Irradiated Bacteria Research Articles

Ultraviolet inducible protein associated with error prone repair in E. coli B.

Escherichia coli is affected in many ways by irradiation with ultraviolet light. New DNA is made containing gaps resulting from synthesis on damaged templates1,2 and postreplication repair seals them by exchanging sections of DNA between daughter duplexes3,4. The induction of mutations by ultraviolet light has been attributed to errors occurring during this type of repair5–7 and so this repair has been called error prone. Ultraviolet radiation also results in pro-phage induction8, inhibition of respiration9, filamentous growth10 and the development of extra capacity to promote the survival11 and mutagenesis12 of irradiated infecting phages. All these events require the rec A+ and exr+ genes2,7,9,13–23 but the ways in which rec A+ and exr+ gene products affect such a diversity of postirradiation events are not known. One proposal is that they act in a single molecular process which in some way initiates all the phenomena described above and that the rec A+ exr+ dependent functions are inducible23 by ultraviolet light and thus require the synthesis of new protein(s) in irradiated bacteria. Here I demonstrate that E. coli does indeed synthesise a protein in response to ultraviolet irradiation and that the synthesis of this protein is dependent on exr+ and rec A+ genes.

Concerning pyrimidine dimers as "blocks" to DNA replication in bacteria and mammalian cells.

Irradiated UV-sensitive bacteria which are mutant in the uvrA6 locus, and cannot excise pyrimidine dimers, are capable of continued DNA replication with the same orientation as that in unirradiated cells. Replication of dimer-containing DNA is also accomplished by irradiated mammalian cells, suggesting that pyrimidine dimers do not act as absolute blocks to DNA synthesis. Thus, the role that dimers play in reducing the amount and size of DNA synthesized after exposure must be reassessed.

The effects of rifampicin on electron- and neutron-irradiated E. coli B/r and BS-1: Survival, DNA degradation and DNA synthesis by membrane fragments.

The effect of rifampicin after irradiation with electrons or neutrons on cell-killing, DNA degradation and the ability of DNA-membrane complex extracted from irradiated bacteria to synthesize DNA was studied in strains E. coli B/r and BS minus 1. Post-irradiation treatment with rifampicin greatly enhanced the lethal effects of both electrons and neutrons on the repair-proficient strain E. coli B/r under anoxic conditions. However, the same post-irradiation treatment of the repair-deficient strain E. coli BS minus 1 with rifampicin gave no consequent increase in lethality. Rifampicin did not alter the extent of DNA degradation in either strain after irradiation with electrons or neutrons. The DNA-synthetic activity of DNA-membrane complex from electron-irradiated E. coli B/r and BS minus 1 was not altered by post-irradiation treatment with rifampicin, nor did this treatment affect synthesis by DNA-membrane complex isolated from neutron-irradiated BS minus 1; but the complex, isolated from E. coli B/r which had been irradiated under anoxic conditions with neutrons, showed a marked decrease in activity. We suggest that post-irradiation sensitization with rifampicin of the repair-proficient E. coli B/r, especially after irradiation under anoxic conditions, indicates that initiation of the synthesis of an RNA molecule is involved. In addition our results suggest that the structure of the membrane complex in E. coli B/r and BS minus 1 is different.

Modification of the response to 8 MeV electrons of auxotrophic strains of Escherichia coli by amino-acid deprivation and rifampicin.

An investigation has been made of the possibility that the accumulation of RNA enriched in messenger can influence the survival of bacteria after exposure to 8 MeV electrons. The multiple amino-acid auxotroph E. coli CP79 RCrel accumulates RNA during inhibition of protein synthesis, and the proportion of RNA present as messenger increases by a factor of 3 during a 2-hour period of amino-acid deprivation. Amino-acid withdrawal led to changes in the shape of the survival curve, which also occurred in strain CP78 RCstr, which synthesizes very little RNA when deprived of amino acids. We conclude that accumulation of RNA enriched in messenger plays little or no part in modification of the survival of irradiated bacteria. We show that rifampicin, an inhibitor of RNA synthesis, increases the sensitivity of both strains equally.

Cell division and DNA synthesis in uvrA recA double mutants of E. coli K12.

