MEMBRANE DAMAGE CAN BE A SIGNIFICANT FACTOR IN THE INACTIVATION OF ESCHERICHIA COLI BY NEAR-ULTRAVIOLET RADIATION

Abstract A DNA repair competent strain of Escherichia coli K-12 showed sensitivity to inorganic salts (at concentrations routinely used in minimal media) after irradiation with broad spectrum near–UV radiation, at fluences that caused little inactivation when plated on complex growth medium. This effect was not observed with cells that had been exposed to 254 nm radiation. This sensitivity to minimal medium was increased by increasing the salt concentration of the medium and by increasing the pH of the medium. This sensitivity was greatly increased by adding to the medium a low concentration of commercial glassware cleaning detergent that had no effect on unirradiated cells or far-UV irradiated cells. These findings may explain the large variability often observed in near-UV radiation survival data, and demonstrate that, at least on minimal medium plates, membrane damage contributes significantly towards cell killing. This phenomenon is largely oxygen dependent.     

POSTREPLICATION REPAIR IN uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 IS INHIBITED BY RICH GROWTH MEDIUM

Abstract Escherichia coli K-12 uvrA or uvrB strains grown to logarithmic phase in minimal medium showed higher survival after ultraviolet (UV) irradiation (254 nm) if plated on minimal medium (MM) instead of rich medium. This'minimal medium recovery'(MMR) was largely blocked by additional recA56 (92% inhibition) or lexA101 (77%) mutations, was partially blocked by additional recB21 (54%), uvrD3 (31%) or recF143 (22%) mutations, but additional polA1 or polA5 mutations had no effect on MMR. When incubated in MM after UV irradiation, the uvrB5 and uvrB5 uvrD3 strains showed essentially complete repair of DNA daughter-strand gaps (DSG) produced after UV radiation fluences up to ∼ 6 J/m² and ∼1 J/m², respectively, and then they accumulated unrepaired DSG as a linear function of UV radiation fluence. However, when they were incubated in rich growth medium after UV irradiation, they did not show the complete repair of DSG and unrepaired DSG accumulated as a linear function of UV radiation fluence. The fluence-dependent correlation observed for the uvrB and uvrB uvrD cells between UV radiation-induced killing and the accumulation of unrepaired DSG, indicates that the molecular basis of MMR is the partial inhibition of postreplication repair by rich growth medium. Rich growth medium can be just MM plus Casamino Acids or the 13 pure amino acids therein in order to have an adverse effect on survival, regardless of whether the cells were grown in rich medium or not before UV irradiation.     

INDUCIBLE POSTREPLICATION REPAIR IS RESPONSIBLE FOR MINIMAL MEDIUM RECOVERY IN UV-IRRADIATED Escherichia coliK–12

Abstract— Ultraviolet (UV)-irradiated Escherichia coli K–12 uvrA cells showed higher survival if plated on minimal growth medium rather than on rich growth medium, i.e., they showed minimal medium recovery (MMR). A 2-hour treatment of UV-irradiated cells with rifampicin inhibited the subsequent expression of MMR, and produced a large reduction in survival. We have recently isolated a new mutant ( mmrA1 ) that does not show MMR. The mmrA mutation protected UV-irradiated uvrA cells from the effect of rich growth medium on survival, but not from the effect of rifampicin on survival. DNA daughter-strand gap (DSG) repair in UV-irradiated (4 J/m²) uvrA cells was inhibited to the same degree whether rich growth medium was added immediately after irradiation or after 10 min of postirradiation incubation in minimal growth medium. However, chloramphenicol added immediately after irradiation greatly reduced this repair; there was less reduction if it was added 10 min after UV irradiation. These findings suggest that MMR is an inducible repair phenomenon, and that rich growth medium inhibits this repair process itself rather than its induction.     

