalase do have a role in NUV radiation damage, it is clear that other factors also influence survival under certain conditions.

Promotion of Septation in Irradiated Escherichia coli by a Cytoplasmic Membrane Preparation

Septation can be promoted in an X-irradiated lon mutant of Escherichia coli K-12 by the addition of an E. coli B/r cytoplasmic membrane preparation to the postirradiation plating medium. The promotion of septation was not associated with an inhibition of growth rate. Two distinct cytoplasmic membrane-associated properties were necessary to promote septation. One of these, the cytochrome-based electron transport system, produced anaerobic conditions by the reduction of oxygen dissolved in the medium. The second system, functioning independently from the first, altered substances found in the peptone and yeast extract components of the postirradiation plating medium. When both systems were operative, significant repair of the cell division mechanism occurred.

Cytoplasmic membrane fraction that promotes septation in an Escherichia coli lon mutant.

A particulate fraction derived from bacterial cells stimulates septation in irradiated Escherichia coli lon mutants when added to postirradiation plating media. It was established that the particles are derived from the cytoplasmic membrane and that they have been partially purified by sucrose density gradient centrifugation. These particles also contain the cytochrome-based respiratory activity of the cell. A variety of experiments established a correlation between the septation-promoting activity of the particles and their ability to remove oxygen from the postirradiation plating medium. It was suggested that the efficient removal of oxygen from the medium allowed the lon cells to repair radiation-induced damage to the septation mechanism.

Frameshift mutagenesis by ultraviolet light effects of broth and caffeine in the post-irradiation plating medium

Ultraviolet-induced back mutation yields were studied in the frameshift strain of Salmonella typhimurium, LT2 hisC3076. The numbers and frequencies per 10 8 survivors of small and large revertant colonies were found to be affected significantly by plating density, but it was possible to detect a considerable enhancement of mutation frequency when broth (2.5%, v/v) was present in the post-irradiation plating medium. Caffeine also significantly enhanced the yields of UV-induced frameshift mutations, but not of γ-induced frameshifts, indicating that the UV-induced pre-mutational lesions which lead to frameshift mutations may be treated in a similar way by the excision-repair system to those which lead to base-pair substitutions.

Effects of ethidium, quinacrine and hycanthone on survival and mutagenesis of UV-irradiated Hcr + and Hcr - strains of E. Coli B/r.

Ethidium, quinacrine or hycanthone when incorporated in the post-irradiation plating medium have all been found to decrease survival and increase str r mutation frequencies in a UV-irradiated E. coli B/r strain. The enhancement of mutational yield was shown, for quinacrine, to apply also to UV-induced trp - → Trp + reversions. Since these three compounds do not enhance mutational yields, and have at best only a weak antimutagenic action, in an Hcr - strain, it seems probable that ethidium, quinacrine and hycanthone are primarily inhibitors of excision repair.

Dark repair inhibitors and pathways for repair of radiation damage in Schizosaccharomyces pombe.

Different compounds known to inhibit dark repair in bacterial and mammalian systems have been tested for a similar effect in the yeast Schizosaccharomyces pombe. A marked inhibition of growth by these different drugs indicated that these were taken up by the cell, yet none except caffeine reduced the colony forming ability when present in the post-irradiation plating medium. The response of 22 different radiation sensitive mutants to the presence of caffeine in the plating medium was tested to investigate the probable number of pathways for repair of radiation damage in this organism. In some of the genetic crosses among single gene mutants showing a differential caffeine-response, supersensitive double mutants were obtained. Strains obtained either by additional crosses or by further mutagenic treatment of the supersensitive double mutants did not show a highly significant increase in radiation sensitivity. These observations indicate that the presently available radiation sensitive mutants represent defects in only two major pathways for repair of radiation damage in the wild type S. pombe.

Pre- and post-irradiation effects upon lethality and reversion in Salmonella typhimurium.