Cell division and incorporation of 3H-thymidine into acid-insoluble fraction were investigated for three uvrA recA double mutants of E. coli K12 irradiated with UV at 1.5 ergs/mm2, producing about ten pyrimidine dimers per genome (about 0.01% survival). Cell division was measured both in M9 medium and in the same medium which was made very viscous by the addition of Metlose (the same product as Methocel used by Lin et al., 1971). It was found that a major fraction of irradiated bacteria continues to divide once or twice and stops thereafter. Incorporation of 3H-thymidine proceeded at a considerable rate for a short period following irradiation and then stopped. During subsequent incubation, the incorporation gradually decreased and after 4 h incubation most of the early incorporated radioactivity disappeared from the acid-insoluble fraction. These results indicate that cell division occurs after irradiation without parallel DNA synthesis as in a recA thy mutant of E. coli K12 deprived of thymine (Inouye, 1971). These results suggest that UV irradiation increases lethal sectoring due to the “reckless” cell division without parallel DNA synthesis. Since DNA synthesis took place only for a short period after irradiation, it may be assumed that the recA gene normally has at least a dual function; 1. elimination of damage induced by UV to support elongation or initiation of DNA, and 2. maintenance of coordination between DNA synthesis and cell division.

Conjugational synthesis of F lac+ and Col I DNA in the presence of rifampicin and in Escherichia coli K12 mutants defective in DNA synthesis.

When F lac+ donors were mixed with irradiated recipient bacteria, there was a stimulation of DNA synthesis in the recipients equivalent to the amount of DNA transferred. Rifampicin had little effect on such conjugational synthesis of F′ and ColI DNA during the first 30 min of mating. In matings of temperature-sensitive (ts) dnaB70 mutants at 43° C, transfer of F lac+ and ColI DNA was associated with an equal amount of conjugational DNA synthesis in the two parents, the amounts being unaffected by the polA1 mutation. Rifampicin abolished conjugational DNA synthesis in dnaB recipients mated with rifampicinresistant F lac+ donors but it stimulated synthesis in equivalent matings involving ColI. Although F lac+ and ColI DNA was transferred to (ts) dnaE486 mutants at 43° C, transfer was not associated with either enhanced DNA synthesis in the recipients or the extensive synthesis in them of DNA which was convertible into covalently closed circular molecules by brief incubation at a permissive temperature. These results imply that DNA polymerase III is essential for synthesis of the strands complementary to the transferred F- and I-like plasmid DNA. In the case of ColI, the process is resistant to rifampicin both in the presence and absence of dnaB function whereas for F lac+, it is sensitive when dnaB gene product is thermally inactivated.

Effect of amino acid starvation on UV sensitivity of Lactobacillus acidophilus cells.

InLactobacillus acidophilus cultures UV irradiated in the exponential phase of growth, the dosesurvival curve was of the simple exponential type, without any shoulder. If the bacteria were subjected to amino acid starvation prior to irradiation, an shoulder corresponding to a quasi-treshold dose (Dq) of about 780 ergs/mm2 appeared in the curve. The administration of protein or RNA-synthesia inhibitors prior to irradiation had the same effect. The effect of pre-irradiation amino acid starvation was abolished by simultaneous thymidine starvation. It was likewise abolished if amino acid starvation was followed by incubation in the presence of amino acids (without thymidine) and then by irradiation of the cells. Post-irradiation amino acid starvation did not lead to the formation of an shoulder but if combined with thymidine starvation it did. It can be concluded from the results that post-irradiation repair processes are facilitated or promoted if, during the post-irradiation interval DNA synthesis is delayed. This delay represents a compensation of the pre-irradiation increase of cellular DNA-content, taking place during inhibition of proteosynthesis. The postirradiation administration of caffeine did not abolish the formation of the shoulder induced by pre-irradiation amino acid starvation; on the contrary, it induced its formation even in exponentially growing, irradiated control bacteria.

