Caffeine-death in Escherichia coli

Summary Growth of a culture of E. coli strain B or 15 in medium containing caffeine resulted in the accumulation of inviable cells in the population. A caffeine concentration of 8 mM caused the death of between 30% and 50% of the cells in 12 independent populations grown for 15 generations or more. The thymine dimer excision-defective strains Bs-1, Bs-8 and Bs-12 and the exr ^– mutant Bs-2 were resistant to this lethal effect. The reckless, hcr ⁺ mutant Bs-11 was more sensitive than the parental B strain. Although 100mM caffeine did not impair DNA synthesis in vitro, concentrations of the drug 8 mM caused a significant decline in DNA synthesis in vivo in E. coli B cells. From the fit of an experimental growth curve to an algebraic model of growth in which a proportion of cells are inactivated at each replication it is suggested that caffeine does not affect the replication rate of the viable cells. The observed impairment of DNA synthesis in vivo is equated with this cell death (caffeine-death). For E. coli 15 or B, 8 mM caffeine induced caffeine-death at a rate of 18% per cell generation. Caffeine-resistant mutants of E. coli B and E. coli 15 were isolated. Of those studied in detail a substantial proportion proved to be U.V. and X-ray sensitive and excision-defective. Others were more U.V. and X-ray resistant than strain B. Yet another class proved highly unstable. A chromosome breakage model of caffeine-death implicating enzymes of the excision-repair process is discussed.     

Induction of DNA breakdown and death in Escherichia coli by phleomycin. Its association with dark-repair processes

Summary Phleomycin, at concentrations above 1 g/ml, induced breakdown of DNA and death in E. coli. Exponentially growing cultures were about 10 times more sensitive to phleomycin than were stationary cultures, and the effect was somewhat dependent on the medium.Excisionless (HCR) mutants of E. coli were insensitive to doses of phleomycin which killed over 99% of wild-type organisms within an hour, while EXR mutants were considerably more sensitive.Mutants of E. coli selected for phleomycin resistance were unable to reactivate U.V. irradiated Tl phage (HCR).It is concluded that the DNA breakdown, inhibition of DNA replication and cell death are a consequence of initial attack by an excision-endonuclease stimulated by the phleomycin.     

Amplification of phleomycin induced death and DNA breakdown by caffeine in Escherichia coli

Summary Caffeine enhanced the degradation of DNA to acid soluble fragments in cultures of Escherichia coli exposed to Phleomycin (2 g/ml). Enhancement was particularly striking with stationary phase cultures, which normally exhibit negligible DNA breakdown when treated with 2 g/ml of Phleomycin. There is little DNA breakdown or death in UVR strains treated with phleomycin (2 g/ml) during exponential growth but when caffeine was present as well as Phleomycin, the kinetics of DNA breakdown and the amounts of DNA degraded were identical in all cultures tested including those of UVR, EXR, B/r type and B strains and equal to the maximum rate observed (with an EXR strain) in the absence of caffeine (ca. 1.7 % per min). High concentrations of Phleomycin (10 g/ml) had the same effect as the caffeine+Phleomycin (2 g/ml) combination and produced a uniform pattern of DNA breakdown in all strains tested. Caffeine did not seem to increase permeability of the bacterial coat. Given to the cells before exposure to Phleomycin it was ineffective in enhancing DNA breakdown. On the other hand, exposure of the bacteria to Phleomycin for a period of 40 min at 37° followed by caffeine was as effective as adding the two drugs together.Caffeine increased the efficiency of Phleomycin as an antibiotic for both growing and stationary phase cultures of e. coli B. It is suggested that caffeine aids the cooperative denaturation of DNA initiated by the attachment of Phleomycin molecules to thymine bases. This would allow single strand-specific endonucleases to attack the DNA and initiate DNA breakdown and cell death.This paper is dedicated to charlotte Auerbach on the occasion of her official retirement.     

Bacteriocin factors responsible for UV-sensitivity and susceptibility to post-irradiation breakdown of DNA

Summary Lack of dose-modifying protection by acriflavine against UV killing of E. coli Bs-2 and Bs-11 is explained by their carrying an inducible colicin. Its induction could account for post-irradiation break-down of DNA.     

