Differential effects of procaine and phenethyl alcohol on excision repair of DNA in u.v.-irradiated Escherichia coli

Experiments were performed to investigate the involvement of the cell membrane in the excision DNA repair process in Escherichia coli. Two membrane-binding drugs, procaine and phenethyl alcohol (PEA), inhibited liquid-holding recovery (LHR) in u.v.-irradiated E. coli wild-type and recA strains. In uvrB and polA strains where, after u.v.-irradiation, LHR was absent the two drugs had no effect. Both drugs markedly reduced the removal of u.v.-induced thymine dimers in the DNA of wild-type cells (H/r30). Analysis by alkaline sucrose gradients revealed that PEA inhibited the incision step in excision repair. In contrast, procaine had no effect on incision but apparently inhibited the late steps in excision repair. PEA dissociated DNA from the cell membrane, whereas procaine did not. The results suggest that the two drugs PEA and procaine inhibit LHR and the excision repair process operating on u.v.-induced damage in E. coli by at least two different mechanisms each of which may involve the cell membrane.     

Characterization of an oxygen-dependent inducible promoter,the Escherichia coli nar promoter,in gram-negative host strains

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.     

Alkyl hydroperoxide reductase dependent on thioredoxin-like protein from Pyrococcus horikoshii

Pyrococcus horikoshii is an obligate anaerobic hyperthermophilic archaeon. In P. horikoshii cells, a hydroperoxide reductase homologue ORF (PH1217) was found to be induced by oxygen. The recombinant protein, which was expressed in E. coli under aerobic conditions, exhibited no activity. However, the recombinant protein prepared under semi-anaerobic conditions exhibited alkyl hydroperoxide reductase activity. Furthermore, it was clarified that it was coupled with the thioredoxin-like system in P. horikoshii. Western blot analysis revealed that the protein was induced by oxygen and hydrogen peroxide. This protein seems to be sensitive to oxygen but forms a thioredoxin-dependent system to eliminate reactive oxygen species in P. horikoshii.     

Characterization of an oxygen-dependent inducible promoter system, the nar promoter, and Escherichia coli with an inactivated nar operon

The nar promoter of Escherichia coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an inducible promoter. To increase the expression level, the nar promoter was expressed in E. coli where active nitrate reductase cannot be expressed from the nar operon on the chromosome. A plasmid with the lacZ gene expressing beta-galactosidase instead of the structural genes of the nar operon was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate and molybdate concentrations maximally inducing the nar promoter, the amount of expressed beta-galactosidase, and induction ratio (specific beta-galactosidase activity after maximal induction/specific beta-galactosidase activity before induction.)The following results were obtained from the experiments: induction of the nar promoter was optimal when E. coli was grown in the presence of 1% nitrate at the beginning of culture; expression of beta-galactosidase was not affected by molybdate; the induction ratio was maximal, approximately 300, when the overnight culture was grown in the flask for 2.5 h (OD(600) is congruent to 1.3) before being transferred to the fermentor; the amount of beta-galactosidase per cell and per medium volume was maximal when E. coli was grown under aerobic conditions to OD(600) = 1.7; then the nar promoter was induced under microaerobic conditions made by lowering dissolved oxygen level (DO) to 1-2%. After approximately 6 h of induction, OD(600) became 3.2 and specific beta-galactosidase activity became 36,000 Miller units, equivalent to 35% of total cellular proteins, which was confirmed from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (c) 1996 John Wiley & Sons, Inc.     

Death effector domain of a mammalian apoptosis mediator, FADD, induces bacterial cell death

FADD is a mammalian pro-apoptotic mediator consisting of the N-terminal death effector domain (DED) and the C-terminal death domain (DD). The N-terminal 88-residue fragment of murine FADD was defined as the stable structural unit of DED, as determined by proteolytic digestion and conformational analysis. This domain induced bacterial as well as mammalian cell death, whereas the full-length or DD of FADD did not. The Escherichia coli cells expressing FADD-DED showed elongated cell morphology and an increased level of nicked chromosomal DNA and mutation. The lethality of FADD-DED was abolished by co-expression of thioredoxin and superoxide dismutase or relieved by the addition of vitamin E as a reducing agent and under anaerobic growth conditions. The toxicity of FADD-DED was genetically suppressed by various oxidoreductases of E. coli. All these results suggest that the death effector domain of mammalian FADD induced bacterial cell death by enhancing cellular levels of reactive oxygen species (ROS).     

