Deoxyribonucleic acid strand breaks during drying of Escherichia coli on a hydorohobic filter membrane.

Cells of Escherichia coli mounted on a hydrophobic filter membrane were dried under various vapor pressures. A mutant defective in deoxyribonucleic acid repair (uvrA recA) was more sensitive to drying at a water activity of 0.53 or below than the parent strain but not at a water activity of 0.75 and above. Sucrose gradient studies showed that single- and double-strand breaks of deoxyribonucleic acid occurred at a water activity of 0.53 or below, but no breaks could be observed at a water activity of 0.75 or above. These results were observed in all cells rehydrated with 0.03 M tris (hydroxymethyl) aminomethane-hydrocholoride buffer solution at 0 or 37 degrees C, in the presence or absence of oxygen, with saturated water vapor or with a hypertonic solution followed by a gradual dilution. Freezable water was detected in the cells only at a water activity above 0.75 by differential scanning calorimetry. Removal of unfreezable water of cells in the drying, therfore, might induce deoxyribonucleic acid strand breaks.     

Mutation induced by drying of Escherichia coli on a hydrophobic filter membrane.

Drying of Escherichia coli to a required cellular water level was conducted on a hydrophobic membrane at the corresponding relative humidity. Mutation from an arginine auxotroph to the prototroph was induced by drying to a water activity (aw) of 0.53 and below, but not to an aw of 0.75 and above. The critical aw below which mutation occurred in the course of drying was similar to that for induction of deoxyribonucleic acid (DNA) strand breakage in the bacteria. Some ultraviolet or gamma-irradiation-sensitive strains, e.g., strains of carrying recA, recB, and uvrA recA were more sensitive to drying than the wild-type strains or strains carrying uvrA and polA. The DNA strand breakage of every strain was observed to be to a similar extent after drying to an aw of less than 0.53. The drying-resistant strains repaired the damaged DNA partially during postdrying incubation in a growth medium but not in phosphate buffer solution, while the drying-sensitive strains could not at all. Significant mutation on drying occurred in the wild-type strains, strains carrying uvrA and polA, but not in strains carrying recA. It is, therefore, concluded that the mutation is caused by errors in rec-dependent repair of the drying-induced breakage in DNA.     

Repair of Alkylation Damage: Stability of Methyl Groups in Bacillus subtilis Treated with Methyl Methanesulfonate

Bacillus subtilis was not inactivated and was able to replicate even though approximately 3 x 10(4) methyl groups added by methyl methanesulfonate (MMS) were bound to the deoxyribonucleic acid (DNA) of each organism. No significant loss of methyl groups from the DNA occurred for several generations upon incubation of methylated wild-type or MMS-sensitive cells. Single-strand breaks were not observed in the DNA from cells treated at this low MMS dose. Higher doses of MMS resulted in significant killing of both wild-type and MMS-sensitive strains, and the DNA extracted from such treated cells sedimented more slowly than control DNA through alkaline sucrose gradients, indicating the presence of breaks or apurinic sites (or both). These breaks were repaired upon incubation of wild-type but not of MMS-sensitive strains. Repair of damage induced by alkylating agents is probably the repair of breaks which occur as a consequence of high levels of alkylation.     

Influence of the interaction of temperature and water activity on the production of ochratoxin A and the growth of Aspergillus niger, Aspergillus carbonarius and Aspergillus ochraceus on coffee-based culture medium

In the present study, the effect of temperature and water activity on fungal growth and ochratoxin production on coffee-based medium was assessed. Optimal growth of three Aspergillus strains was observed in the same ecological conditions, namely 30 degrees C and 0.99 water activity. Maximal daily growth is 11.2, 6.92, and 7.22 mm/day for Aspergillus niger, Aspergillus carbonarius, and Aspergillus ochraceus, respectively. However, ecological conditions for optimal ochratoxin production vary according to the toxinogenic strain, with water activity as a limiting factor. Such an ochratoxin A production is inhibited at 42 degrees C and 0.75 water activity. Correspondence between laboratory tested water activity and that measured on a sun-dried ripe cherry batch shows that the first 5 days of drying are critical for fungal growth and ochratoxin A production. Accordingly, artificial drying of cherries at temperatures above 42 degrees C will impede not only fungal growth but also contamination with ochratoxin A.     

Repair of hydrogen peroxide-induced single-strand breaks in Escherichia coli deoxyribonucleic acid.

Near-ultraviolet (300 to 400 nm) irradiation of L-tryptophan yielded H2O2 (a toxic photoproduct) that was selectively lethal for rec and polA1 Escherichia coli mutants. H2O2 treatment of cells resulted in the induction of single-strand deoxyribonucleic acid breaks. These breaks were repaired to only a small extent in polA1, recA recB, and recA mutants, but were efficiently repaired in wild-type strains. We conclude that H2O2 deoxyribonucleic acid lesions require both the polA+ and recA+ pathways for repair.     

