Incorporation of 5-Diazouracil-2-14C into Ribonucleic Acids of Escherichia coli During Division Inhibition

Radioactive diazouracil (DZU-2-(14)C) was rapidly incorporated into acid-insoluble material of Escherichia coli B. Isolated ribonucleic acid contained essentially all of the incorporated label, and this was solubilized by ribonucleases but not by deoxyribonuclease. A maximum of 45 to 50% of added label was incorporated at division-inhibitory and subinhibitory concentrations. Incorporation levels and filament-forming capacity were concomitantly depressed by preincubation of DZU in various medium components. The lower levels of incorporation brought about by preincubation were apparently related to an inherent instability or high reactivity of the DZU. The limit on incorporation of fresh DZU suggests the presence of geometrical isomers in the original DZU. The nature of these isomers and the reactivity of DZU are discussed in relation to its use in future cell division studies.     

Effects of the formaldehyde releasing preservatives dimethylol urea and diazolidinyl urea in several short-term genotoxicity tests

The two formaldehyde (FA)-releasers dimethylol urea (DMU) and diazolidinyl urea (DZU) are widely used as preservatives or additives. They were tested for genotoxicity in three short-term test systems, i.e. in the Salmonella typhimurium mutagenicity assay, in the in vitro micronucleus test with V79 Chinese hamster cells and in the in vitro tubulin assembly assay using isolated tubulin from pig brains. The polymerization products obtained in the tubulin assembly assay were examined additionally by electron microscopy.In the S. typhimurium mutagenicity assay with the pre-incubation assay both FA-releasers tested show a clear and concentration-dependent increase in the number of revertants in strains TA98, TA100 and TA102 with and without metabolic activation (rat liver S9 mix). In all cases, a biologically relevant increase in the number of revertants was achieved within the concentration range tested (DZU: 0.04-1.8 micromol per plate, DMU: 0.21-8.33 micromol per plate). FA was tested at 0.06-2.5 micromol per plate and lead to similar effects.Both compounds induce the formation of micronuclei (concentration range tested: DZU: 2.5-50 micromol/l, DMU: 3.3-333 micromol/l). However, DMU shows a comparatively weaker effect exclusively in the absence of the metabolizing enzymes. By contrast, DZU yields a distinct increase of the micronucleus rate in the absence and in the presence of S9. In addition, DZU predominantly causes an increase of large micronuclei, which suggests that this compound has a marked aneugenic potential. Cytotoxic effects accompany the clastogenic effects of both DMU and DZU.The examination of DMU and DZU in view of a possible aneugenic potential in the tubulin assembly assay yielded the following results: DMU at concentrations up to 10 mmol/l did not influence the formation of microtubuli, whereas DZU inhibited this process completely at 3 mmol/l. FA at 6 mmol/l completely inhibited the tubulin assembly. These results could clearly be confirmed by electron microscopy examination. The different potential of the two compounds with respect to the inhibition of tubulin formation is apparently due to a significant difference in the degree of FA release.According to these results, both compounds have to be considered as genotoxic in vitro. On account of these data and because of the widespread use of these two compounds in various products used in daily life, a reevaluation of the risk associated with these compounds seems to be necessary.     

Phospholipid Alterations During Growth of Escherichia coli

As cultures of Escherichia coli progressed from the exponential growth phase to the stationary growth phase, the phospholipid composition of the cell was altered. Unsaturated fatty acids were converted to cyclopropane fatty acids, and phosphatidyl glycerol appears to have been converted to cardiolipin. With dual isotope label experiments, the kinetics of synthesis of cyclopropane fatty acid for each of the phospholipids was examined in vivo. The amount of cyclopropane fatty acid per phospholipid molecule began to increase in phosphatidyl ethanolamine at a cell density below the density at which this increase was observed in phosphatidyl glycerol or cardiolipin. The rate of this increase in phosphatidyl glycerol or in cardiolipin was faster than the rate of increase in phosphatidyl ethanolamine. After a few hours of stationary-phase growth, all the phospholipids were equally rich in cyclopropane fatty acids. It is suggested that the phospholipid alterations observed are a mechanism to protect against phospholipid degradation during stationary phase growth. Cyclopropane fatty acid synthetase activity was assayed in cultures at various stages of growth. Cultures from all growth stages examined had the same specific activity in crude extracts.     

