The Role of Antioxidant Systems in the Response of Escherichia colito Heat Shock

Shifting the temperature from 30 to 45°C in an aerobic Escherichia coliculture inhibited the expression of the antioxidant genes katG, katE, sodA, and gor.The expression was evaluated by measuring -galactosidase activity in E. colistrains that contained fusions of the antioxidant gene promoters with the lacZoperon. Heat shock inhibited catalase and glutathione reductase, lowered the intracellular level of glutathione, and increased its extracellular level. It also suppressed the growth of mutants deficient in the katG-encoded catalase HPI, whereas the sensitivity of the wild-type andsodA sodBmutant cells to heat shock was almost the same. In the E. coliculture adapted to growth at 42°C, the content of both intracellular and extracellular glutathione was two times higher than in the culture grown at 30°C. The temperature-adapted cells grown aerobically at 42°C showed an increased ability to express the fused katG–lacZgenes.     

Effects of <Emphasis Type="Italic">Escherichia coli</Emphasis> RraA Orthologs of <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> on the Ribonucleolytic Activity of RNase E In Vivo

RraA is a recently discovered protein inhibitor of RNase E that catalyzes the initial step in the decay and processing of numerous RNAs in Escherichia coli. In the genome of Vibrio vulnificus, two open reading frames that potentially encode proteins homologous to E. coli, RraA-designated RraAV1 and RraAV2, have respectively 80.1% and 59.0% amino acid identity to RraA. The authors report that coexpression of RraAV1 protein in E. coli cells overproducing RNase E rescued these cells from growth arrest and restored their normal growth, whereas coexpression of RraAV2 protein further inhibited the growth of E. coli cells, whose growth was already impaired by overproduction of RNase E. Analyses of the steady-state level of various RNase E substrates indicated that the coexpression of RraAV1 more efficiently inhibited RNase E action than coexpression of RraA, and consequently resulted in the more increased abundance of each RNA species tested in vivo. The inhibitory effect by RraAV2 coexpression on RNase E was observed only in the case of trpA mRNA, indicating the possibility of RNA substrate-dependent inhibition of RraAV2 on RNase E. The findings suggest that these regulators of ribonuclease activity have both a conserved inhibitory function and a differential inhibitory activity on RNase E-like enzymes across the species.     

Production of a New Ammonucleoside, 9-(2′-Amino-2′-deoxypentofuranosyl) Guanine,by Aerobacter sp.

A species of Aerobacter KY 3071 isolated from soil was found to produce a guanosine analog. It was isolated in a crystalline form from the broth culture through chromatographies on ion-exchange resins and porous resin, and characterized as 9-(2′-amino-2′-deoxypentofuranosyl) guanine by paper chromatographies, UV, NMR, IR spectra and chemical analysis.

This compound inhibited the growth of E. coli KY 8323 but not of other bacteria including most of the other strains of E. coli. It also showed antitumor activity against HeLa cell and Sarcoma 180.     

Monitoring growth of Escherichia coli using a two-channel optical sensor

Summary The growth of Escherichia coli strain TG 1 was monitored, measuring simultaneously the culture fluorescence and the 360° reflection at 578 nm with a two-channel optical sensor. It was observed that the culture fluorescence at 366 nm excitation was approximately three times higher than the NADH fluorescence of washed E. coli cells whereas the 360° reflection at 578 nm was comparable. The reason for this effect was found to be the accumulation of riboflavin in the cultivation liquid of the E. coli cells being equal to approximately 0.05 mg/g biomass. In shaken batch cultivations of the same strain the amount of riboflavin in the cell-free cultivation liquid correlated with the biomass being a very sensitive indicator of E. coli growth.Correspondence to: W. S. Kunz     