DNA damage and repair in the steps for obtaining biopreparations

The change in the number of single-strand breaks in the genome of E. coli cells was being studied during centrifugation and drying of biomass used as raw material for producing biopreparations. The results obtained allowed the authors: (1) to establish the value of DNA damage at the two stages mentioned above and to reveal that structural integrity of the genome is mostly damaged if the humidity of dried biomass is below 15%; (2) to demonstrate that the repair of bacterial genome damages occurs after centrifugation and superficial dehydration, but it is not observed after deep dehydration.     

Effects of chloramine on Bacillus subtilis deoxyribonucleic acid.

The lesions induced in Bacillus subtilis deoxyribonucleic acid (DNA) after treating bacterial cells (in vivo) and bacterial DNA (in vitro) with chloramine were studied biologically and physically. Single-strand breaks and a few double-strand scissions (at higher chloramine doses) accompanied loss of DNA-transforming activity in both kinds of treatments. Chloramine was about three times more efficient in vitro than in vivo in inducing DNA single-strand breaks. DNA was slowly chlorinated; the subsequent efficiency of producing DNA breaks was high. Chlorination of cells also reduced activity of endonucleases in cells; however, chlorinated DNA of both treatments was sensitized to cleavage by endonucleases. The procedure of extracting DNA from cells treated with chloramine induced further DNA degradation. Both treatments introduced a small fraction of alkali-sensitive lesions in DNA. DNA chlorinated in vitro showed further reduction in transforming activity as well as further degradation after incubation at 50 C for 5 h whereas DNA extracted from chloramine-treated cells did not show such a heat sensitivity.     

Effects of pH in the in vitro chromosomal aberration test

The relation between the pH of the medium and clastogenic activity was studied in Chinese hamster ovary (CHO) K1 cells in vitro. The pH was adjusted with NaOH, KOH, HCl or H2SO4. No clastogenic activity was observed over the initial pH range of 7.3-10.9 without S9 mix, but a few chromosomal aberrations were induced at pH 10.4 with S9 mix. The frequency of aberrations increased with the increase in amount of S9. At acidic pH, many chromatid breaks were induced at initiatial pH 5.5 or below without S9 mix, and aberrations such as chromatid breaks and chromatid exchanges were induced at initial pH 6.2 or below with S9 mix. Using MES and Bis-Tris as buffers instead of sodium bicarbonate, we observed that aberrations of the chromatid break type were inducible at pH 6.2 or below. These results show that the combination of strong alkalinity and S9 is clastogenic to CHO-K1 cells, and also that weakly acidic media are genetically active. The results indicate that incubations at non-physiological pH might give false-positive responses.     

Similarity in Several Properties of Psychrophilic Bacteria Grown at Low and Moderate Temperatures

Several properties of psychrophilic pseudomonads were studied with cells grown in batch culture in nutrient broth at 2 and 30 C. No differences were observed in the size, catalase activity, deoxyribonucleic acid, ribonucleic acid, or protein content of cells grown at either temperature. The importance of comparing physiologically similar cells is discussed.     

Tryptophan photoproduct(s): sensitized induction of strand breaks (or alkali-labile bonds) in bacterial deoxyribonucleic acid during near-ultraviolet irradiation.

Long-wavelength ultraviolet light (300 to 400 nm) converts L-tryptophan to a photoproduct that is toxic for bacterial cells in dark conditions. We now report that similar photoproducts of l-tryptophan sensitize bacterial deoxyribonucleic acid to 365-nm radiation, increasing the yield of deoxyribonucleic acid strand breaks (or alkali-labile bonds) by approximately 11.5-fold. Evidence is also presented which indicates that thse sensitized deoxyribonucleic acid lesions contribute to lethality for Escherichia coli irradiated with 365-nm ultraviolet light in suspensions of tryptophan photoproducts.     

Effect of challenge temperature and solute type on heat tolerance of Salmonella serovars at low water activity