Fed-batch cultivation of an oxygen-dependent inducible promoter system, the nar promoter in Escherichia coli with an inactivated nar operon

The nar promoter of Escherichia coli is maximally induced under anaerobic or microaerobic conditions in the presence of nitrate. We previously demonstrated in batch experiments that the intact nar promoter of E. coli cloned into a pBR322-based plasmid serves as a high-level expression system in a nar mutant of E. coli (Lee et al., 1996b). In this study, we extend characterization of the nar promoter expression system to the fed-batch culture mode, which is widely used in industrial-scale fermentation. From these experiments, it was found that the specific beta-galactosidase activity expressed from the lacZ gene fused to the nar promoter was maximal when host cells were grown under aerobic conditions [dissolved oxygen, (DO) = 80%] to absorbance at 600 nm (OD600) = 35 before induction of the nar promoter by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. Approximately 15 h after induction, the OD600 of the culture reached 135 and the specific beta-galactosidase activity increased to 40,000 Miller units, equivalent to approximately 35% of the total cellular proteins. The specific beta-galactosidase activity before induction was approximately 1,000 Miller units, giving an induction ratio of approximately 40. Based on these results, we conclude that the nar promoter provides a convenient and effective high level expression system under conditions of fed-batch culture.     

Characterization of a novel tetracycline resistance that functions only in aerobically grown Escherichia coli.

A tetracycline resistance (Tcr) gene that was found originally on two Bacteroides plasmids (pBF4 and pCP1) confers tetracycline resistance on Escherichia coli, but only when it is grown aerobically. Using maxicells, we have identified a 44-kilodalton protein which is encoded by the region that carries the Tcr gene and which may be the Tcr gene product. Localization experiments indicate that this 44-kilodalton protein is cytoplasmic. To determine whether the tetracycline resistance gene is expressed under anaerobic conditions, we have constructed a protein fusion between the Tcr gene and lacZ. In strains of E. coli carrying the fusion, beta-galactosidase activity was the same when the cells were grown under anaerobic conditions as when the cells were grown under aerobic conditions. This indicates that the tetracycline resistance gene product is made under anaerobic conditions but does not work. The failure of the Tcr protein to function under anaerobic conditions was not due to a requirement for function of the anaerobic electron transport system, because neither nitrate nor fumarate added to anaerobic media restored tetracycline resistance. Inhibition of the aerobic electron transport system with potassium cyanide did not prevent growth on tetracycline of cells containing the Tcr gene. A heme-deficient mutant, E. coli SHSP19, which carries the Tcr gene, was still resistant to tetracycline even when grown in heme-free medium. These results indicate that functioning of the Tcr gene product is not dependent on the aerobic electron transport system. Thus the requirement for aerobic conditions appears to reflect a requirement for oxygen. Spent medium from an E. coli strain carrying the Tcr gene, which was grown in medium containing tetracycline (50 micrograms/ml), did not inhibit growth of a tetracycline-susceptible strain of E. coli. Thus, the Tcr gene product may be detoxifying tetracycline.     

Interaction of E. coli cells with ascorbic acid and sodium nitrite studied by ESR method]

Ascorbic acid, an effective modulator and regulator of cell metabolism, was shown to induce the production of nitric oxide in E. coli cells. This process was detected by EPR spectroscopy as the generation of a spectral signal typical of nitrosyl-iron-sulfur centers (Fe-S-NO) under anaerobic conditions. Incubation of E. coli cells in the presence of ascorbic acid under aerobic conditions was shown to be accompanied by sodium nitrite formation. It is suggested that ascorbic acid is capable of supporting the system of energy supply to cells in hypoxia caused by reduced oxygen content or treatment with sodium nitrite.     