Apolipoprotein E: A potent inhibitor of endothelial and tumor cell proliferation

Recombinant human apolipoprotein E3 (apoE), purified from E. coli, inhibited the proliferation of several cell types, including endothelial cells and tumor cells in a dose- and time-dependent manner. ApoE inhibited both de novo DNA synthesis and proliferation as assessed by an increase in cell number. Maximal inhibition of cell growth by apoE was achieved under conditions where proliferation was dependent on heparin-binding growth factors. Thus, at low serum concentrations (0–2.5%) basic fibroblast growth factor (bFGF) stimulated the proliferation of bovine aortic endothelial (BAE) cells severalfold. The bFGF-dependent proliferation was dramatically inhibited by apoE with an IC₅₀ ≈ 50 nM. Under conditions where cell proliferation was mainly serum-dependent, apoE also suppressed growth but required higher concentrations to be effective (IC₅₀ ≈ 500 nM). ApoE also inhibited growth of bovine corneal endothelial cells, human melanoma cells, and human breast carcinoma cells. The IC₅₀ values obtained with these cells were generally 3–5 times higher than with BAE cells. Inhibition of cell proliferation by apoE was reversible and dependent on the time of apoE addition to the culture. In addition, apoE inhibited the chemotactic response of endothelial cells that were induced to migrate by a gradient of soluble bFGF. Inhibition of cell proliferation by apoE may be mediated both by competition for growth factor binding to proteoglycans and by an antiadhesive activity of apoE. The present results demonstrate that apoE is a potent inhibitor of proliferation of several cell types and suggest that apoE may be effective in modulating angiogenesis, tumor cell growth, and metastasis.     

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L-180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell-associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.     

An adhesive factor found in strains ofEscherichia coli belonging to the traditional infantile enteropathogenic serotypes

Escherichia coli strains isolated from outbreaks of diarrheal disease were tested for the presence of adhesive factors. Fifty-one of these strains belonged to traditional infantile entero-pathogenic serotypes (EPEC) and 17 belonged to other serotypes. None of these strains were enterotoxigenic and none possessed colonization factors CFA/I or CFA/II, which have been described among strains of enterotoxigenicE. coli (ETEC). EnterotoxigenicE. coli strains from patients with diarrhea and strains which were neither EPEC nor ETEC from subjects without diarrhea were also examined. By means of a tissue culture technique using HEp-2 cells, a new adhesive factor was found to occur with greater frequency in EPEC strains. The adhesive factor was found less frequently in the other groups ofE. coli studied. It was distinct from type 1 pili and was not inhibited by the presence ofD-mannose.     

Growth Stimulating Substance for Microorganisms Produced by Escherichia coli Causing the Reduction of the Lag Phase in Microbial Growth and Identity of the Substance with Pyrroloquinoline Quinone

The finding that most strains of microbes produce a growth stimulating substance for microorganisms was demonstrated and confirmed with the culture broth of Escherichia coli grown on a glucose-mineral medium. Addition of culture broth of E. coli to the culture media of the others markedly reduced the lag phase in microbial growth but not growth rate in the subsequent exponential phase nor the total cell yield in the stationary phase. The growth stimulation causing reduction of the lag phase was dependent on the amount of culture broth added. Occurrence of cell growth was essential for the excretion of the growth stimulating substance by E. coli. Under identical inoculum size, even with a heavy inoculum, a further reduction of the lag phase was observed by the addition of culture broth of E. coli. The substance was only effective at the initial growth phase but inert when the substance was added to a growing culture at the exponential phase. Finally, the substance was identified as pyrroloquinoline quinone, a newly established coenzyme, through chromatographic, spectroscopic and enzymatic criteria.     

The effect of growth rate and other growth conditions on the lipid composition of Escherichia coli

Escherichia coli was grown as a continuous culture at various defined conditions of temperature, pH, aeration rate and dilution rate. The lipids were extracted from disrupted cells and the relative fatty acid content of the individual and total phospholipids was determined. The lipid composition of E. coli was shown to change with the fermentation conditions. Interestingly, E. coli adapted to high growth rates and to low oxygen tension by changing the lipid composition of the membrane in exactly the same way, thus indicating a common effect.     