Regulation of the Synthesis of the Lactose Repressor

Measurements of the lactose repressor over a tenfold range of cell growth rates were made on protein extracts from Escherichia coli cultures grown in media with various carbon energy sources. The concentration of lactose repressor varied with the number of genome equivalents per cell over this range in growth rates, suggesting that the number of lactose molecules within the cell is determined by the number of I gene copies present. The timing of repressor synthesis during the cell division cycle and its correlation with deoxyribonucleic acid synthesis was examined by synchronizing the cell division cycle of E. coli ED1039, in which the Lac region has been transposed from 10 to 36 min on the genetic map. Measurements of lactose repressor in the synchronized culture revealed a burst of repressor synthesis at the time of I gene duplication. The concentration of lactose repressor was found to decrease as a function of total cell protein during the division cycle until an increase in synthesis occurred, suggesting that repressor synthesis probably does not occur throughout the division cycle. A model for I gene regulation is proposed.     

Isolation and Characterization of an Escherichia coli ruv Mutant Which Forms Nonseptate Filaments After Low Doses of Ultraviolet Light Irradiation

Two ultraviolet light (UV)-sensitive mutants have been isolated from Escherichia coli K-12. These mutants, designated RuvA(-) and RuvB(-), were controlled by a gene located close to the his gene on the chromosome map. They were sensitive to UV (10- to 20-fold increase) and slightly sensitive to gamma rays (3-fold increase). Host cell reactivation, UV reactivation and genetic recombination were normal in these mutants. Irradiation of the mutants with UV resulted in the production of single-strand breaks in deoxyribonucleic acid, which was repaired upon incubation in a growth medium. After UV irradiation, these mutants resumed deoxyribonucleic acid synthesis at a normal rate, as did the parent wild-type bacteria, and formed nonseptate, multinucleate filaments. From these results we concluded that the mutants have some defect in cell division after low doses of UV irradiation, similar to the lon(-) or fil(+) mutant of E. coli. The ruv locus was divided further into ruvA and ruvB with respect to nalidixic acid sensitivity and the effect of minimal agar or pantoyl lactone on survival of the UV-irradiated cell. The ruvB(-)mutant was more sensitive to nalidixic acid than were ruvA(-) and the parent strain. There was a great increase in the surviving fraction of the UV-irradiated ruvB(-) mutant when it was plated on minimal agar or L agar containing pantoyl lactone. No such increase in survival was observed in the ruvA(-) mutant.     

Effects of lemongrass oil on the morphological characteristics and peptidoglycan synthesis of Escherichia coli cells

The antibacterial effect of lemongrass oil, obtained from the aerial part of Cymbopogon citratus, on cells of Escherichia coli was investigated by electron microscopy and by measuring cell wall formation. Two strains of E. coli K-12 were used, one of which required diaminopimelic acid in the growth medium for its murein formation. Lemongrass oil was found to elicit morphological changes like filamentation, inhibition of septum formation, spheroplast formation, production of 'blisters', 'bulges' or mesosomes, as well as lysis and development of abnormally shaped cells. The incorporation of radioactively labelled diaminopimelic acid into the cell wall murein of strain W7, was inhibited by lemongrass oil in a dose dependent way. The sequence of changes induced by lemongrass oil on bacterial cell morphology and also its interference with murein synthesis in E. coli cells were interpreted to involve the penicillin binding proteins PBP 2 and PBP 3.     

加压CO2对大肠杆菌细胞膜的损伤作用

[目的]细菌细胞膜的损伤可以表现在细菌细胞内物质泄漏和细菌细胞吸收染料.与巴氏杀菌(63℃C、30 min)比较,研究加压CO2对大肠杆菌细胞膜的损伤作用,目的是分析出大肠杆菌死亡与细胞膜损伤的关系.[方法]检测大肠杆菌细胞膜通透性的改变情况,大肠杆菌内蛋白质和核酸的泄漏程度,并通过透射电镜观察大肠杆菌形态的改变情况.[结果]在研究范围内,加压CO2处理使大肠杆菌细胞膜通透性发生改变;加压CO2处理时虽然发生了胞内蛋白质泄漏,但发生泄漏的时间明显滞后于99％以上菌体死亡时间,因此并不是大肠杆菌死亡的原因,只是大肠杆菌死亡后的继发现象;大肠杆菌死亡与加压CO2处理导致的胞内核酸泄漏有关;大肠杆菌死亡与加压CO2处理导致的菌体形态改变有关.[结论]加压CO2对大肠杆菌细胞膜的损伤作用与菌体死亡有直接关系.     