DECREASED SURVIVAL RESULTING FROM LIQUID-HOLDING OF U.V.-IRRADIATED ESCHERICHIA COLI C AND SCHZZOSACCHAROMYCES POMBE

Abstract— Ultraviolet-irradiated cells of E. coli C and of haploid wild type yeast Schizosac-charomyces pombe , held in buffer at 22°-25°C for various periods of time prior to plating, show a lower survival than those plated immediately after irradiation. This 'negative liquid-holding effect' (NLHE) contrasts 'liquid-holding recovery' (LHR), found in a number of other E. coli strains and in Saccharomyces cerevisiae . NLHE was observed at all u.v. doses tested. The effect is maximal at holding temperatures in the range 25–30°C, it is very small at 5°C and (in E. coli C) at 44°C. NLHE and LHR resemble each other in several respects. In E. coli both effects are inhibitable by the dark repair inhibitors acriflavine, caffeine and potassium cyanide. They do not occur in nutrient broth, and they are much reduced if the irradiated cells were illuminated with photoreactivating light before holding. NLHE in S. pombe shows characteristics similar to those observed in E. coli C . Mutations leading to increased u.v. sensitivity in E. coli C and S. pombe can alter the liquid-holding response so that LHR is observed. Tetrad analysis of crosses between u.v.-sensitive and u.v.-resistant S. pombe strains indicates that a single chromosome region can control both u.v. sensitivity and liquid-holding response. Several possibilities explaining NLHE are discussed. From current knowledge about dark repair processes and from the similarities between NLHE and LHR in E. coli it seems likely that the two effects reflect slight changes in the efficiency of dark repair.     

The effect of A uvr mutation on rescue of U.V. irradiated Lon strains of Escherichia coli

Abstract— Treatments such as plating at elavated temperature, plating in the presence of pantoyl lactone, plating on minimal medium, plating on complete medium after intense white illumination, or plating on complete medium after holding in buffer, are known to increase survival of U.V. irradiated Lon strains of Escherichia coli. The effect of these treatments on Hcr Lon⁺ and Hcr Lon strains was studied. Pantoyl lactone and elavated temperature recovery seem specifically related to the Lon defect. Holding in buffer does not enhance survival in an Hcr strain, and only exerts a small effect in an Hcr Lon strain. White irradiation and minimal medium may greatly enhance recovery of both Hcr and Lon strains. However, an Hcr Lon strain is sensitive to white irradiation itself.     

A mutant of Escherichia coli K12 exhibiting varying ultraviolet sensitivities depending on the temperature of incubation after irradiation

Abstract— A mutant, URT-43, was isolated from E. coli C600 dar⁺. The mutant has a characteristic feature in that its sensitivity to ultraviolet (u.v.) light is greatly influenced by the temperature at which irradiated bacteria are incubated. On the basis of dose-reduction factor, URT-43 is approximately ten times more sensitive at 42° than at 30°C, even though unirradiated bacteria are not thenno-sensitive, The mutant could not repair u.v.-irradiated bacteriophage Λ_vir in the dark either at 30° or at 42°C, indicating that it is defective in host-cell reactivation. In contrast, the same bacteriophage was reactivated in preirradiated URT-43 if the host-bacteriophage complex was plated at 30° but there was no reactivation at 42°C. Therefore u.v.reactivation was positive at 30° but negative at 42°C. The induction of prophage by URT-43(Λ_h) was achieved by much lower doses of U.V. light than that required for the induction of lysogenic wild type bacteria. Experiments were performed in which irradiated URT-43 was first incubated for various periods in liquid media and plated both at 30° and 42°C. It was found that irradiated bacteria came to be resistant to subsequent plating at 42° only when they were preincubated in the liquid medium containing necessary amino acids and at 30°C. Since this phenomenon was completely inhibited by chloramphenicol, the process seemed to require de novo protein synthesis. An hypothesis was proposed that there are at least two independent dark-repair mechanisms in E. coli; one is responsible for host-cell reactivation and the other is responsible for U.V. reactivation.     

U.V. INDUCED CONFORMATIONAL CHANGES IN TRANSFER RNA

Abstract— The quantum yields for the u.v. inactivation of the amino acid acceptor function of E. coli transfer RNA (for val, phe and lys) and for the loss of its conformation, as a function of exposure, have been determined following irradiation at 280, 265 and 254 nm. Our results suggest that u.v. damage produces a change in the conformation of transfer RNA which in turn inactivates it, and that the anticodon is not the u.v. sensitive site. Calculations indicate that a small number of photoproducts inactivate the transfer RNA.     

PHOTOPROTECTION OF E. COLI B/r: RESPIRATION,GROWTH, MACROMOLECULAR SYNTHESIS AND REPAIR OF DNA*

Abstract— Photoprotecting effects of near UV radiations (300–400 nm, maximum at 360 nm) against far UV radiations (primarily 254 nm) have been studied in Escherichia coli B/r cells in minimal medium with glycerol as a carbon source. Near UV light (10⁵ Jm^-2) has a negligible effect on survival, but causes transitory inhibition of respiration, growth, DNA, RNA, and protein syntheses and cell division. Far UV (52 J m^-2) reduces survival to about 0.5 per cent; respiration, growth and RNA and protein syntheses proceed for about 60 min, after which they nearly cease for several hours. Near UV given before this fluence of far UV increases survival 10-fold and the above processes resume at times and with kinetics characteristic of those produced by lower fluences of far UV. Single-strand breaks appear in the DNA of both unprotected and photoprotected cells; repair of the breaks is essentially complete in protected but not unprotected cells. The viability kinetics for far-UV-irradiated cells with and without photoprotecting treatment are identical except that the curve for the latter is displaced upward about 1 log; exponential increases (cell division) begin at 120 min in each case. The data suggest that, in B/r cells grown under our particular conditions, namely in minimal medium with glycerol, photoprotection is not the result of growth or division delays, but reflects an increased repair capability due to continued respiration.     