Degradation of trichloroethylene by recombinant E. coli in continuous culture

Trichloroethylene (TCE) degradation by the recombinant E. coli JM109 harboring a TCE-degradative plasmid (pIO720 or pIO72K) in continuous culture was studied. The ampicillin-resistant plasmid, pIO720, contained the cumene dioxygenase genes and the dimethyl sulfide monooxygenase genes. pIO72K was constructed according to replacement of an ampicillin resistance gene on pIO720 by a kanamycin resistance gene. In the case of E. coli JM109 (pIO720) in continuous culture, TCE degradation activity decreased rapidly after continuous culture started, and the remaining number of host cells harboring pIO720 also decreased rapidly. In the case of E. coli JM109 (pIO72K) in continuous culture, TCE degradation activity was stable during continuous culture for at least 300 h and the number of the host cells harboring pIO72K did not decrease. TCE degradation activity of E. coli JM109 (pIO72K) was the highest at a dilution rate of 0.2 h^–1.     

Antibacterial effect of mesenchymal stem cells against Escherichia coli is mediated by secretion of beta‐ defensin‐ 2 via toll‐ like receptor 4 signalling

Recently, we demonstrated that intratracheal transplantation of human umbilical cord blood‐ derived mesenchymal stem cells (MSCs) attenuates Escherichia (E) coli‐ induced acute lung injury primarily by down‐ modulating inflammation and enhancing bacterial clearance iQn mice. This study was performed to elucidate the mechanism underlying the antibacterial effects of MSCs. The growth of E. coli in vitro was significantly inhibited only by MSCs or their conditioned medium with bacterial preconditioning, but not by fibroblasts or their conditioned medium. Microarray analysis identified significant up‐ regulation of toll‐ like receptors (TLR)‐ 2 and TLR‐ 4, and β‐ defensin 2 (BD2) in MSCs compared with fibroblasts after E. coli exposure. The increased BD2 level and the in vitro antibacterial effects of MSCs were abolished by specific antagonist or by siRNA‐ mediated knockdown of TLR‐ 4, but not TLR‐ 2, and restored by BD2 supplementation. The in vivo down‐ modulation of the inflammatory response and enhanced bacterial clearance, increased BD2 secretion and the resultant protection against E. coli‐ induced pneumonia observed only with MSCs, but not fibroblasts, transplantation in mice, were abolished by knockdown of TLR‐ 4 with siRNA transfection. Our data indicate that BD2 secreted by the MSCs via the TLR‐ 4 signalling pathway is one of the critical paracrine factors mediating their microbicidal effects against E. coli, both in vitro and in vivo. Furthermore, TLR‐ 4 from the transplanted MSCs plays a seminal role in attenuating in vivo E. coli‐ induced pneumonia and the ensuing acute lung injury through both its anti‐ inflammatory and antibacterial effects.     

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

The mechanism whereby Escherichia coli K12 accumulates orotic acid in culture fluid was studied. Pyrimidine compounds were incorporated effectively into cells of E. coli K12, stimulated the growth, and depressed the accumulation; while purine compounds were not so much consumed by the microorganism for its growth, and affected the accumulation to a lesser extent. On the other hand, E. coli B unable to accumulate orotic acid utilized less effectively pyrimidine compounds for its growth than strain K12.

It is supposed, therefore, that in the de novo pathway for pyrimidine synthesis in E. coli K12 the step from orotic acid to 5′-UMP is genetically depressed so that orotic acid is accumulated when pyrimidine compounds, that would cause a feedback inhibition of orotic acid synthesis upon incorporation, are not supplemented.     

Amine neuromediators,their precursors,and oxidation products in the culture of <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

The growth dynamics of the synthesis of monoamine neuromediators serotonin, norepinephrine, and dopamine in Escherichia coli K-12 was investigated for the first time using high performance liquid chromatography with electrodetection. Maximum (micromolar) concentrations of these compounds were detected in E. coli cells during the early growth phases; their intracellular content decreases after the transition to late growth phases. E. coli biomass contains (i) the substances DOPA and 5-hydroxytryptamine that serve in animal cells as neuromediator precursors and (ii) the products of their oxidative deamination. Presumably, the biosynthesis and degradation of monoamine neuromediators in bacterial cells involves enzyme systems analogous to those typical of animals. The culture fluid of E. coli contains micromolar concentrations of DOPA and nanomolar of serotonin, dopamine, and norepinephrine during the late growth phase. These concentrations are sufficient for animal/human receptors to bind them. This article deals with the potential biotechnological applications of the data obtained.     