A Functional Homolog of Escherichia coli NhaR in Vibrio cholerae

Escherichia coli NhaR controls expression of a sodium/proton (Na⁺/H⁺) antiporter, NhaA. The Vibrio cholerae NhaR protein shows over 60% identity to those of Escherichia coli and Salmonella enteritidis. V. cholerae NhaR complements an E. coli nhaR mutant for growth in 100 mM LiCl–33 mM NaCl, pH 7.6, and enhances the Na⁺-dependent induction of an E. coli chromosomal nhaA::lacZ fusion. These findings indicate functional homology to E. coli NhaR. Two V. cholerae nhaR mutants were constructed by using kanamycin resistance cartridge insertion at different sites to disrupt the gene. Both mutants showed sensitivity to growth in 120 mM LiCl, pH 9.2, compared with the wild-type strain and could be complemented by the introduction of V. cholerae nhaR on a low-copy-number plasmid. An nhaR mutation had no detectable effect on the virulence of the V. cholerae strain in the infant mouse model, suggesting that the antiporter system involved is not required in vivo, at least in this animal model.     

Effects of puromycin aminonucleoside on excision repair and recombination repair inescherichia coli

Ultraviolet (UV) lethality was increased when puromycin aminonucleoside (PAN) (3.0 mM) was added to the postirradiation medium ofEscherichia coli strains. The extent of repair inhibition differed greatly for strains WP-2hcr ⁺, B/r()hcr ⁺, WP-2hcr ^–, and Bs-1hcr ^–. The interaction between PAN and UV was synergistic in thehcr ⁺ strains. PAN enhanced UV lethality in strain B/r () to a greater degree than in WP-2hcr ⁺. There was no UV lethality enhancement by PAN (3.0 mM) in thehcr ^– strains, but the interaction of PAN (8.0 mM) with UV was synergistic. PAN decreased plaque formation of T1 UV-irradiated phage plated onE. coli Bhcr ⁺ but had no effect on phage plated on Bs-1 or WP-2hcr ^– strains. These results suggest that PAN interferes with thehcr function in UV-irradiated bacteria.     

Differential antibacterial activity of genistein arising from global inhibition of DNA, RNA and protein synthesis in some bacterial strains

Antibacterial activities of various flavonoids have been reported previously, but mechanism(s) of their action on bacterial cells remain(s) largely unknown. Here, we investigated effects of genistein, an isoflavone, and representatives of other flavonoids: daidzein (another isoflavone), apigenin (a flavone), naringenin (a flavanone) and kaempferol (a flavonol), on commonly used laboratory strains of model bacterial species: Escherichia coli, Vibrio harveyi and Bacillus subtilis. We found that E. coli was resistant to all tested flavonoids at concentrations up to 0.1 mM, while high sensitivity of V. harveyi to most of them (except daidzein, which exhibited significantly less pronounced effect) was observed. Effects of the flavonoids on B. subtilis were relatively intermediate to the two extremes, i.e., E. coli and V. harveyi. Action of genistein on bacterial cells was investigated in more detail to indicate changed cell morphology (formation of filamentous cells) of V. harveyi and drastic inhibition of global synthesis of DNA and RNA as shortly as 15 min after addition of this isoflavone to a bacterial culture to a final concentration of 0.1 mM. Protein synthesis inhibition was also apparent, but delayed. Both cell morphology and synthesis of nucleic acids and proteins were unaffected in E. coli cultures under analogous conditions. Studies on cell survival suggest that genistein is a bacteriostatic agent rather than a bactericidal compound.     

Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli

A genomic library of Zymomonas mobilis DNA was constructed in Escherichia coli using cosmid vector pHC79. Immunological screening of 483 individual E. coli strains revealed two clones expressing pyruvate decarboxylase, the key enzyme for efficient ethanol production of Z. mobilis. The two plasmids, pZM1 and pZM2, isolated from both E. coli strains were found to be related and to exhibit a common 4.6 kb SphI fragment on which the gene coding for pyruvate decarboxylase, pdc, was located.The pdc gene was similarily well expressed in both aerobically and anaerobically grown E. coli cells, and exerted a considerable effect on the amount of fermentation products formed. During fermentative growth on 25 mM glucose, plasmid-free E. coli lacking a pdc gene produced 6.5 mM ethanol, 8.2 mM acetate, 6.5 mM lactate, 0.5 mM succinate, and about 1 mM formate leaving 10.4 mM residual glucose. In contrast, recombinant E. coli harbouring a cloned pdc gene from Z. mobilis completely converted 25 mM glucose to up to 41.5 mM ethanol while almost no acids were formed.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Decolorization kinetics of a recombinant Escherichia coli strain harboring azo-dye-decolorizing determinants from Rhodococcus sp.

A 6.3 kb DNA fragment containing genes responsible for azo-dye decolorization was cloned and expressed in Escherichia coli. The resulting recombinant strain E. coli CY1 decolorized 200 mg azo dye (C.I. Reactive Red 22) l^–1 at 28 °C at 8.2 mg g cell^–1 h^–1, while the host (E. coli DH5) had no color-removal activity. Addition of 0.5 mM isopropyl--d-thiogalacto-pyranoside (IPTG) increased the decolorization rate 3.4-fold. The dependence of the decolorization rate on initial dye concentration essentially followed Monod-type kinetics and the maximal rate occurred with the dye at 600 mg l^–1. The decolorization rate of E. coli CY1 was optimal at 40 °C and pH 11. Aeration (increased dissolved O₂ level) strongly inhibited the decolorization, but decolorization occurred effectively under static incubation conditions (no agitation was employed). The CY1 strain also exhibited excellent stability during repeated-batch operations.     

Genetic control of DNA breakdown and repair inE. coli K-12 treated with mitomycin C or ultraviolet light

Summary Ultraviolet light sensitive mutants ofE. coli defective at theuvrA,uvrB oruvrC locus showed increased sensitivity to the lethal effects of mitomycin C when compared with theuvr ⁺ parental strain. In addition, DNA breakdown after treatment of cells with either mitomycin C or with ultraviolet light was greater in the parental strain carrying the activeuvr ⁺ genes than inuvr mutants. Thus, injuries produced by either mitomycin C or by ultraviolet light may be repaired by the same molecular mechanism which has been proposed and which involves defect excision, single strand breakdown and reconstruction of the DNA.With 2 Figures in the Text     

Cloning of the gene coding for aromatic amino acid aminotransferase from anE. Coli B strain

Summary AnE. coli B strain showing high activity in the transamination of phenylpyruvate to phenylalanine was used as the DNA source for the construction of a cosmid library inE. coli DG30, a strain which is known to be defective in all three major transaminase genes (aspC, ilvE andtyrB). By complementation analysis, cosmid clones could be identified with inserts carrying atyrB gene. The DNA inserts were further subcloned into pAT153 and thetyrB gene fromE. coli B was found to be similar to the gene reported forE. coli K12. Plasmids containing theE. coli BtyrB gene were transformed into the originalE. coli B strain and the recombinant strains assayed for transaminase activity and plasmid stability.Dedicated to Prof. Dr. Heinz Harnisch on the occasion of his 60th birthday.     

Mechanism of Caffeine Enhancement of Mutations Induced by Sublethal Ultraviolet Dosages

Certain chemical compounds increase mutation frequency of Escherichia coli B/r significantly when used in conjunction with nonlethal ultraviolet (UV) dosages. Studies were done to elucidate the mechanism of this enhancing mutational effect. Dark survival curves showed that 500 μg of caffeine per ml in the postirradiation medium markedly decreased survival to 60 ergs/mm² of UV in strain B/r. Caffeine did not markedly decrease survival to UV in strain B/r WP-2 hcr⁻. At least 90% of the mutations induced to streptomycin resistance by UV and 85% of those induced by UV with caffeine could be photoreversed. Experiments with thymine analogues suggested that thymine dimerization at the streptomycin locus was the primary premutational photoproduct induced by sublethal UV dosages. Caffeine did not interfere with the photoreversal of induced mutants, indicating that it probably does not bind to the photoreactivating enzyme or to a UV-induced lesion in the DNA. Addition of DNA or irradiated DNA with 500 μg of caffeine per ml resulted in no loss of the caffeine activity. The excision of UV-induced thymine-containing dimers from E. coli B/r T⁻ was investigated in the presence and absence of caffeine. Our results indicated that caffeine prevents excision of thymine dimers, presumably by binding to the excising enzyme. This binding results in an impairment of repair, which produces the increase in mutant numbers.     