Characterisation of a Pasteurella multocida esterase gene which confers a hemolytic phenotype in Escherichia coli under anaerobic conditions

Investigation of the hemolytic phenotype under anaerobic growth conditions of an avian Pasteurella multocida strain, PBA100, resulted in the identification and characterisation of a gene encoding an esterase enzyme, mesA, that conferred a hemolytic phenotype in Escherichia coli under anaerobic conditions. MesA appeared to be expressed and functional under anaerobic and aerobic conditions in both E. coli and P. multocida. A P. multocida mesA mutant was generated which resulted in the loss of acetyl esterase activity under anaerobic conditions. However, this mutation did not cause any attenuation of virulence for mice nor a detectable change to the anaerobic hemolytic phenotype of P. multocida. In E. coli MesA appeared to cause hemolysis indirectly by the induction of the latent E. coli K-12 cytolysin, sheA.     

Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157

A strain of Escherichia coli serotype O157 was grown in steady state chemostat culture under aerobic, oxygen-limited and anaerobic conditions. The growth and metabolic efficiency of oxygen-limited and anaerobic cultures was impaired, with biomass yield and the molar growth yield for glucose, Yglucose, reduced markedly in comparison with aerobic cultures. Steady state cells were typically short rods 2-3 microns long, and were encapsulated by a layer of extracellular material. The majority of cells were non-flagellated and fimbriae were not observed. Chemostat-grown cells were significantly more adhesive for HEp-2 monolayers than cells grown in aerobic batch culture. Furthermore, oxygen-limited and anaerobic cultures were significantly more adhesive for Hep-2 cells when compared with cells grown in aerobic chemostat culture, possibly reflecting increased pathogenicity associated with the induction of novel adhesins. Type 1 pili were not responsible for increased adherence. Verocytotoxins, VT1 and VT2, were expressed constitutively and were not influenced by oxygen availability. This study demonstrates that E. coli O157 is a versatile micro-organism, which responds to environmental conditions likely to be encountered during infection by inducing a phenotype which is more adhesive for human epithelial cells.     

In vivo evidence for the iron-binding activity of an iron-sulfur cluster assembly protein IscA in Escherichia coli

IscA is a key member of the iron-sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. In the present paper we report the in vivo evidence demonstrating the iron-binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1 μM) in M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron-sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron-binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA, and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron-binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralogue SufA are essential for the iron-sulfur cluster assembly in E. coli cells under aerobic growth conditions, but not under anaerobic growth conditions. The results provide in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron-sulfur clusters in E. coli cells under aerobic conditions.     

The role of redox in the regulation of manganese-containing superoxide dismutase biosynthesis in Escherichia coli

The manganese-containing superoxide dismutase in Escherichia coli is an inducible enzyme that protects cells against oxygen toxicity. The manganese-enzyme is induced by oxygen, nitrate, redox active compounds that react with oxygen to generate superoxide radicals, as well as iron chelators. In order to test the hypothesis that the redox state of the cell is involved in regulating manganese-superoxide dismutase biosynthesis, we studied the effects of several oxidants on growth and superoxide dismutase biosynthesis. The data showed, that under anaerobic conditions, the active manganese-enzyme is induced in the presence of potassium ferricyanide, copper-cyanide complex, ammonium persulfate, and hydrogen peroxide. Western blot analysis revealed that the induction of manganese-superoxide dismutase by the oxidants is associated with de novo protein biosynthesis. Potassium ferricyanide and hydrogen peroxide induced the enzyme under aerobic conditions as well. It is concluded that the redox state of the cell possibly influences the biosynthesis and/or activity of an iron-containing repressor protein that serves to negatively regulate manganese-superoxide dismutase biosynthesis.     

Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli.