Study of the Action of Se and Cu on the Growth Metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> by Microcalorimetry

The biological effect of Se and Cu²⁺ on Escherichia coli (E. coli) growth was studied by using a 3114/3236 TAM Air Isothermal Calorimeter, ampoule method, at 37°C. From the thermogenesis curves, the thermokinetic equations were established under different conditions. The kinetics showed that a low concentration of Se (1–10 μg/mL) promoted the growth of E. coli, and a high concentration of Se (>10 μg/mL) inhibited the growth, but the Cu²⁺ was always inhibiting the growth of E. coli. Moreover, there was an antagonistic or positive synergistic effect of Se and Cu²⁺ on E. coli in the different culture medium when Se was 1–10 μg/ml and Cu²⁺ was 1–20 μg/ml. There was a negative synergistic effect of Se and Cu²⁺ on E. coli when Se was higher than 10 μg/ml and Cu²⁺ was higher than 20 μg/ml. The antagonistic or synergistic effect between Se and Cu²⁺ on E. coli was related to the formation of Cu–Se complexes under the different experimental conditions chosen.     

Antimikrobielle Inhaltsstoffe in Algen

Zusammenfassung 1. Acrylsäure ist innerhalb der einfachen ungesättigten Carbonsäuren ein relativ spezifischer Hemmstoff fürEscherichia coli, Staphylococcus aureus und andere Bakterien.2. Acrylsäure hat nur in sehr hohen Konzentrationen bei sehr langen Inkubationszeiten schwach bakterizide Eigenschaften.3. Sie hemmt besonders bei älterenE. coli-Zellen die Atmung sehr viel stärker als das Wachstum. Bei jungenE. coli-Kulturen und beiSt. aureus sind Wachstums- und Atmungshemmung etwa gleich stark.4. Die antibakteriallen Eigenschaften der Acrylsäure gegenE. coli werden durch SH-haltige Verbindungen aufgehoben. 2,4-Dinitrophenol wirkt dagegen stark synergistisch. BeiSt. aureus sind diese Wirkungen nur schwach ausgeprägt.5. Der Einbau von Leucin in Protein wird durch Acrylsäure beiE. coli weniger als das Wachstum, der Einbau von Thymidin in DNS fast ebenso stark wie das Wachstum und der Einbau von Uracil in RNS von Anfang an sehr viel stärker gestört. DNP steigert die Hemmung des Uracil-Einbaues durch Acrylsäure.6. Die Synthese der r-RNS und s-RNS wird in etwa gleichem Umfang gestört; die Synthese der m-RNS scheint nicht beeinflußt zu werden.

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Antimicrobial components in algae4th contribution. Effect of acrylic acid on respiration and macromolecular synthesis inStaphyloccus aureus andEscherichia coli

Among the simple unsaturated carbonic acids, acrylic acid is a comparatively specific inhibitor forStaphylococcus aureus, Escherichia coli and other bacteria. Insignificant bactericidal action can only be observed at high concentrations and long incubation time of acrylic acid. In olderE. coli cells, acrylic acid inhibits respiration more strongly than growth. On the other hand, in youngerE. coli cells and inSt. aureus, inhibition of growth is more pronounced than that of respiration. Antibacterial activities of acrylic acid againstE. coli can be reduced by compounds with thiol groups; they can be increased by 2.4-dinitrophenol (DNP). The effect of acrylic acid and DNP is synergistic; such effects are very small inSt. aureus. Incorporation of leucine into the protein ofE. coli cells is less inhibited by acrylic acid than their growth rate; incorporation of thymidine into DNA is inhibited to the same extent as growth; incorporation of uracil into RNA is inhibited to a much higher extent right from the beginning of the experiment. DNP is capable of increasing the degree of inhibition of uracil incorporation caused by acrylic acid. Syntheses of r-RNA and s-RNA are inhibited to nearly the same extent; there seems to be no influence on the synthesis of m-RNA.

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Teil der Dissertation vonR. Heyser, Bonn, 1970 (D5).     