Characterization of Labeled Residues from 5-Diazouracil-2-C Incorporated into Ribonucleic Acids of Filamentous Escherichia coli

E. coli B, filamented with 5-diazouracil (DZU)-2-¹⁴C, yielded ribonucleic acid (RNA)-(DZU-2-¹⁴C) which was converted by pancreatic ribonuclease to ¹⁴C-mono-and oligo-nucleotides. The mixed ¹⁴C-mononucleotides isolated by diethylaminoethyl-cellulose fractionation were identified as cytidylic, uridylic, and hydroxyuridylic acids, by using a combination of paper chromatography and treatment with alkaline phosphatase and cytidine deaminase. Rifampin blocked incorporation of DZU-2-¹⁴C under conditions which inhibit RNA synthesis. Division inhibition by DZU-2-¹⁴C and the incorporation into Escherichia coli B were retarded by uracil but not by other RNA bases. In a pyrimidine-requiring E. coli, DZU substituted for uracil or cytosine to an extent limited by toxic effects. Cytosine and uracil retarded these effects and retarded the incorporation of DZU-2-¹⁴C into the pyrimidineless strain. A small proportion of DZU-2-¹⁴C was converted by the latter strain into hydroxyuridylic acid, but the bulk of the incorporated label was in cytidylic and uridylic acid, as in the wild strain.     

Availability of glutamate and arginine during acid challenge determines cell density-dependent survival phenotype of Escherichia coli strains

The cell density-dependent acid sensitivity phenotypes of Escherichia coli strains K-12 and O157:H7 were examined with reference to three possible mechanisms of acid resistance. There was no evidence of any diffusible substance released from dead cells which could influence the cell density-dependent acid survival phenotype. Instead, cell density-dependent acid survival phenotype was associated with induction of glutamate- and arginine-decarboxylase acid survival pathways and concomitant availability of glutamate and arginine during acid challenge.     

Effects of concentrated supernatants recovered from Lactobacillus plantarum on Escherichia coli growth and on the viability of a human promyelocytic cell line

Aims:  The ability of concentrated supernatants from Lactobacillus plantarum to produce a disruption of plasma membrane in eukaryotic and prokaryotic cells has been examined.
Methods and Results:  A strain of Lact. plantarum (tolerant to acid and bile salts and resistant to several antibiotics) was used. It inhibited the growth of pathogenic Escherichia coli and L. monocytogenes . Supernatants from Lact. plantarum were concentrated by centrifugation. Either E. coli or HL-60 cells (a human promyelocytic cell line) were treated in the presence of the concentrated supernatants. The effect of concentrated supernatants from Lact. plantarum on E. coli growth demonstrated a bacteriostatic activity and a loss of cell viability measured by sytox green staining. Concentrated supernatants were capable of disturbing plasma membrane in E. coli and of promoting a cytotoxic and lyctic action on HL-60 cells and on human erythrocytes, respectively.
Conclusions:  These results suggest that Lact. plantarum release an effective compound responsible for an important effect in the disruption of E. coli plasma membrane and for a cytototoxic activity on promyelocytic leukaemia cells.
Significance and Impact of the Study:  This is the first in vitro study about the antimicrobial and biological activities of concentrated supernatants from Lact. plantarum .     

Effects of ethanol on the Escherichia coli plasma membrane.

The effects of ethanol on the fluidity of Escherichia coli plasma membranes were examined by using a variety of fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene, perylene, and a set of n-(9-anthroyloxy) fatty acids. The anthroyloxy fatty acid probes were used to examine the fluidity gradient across the width of the plasma membrane and artificial membranes prepared from lipid extracts of plasma membranes. Ethanol caused a small decrease in the polarization of probes primarily located near the membrane surface. In comparison, hexanol decreased the polarization of probes located more deeply in the membrane. Temperature had a large effect on probes located at all depths. The effects of ethanol on E. coli membranes from cells grown with or without ethanol were also examined. Plasma membranes isolated from cells grown in the presence of ethanol were more rigid than those from control cells. In contrast to plasma membranes, artificial membranes prepared from lipid extracts of ethanol-grown cells were more fluid than those from control cells. These differences are explained by analyses of membrane composition. Membranes from cells grown in the presence of ethanol are more rigid than those from control cells due to a decrease in the lipid-to-protein ratio. This change more than compensates for the fluidizing effect of ethanol and the ethanol-induced increase in membrane C18:1 fatty acid which occurs during growth. Our results suggest that the regulation of the lipid-to-protein ratio of the plasma membrane may be an important adaptive response of E. coli to growth in the presence of ethanol.     