Screening of high-yielding biocontrol bacterium Bs<Emphasis Type="Italic">-</Emphasis>916 mutant by ion implantation

Bacillus subtilis 916 was an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. To further improve its antagonistic ability, low-energy ion implantation was applied in Bs-916. We studied the effects of different doses of N⁺ implantation. The optimum dose of ion implantation for the Bs-916 was from 15 × 2.6 × 10¹⁴ N⁺/cm² to 25 × 2.6 × 10¹⁴ N⁺/cm². The mutant strain designated as Bs-H74 was obtained, which showed higher inhibition activity in the screening plate. Its inhibition zone against the indicator organism increased by 30.7% compared to the parental strain. The control effect of rice sheath blight was improved by 14.6% over that of Bs-916. Thin-layer chromatography and high-performance liquid chromatography analysis indicated that lipopeptides produced by Bs-916 and the mutant strains belonged to the surfactin family. Bs-H74 produced approximately 3.0-fold surfactin compared to that of Bs-916. To determine the role of surfactin in biocontrol by Bs-916, we tested another mutant strain, Bs-M49, which produced lower levels of surfactin significantly, and found that Bs-M49 had no obvious effects against R. solani. These results suggested that the surfactin produced by Bs-916 plays an important role in the suppression of sheath blight. These observations also showed that the Bs-H74 mutant strain is a better biocontrol agent than the parental strain.     

Evidence that a system similar to the recA system of Escherichia coli exists in Vibrio cholerae

Summary Two lines of evidence suggest that a gene analogous to the recA gene of Escherichia coli exists in Vibrio cholerae and that its product serves a proteolytic function in the SOS response. Firstly, Southern blot hybridization using the recA gene of E. coli as a probe revealed a genomic sequence in V. cholerae which hybridized with the probe. Secondly, the SOS-like response in V. cholerae (as measured by beta phage induction) triggered by DNA damaging agents like Furazolidone could be blocked by Antipain, a protease inhibitor known to inhibit RecA protease action in E. coli. Maximal blocking effect of Antipain on beta phage induction occurred at 1 mM. At this concentration neither the viability of the host bacterium nor the lytic growth of a clear plaque mutant of the phage was affected by Antipain.     

Escherichia coli-induced productions of pro-inflammatory cytokines are regulated by MAP kinases and G-protein but not by Akt: Relationship with phylogenetic groups and resistance patterns

Introduction

We investigated the role of PI3-K, MAP kinases, and heterotrimeric G proteins in inducing cytokines production in human whole blood cultures stimulated by viable Escherichia coli (E. coli) clinical strains.

Materials and methods

We used eight E. coli strains that belong to different phylogenetic groups and presented by different antibiotic resistance patterns. Whole blood from healthy volunteers was incubated at 37 °C for 150 min, with lipopolysaccharide (LPS) from E. coli O111:B4 or selected viable E. coli clinical strains, with or without SB202190 (p38 inhibitor), PD98059 (ERK inhibitor), PTX (pertussis toxin; heterotrimeric G proteins inhibitor), wortmaninn (PI3-K inhibitor). The TNF-α, IL-1β, IL-10 and IFN-γ concentrations were measured in culture supernatants (ELISA).