Summary: Lag, exponential and stationary phase cells of Salmonella typhimurium LT-2 trpC3) grown in nutrient broth or tryptophan-supplemented minimal medium were irradiated with doses of u.v. up to 570 ergs/mm2. Casein hydrolysate supplementation of the post-irradiation plating medium causes enhanced survival only for lag and exponential phase broth-grown and exponential phase minimal-grown cells. Caffeine invariably leads to decreased survival levels and abolition of any casein hydrolysate enhancement of survival. Cells of all six growth conditions give induced Trp+ reversions, often due to suppressor mutations, on plating media containing casein hydrolysate. Only for minimal-grown lag and exponential phase cells do any induced Trp+ appear on media devoid of casein hydrolysate supplementation. In these cases too, caffeine has a definite antimutagenic action. Mutation frequency decline (MFD) experiments revealed that those cells exhibiting a casein hydrolysate enhancement of survival on plates also show a delayed onset of MFD and a fall of survival, after an initial delay, in liquid minimal medium. MFD experiments in liquid do not give a complete quantitative explanation for the Trp+ revertant yields found on plates. We suggest that intracellular free amino acid pool sizes may be a common factor in the correlations which we have observed.

Caffeine- and amino acid-effects upon try(plus) revertant yield in U.V.-irradiated hcr(plus) and hcr (minus) mutants of E. coli B-r.

The influence of supplementation of the post-irradiation plating medium with caffeine and/or amino acids, upon both U.V.-induced killing and try+ reversion yield, has been studied in hcr+ and hcr- derivatives of E. coli B/r tryptophan auxotroph WWP-2. All experiments have been carried out with stationary phase cells grown in aerated nutrient broth. In the hcr- strain, caffeine causes no enhancement of either killing or try+ revertant yield. There is mutational enhancement by supplementation with a low level of tryptophan, and an even greater effect when supplementation is with tryptophan plus an additional pool of other amino acids. In the hcr+ strain, tryptophan and/or a pool of other amino acids cause an enhancement of try+ yield, as in the hcr- strain. Caffeine causes lethal enhancement on several different media. There is an apparently straightforward dose enhancement by caffeine, of lethality and try+ revertant frequency, on media containing no, or only, tryptophan. On media containing, however, both a low level of tryptophan and an additional pool of other amino acids, caffeine causes a preferential enhancement of try+ revertant frequency over and above pure dose enhancement. These results suggest that U.V. may cause two types of lesion. One type may lead only to mutation, and is repaired by both the caffeine-insensitive MFD, and the caffeine-sensitive hcr, dark repair systems. This first type of lesion is protected from repair by the presence of a pool of amino acids. The second type of lesion is hypothesised to be capable of causing either lethality or mutation, is repaired only by the hcr dark-repair system, and is not subject to protection by an amino acid pool.

THE EFFECT OF ACRIFLAVINE ON PHOTOREVERSAL OF LETHAL AND MUTAGENIC DAMAGE PRODUCED IN BACTERIA BY ULTRAVIOLET LIGHT.

ABS>The addition of acriflavine to the postirradiation plating medium has the following effects in strain WU36, a tyrosine-requiring substrain of E. coli B/r: it enhances both the lethal and mutagenic effects of uv, decreasing survival and increasing the yield of tyr+ mutations; it reduces the efficiency of photoreversal of induced prototrophs, increasing the amount of light required to accomplish maximal photoreversal about fourfold, while having no such effect on the photoreversal of uv killing; it reduces the efficiency of photoprotection against induced prototrophy, while having no such effect on photoprotection against uv killing. Mutations to prototrophy are reducible in frequency by pretreatment with visible light to the same extent as by posttreatment with visible light, although maximal photoprotection requires about four times as much light as maximal photoreversal. In contrast, very little photoprotection against uv killing is obtained in this strain. The results are interpreted as evidence that the lesions leading to photoreversible uv killing and those leading to photoreversible uvinduced prototrophy are different, either in their primary nature or in some unknown secondary feature. (auth)