The role of oxygen radicals in dye-mediated photodynamic effects in Escherichia coli B

Photosensitive dyes representative of the thiazines, xanthenes, acridines, and phenazines mediated phototoxicity in Escherichia coli B. The observed phototoxicity was sensitizer-, light-, and oxygen-dependent and is therefore a photodynamic effect. Hydroxyl radical scavengers conferred protection against the photodynamic action of all of the representative dyes. The extent of protection was dependent on the concentration of scavenger and on the in vitro reactivity of the scavenger with the hydroxyl radical. Exogenous superoxide dismutase and catalase partially protected the cells against the dye-mediated phototoxicity, and prior induction of intracellular superoxide dismutase and catalase by growth in glucose minimal medium containing manganese and paraquat substantially protected E. coli B against the photodynamic action of all of the dyes examined. Combinations of protective treatments against the phototoxicity of all four classes of dyes, including superoxide dismutase and catalase preinduction and addition of extracellular superoxide dismutase and catalase or the addition of hydroxyl radical scavengers, provided nearly complete protection against the oxygen-dependent component of dye-mediated lethality. E. coli B grown in glucose minimal medium containing manganese and photosensitive dyes induced manganese superoxide dismutase. The extent of induction was correlated with the dyes' ability to photooxidize NADH in vitro. Thus, oxygen radicals are primarily responsible for the oxygen-dependent toxicity of the photosensitive dyes examined, and one adaptive response of E. coli B to a dye-mediated oxidative threat is to induce superoxide dismutase.     

Mutations in Escherichia coli that effect sensitivity to oxygen

Fifteen oxygen-sensitive (Oxys) mutants of Escherichia coli were isolated after exposure to UV light. The mutants did not form macroscopic colonies when plated aerobically. They did form macroscopic colonies anaerobically. Oxygen, introduced during log phase, inhibited the growth of liquid cultures. The degree of inhibition was used to separate the mutants into three classes. Class I mutants did not grow after exposure to oxygen. Class II mutants were able to grow, but at a reduced rate and to a reduced final titer, when compared with the wild-type parent. Class III mutants formed filaments in response to oxygen. Genetic experiments indicated that the mutations map to six different chromosomal regions. The results of enzymatic assays indicated that 7 of the 10 class I mutants have low levels of catalase, peroxidase, superoxide dismutase, and respiratory enzymes when compared with the wild-type parent. Mutations in five of the seven class I mutants which have the low enzyme activities mapped within the region 8 to 13.5 min. P1 transduction data indicated that mutations in three of these five mutants, Oxys-6, Oxys-14, and Oxys-17, mapped to 8.4 min. The correlation of low enzyme levels and mapping data suggests that a single gene may regulate several enzymes in response to oxygen. The remaining three class I mutants had wild-type levels of catalase, peroxidase, and superoxide dismutase, but decreased respiratory activity. The class II and III mutants had enzyme activities similar to those of the wild-type parent. Our results demonstrate that mutations in at least six genes can be expressed as oxygen sensitivity. Some of these genes may be involved in respiration or cell division or may regulate the expression of several enzymes.     

Effect of ascorbate on oxygen uptake and growth of Escherichia coli B

The addition of ascorbate to aerobically growing cultures of Escherichia coli B caused only a short pause in growth and no subsequent change in the rate or extent of growth. The effect of ascorbate on oxygen uptake varied from inhibition in minimal medium to stimulation in rich medium. Cyanide-resistant growth and oxygen uptake were stimulated by ascorbate. Both the rate and extent of anaerobic growth were stimulated in proportion to the amount of ascorbate added when fumarate was the terminal electron acceptor. Ascorbate had no effect on any aspect of anaerobic growth in the absence of a terminal electron acceptor or in the presence of nitrate.     

Role of oxygen radicals in the phototoxicity of tetracyclines toward Escherichia coli B.

