R. J. Pinney

R factor-mediated resistance to ultraviolet light in strains of Escherichia coli deficient in known repair functions.

The expression of resistance to u.v. irradiation mediated by R factor R46 has been studied in strains deficient in excision repair and recombination repair. The R factor protected wild-type bacteria and also wild-type cells in which repair had been inhibited by the substitution of bromouracil for chromosomal thymine. It increased the survival of strains defective in the endonucleolytic (uvr), repolymerizing (pol) and joining (lig) stages of the excision repair process. Recombination deficient bacteria mutant at the recB or recC loci were protected by R46, but the R factor had little effect on the survival of a recA strain or a recA recB double mutant. R46 increased the survival of cells that had been treated with chloramphenicol before u.v. irradiation, but did not protect cultures treated with chloramphenciol after irradiation. It is concluded that R46 confers resistance to the lethal effects of u.v. irradiation by a mechanism that is independent of excision repair. Resistance appears to be mediated by an inducible gene product, which is possibly a nuclease and dependent on a functional host recA gene for expression.

Function of the SOS Process in Repair of DNA Damage Induced by Modern 4‐Quinolones

Abstract— The recA13 mutant of Escherichia coli strain K‐12, which lacks recombination and SOS error‐prone DNA repair is hypersensitive to nalidixic acid and to the newer 4‐quinolones ciprofloxacin, norfloxacin and ofloxacin. However, whereas recombination‐proficient but SOS repair‐deficient strains, such as those carrying the lexA3 or recA430 alleles are no more sensitive to nalidixic than the lexA+ recA+ parent, they are more sensitive to the newer quinolones, although not as sensitive as the recA13 derivative. Nalidixic acid possesses only bactericidal mechanism A (which requires RNA and protein synthesis and is only effective on actively dividing cells), whereas the newer 4‐quinolones exhibit additional mechanisms B (which does not require RNA and protein synthesis and is effective on bacteria unable to multiply) and C (which requires RNA and protein synthesis but does not depend on cell division). Results obtained with bacteria suspended in phosphate‐buffered saline, which inhibits mechanism A, and with bacteria suspended in nutrient broth plus rifampicin, which inhibits mechanisms A and C, showed that the lexA3 mutant was still more sensitive than the lexA+ parent under these conditions. The results suggest that, unlike bactericidal mechanism A, DNA damage that results from bactericidal mechanisms B and C of the newer 4‐quinolones is subject to SOS error‐prone (mutagenic) repair.

Inhibition of bacterial DNA cytosine-5-methyltransferase by S-adenosyl-l-homocysteine and some related compounds†

S‐Adenosyl‐l‐homocysteine and five related compounds have been evaluated as inhibitors of a DNA cytosine‐5‐methyltransferase. DNA methylation was assayed in cell extracts from E. coli strain J6‐2 dcm+, proficient in DNA cytosine‐5‐methyltransferase activity, containing substrate DNA isolated from E. coli strain J6–2 dcm−, a strain deficient in DNA cytosine‐5‐methyltransferase. S‐Adenosyl‐l‐homocysteine and its 7‐deaza analogue, S‐tubercidinylhomocysteine, were competitive inhibitors of DNA cytosine‐5‐methyltransferase with Kis of 14·2 and 17·6 μm, respectively, in the above enzyme assay.

Survival of plasmid-containing strains of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus in phenylmercuric nitrate and thiomersal.

Strains of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus harbouring plasmids that confer mercury resistance grew in nutrient broth containing concentrations of phenylmercuric nitrate (PMN) that were inhibitory to isogenic plasmid‐less strains. Decimal reduction times of P. aeruginosa and S. aureus in aqueous PMN solution were also increased by the presence of plasmids. The viable count of a plasmid‐containing P. aeruginosa strain in Davis and Mingiolis minimal medium (DM) containing 10 μg ml−1 PMN fell by approximately 99% after 5 h. The count then remained constant for two weeks, when growth recommenced. This pattern of death followed by growth was also observed with the P. aeruginosa strain in DM + 10 μg ml−1 thiomersal.

Contributions of post-antibiotic lag and repair-recovery to the post-antibiotic effects of ciprofloxacin on Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus pyogenes.

A viable counting technique was used to determine the post-antibiotic effect (PAE) of ciprofloxacin against four bacterial species, treated with either one or four times the minimum inhibitory concentration for 1 or 3 h. PAE were demonstrated with Escherichia coli, Staphylococcus aureus or Streptococcus pyogenes after exposure to either concentration for both times. No clear PAE was demonstrated for Klebsiella pneumoniae after any treatment. In some cases, PAE was due to an increased post-treatment lag phase, which was followed by nearly normal multiplication, whereas in other cases a long lag was followed by abnormally rapid cell division, with the generation times of treated cultures being much less than those of the corresponding drug-free controls. This is evidence of recovery of cells that have completed DNA repair. S. aureus, E. coli and K. pneumoniae all exhibited evidence of this type of repair even though K. pneumoniae gave no significant PAE. However, the post-treatment generation times of S. pyogenes, which produced the greatest PAE, gave no evidence of such repair. It is concluded that PAEs may result from a variety of factors.