Endonuclease-sensitive sites in the DNA of irradiated bacteria: A rapid and sensitive assay

The endonucleolytic activity from an extract of Micrococcus luteus is used to detect small amounts of damage in the DNA of bacteria exposed to ultraviolet light or γ rays. The extract is added directly to a lysate of the bacteria, and after a short period of incubation a sample of the bacterial DNA is assayed for endonuclease-sensitive sites by sedimentation through alkaline sucrose gradients. As few as one or two sites per 10 8 daltons of DNA can be detected by this method.

A Posteffect of Oxygen in Irradiated Bacteria: A Submillisecond Fast Mixing Study

time scale of the oxygen effect in irradiated Serratia marcescens. The bacteria were held on a Millipore filter inside a chamber which was usually flushed with humidified nitrogen and were exposed to an explosion of oxygen released into the chamber through a fastacting solenoid-operated valve. The bacteria were irradiated with a single 2-psec pulse of 1.8 MeV electrons from a linear accelerator. The arrival of oxygen at the bacteria was timed to occur at a preset interval before or after the electron pulse. The time resolution was 100 ,sec. After fixed doses of 10, 19, and 28 krads per pulse, the bacterial survival was measured as a function of the oxygen contact time. It was found that, when oxygen contact occurred at any instant before or up to the time of the electron pulse, full sensitization was obtained. However, bacteria exposed to oxygen at times after the electron pulse showed decreasing sensitization as the time interval increased, the survival approaching the anoxic control value at times greater than 2 msec. The data show that, in Serratia marcescens irradiated under anoxic conditions, oxygen-dependent damage decays approximately exponentially with a half-life of about 500 ,usec.

Evidence for differing modes of interaction of acriflavine with ultraviolet-induced lesions in an Hcr + bacterial strain.

In buffer suspensions of UV-irradiated Escherichia coli B/r WP2 Hcr+ (auxotrophic for tryptophan) acriflavine binds to DNA, but this treatment has little effect on killing and results in the appearance of fewer prototrophs on tryptophan-supplemented minimal agar. If plates contain a broth supplement, however, the buffer-acriflavine treatment greatly increases the yield of UV-induced prototrophs; but this increase does not depend on complete binding of acriflavine to the DNA as a whole, since it is observed with contact times too short for this to occur (as short as 20 seconds). The incorporation of acriflavine in both kinds of plating medium increases the yields of prototrophs. The maximum yield is observed when irradiated bacteria are exposed to acriflavine in buffer before they are plated on medium containing both acriflavine and a broth supplement. Thus post-irradiation effects of acriflavine cannot be accounted for in terms of a single mechanism of action. Our results support the suggestion that phenomena classed together as “mutation frequency decline” may not represent a single specific repair system.

Ultraviolet reactivation of lambda phage: Assay of infectivity of DNA molecules by spheroplast transfection

Prior irradiation of non-lysogenic bacteria by ultraviolet light leads to an increase in the viability of infecting irradiated λ phage (ultraviolet reactivation). Similarly, u.v. irradiation of wild type or uvrD bacteria lysogenic for λcIind − increased the fraction of closed circular duplex phage DNA molecules formed after infection with u.v.-irradiated λ phage. The closed circular molecules isolated from the irradiated lysogens were shown to be free from u.v. damage by a spheroplast transfection assay. The increase of closed circular molecules is sufficient to explain the ultraviolet reactivation observed by the increase of viability of irradiated phage. In ultraviolet reactivation, damage must be erased on irradiated DNA molecules and the repair is independent of total replication of phage genomes, exchange of sister chromatids or recombination between phage genomes. Protein synthesis is necessary to increase the level of closed circular molecules of irradiated λ phage after irradiation of bacteria.

Toxicity of Irradiated 1-Naphthol

Irradiation of bacteria in the presence of 1-naphthol produces a radiosensitization. The magnitude of the response is dependent upon cell concentration and type, and requires an extended incubation...

Minimal medium recovery of heated Salmonella typhimurium LT2.

SUMMARY: Exponential phase Salmonella typhimurium lt2, S. thompson and S. heidelberg grown in a glucose-salts medium (M-9), harvested at 37°C and heat-treated at 50°C exhibit a higher recovery on M-9 agar than on Trypticase Soy agar with yeast extract. This phenomenon is analogous to minimal medium recovery of irradiated bacteria. Supplementation of M-9 agar with yeast extract or Casamino acids resulted in lower viable counts of heat-treated bacteria than on M-9 agar alone. Heat-treated bacteria incubated at 37°C in M-9 broth or distilled water recovered their ability to grow on TSY agar. This recovery is inhibited by hydroxyurea.