Salmonella spp. are reported to have an increased heat tolerance at low water activity (a(w); measured by relative vapor pressure [rvp]), achieved either by drying or by incorporating solutes. Much of the published data, however, cover only a narrow treatment range and have been analyzed by assuming first-order death kinetics. In this study, the death of Salmonella enterica serovar Typhimurium DT104 when exposed to 54 combinations of temperature (55 to 80 degrees C) and a(w) (rvp 0.65 to 0.90, reduced using glucose-fructose) was investigated. The Weibull model (LogS = -bt(n)) was used to describe microbial inactivation, and surface response models were developed to predict death rates for serovar Typhimurium at all points within the design surface. The models were evaluated with data generated by using six different Salmonella strains in place of serovar Typhimurium DT104 strain 30, two different solutes in place of glucose-fructose to reduce a(w), or six low-a(w) foods artificially contaminated with Salmonella in place of the sugar broths. The data demonstrate that, at temperatures of > or =70 degrees C, Salmonella cells at low a(w) were more heat tolerant than those at a higher a(w) but below 65 degrees C the reverse was true. The same patterns were generated when sucrose (rvp 0.80 compared with 0.90) or NaCl (0.75 compared with 0.90) was used to reduce a(w), but the extent of the protection afforded varied with solute type. The predictions of thermal death rates in the low-a(w) foods were usually fail-safe, but the few exceptions highlight the importance of validating models with specific foods that may have additional factors affecting survival.     

Rejoining of radiation-induced single-strand breaks in deoxyribonucleic acid of Escherichia coli: effect of phenethyl alcohol.

Single-strand breaks in deoxyribonucleic acid of Escherichia coli B/r cells exposed to 20 krads of gamma radiation could be rejoined by incubation of irradiated cells in growth medium. In the presence of 0.25% phenethyl alcohol, this repair was completely inhibited although deoxyribonucleic acid and protein syntheses were suppressed only partially.     

Deoxyribonucleic Acid Damage by Monofunctional Mitomycins and Its Repair in Escherichia coli

Exposure of Escherichia coli to the antibiotic mitomycin C (MTC) at a concentration of 0.5 mug/ml caused cross-linkage between complementary strands of deoxyribonucleic acid (DNA). Derivatives of mitomycin, 7-methoxymitosene (7-MMT) and decarbamoyl mitomycin C (DCMTC), at a level as high as 20 mug/ml formed no cross-links between DNA strands. Ultraviolet light-sensitive mutants of E. coli K-12 bearing uvrA, uvrB, uvrC, or recA mutations were more sensitive to the lethal action of 7-MMT and of DCMTC than was the wild-type strain. Treatment of wild-type cells with these antibiotics resulted in the production of single-strand breaks in DNA, which were repaired upon incubation in a growth medium. Such breaks in DNA were not produced in the uvrA and the uvrB mutants. In the uvrC mutant, single-strand breaks were produced by 7-MMT or by DCMTC, but these breaks were not repaired upon incubation. These results are discussed in connection with the mechanism for removal of pyrimidine dimers in ultraviolet-irradiated bacteria.     

Time Scale for Rejoining of Bacteriophage λ Deoxyribonucleic Acid Molecules in Superinfected pol+ and polA1 Strains of Escherichia coli After Exposure to 4 MeV Electrons

The time scale for rejoining of radiation-induced deoxyribonucleic acid (DNA) single-strand breaks was measured in the presence and absence of oxygen. The involvement of DNA polymerase I in this repair process was studied. Formation and rejoining of DNA strand breaks were measured in lambda DNA infecting lysogenic pol(+) and polA1 strains of Escherichia coli irradiated by 4 MeV electrons under identical conditions. Irradiation and transfer to alkaline detergent could be completed in less than 180 ms. The initial yields of DNA strand breaks were identical in pol(+) and polA1 host cells and four- to fivefold higher in the presence of oxygen than in nitrogen anoxia. Evidence for the existence of a very fast repair process, independent of DNA polymerase I, was not found, since no rejoining of radiation-induced DNA strand breaks was observed during incubation from 45 ms to 3 s. In pol(+) host cells most of the strand breaks produced in the presence of oxygen were rejoined within the first 30 to 40 s of incubation, whereas no rejoining could be detected within the same period of time in anoxic cells. Since no rejoining of broken lambda DNA molecules was observed in polA1 host cells, it is concluded that the synthetase activity of DNA polymerase I is involved in the rejoining of DNA breaks induced by radiation in the presence of oxygen.     

Role of exonucleases V and VIII in adenosine 5''-triphosphate- and deoxynucleotide triphosphate-dependent strand break repair in toluenized Escherichia coli cells treated with X-rays.

The repair of X-ray-induced strand breaks was studied in permeabilized Escherichia coli recBC cells deficient for the adenosine 5'-triphosphate (ATP)-dependent exonuclease V and in recBC sbcA cells that possess the ATP-independent exonuclease VIII. It is shown that repair induced by additon of ATP does not take place in recBC and recBC sbcB cells and is limited in recBC sbcA cells. ATP-dependent repair is nevertheless observable if together with ATP a mixture of deoxynucleotide monophosphates is supplied to the cells. These data fit with the assumption that in wild-type cells ATP-dependent repair involves exonuclease V-induced deoxyribonucleic acid degradation and rephosphorylation of the degradation products which are reused for deoxyribonucleic acid polymerase I-dependent break closure. Repair in the presence of deoxynucleotide triphosphates rejoins a similar fraction of breaks in all strains tested irrespective of the amount of postirradiation degradation resulting from exonuclease V and exonuclease VIII activities. Thus, exonuclease V is dispensable for deoxynucleotide triphosphate-dependent repair, i.e., does not "clean" the ends of breaks produced by X-irradiation. ATP- and deoxynucleotide triphosphate-dependent repair are not additive and seem to repair the same population of deoxyribonucleic acid molecules damaged by X-irradiation.     