THE INFLUENCE OF NUTRITION ON THE DNA CONTENT OF ESCHERICHZA COLI AND ITS RESPONSE TO ULTRAVIOLET LIGHT

Abstract— The influence of nutrition on the sensitivity of Escherichia coli 15 T- to ultraviolet light (u.v.) and the synthesis of DNA has been studied. Growth in media containing glucose or NH,⁺ has been found to endow cells with a greater resistance to lethal u.v. damage than those grown in media containing succinate or amino acids, respectively. In addition, the sensitivity of the lactose ( lac ) locus of the DNA to mutagenic damage has been found to be altered by changes in the carbon supply but not by changes in the nitrogen source, while the sensitivity of loci controlling amino acid synthesis was altered by changes in the nitrogen source but not in the carbon source. Cells fed with glucose or NH₄⁺ have been found to possess more DNA than cells fed with succinate or amino acids, respectively. The data indicate that the type of carbon and nitrogen supplied to the cells will determine whether or not set regions of the DNA will undergo more than one round of replication. The presence in the cell of identical genetic loci either in duplicate or in multiples, directed by the particular types of carbon and nitrogen supplied, is suggested to be, in part, the reason why an alteration in nutrition is able to influence the sensitivity of bacterial cells to radiation.     

Production of coal-solubilizing activity byPaecilomyces Sp. during submerged growth in defined liquid media

Paecilomyces TLi, a fungus isolated from coal, had previously been shown to transform solid coal into a water-miscible liquid during surface growth on coal or on a complex, solid microbiological medium. Coal solubilization has now been demonstrated in submerged cultures grown in defined liquid minimal media. Activity under these conditions is less than that observed in surface culture, and is affected by both nitrogen and carbon nutrition. Coal solubilization occurs at low pH (≤ 4) but may be associated with pH increases during the assay period. Spectroscopic data suggest a role for alkaline catalysis in coal solubilization by this organism.

     

THE ROLE OF HOST-CELL REPAIR IN LIQUID-HOLDING RECOVERY OF U.V.-IRRADIATED ESCHERICHIA COLI

Abstract— The experiments reported give evidence that liquid-holding recovery (LHR) of u.v. irradiated E. coli cells involves basically the same type of dark repair which causes reactivation of phage and which results in much increased survival of the cells themselves [host-cell reactivation (HCR)]. LHR is very small in the two HCR(-) strains B syn^- and B_s-1, but occurs to larger but different extents in the three HCR(+) strains B, B/r, and B/r (Λ). LHR is inhibited if the liquid contains caffeine or acriflavine, both of which are known to inhibit HCR. The results indicate that most of the LHR effect, if not all, occurs during the liquid holding, rather than under growth conditions after liquid holding. It is assumed that the holding itself allows a prolonged time for, and therefore an enhancement of, HCR. It is thus implicit that LHR can be observed only where otherwise HCR of repairable u.v. damage would be incomplete, and that different extents of LHR, as observed in the three HCR(+) strains, reflect different extents of incompleteness of HCR. It is concluded that the repairable u.v. hits which are not fully repaired by HCR are predominantly those concerned with the extra u.v. sensitivity of the strains B and B/r (Λ), relative to B/r.     

THE U.V. SENSITIVITY OF BACTERIA: ITS RELATION TO THE DNA REPLICATION CYCLE

Abstract— A striking increase in the shoulder of the u.v. survival curve but no change in the limiting slope is obtained when cultures of Escherichia coli strain TAU complete the DNA replication cycle in the absence of concommitant protein synthesis prior to irradiation. The u.v. sensitivity of protein synthesis or RNA synthesis is not altered significantly by this treatment.
In contrast to the result for strain TAU, there is no significant change in the u.v. survival curve for the u.v. sensitive E. coli B_s-1 when its DNA replication cycle is completed under similar conditions.
Following a period of inhibited protein synthesis there is a delay in the reinitiation of the normal DNA replication cycle when protein synthesis resumes. This delay would allow time for an intracellular repair system to operate before the attempted resumption of normal replication. Strain B_s-1, which is deficient in this repair system, would not be expected to benefit from such a delay, as consistent with the observed results. A model is presented to account for lethality due to attempted DNA replication during a period of repair synthesis. The maximum survival for a given u.v. dose would be predicted for a culture which has completed the normal DNA replication cycle prior to irradiation and which is not permitted to reinitiate the cycle until all possible repair synthesis is completed.     