Factors influencing potent antagonistic effects of Escherichia coli and Bacteroides ovatus on Staphylococcus aureus in anaerobic continuous flow cultures

We examined factors related to the potent antagonistic effect of Escherichia coli and Bacteroides ovatus on Staphylococcus aureus in anaerobic continuous flow cultures. In the presence of sugars fermentable by E. coli alone or both E. coli and S. aureus, motile E. coli strains exerted a potent antagonistic effect and S. aureus was expelled from the culture vessel within a few days. Conversely, in the presence of a sugar fermentable by S. aureus alone, the antagonistic effect of E. coli was diminished and S. aureus persisted at ca. 5 x 10(5) cfu/mL. B. ovatus alone exerted only a weak antagonistic effect on S. aureus in any culture conditions; however, when B. ovatus was cocultivated with E. coli and S. aureus, even in the presence of a sugar fermentable by S. aureus but not by E. coli, the potent antagonistic effect was restored. Escherichia coli showed the same level of antagonistic effect either in the presence of acetic acid (ca. 32 mM), propionic acid (4 mM), butyric acid (17 mM) and hydrogen sulfide (5 x 10(-1) mM) or when these metabolic products, except for a small amount of acetic acid (1.2 mM) were not present. In these culture conditions, S. aureus populations were lost at rates much higher than theoretical wash out rates of resting cells. These results indicate the presence of some bactericidal factors other than the volatile fatty acids and hydrogen sulfide. The bactericidal factors were not found in cultures of E. coli heated in boiling water for 10 min and in cell-free culture filtrates. Thus, the bactericidal factors seem to be associated with live E. coli cells. The nature of the bactericidal factors is not clear at present.     

Cell surface properties of organic solvent-tolerant mutants of Escherichia coli K-12.

In this study, we examined cell surface properties of mutants of Escherichia coli for which organic solvent tolerance levels were elevated. The cell surface of each mutant was less hydrophobic than that of the parent, probably due to an increase in lipopolysaccharide content. OmpF synthesis was repressed in the mutants. Organic solvent bound readily to viable E. coli cells in response to the polarity of the solvent. The mutants were bound less abundantly with the organic solvent than was the parent.     

Influence of amino acids on low-density Escherichia coli responses to nutrient downshifts

A vast bibliography on nutrient effects on high-density cultures exists, while it has been overlooked that low densities of starving cells are often the rule in natural environments. By means of a novel sensitive beta-galactosidase assay, we examined Escherichia coli transitions to minimal media when the cell concentration was 100 to 10,000 cells per ml. As in high-density cultures, the enzyme activity depended on amino acid availability and was subject to catabolite repression and stringent control. In all conditions tested, despite the presence of other nutrient sources, the relationship between beta-galactosidase activity and the l-amino acid pool was hyperbolic. The affinity constant when the amino acid pool was the only nutrient source averaged 14 muM after 90 min and increased up to 222 muM after 4.5 h. While investigating the transition from lag phase to exponential phase, we observed that the cells did not enter into starvation mode in the presence of amino acids, even when the nutrient amount was insufficient to support full survival. Based on these premises, the switch from starvation to hunger was investigated in relation to the amino acid pools. A critical range of concentrations at which E. coli linearly synthesized beta-galactosidase despite, at the same time, suffering a large decrease in cell viability was then recognized. Since both beta-galactosidase production and the dilution rate were reduced by more than half in the absence of leucine, we examined the contribution of leucine to cell recovery capabilities.     

Genetic analysis of the 5-azacytidine sensitivity of Escherichia coli K-12.

DNA containing 5-azacytidine (5-azaC) has been shown to form stable detergent-resistant complexes with cytosine methylases. We reasoned that if 5-azaC treatment causes protein-DNA cross-links in vivo, then mutations in DNA repair and recombination genes may increase the sensitivity of a cell to 5-azaC. We found that although recA (defective) and lexA (induction-negative) mutants of Escherichia coli were very sensitive to the drug, mutations in uvrA and ung genes had little effect on cell lethality. The sensitivity of recA strains to 5-azaC was dose dependent and was enhanced by the overproduction of a DNA cytosine methylase in the cell. Unexpectedly, a strain of E. coli carrying a recA mutation and a deletion of the DNA cytosine methylase gene (dcm) was found to be significantly sensitive to 5-azaC. Study of mutations in the pyrimidine salvage pathway of E. coli suggests that direct phosphorylation of 5-azaC, rather than phosphorylation of its degradation products, is largely responsible for the lethal effects of the drug. The addition of uracil to the growth medium has little effect on cell lethality of recA mutants, but it partially reversed the inhibition of cell growth caused by 5-azaC. This reversal of the bacteriostatic effects of the drug could not be achieved by adding cytosine or orotic acid to the growth medium and required the presence of functional UMP-pyrophosphorylase (gene upp) in the cell.     

