Effect of Metallic Cations on the Viability of Phenol-treated Escherichia coli

Five metallic cations (Fe³⁺, Cr³⁺, Ca²⁺, Mg²⁺, Mn²⁺; concentration range, 1.85 × 10^-4 to 37 × 10^-4m) were incorporated individually as chlorides into nutrient broth and agar media used for the recovery of phenol-treated Escherichia coli. The effects observed varied with the concentration and the ionic species. In nutrient agar, Fe³⁺ and Cr³⁺ were generally beneficial but were toxic at 37 × 10^-4m. Of the divalent ions tested, Ca²⁺ and Mg²⁺ usually gave higher counts in nutrient broth, except at a concentration of 9.25 × 10^-4m, whereas the effect of Mn²⁺ was rather variable. Two possible explanations are suggested to explain these effects. Toxic materials may be removed from the media by the precipitates formed on the addition of Fe³⁺ or Cr³⁺, or, in the case of the divalent ions, the integrity of the bacterial cell membranes may be maintained.     

Effect of Dichlorodifluoromethane on the Appearance, Viability, and Integrity of Escherichia coli

Cultures of Escherichia coli H52 were treated with liquid dichlorodifluoromethane (fluorocarbon-12 [f-12]) for 2 h at 22 C and then examined microscopically. Treated cells tended to clump, and their cytoplasms were generally less dense and less uniform in appearance than those of control cells. E. coli ML30 was exposed to f-12 at a concentration of 1.25 × saturation for times up to 1,200 min at 22 C. Cells were examined for changes in viability (plate count), permeability (as measured by exit of α-[¹⁴C]methylglucoside or uptake of o-nitrophenyl-β-D-galactopyranoside), release of compounds absorbing at 260 nm, and lysis (changes in absorbance at 420 nm). Large losses of α-methylglucoside and of percentage of viability occurred after brief exposure to f-12. Release of compounds absorbing at 260 nm occurred more slowly than the aforementioned events, possibly because these molecules are larger than α-methylglucoside. During 1,200-min exposure to f-12, the number of survivors decreased from 10⁹ to 10⁴ organisms/ml, the loss of compounds absorbing at 260 nm amounted to 50% and 32% lysis occurred. Most of these changes occurred during the first 300 min of treatment. Loss of α-methylglucoside was almost complete after 1-min exposure to f-12. These results suggest that death of the cell involves several stages, with a change of permeability occurring first, followed by leakage of compounds of increasing size and, finally, lysis.     

Factors affecting the Recovery of Gamma Irradiated Escherichia coli

S ummary . Two methods of viable counting have been compared for unirradiated and γ irradiated Escherichia coli NCTC 5933. The spread plate method has been shown to be more reproducible than a serial dilution method in a liquid medium, but the survivor-dose curves of γ irradiated bacteria obtained by the two methods were identical. The spread plate method was used to compare the survival of γ irradiated E. coli on peptone agar and on a synthetic glucose-salts agar over a range of survivor levels of c. 9 log cycles. The log % survivor-dose curve was linear for the synthetic medium but had a double exponential shape for the peptone agar. Recovery was much higher on peptone than on a synthetic medium. Using a tube dilution method in a liquid glucose-salts medium, 19 l -amino acids were tested for their effect on recovery. The slight increases in number of survivors attributable to some amino acids were associated with a change in the first part (100–1% survivors) of the curve rather than an alteration in final slope. The phosphate concentration of the solid synthetic medium markedly affected its ability to recover γ irradiated cells.     

Toxicity of Irradiated Medium for Repair-Deficient Strains of Escherichia coli

Nutrient agar medium is made highly toxic to certain repair-deficient strains of Escherichia coli by exposure of the uninoculated plates to radiation from cool-white fluorescent lamps or black-light fluorescent lamps. This sensitivity is associated with the genetic deficiencies, fil, phr, and recA. Repair-sufficient and uvr strains are only slightly affected by the irradiated media. The poor growth and reduced plating efficiency frequently associated with BPhr and recA strains are very likely caused by inadvertent exposure of the medium to fluorescent light.     

Structure of Escherichia coli After Freeze-Etching

Survival of Escherichia coli, quick-frozen under conditions similar to those employed for freeze-etching, is close to 100%. For determination of cell shrinkage, the diameters of freeze-etched E. coli cells (average, 0.99 mum) were compared with those of preparations after negative staining and after ultrathin sectioning. Negatively stained cells measured from 0.65 to 1.0 mum in diameter, and ultrathin sections showed average cell diameters of 0.70 mum. Freeze-etched replicas of logarithmically growing, as well as stationary, E. coli B cells revealed a smooth, finely pitted cell surface in contrast to cell surfaces seen with other preparative methods. The frozen cell wall may cleave in two planes, exposing (i) a smooth fracture face within the lipid layer and (ii) in rare instances an ill-defined particulate layer. Most frequently, however, cleavage of the envelope occurred between wall and protoplasmic membrane; large areas of the membrane were then exposed and showed a surface studded with predominantly spherical particles, an appearance which did not significantly change when the cells were fixed in formaldehyde and osmium tetroxide before freeze-etching. The distribution of these particles differed between logarithmically growing cells and stationary cells.     