Deoxyribonuclease-susceptible Floc Forming Pseudomonas sp.

A bacterium belonging to Pseudomonas which was isolated from activated sludge formed flocs in glycerol-containing medium. The flocs were deflocculated by deoxyribonuclease treatment in the presence of magnesium ions. Flocs were also deflocculated by 2 m NaCl, heating at temperatures higher than 50°C, and at pH below 1 or above 11. The observations suggest that deoxyribonucleic acid is directly involved in the association of cells and that ionic bonds are responsible for the flocculation of cells.     

Effect of pH on competence development and deoxyribonucleic acid uptake in Streptococcus sanguis (Wicky).

Streptococcus sanguis (Wicky) cells, strain WE4, developed little or no competence and failed to autolyze in permissive conditions when treated with competence factor (CF) below PH 7.0. This lack of activity was directly correlated with the inability of the cells to bind or take up CF at pH values of 5.5, 6.0, and 6.5. On the other hand, competent cells bound deoxyribonucleic acid molecules maximally below pH 7.0 and transformed maximally at pH 6.5. Deoxyribonucleic acid was optimally bound to cells in a deoxyribonuclease-resistant form at pH values between 7.0 and 8.5. Concomitant with this binding, undefined acid-soluble DNA fragments appeared in the culture menstrua. CF binding and uptake by cells was not only influenced by low pH but also by low temperature. At 0 C, WE4 cells bound only 4% of the input CF and took up less than 1% into a trypsin-insensitive state compared to cells treated at 37 C. Cells treated with CF at 0 C did not autolyze when transferred to permissive conditions. The results presented in this report extend earlier findings that showed that competence development and autolysis are related to the uptake of CF.     

Effect of aeration on minimal medium recovery of heated Salmonella typhimurium.

The effect of presence or absence of air on minimal medium recovery of heated Salmonella typhimurium was investigated. It was determined that the expression of minimal medium recovery is not only dependent on heat and a nutritionally complex medium but also on air. Unlike in the presence of air, in the presence of nitrogen, cells were able to recover their ability to grow on Trypticase soy agar enriched with 0.5% yeast extract (TSY) when incubated in TSY broth. It was established that in the presence of nitrogen the number of heat-TSY- induced, single-straneded breaks in deoxyribonucleic acid (DNA) were less than in the presence of air. Furthermore, the DNA breaks in nitrogen were repaired, whereas DNA breaks in air were not. The ability of cells to grow on TSY agar corresponded well with their ability to repair damage to DNA.     

Cytological Studies of Deoxyribonucleic Acid Replication in Escherichia coli 15T−: Replication at Slow Growth Rates and After a Shift-Up into Rich Medium

We examined the gross nuclear morphology of Escherichia coli 15T⁻ grown in different media with doubling times ranging from 22 to 270 min. In slowly growing cells, deoxyribonucleic acid synthesis was measured by autoradiography and shown to occur with greatest probability during the first two-thirds of the division cycle. In such cells, segregation occurred later, at the end of the division cycle rather than at the end of deoxyribonucleic acid replication. Nuclear regions in L-broth cells (22-min doubling time) cannot correspond to separate chromosomes but probably represent regions of replication activity. Segregation of template nucleotide strands was measured after a shift-up from proline M9 or glucose M9 media into L broth. A model is presented to account for the pattern of segregation observed.     

Nature of the Repair of Methyl Methanesulfonate-Induced Damage in Bacillus subtilis

A nuclease present in extracts of Bacillus subtilis inserts breaks in deoxyribonucleic acid (DNA) treated with the monofunctional alkylating agent, methyl methanesulfonate (MMS), but the nature of the sites within the alkylated macromolecule at which these breaks occur is not known. DNA extracted from B. subtilis cells that have recovered from MMS damage has lost its susceptibility to enzyme action. The recovery process is accompanied by some DNA breakdown and by the incorporation of thymidine. Some recovery from ultraviolet irradiation (UV) and MMS occurred in organisms starved for thymine or adenine, but UV recovery was stimulated by their addition. It is possible that MMS recovery proceeds by a process of excision and repair similar to, but not identical with, UV repair.