CONTROL OF CLOSTRIDIUM BOTULINUM AND STAPHYLOCOCCUS AUREUS IN SEMI-PRESERVED MEAT PRODUCTS

Clostridium botulinum and Staphylococcus aureus are naturally occurring contaminants in semi-preserved meat products. They can be inhibited by (a) storage below 3 C, (b) 10% sodium chloride (brine concentration), (c) pH values below 4.5, or (d) proper combinations of these factors. However, most meat products do not have the pH values and brine concentrations required to completely inhibit C. botulinum and S. aureus and there is always a risk of temperature abuse. Improved safety can be achieved by adding 1% or more glucose to the product. The glucose will, in the event of temperature abuse, generally be fermented to lactic acid by the indigenous microflora in the product. As a result, the pH value drops to a level at which the brine concentration is sufficient to inhibit C. botulinum and S. aureus. A better approach to safety is to add, together with glucose, a radiation-killed preparation of lactic acid bacteria, e.g., Pediococcus cerevisiae. Such preparations cause a rapid decline in pH only when the product is exposed to a high temperature, and they are stable during storage of meat products. Addition of irradiated lactic acid bacteria to meat products has not yet been officially approved.Another way to improve the safety of semi-preserved meat is to add sufficient glucono-delta-lactone to reduce the initial pH of the product to a level at which the salt concentration is inhibitory. Use of larger amounts of glucono-delta-lactone may result in flavor and color problems even when the meat product is kept at refrigeration temperatures.

Protection of normal, lysogenic, and pyocinogenic strains against ultraviolet radiation by bound acriflavine.

The presence of bound acriflavine protects bacteria against the lethal effects of ultraviolet (UV) light, presumably because pyrimidine dimer formation is inhibited. Although acriflavine present in plating medium usually results in reduced viable counts from irradiated bacteria, no enhancement of lethal effects is observed when acriflavine is added to irradiated bacteria left in suspending buffer for 45 min before plating. Acriflavine remaining bound to the deoxyribonucleic acid of irradiated bacteria at the time they are plated likewise does not affect their survival. Protection is precisely dose-modifying unless some killing of bacteria by UV results from induction of prophage, against which bound acriflavine is less protective, or from induction of pyocin, against which there is no protection at all. It is inferred that prophage induction proceeds in part, and pyocin induction wholly, by virtue of effects of UV other than pyrimidine dimerization. The response of Escherichia coli strain B to radiation has been postulated to be attributable in part to induction of a prophage or a lethal protein; but exact dose modification was observed for this strain, to about the same extent, whether or not the irradiated organisms were grown in conditions thought to enhance the expected contribution to killing if such a mechanism were involved. Our results support the hypothesis that the inhibition by acriflavine of dimer formation is attributable to energy transfer mechanisms. They fail to support the hypothesis that shapes of survival curves (in particular the manifestation of "shoulders") can be attributed to inactivation by radiation of repair enzymes.

Semi- in vitro repair of radiation-induced damage in transforming DNA of Bacillus subtilis

Toluene-treatment at 0 °C was effective in making Bacillus subtilis cells accessible to substrates and co-factors of enzymes involved in the repair of damage induced by ionizing radiation. Transforming activity in the lysate of γ-ray irradiated and toluene-treated bacteria increased remarkably in the presence of four deoxy-nucleoside triphosphates, nicotinamide adenine dinucleotide and Mg 2+ at 37 °C. Under similar conditions, γ-ray induced DNA single-strand breaks were rejoined to a significant extent. Involvement of DNA-polymerase and DNA-ligase functions was strongly suggested in the repair of DNA damage induced by ionizing radiation.

Effects of ionizing radiation on the capacity of Escherichia coli to support bacteriophage T4 growth.