Dark-Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light III. Effect of rec Mutations on Recovery of Excision-Deficient Mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 unable to excise pyrimidine dimers from their deoxyribonucleic acid (DNA) because of a uvr mutation show a higher survival when plated on a minimal salts medium after exposure to ultraviolet radiation than when plated on a complex medium such as nutrient agar containing yeast extract. This response has been called minimal medium recovery (MMR). Recovery of uvr mutants can take place in liquid as well as on solid medium, but not in buffer or under conditions of amino acid starvation that do not permit cell growth and normal DNA replication. MMR can thus be distinguished from the recovery of recombination-deficient (rec(-)uvr(+)) derivatives of K-12 which can occur under conditions where growth is not possible. Because MMR is characteristic of excision-defective mutants, it evidently reflects a type of repair independent of excision. We have obtained genetic evidence that MMR is determined by the rec genes, which also control recombination in K-12. Cells carrying a uvr mutation together with recA13, recA56, recB21, or recC22 failed to show MMR and were more sensitive to ultraviolet radiation than either their rec(+)uvr(-) or rec(-)uvr(+) parents. The rec(+)uvr(-) derivatives obtained from recA uvr(-) strains by transduction or by reversion regained the capacity for MMR. Our results indicate that inactivation of any one of the three genes, recA, recB, or recC, prevents cells from showing MMR.     

Comparative Effects of Anesthetics on the Viability and Integrity of Escherichia coli ML30

Cells of Escherichia coli ML30 in a mineral salts medium were exposed to dichlorodifluoromethane (f-12), cyclopropane, halothane, or Ethrane at concentrations of 1.25, 0.2, 0.04, and 0.008× saturation for times up to 1,200 min, and at temperatures in the range of 2 to 37 C. When any of these anesthetics were applied for 300 min at 1.25× saturation, a substantial decrease in number of survivors occurred. Halothane was most bactericidal, cyclopropane and Ethrane were moderately bactericidal, and f-12 was least bactericidal. At saturation values of less than 1.0, none of the four anesthetics had an appreciable effect on viability of E. coli. Greatest increases in cell permeability occurred when anesthetics were used at saturation values of 1.25, and permeability changes generally decreased as the concentrations of the chemicals were reduced. In many instances, anesthetics in the vapor state caused significant increases in cell permeability but little or no loss of viability. This indicated that a close relationship did not exist between loss of viability and increased permeability. All four anesthetics caused E. coli to lose substantial and similar amounts of compounds absorbing at 260 nm. Release of compounds absorbing at 260 nm generally increased as the saturation value of a given chemical was increased. Halothane, Ethrane, and cyclopropane but not f-12 caused lysis of E. coli ML30. Considering all results, E. coli ML30 was damaged more by halothane or cyclopropane than by f-12 or Ethrane. When f-12 was applied at a saturation value of 1.25, the bactericidal effect on E. coli was much greater at 37 or 22 C than at 12 or 2 C.     

Viability of Escherichia coli topA mutants lacking DNA topoisomerase I

The viability of the topA mutants lacking DNA topoisomerase I was thought to depend on the presence of compensatory mutations in Escherichia coli but not Salmonella typhimurium or Shigella flexneri. This apparent discrepancy in topA requirements in different bacteria prompted us to reexamine the topA requirements in E. coli. We find that E. coli strains bearing topA mutations, introduced into the strains by DNA-mediated gene replacement, are viable at 37 or 42 degrees C without any compensatory mutations. These topA(-) cells exhibit cold sensitivity in their growth, however, and this cold sensitivity phenotype appears to be caused by excessive negative supercoiling of intracellular DNA. In agreement with previous results (Zhu, Q., Pongpech, P., and DiGate, R. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9766-9771), E. coli cells lacking both type IA DNA topoisomerases I and III are found to be nonviable, indicating that the two type IA enzymes share a critical cellular function.     

Effect of Sodium Fluorescein and Plating Medium on Recovery of Irradiated Escherichia coli and Serratia marcescens from Aerosols

Irradiation of aerosols of either Escherichia coli or Serratia marcescens with simulated solar (xenon) radiation caused a significant decrease in viability. When sodium fluorescein was employed to determine the physical loss of organisms from the aerosol, an additional adverse effect upon survival was noted. The decay curves indicated that at least two mechanisms of inactivation were operative, one due to aerosolization, the other to irradiation. After collection from aerosols, both species of microorganisms grew better on blood agar base than on Casitone agar, but this finding did not appear to be related to the effect of irradiation.     