Accumulation of Xanthosine by Escherichia coli in the Presence of Angustmycin C

Effect of angustmycin C, an adenosine analogue having an unusual sugar linked to adenine, on Escherichia coli was studied. It inhibited biosynthesis of RNA and DNA preferentially and xanthosine was excreted from the inhibited cells into the medium. During the course of its growth with addition of the antibiotic, the specific activity of inosine-5′-phosphate dehydrogenase of E. coli cells increased six times. These effects could be explained by the inhibitory effect of angustmycin C on xanthosine-5′-phosphate aminase which resulted in decreased level of IMP-dehydrogenase. Accumulation of xanthosine induced by the antibiotic reached the concentration of 940 μg/ml under an appropriate condition.     

Nutritional Study of Three Strains of Naegleria gruberi*

Naegleria gruberi strains cloned from amebas isolated from a Vero cell culture (“TS”), a sewer drainage ditch (“PD”), and an established laboratory line (“S”) were morphologically identical except for differences in size and flagellate transforming ability. Cultivation on a Trypticase-yeast extract-glucose medium (“TYG”) fortified with autoclaved E. coli resulted in increased cell size of 2 strains. Differences also were noted in growth rates and optimal growth temperatures. The autoclaved E. coli in TYG medium was replaceable with serum only for strains TS and PD. A basal salts medium + autoclaved E. coli supported growth of all 3 strains, but the basal salts medium + serum would not support growth of any of the strains.     

Biological Activity and Mode of Action of a Specific Antiphage Substance,AFS

Purified AFS (anti-filamentous phage substance) produced by Streptomyces lavendulae AM–7a showed specific antiphage activity against the male specific, deoxyribonucleic acid-containing filamentous phages of Escherichia coli without any activity against other DNA-phages nor the male-specific ribonucleic acid-containing phages of E. coli. AFS brought about no inactivation of free particles of filamentous phage, fl, nor the receptor of the host cells for the phage, while it showed strong killing effect against the fl-infected host cells at the concentration below 0.01 μg/ml. Antiphage activity of AFS might be due to its highly specific killing effect only on the E. coli cells infected with the filamentous DNA phages, while it exerted no effect on the growth of the unifected E. coli nor other microorganisms. Killing by AFS seemed to require the energy metabolism of the phage-infected host cells. Macro-molecular synthesis and respiration of the infected host cells were inhibited soon after the addition of small amounts of AFS without any cell lysis.     

Significantly enhanced production of recombinant nitrilase by optimization of culture conditions and glycerol feeding

The production of a recombinant nitrilase expressed in Escherichia coli JM109/pNLE was optimized in the present work. Various culture conditions and process parameters, including medium composition, inducer, induction condition, pH and temperature, were systematically examined. The results showed that nitrilase production in E. coli JM109/pNLE was greatly affected by the pH condition and the temperature in batch culture, and the highest nitrilase production was obtained when the fermentation was carried out at 37°C, initial pH 7.0 without control and E. coli was induced with 0.2 mM isopropyl-β-d-thiogalactoside at 4.0 h. Furthermore, enzyme production could be significantly enhanced by adopting the glycerol feeding strategy with lower flow rate. The enzyme expression was also authenticated by sodium dodecyl phosphate polyacrylamide gel electrophoresis analysis. Finally, under the optimized conditions for fed-batch culture, cell growth, specific activity and nitrilase production of the recombinant E. coli were increased by 9.0-, 5.5-, and 50-fold, respectively.     

3′, 5′-Cyclic Adenosine Monophosphate Dependent Xylose Lethal Phenomenon in Escherichia coli

The growth of Escherichia coli W2252 was found to be inhibited when xylose and cAMP coexisted in the medium such as peptone or nutrient broth. Among other sugars, only arabinose imposed weaker effect. cAMP could not be replaced by adenine, adenosine, 5′-AMP, 3′-AMP and other 3′,5′-cyclic nucleoside monophosphates. Dose response was observed with reference to either xylose or cAMP. In the presence of both 1% xylose and 10 mm cAMP in peptone broth, 90% of logarithmic phase cells of E. coli W2252 were killed within 6 hr at 37°C. We call this phenomenon as cAMP dependent xylose lethal. This phenomenon was also observed with many substrains of E. coli K–12, E. coli C, Aerobacter aerogenes and Salmonella typhimurium, but not with their xylose negative mutants.