Photoillumination of tetracycline derivatives with low-intensity (320- to 400-nm) light and visible light generated superoxide, observed as the reduction of ferricytochrome c. The rate of reduction was dependent on the tetracycline concentration and on the derivative being examined, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. Tetracycline-mediated cytochrome c reduction was oxygen dependent and inhibited up to 70% by superoxide dismutase. Illuminated tetracyclines were lethal to Escherichia coli B incubated in a glucose minimal medium containing chloramphenicol. This lethality was light dependent, oxygen dependent, and dependent on the concentration of tetracycline. Kill rates also varied according to the derivative under study, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. The addition of superoxide dismutase and catalase to the incubation medium partially protected E. coli B against the light-dependent lethality. Preinduction of intracellular superoxide dismutase and catalase substantially protected E. coli B against the phototoxicity of tetracyclines. Iron EDTA augmented the phototoxicity of tetracyclines, while diethylenetriaminepentaacetic acid protected against their lethality. Hydroxyl radical scavengers also conferred protection against tetracycline phototoxicity. The extent of protection was in order of the in vitro reactivity of the scavengers with the hydroxyl radical. These results indicate that superoxide, hydrogen peroxide, and the hydroxyl radical are generated by illuminated tetracyclines and are molecular agents of tetracycline phototoxicity in E. coli B.     

The effect of proflavine on pyruvate kinase I of Escherichia coli B.

Proflavine (PF) inhibited glucose use in sensitive but not resistant Escherichia coli B. Glucose transport (as measured by alpha-methylglucoside accumulation) was only partly inhibited by PF concentration that completely blocked glucose use. Fructose 1,6-diphosphate-(FDP)-regulated pyruvate kinase (PK1) (EC 2.7.1.40), the only glycolytic enzyme affected by PF, was completely inhibited by a dye concentration of 0.8 mM. The inhibition curve for PF was sigmoidal, suggesting that PF was acting as an allosteric inhibitor. PF increased the K 1/2 for phosphoenolpyruvate (PEP) and lowered the V; however, it had no effect on the Hill number for PEP. PF inhibition was partially reversed by FDP but not by cyclic AMP, AMP, ATP, fuctose 6-phosphate, or dithiothreitol. Studies with a variety of acridines indicated that those substituted at the 3-position are the most effective inhibitors and also that hydrophobic interactions may be involved in PF inhibition of PK I. PK I for E. coli B/Pr was also strongly inhibited by PF, indicating that PF resistance does not lie at the level of this enzyme. Ribose-5-phosphate-regulated pyruvate kinase (EC 2.7.1.40) was much less sensitive that PK I to the inhibitory effects of PF. A role for PF as a molecular probe for PK I has been proposed.     

The long-term survival of Escherichia coli in river water

Escherichia coli introduced into autoclaved filtered river water survived for up to 260 d at temperatures from 4 degrees to 25 degrees C with no loss of viability. Survival times were less in water which was only filtered through either a Whatman filter paper or a 0.45 micron Millipore filter or in untreated water, suggesting that competition with the natural microbial flora of the water was the primary factor in the disappearance of the introduced bacteria. Survival was also dependent upon temperature with survival at 4 degrees C greater than 15 degrees C greater than 25 degrees C greater than 37 degrees C for any water sample. Direct counts showed that bacterial cells did not disappear as the viable count decreased. The possession of the antibiotic resistance plasmids, R1drd-19 or R144-3, did not enhance survival nor cause a faster rate of decay, indicating that the metabolic burden imposed by a plasmid was not a factor in survival under starvation conditions. There was no evidence of transfer of either plasmid at 15 degrees C or of loss of plasmid function during starvation.     

The long-term survival of Escherichia coli in river water

Escherichia coli introduced into autoclaved filtered river water survived for up to 260 d at temperatures from 4° to 25°C with no loss of viability. Survival times were less in water which was only filtered through either a Whatman filter paper or a 0.45 μm Millipore filter or in untreated water, suggesting that competition with the natural microbial flora of the water was the primary factor in the disappearance of the introduced bacteria. Survival was also dependent upon temperature with survival at 4°C > 15°C > 25°C > 37°C for any water sample. Direct counts showed that bacterial cells did not disappear as the viable count decreased. The possession of the antibiotic resistance plasmids, R 1 drd -19 or R144-3, did not enhance survival nor cause a faster rate of decay, indicating that the metabolic burden imposed by a plasmid was not a factor in survival under starvation conditions. There was no evidence of transfer of either plasmid at 15°C or of loss of plasmid function during starvation.