The Inhibitory Effect of Sodium Deoxycholate on Escherichia coli

SUMMARY: Sodium deoxycholate (0.2%) inhibited growth of Escherichia coli in semi-digested meat and milk, but had very little effect in media in which the source of nitrogen was amino acids or ammonia. The addition of more digested forms of protein (peptone and casein hydrolysate) to a less digested form (beef infusion) overcame the inhibitory effect. The significance which this phenomenon may have in controlling the population of E. coli in the small intestine is discussed.     

Heterozygous Effects of Irradiated Chromosomes on Viability in DROSOPHILA MELANOGASTER

Two large experiments were conducted in order to evaluate the heterozygous effects of irradiated chromosomes on viability. Mutations were accumulated on several hundred second chromosomes by delivering doses of 2,500r over either two or four generations for total X-ray exposures of 5,000r or 10,000r. Chromosomes treated with 5,000r were screened for lethals after the first treatment, and surviving nonlethals were used to generate families of fully treated chromosomes. The members of these families shared the effects of the first irradiation, but differed with respect to those of the second. The chromosomes treated with 10,000r were not grouped into families since mutations were accumulated independently on each chromosome in that experiment. Heterozygous effects on viability of the irradiated chromosomes were tested in both isogenic (homozygous) and nonisogenic (heterozygous) genetic backgrounds. In conjunction with these tests, homozygous viabilities were determined by the marked-inversion technique. This permitted a separation of the irradiated chromosomes into those which were drastic when made homozygous and those which were not. The results indicate that drastic chromosomes have deleterious effects in heterozygous condition, since viability was reduced by 2–4% in tests performed with the 10,000r chromosomes, and by 1% in those involving the 5,000r material. Within a series of tests, the effects were more pronounced when the genetic background was homozygous. Nondrastic irradiated chromosomes did not show detectable heterozygous effects. They also showed no homozygous effects when compared to a sample of untreated controls. In addition, there was no evidence for an induced genetic component of variance with respect to viability in these chromosomes. These results suggest that the mutants induced by high doses of X-rays are principally drastic ones which show deleterious effects on viability in heterozygous condition.     

Effect of Lactobacillus rhamnosus NCDC 298 with FOS in Combination on Viability and Toxin Production of Enterotoxigenic Escherichia coli

Probiotics and Antimicrobial Proteins - The present study was to investigate the utilization of prebiotics by Lactobacillus rhamnosus NCDC 298 and its synergistic adversary effect on both...     

Dark Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light II. Effect of uvr Genes on Liquid Holding Recovery

The uvr mutations of Escherichia coli K-12 decrease the ability of cells to survive ultraviolet light (UV), to excise pyrimidine dimers from their deoxyribonucleic acid and to reactivate bacteriophage exposed to UV. The rec mutations decrease the ability of the cells to survive UV and to undergo genetic recombination. Certain rec mutations, including recA1, rec-12, recA13, and rec-56, are necessary for the expression of liquid-holding recovery (LHR), observed as an increase in colony-forming ability when irradiated cells are held in buffer in the dark. These rec mutations appear to act indirectly to permit the detection of LHR rather than to affect its occurrence directly. We have tested the effect of uvr markers on LHR in cells containing one of these rec mutations. Recombinants containing rec-56 together with a uvr marker were constructed and tested for LHR. None of the 39 recombinants examined, carrying uvrA6, uvrB5, or uvrC34, showed LHR. Three rec(-)uvr(-) strains were also tested for photoreactivation. In all three, photoreactivation was observed, indicating that they contained detectable amounts of pyrimidine dimers. Our results are consistent with the idea that uvr mutations inactivate LHR, and suggest that LHR reflects excision-dependent repair of pyrimidine dimers.     

The Effect of thecrp Genotypes on the Transformation Efficiency in Escherichia coli

We have found that a significant difference exists in transformationefficiency between the crp⁺/crp^– isogenic pair of strainsof Escherichia coli, with the efficiency being much higher incrp^– than in crp⁺. The ratio of transformation efficiencybetween crp⁺ and crp^– strains depends very little on theplasmid size. This observation suggests that the differenceof the transformation efficiency is due to mechanisms otherthan a crp-regulated endonuclease. The crp gene is one of thefirst specific genes that have been shown to affect transformationefficiency.     

Effect of Nalidixic Acid on Semiconservative Replication and Repair Synthesis After Ultraviolet Irradiation in Escherichia coli

Both semiconservative deoxyribonucleic acid replication and "extensive repair" synthesis, after ultraviolet irradiation, appear to be blocked by nalidixic acid. These findings suggest that the agent(s) responsible for both of these modes of replication, or some necessary common process or structure, is affected by this drug.