The loss in capacity of irradiated bacteria to support the growth of T4 phage has been studied for two strains, Escherichia coli B and E. coli B(s-1). Following a dose of 25 krads, the capacity is quite rapidly lost during postirradiation incubation so that after two hr of such incubation at 37 C only 12% remains in strain B and 3% in strain B(s-1). Evidence that capacity is lost in an all-or-none fashion was provided by two types of experiments: (i) a single-burst analysis of those cells which survived to give a burst, and (ii) an analysis of the regulation of an early phage enzyme. Several processes were examined to try to determine the cause of the loss in capacity. These were the ability of the irradiated and postirradiation incubated cells to respire, to allow phage adsorption and injection of phage deoxyribonucleic acid, to support the transcription and translation of an early gene, and to support replication of phage deoxyribonucleic acid. Of these processes, transcription and translation appeared to be the most closely associated with the capacity loss, although respiration was reduced to 50% after 2 hr.

Effects of coumarin, pyronin Y, 6,9-dimethyl 2-methylthiopurine and caffeine on excision repair and recombination repair in Escherichia coli.

SUMMARY: Pyronin Y (0.06 and 0.16 mM), coumarin (2 and 4 mM), 6,9-dimethyl 2-methyl-thiopurine (2 mM) and caffeine (8 mM), strongly inhibit Uvr repair (presumed excision repair) of u.v. lesions in Escherichia coli. With 0.16 mM-pyronin Y or 4 mM-coumarin, one thymine dimer was a lethal event in the K12 strain JC 2926 rec A-13 and one to three dimers in the B/r derivative WP-2 rec A-13 and the B exr A derivative BS-2. Neither these compounds nor 8 mM-caffeine had a significant effect on the survival of irradiated bacteria of the corresponding uvr strains WP-2 Hcr-, BS-12, and BS-8. I suggest that 8 mM-caffeine, 0.16 mM-pyronin Y, 4 mM-coumarin and 2 mM-6,9-dimethyl 2-methylthiopurine selectively block excision repair without substantially affecting recombination repair. Caffeine at 12 mM only slightly depressed the viability of a u.v.-irradiated culture of WP-2 Hcr- or BS-12, but at 16 mM there was a much greater effect, particularly at low u.v. doses (< 40 ergs/mm2). Since excision repair appeared to be effectively blocked by 8 mM-caffeine, the depression of u.v. survival of the uvr mutants by 16 mM-caffeine was presumed to be due to interference with recombination repair (Witkin & Farquharson, 1969).

Methylation of DNA in ultraviolet-irradiated bacteria

Methylation of DNA continues in Escherichia coli K12, W, and B strains in which net synthesis of the polymer has been eliminated by ultraviolet irradiation. This appears to be independent of dimer excision, yet degradation of DNA in the post-irradiation period does modify the ultimate level of overmethylation. DNA methylated in vivo after irradiation has a decreased ratio of 6 methyl adenine to 5-methyl-cytosine relative to that found when the labeling occurs during logarithmic growth. In addition, a decrease in the activities of the DNA methylases in crude extracts is evident 8 to 12 min after irradiation; heterologous DNA reisolated from such reaction mixtures exhibits a drop in both 6-methyladenine and 5-methylcytosine label with distortion of the 6- methyladenine 5- methylcytosine ratio. Since the methylating capacity falls, the overmethylation observed in irradiated bacteria may not be attributable to new enzyme specificities. We suggest that a relaxation of the restrictions on DNA methylation which usually prevail occurs as a result of ultraviolet irradiation damage.

Studies on the digestibility and utilization of the nitrogen of irradiated rumen bacteria by rats

SUMMARYRumen bacteria were isolated from the rumen contents of eight Friesian cattle that had been fed a barley-soyabean diet. These preparations were freeze-dried, irradiated with 1 Mrad and analysed for various nitrogenous components.In nitrogen utilization studies involving both young and adult rats it was observed that the ‘true’ digestibility of the bacterial nitrogen was about 80%, while the net protein utilization (NPU) value was approximately 57.It was concluded that about 20% of the bacterial nitrogen, mainly incorporated in the cell wall, was not absorbed by the rat. On the other hand, about 71% of the absorbed nitrogen was utilized by the growing rat for tissue synthesis.Irradiation with 1 Mrad killed virtually all the rumen bacteria without adversely influencing the nutritional value of the bacterial protein.