The inhibition of phospholipid synthesis in escherichia coli by phenethyl alcohol.

The kinetics of lipid metabolism during phenethyl alcohol treatment of Escherichia coli were examined. Phenethyl alcohol at a non-bacteriostatic concentration reduces the accumulation of [32-P] phosphate into phospholipids and alters the phospholipid composition of the cell membrane. The changes in phospholipid composition are a result of the inhibitory effect of phenethyl alcohol on the rates of synthesis of the individual phospholipids. The inhibition in the rate of phosphatidylethanolamine synthesis by phenethyl alcohol was twice the inhibition in the rate of phosphatidyglycerol synthesis. The de novo rate of cardiolipin synthesis was only slightly inhibited. However, net cardiolipin accumulation increased during phenethyl alcohol treatment due to a more rapid turnover of phosphatidylglycerol to cardiolipin. Phenethyl alcohol also altered the fatty acid composition of the cell as a result of its inhibitory effect on the rate of individual fatty acid synthesis. However, the inhibition of phospholipid synthesis was not reversed by fatty acid supplementation of phenethyl alcohol treated cells. This result indicates that phenethyl alcohol does not inhibit phospholipid synthesis solely at the level of fatty acid synthesis.     

Influence of Thymine Starvation on the Integrity of Episomal and Chromosomal Deoxyribonucleic Acids in Escherichia coli CR34 (λ ind−)

The effect of thymine deprivation on the integrity of phage lambda, sex factor, and chromosomal deoxyribonucleic acid (DNA) in Escherichia coli CR34 (lambda ind(-)) was examined by sedimenting cell lysates through alkaline sucrose gradients. Both sex factor and chromosomal DNAs showed evidence of being degraded during the starvation period. In contrast, no loss of closed circular lambda DNA was observed.     

Increase in negative supercoiling of plasmid DNA in Escherichia coli exposed to cold shock

Negative supercoiling of plasmid DNA in Escherichia coli cells can decrease transiently when exposed to heat shock. The effect of cold shock on DNA supercoiling was examined, and analysis by agarose gel electrophoresis in the presence of chloroquine revealed that negative supercoiling of plasmid DNA in cells increased when cells were exposed to cold shock. This increase was transient and was nil when the cells were pretreated with nalidixic acid, an inhibitor of DNA gyrase. In a mutant deficient in expression of HU protein, the increase in negative supercoiling of DNA by cold shock is less apparent than in wild-type cells. It is proposed that DNA gyrase and HU protein have a role in the DNA supercoiling reaction seen with cold shock.     

Modeling the effects of sodium chloride, acetic acid, and intracellular pH on survival of Escherichia coli O157:H7

Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primary antimicrobial compounds in these products are acetic acid and NaCl, which can alter the intracellular physiology of E. coli O157:H7, leading to cell death. For combinations of acetic acid and NaCl at pH 3.2 (a pH value typical for non-heat-processed acidified vegetables), survival curves were described by using a Weibull model. The data revealed a protective effect of NaCl concentration on cell survival for selected acetic acid concentrations. The intracellular pH of an E. coli O157:H7 strain exposed to acetic acid concentrations of up to 40 mM and NaCl concentrations between 2 and 4% was determined. A reduction in the intracellular pH was observed for increasing acetic acid concentrations with an external pH of 3.2. Comparing intracellular pH with Weibull model predictions showed that decreases in intracellular pH were significantly correlated with the corresponding times required to achieve a 5-log reduction in the number of bacteria.     

Engineering of a novel biochemical pathway for the biosynthesis of L-2-aminobutyric acid in Escherichia coli K12

L-2-Aminobutyric acid was synthesised in a transamination reaction from L-threonine and L-aspartic acid as substrates in a whole cell biotransformation using recombinant Escherichia coli K12. The cells contained the cloned genes tyrB, ilvA and alsS which respectively encode tyrosine aminotransferase of E. coli, threonine deaminase of E. coli and alpha-acetolactate synthase of B. subtilis 168. The 2-aminobutyric acid was produced by the action of the aminotransferase on 2-ketobutyrate and L-aspartate. The 2-ketobutyrate is generated in situ from L-threonine by the action of the deaminase, and the pyruvate by-product is eliminated by the acetolactate synthase. The concerted action of the three enzymes offers significant yield and purity advantages over the process using the transaminase alone with an eight to tenfold increase in the ratio of product to the major impurity.