Investigation of Potential Genotoxic Effects of Low Frequency Electromagnetic Fields on Escherichia coli

Abstract— Exposure of growing cells of Escherichia coli strain AB1157 to a frequency of 1 Hz with field strengths of 1 or 3 kV m−1 did not affect spontaneous or ultraviolet light (UV)‐induced mutation frequencies to rifampicin resistance. Neither did growth in the presence of charge alter the sensitivities of strains AB1157, TK702 umuC or TK501 umuC uvrB to UV. Similarly, although the resistance of strains TK702 umuC and TK501 umuC uvrB to UV was increased by the presence of plasmid pKM101, which carries DNA repair genes, pregrowth of plasmid‐containing strains in electric fields did not increase UV resistance. Finally, growth in a low frequency field in the presence of sub‐inhibitory concentrations of mitomycin C did not affect mitomycin C‐induced mutation frequencies. It is concluded that low frequency electromagnetic fields do not increase spontaneous mutation, induce DNA repair or increase the mutagenic effects of UV or mitomycin C.

The relation between the bactericidal activities and certain physico-chemical properties of some fluorophenols

The antibacterial activities, as demonstrated by the Rideal‐Walker coefficients and viable counts, of mono‐, 2,4,5– tri‐, 2,3,5,6‐ tetra‐ and pentafluorophenol increase with the number of substituent fluorine atoms. The thermodynamic activities (Ferguson values) range from 0·083 to 0·022, and indicate a non‐specific physical mode of action. Linear correlations occur between the equitoxic concentrations of the compounds to Escherichia coli (99·9% mortality after 50 min contact) and their molecular weights, number of fluorine substituents and water solubilities. A similar relation exists between the molar concentrations at the Rideal‐Walker end points and the oleyl alcohol‐water partition coefficients; no correlation occurs between toxicity and the cyclohexane‐water partition coefficients. The surface tensions of equitoxic solutions vary between 57·5 and 63·2 mNm−1 (dynes/cm).

PLASMID PGW16, A DERIVATIVE OF PKM101, INCREASES POST-UV DNA SYNTHESIS, BUT SENSITISES SOME STRAINS OF ESCHERICHIA COLI TO UV

Plasmid pKM101, which carries muc genes that are analogous in function to chromosomal umu genes, protected Escherichia coli strains AB1157 uvrB+ umuC+, JC3890 uvrB umuC+, TK702 uvrB+ umuC and TK501 uvrB umuC against ultraviolet irradiation (UV). Plasmid pGW16, a derivative of pKM101 selected for its increased spontaneous mutator effect, also gave some protection to the UmuC-deficient strains, TK702 and TK501. However, it sensitised the wild-type strain AB1157 to low, but protected against high doses of UV, whilst sensitising strain JC3890 to all UV doses tested. Even though its UV-protecting effects varied, pGW16 was shown to increase both spontaneous and UV-induced mutation in all strains. Another derivative of pKM101, plasmid pGW12, was shown to have lost all spontaneous and UV-induced mutator effects and did not affect post-UV survival. Plasmids pKM101 and pGW16 increased post-UV DNA synthesis in strains AB1157 and TK702, whereas pGW12 had no effect. Similarly, the wild-type UV-protecting plasmids R46, R446b and R124 increased post-UV DNA synthesis in strain TK501, but the non-UV-protecting plasmids R1, RP4 and R6K had no effect. These results accord with the model for error-prone DNA repair that requires umu or muc gene products for chain elongation after base insertion opposite non-coding lesions. They also suggest that the UV-sensitizing effects of pGW16 on umu+ strains can be explained in terms of overactive DNA repair resulting in lethal, rather than repaired UV-induced lesions.

Increase in susceptibility to EcoRII restriction of bacteriophage λ produced by propagation on host cells growing in 5-azacytidine : a new in-vivo method for demonstration of DNA-methylation inhibition

Abstract— The efficiency of plating on EcoRII‐restricting cells of bacteriophage λvir propagated on an Escherichia coli K‐12 dcm+ host decreased with increase in concentration of 5‐azacytidine (5‐azaC) in the propagating medium. This illustrates, in‐vivo, the inhibition of DNA‐cytosine methylation induced by 5‐azaC and provides a simple system for the detection of DNA‐methylation inhibitors.

R‐factor mediated resistance to ultraviolet light

organism, as judged by increase in generation time caused by concentrations of 2 and 4 mg ml-l whereas P. niirabilis F67 (TEM) showed a marked sensitivity to this agent, cell lysis occurring at a concentration of 2 mg ml-l. Evidence that R-factor DNA is associated with a cellular component, presumed to be the cytoplasmic membrane has been presented (Hershfield, LeBlanc & Falkow, 1973). In an attempt to elucidate the nature of the R-factor induced effect, spheroplasts were produced by treating exponential cells in DM medium containing 0.4 M sucrose with benzylpenicillin (1000 units ml-l). Spheroplast formation, as judged by phase contrast microscopy was virtually complete after 4 h. The sensitivity of the spheroplasts from each strain to lysis by sodium desoxycholate was similar, suggesting that the R-factors have not caused a gross change in the cytoplasmic membrane and furthermore that the different response of whole cells to sodium desoxycholate is associated with modification to a cell envelope component external to the cytoplasmic membrane resulting in an increased permeability.