Results

IL-10 and IFN-γ were not detectable. Susceptible strains induced higher TNF-α and IL-1β productions than β-lactam resistant strains (p < 0.05), with no difference between phylogenetic groups. A transformed strain carrying a plasmid-mediated AmpC-β-lactamase gene (CMY-2) induced lower TNF-α and IL-1β production than the parent wild type strain (p < 0.05). SB202190 (p38 inhibitor) and PD98059 (ERK inhibitor) reduced TNF-α concentrations by, respectively, 80% (p < 0.05) and 50% (p < 0.05). Wortmaninn (PI3-K inhibitor) had no significant effect. PTX (heterotrimeric G proteins inhibitor) altered TNF-α production after viable bacteria stimulation (1.7-fold increase; p < 0.05) but not after LPS (TLR-4) stimulation. Regarding IL-1β, wortmaninn, SB202190 and PTX had no significant effect whereas PD98059 significantly decreased production in whole cell cultures (p < 0.05).

Conclusion

Susceptible strains induce greater TNF-α and IL-1β productions than resistant strains. ERK kinase plays a major role in viable E. coli strains inducing TNF-α and IL-1β production. E. coli exerts an effect on the pertussis toxin-sensitive G-protein through a TLR-4-independent mechanism.     

Caffeine- and amino acid-effects upon try(plus) revertant yield in U.V.-irradiated hcr(plus) and hcr (minus) mutants of E. coli B-r

Summary The influence of supplementation of the post-irradiation plating medium with caffeine and/or amino acids, upon both U.V.-induced killing and try⁺ reversion yield, has been studied in hcr⁺ and hcr^- derivatives of E. coli B/r tryptophan auxotroph WWP-2. All experiments have been carried out with stationary phase cells grown in aerated nutrient broth.In the hcr^- strain, caffeine causes no enhancement of either killing or try⁺ revertant yield. There is mutational enhancement by supplementation with a low level of tryptophan, and an even greater effect when supplementation is with tryptophan plus an additional pool of other amino acids.In the hcr⁺ strain, tryptophan and/or a pool of other amino acids cause an enhancement of try⁺ yield, as in the hcr^- strain. Caffeine causes lethal enhancement on several different media. There is an apparently straightforward dose enhancement by caffeine, of lethality and try⁺ revertant frequency, on media containing no, or only, tryptophan. On media containing, however, both a low level of tryptophan and an additional pool of other amino acids, caffeine causes a preferential enhancement of try⁺ revertant frequency over and above pure dose enhancement.These results suggest that U.V. may cause two types of lesion. One type may lead only to mutation, and is repaired by both the caffeine-insensitive MFD, and the caffeine-sensitive hcr, dark repair systems. This first type of lesion is protected from repair by the presence of a pool of amino acids. The second type of lesion is hypothesised to be capable of causing either lethality or mutation, is repaired only by the hcr dark-repair system, and is not subject to protection by an amino acid pool.This work was initiated at the Radiological Research Laboratories, Columbia University, New York.     

Across Genus Plasmid Transformation Between <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and the Effect of <Emphasis Type="Italic">Escherichia coli</Emphasis> on the Transforming Ability of Free Plasmid DNA

The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.     

Replication of the mini-R1 plasmid Rsc11 and Rsc11 hybrid plasmids

Summary The DNA polymerase induced by bacteriophage T7 is composed of a phage-specified subunit, the gene 5 protein, and a host-specified subunit, the 12,000 dalton thioredoxin of Escherichia coli. tsnC mutants of E. coli B (Chamberlin, 1974) have no detectable thioredoxin, and thus cannot support the growth of phage T7, although they are killed by phage infection. A mutant of E. coli K12 affecting thioredoxin has been isolated by a modification of the procedure used by Chamberlin (1974) to isolate tsnC mutants of E. coli B. The gene affecting thioredoxin has been designated trxA. This mutant, E. coli JM109, shows the TsnC phenotype in that it is killed by, but cannot support the growth of, bacteriophage T7. T7 DNA replication does not occur in mutantinfected cells. These phenotypic expressions of the tsnC mutation have enabled us to screen recombinants for the trxA allele in HfrxF^- crosses and F-ductants in episome transfer experiments. Extracts of transductants in generalized transduction by P1 phage were screened for their ability to complement partially purified phage T7 gene 5 protein to form T7 DNA polymerase. The trxA gene is located at 84 min on the E. coli linkage map, between uvrE and metE; trxA is 34% co-transducible with metE.