The biosynthetic origin of the oxygen atoms of ubiquinone 8 from aerobically grown Escherichia coli was studied by 18O labeling. An apparatus was developed which allowed the growth of cells under a defined atmosphere. Mass spectral analysis of ubiquinone 8 from cells grown under highly enriched 18O2 showed that three oxygen atoms of the quinone are derived from molecular oxygen. It was established that the molecular oxygen is incorporated into the two methoxyl groups (at C-5 and C-6) and one of the carbonyl positions of the ubiquinone molecule by demonstrating that only one of the incorporated oxygens will exchange with water under acidic conditions that specifically catalyze the exchange of carbonyl, but not methoxyl, oxygens. That the C-4 carbonyl oxygen is derived from molecular oxygen was shown by the incorporation of three atoms of 18O2 into ubiquinone 8 biosynthesized from added 4-hydroxybenzoic acid. Comparison of ubiquinone 8 and menaquinone 8 from E. coli grown under 18O2 confirmed that the labeled carbonyl oxygen of the [18O2]ubiquinone 8 is incorporated biosynthetically and not by chemical exchange in the cell. It is concluded that the three hydroxylation reactions involved in the pathway for the aerobic biosynthesis of ubiquinone are all catalyzed by monooxygenases. The implications of this study for the anaerobic biosynthesis of ubiquinone 8 in E coli are discussed.     

Effect of oxygen on survival of faecal pollution indicators in drinking water

AIMS: The aim of this study was to determine the effect of oxygen on the survival of faecal pollution indicators including Escherichia coli in nondisinfected drinking water. METHODS AND RESULTS: Aerobic and anaerobic drinking water microcosms were inoculated with E. coli ATCC 25922 or raw sewage. Survival of E. coli was monitored by membrane filtration combined with cultivation on standard media, and by in situ hybridization with 16S rRNA-targeted fluorescent oligonucleotide probes. Anaerobic conditions significantly increased the survival of E. coli in drinking water compared with aerobic conditions. Escherichia coli ATCC 25922 showed a biphasic decrease in survival under aerobic conditions with an initial first-order decay rate of -0.11 day(-1) followed by a more rapid rate of -0.35 day(-1). In contrast, the first-order decay rate under anaerobic conditions was only -0.02 day(-1). After 35 days, <0.01% of the initial E. coli ATCC 25922 population remained detectable in aerobic microcosms compared with 48% in anaerobic microcosms. A poor survival was observed under aerobic conditions regardless of whether E. coli ATCC 25922 or sewage-derived E. coli was examined, and regardless of the detection method used (CFU or fluorescent in situ hybridization). Aerobic conditions in drinking water also appeared to decrease the survival of faecal enterococci, somatic coliphages and coliforms other than E. coli. CONCLUSIONS: The results indicate that oxygen is a major regulator of the survival of E. coli in nondisinfected drinking water. The results also suggest that faecal pollution indicators other than E. coli may persist longer in drinking water under anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of oxygen should be considered when evaluating the survival potential of enteric pathogens in oligotrophic environments.     

Modeling of the biotransformation of crotonobetaine into L-(-)-carnitine by Escherichia coli strains

A simple unstructured model, which includes carbon source as the limiting and essential substrate and oxygen as an enhancing substrate for cell growth, has been implemented to depict cell population evolution of two Escherichia coli strains and the expression of their trimethylammonium metabolism in batch and continuous reactors. Although the model is applied to represent the trans-crotonobetaine to L-(-)-carnitine biotransformation, it is also useful for understanding the complete metabolic flow of trimethylammonium compounds in E. coli. Cell growth and biotransformation were studied in both anaerobic and aerobic conditions. For this reason we derived equations to modify the specific growth rate, mu, and the cell yield on the carbon source (glycerol), Y(xg), as oxygen increased the rate of growth. Inhibition functions representing an excess of the glycerol and oxygen were included to depict cell evolution during extreme conditions. As a result, the model fitted experimental data for various growth conditions, including different carbon source concentrations, initial oxygen levels, and the existence of a certain degree of cell death. Moreover, the production of enzymes involved within the E. coli trimethylammonium metabolism and related to trans-crotonobetaine biotransformation was also modeled as a function of both the cell and oxygen concentrations within the system. The model describes all the activities of the different enzymes within the transformed and wild strains, able to produce L-(-)-carnitine from trans-crotonobetaine under both anaerobic and aerobic conditions. Crotonobetaine reductase inhibition by either oxygen or the addition of fumarate as well as its non-reversible catalytic action was taken into consideration. The proposed model was useful for describing the whole set of variables under both growing and resting conditions. Both E. coli strains within membrane high-density reactors were well represented by the model as results matched the experimental data.