Optimum conditions for transformed Panax ginseng hairy roots in flask culture

Panax ginseng hairy roots were transformed by Agrobacterium rhizogenes KTCT 2744. They showed an active branching pattern and fast growth in hormone-free medium, and good growth at 23°C, pH 5.8, 1/2 MS medium, and 3% sucrose. Sucrose provided the highest growth among seven carbon sources tested. Six complex media were also tested. In the combined sugar study, hairy roots grew better on sucrose without glucose or fructose than with glucose or fructose. In the 1/2 MS basal medium, 30 mM in nitrogen and 0.62 mM phosphate salt concentration was the optimum. The growth ratio was maximal at an inoculum size of 0.4% (w/v). Crude saponin and polysaccharide levels were also measured.     

THE EXPRESSION OF LIQUID HOLDING RECOVERY IN ULTRAVIOLET-IRRADIATED ESCHERICHIA COLI REQUIRES A DEFICIENCY IN GROWTH MEDIUM-DEPENDENT DNA REPAIR

Ultraviolet (UV) irradiated Escherichia coli K-12 recA cells (but not rec⁺ cells) show enhanced survival if they are held in buffer prior to plating for viability. To understand the role of the recA mutation in this liquid holding recovery (LHR) phenomenon, we have studied LHR in a temperature sensitive recA 200 mutant. The detection of LHR requires that the irradiated cells be recA when they are plated on growth medium, but the recA deficiency plays no role during liquid holding (LH). We conclude that it is the extreme sensitivity of recA cells in growth medium to unrepaired DNA daughter-strand gaps that magnifies the beneficial effects of the excision repair of DNA lesions during LH. Furthermore, we demonstrate a correlation between a strain's inability to perform growth medium dependent repair and its ability to express LHR. The relative amount of LHR was: recA > recF > lexA > recB > wild type (with the recB and wild-type strains showing negative LHR). Two strains did not show this correlation; the uvrD strain showed less LHR than expected from its UV radiation sensitivity, while the polA strain showed more. The molecular bases for these exceptions are explored.     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

Thermokinetic responses of the metabolic activity of <Emphasis Type="Italic">Staphylococcus lentus</Emphasis> cultivated in a glucose limited mineral salt medium

Biocalorimetric experiments were performed to investigate the metabolic thermal responses of the halotolerant species Staphylococcus lentus in glucose limited mineral salt medium. Growth factors were optimized in both shaker flask and calorimetric experiments. A limiting value of 5 g/L glucose was found to be the optimum for S. lentus growth. The heat flux profiles, OUR, biomass growth, and substrate depletion profiles were compared to deduce the metabolic activity of S. lentus. Shifts in heat flux due to the shifts in substrate uptake and three distinct phases of growth are noticeable in heat profile curves. Respirogram (OUR) and heat profiles are seen to follow the biomass growth curve. Oxycalorific coefficient is validated with the theoretical studies and those noticed in published literature.     

EFFECT OF OXYGEN ON PHOTO AUTOTROPHIC and HETEROTROPHIC GROWTH OF Chlamydomonas reinhardtii IN AN ANOXIC ATMOSPHERE

Photoautotrophic growth of Chlamydomonas reinhardtii was shown to be independent of the presence of atmospheric oxygen. Under constant light and photoautotrophic conditions, C. reinhardtii grew equally well in either air or 367 PPM CO2-in-He. During 12-h light-dark cycles, the cells in air grew substantially faster than those grown in CO2-in-He, indicating a significant role for O2 in dark metabolism. Although cells grown under CO2-in-He were not supplied any exogenous O2, photosynthetic water splitting resulted in the liberation of O2. The effect of photoevolved O2 on the growth of C. reinhardtii was examined (1) by measuring the amount of O2 consumed by photosynthesizing algae, (2) by growing the algae heterotrophically on acetate in the dark and supplied with O2 generated by photoautotrophically grown cells, and (3) by determining the minimum level of O2 needed to stimulate CO2 evolution from cells suspended in minimal medium supplemented with acetate. The results from these investigations indicated that exogenous O2 was not required for photoautotrophic growth by C. reinhardtii and that this alga grew in an anoxic environment if supplied with CO2 and light.