The use of EDTA or polymyxin with lysozyme for the recovery of intracellular products fromEscherichia. coli

Summary Escherichia coli cells taken from exponential and late stationary (or decline) phases of culture were very susceptible to lysis by EDTA/lysozyme. Log phase cells were most susceptible to lysis by polymyxin/lysozyme. Treatment ofE. coli with EDTA and lysozyme compared favourably with sonication as a method for release of intracellular protein. Concentration ranges for optimal lysis were 100–800 μg/ml for EDTA and 25–50 μg/ml for lysozyme.     

A highly efficient procedure for the quantitative formation of intact and viable lysozyme spheroplasts from escherichia coli

This paper describes a highly efficient procedure for the quantitative conversion of Escherichia coli cells to spheroplasts utilizing 100- to 1000-fold less lysozyme than in the most efficient procedures used to date. The resulting spheroplasts have intact outer and inner membranes and are fully viable on agar plates. The spheroplasting procedure is a refinement of earlier procedures and enables regulation of the translocation of minute amounts of lysozyme into the periplasmic space of E. coli cells, based on a Ca²⁺ pretreatment, an EDTA incubation, and a heat shock. About 1000 lysozyme molecules per cell are sufficient for complete spheroplast formation (>98%). Some of the characteristics of these spheroplasts prior to and after recovery are described. It is anticipated that such viable spheroplasts will be useful in the study of fusion of gram-negative cells and other membrane systems, in the introduction of DNA and proteins into refractory gram-negative cell, and in investigating envelope-related synthesis and assembly processes.     

Sterile Culture of Rotylenchulus reniformis on Tomato Root with Gellan Gum as a Supporting Medium

Rotylenchulus reniformis was repeatedly propagated in sterile excised tomato roots growing on modified White''s medium with gellan gum as the support. Gellan gum provided an optically clear support medium that could be liquified by adding 5 mM disodium ethylenediaminetetraacetate (EDTA) to facilitate nematode extraction. Liquefaction of the gellan-gum medium by EDTA allowed efficient recovery of eggs and vermiform stages of R. reniformis. Extraction efficiency was quantified with Radopholus similis as a test organism. The efficiency of extracting R. similis from the gellan gum did not vary with the concentrations of EDTA tested.     

Role of Lysozyme Inhibitors in the Virulence of Avian Pathogenic Escherichia coli

Lysozymes are key effectors of the animal innate immunity system that kill bacteria by hydrolyzing peptidoglycan, their major cell wall constituent. Recently, specific inhibitors of the three major lysozyme families occuring in the animal kingdom (c-, g- and i-type) have been discovered in Gram-negative bacteria, and it has been proposed that these may help bacteria to evade lysozyme mediated lysis during interaction with an animal host. Escherichia coli produces two inhibitors that are specific for c-type lysozyme (Ivy, Inhibitor of vertebrate lysozyme; MliC, membrane bound lysozyme inhibitor of c-type lysozyme), and one specific for g-type lysozyme (PliG, periplasmic lysozyme inhibitor of g-type lysozyme). Here, we investigated the role of these lysozyme inhibitors in virulence of Avian Pathogenic E. coli (APEC) using a serum resistance test and a subcutaneous chicken infection model. Knock-out of mliC caused a strong reduction in serum resistance and in in vivo virulence that could be fully restored by genetic complementation, whereas ivy and pliG could be knocked out without effect on serum resistance and virulence. This is the first in vivo evidence for the involvement of lysozyme inhibitors in bacterial virulence. Remarkably, the virulence of a ivy mliC double knock-out strain was restored to almost wild-type level, and this strain also had a substantial residual periplasmic lysozyme inhibitory activity that was higher than that of the single knock-out strains. This suggests the existence of an additional periplasmic lysozyme inhibitor in this strain, and indicates a regulatory interaction in the expression of the different inhibitors.     

Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl₂. Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.     

Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 μg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.     

How does lysozyme penetrate through the bacterial outer membrane?

Lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells. Lysozyme penetrates the outer membrane only when these cells are exposed to a mild osmotic shock in the presence of EDTA and lysozyme.In the presence of Mg²⁺, the outer membrane is stabilized sufficiently so that there is no lysozyme penetration during osmotic shock. If Mg²⁺ is added after an osmotic shock has been used to cause lysozyme to penetrate a destabilized outer membrane, the outer membrane is stabilized once again. In this case however, cells are converted to spheroplasts by the lysozyme which has gained access to the murein layer prior to the addition of Mg²⁺. Mg²⁺ stabilizes the outer membranes of these spheroplasts sufficiently so that they remain immune to lysis even in the absence of osmotic stabilizers such as sucrose.These results are discussed in terms of current information on the structure of the murein layer and the outer membrane.     

Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.     

Presence of growth factors for Methanobacterium ruminantium in both gram-positive and gram-negative bacteria

Autoclaved cells of gram-positive bacteria or mixed rumen organisms promote the growth of rumen strains of Methanobacterium ruminantium, but cells of E. coli were only stimulatory to growth after treatment with lysozyme plus EDTA or with EDTA alone.N-acetylglucosamine is identified as one of the growth factors for rumen strains of Mb. ruminantium.     

Assimilation of E. coli cells by Daphnia magna on the whole organism level

Consumption of E. coli cells by Daphnia magna was studied. It was found that this organism not only ingested E. coli cells but digested them as demonstrated by the release of ¹⁴CO₂ originating from E. coli grown on ¹⁴C-glucose, and by the transfer of the radioactive label from parental Daphnia to their progenies. In addition the effect of antibiotics on the consumption of E. coli cells by Daphnia magna was studied. In long incubation times, antibiotics inhibited bacterial uptake by Daphnia. The microflora isolated from Daphnia was found to be capable of causing leakage of enzymes out of E. coli cells thus playing at least a partial role in the digestion of E. coli cells by Daphnia.     

Lysis of lyophilizedEscherichia coli cells with egg-white lysozyme without ethylenediaminetetraacetic acid

A simple method of lysis of lyophilized cells ofEscherichia coli is described, using egg-white lysozyme in the absence of ethylenediaminetetraacetic acid (EDTA).     

Murein hydrolase (N-acetyl-muramyl-l-alanine amidase) in human serum

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and l-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetylmuramyl-l-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed.The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.     

Corrected Identification of a Test Organism (ATCC 4352) Previously Thought to be Escherichia coli

A gram-negative test organism (ATCC 4352) previously identified as Escherichia coli was found to be Klebsiella pneumoniae.     

Metal Ions,Not Metal-Catalyzed Oxidative Stress,Cause Clay Leachate Antibacterial Activity

Aqueous leachates prepared from natural antibacterial clays, arbitrarily designated CB-L, release metal ions into suspension, have a low pH (3.4–5), generate reactive oxygen species (ROS) and H₂O₂, and have a high oxidation-reduction potential. To isolate the role of pH in the antibacterial activity of CB clay mixtures, we exposed three different strains of Escherichia coli O157:H7 to 10% clay suspensions. The clay suspension completely killed acid-sensitive and acid-tolerant E. coli O157:H7 strains, whereas incubation in a low-pH buffer resulted in a minimal decrease in viability, demonstrating that low pH alone does not mediate antibacterial activity. The prevailing hypothesis is that metal ions participate in redox cycling and produce ROS, leading to oxidative damage to macromolecules and resulting in cellular death. However, E. coli cells showed no increase in DNA or protein oxidative lesions and a slight increase in lipid peroxidation following exposure to the antibacterial leachate. Further, supplementation with numerous ROS scavengers eliminated lipid peroxidation, but did not rescue the cells from CB-L-mediated killing. In contrast, supplementing CB-L with EDTA, a broad-spectrum metal chelator, reduced killing. Finally, CB-L was equally lethal to cells in an anoxic environment as compared to the aerobic environment. Thus, ROS were not required for lethal activity and did not contribute to toxicity of CB-L. We conclude that clay-mediated killing was not due to oxidative damage, but rather, was due to toxicity associated directly with released metal ions.     

Hydrophobicity of the cell surface and drug susceptibility of Escherichia coli cultivated at high pressures up to 30 MPa

Summary Escherichia coli was cultivated under hydrostatic pressures up to 30 MPa (300 bar) and then partitioned between an aqueous phase (physiological saline) and oil phase (n-hexadecane). The partition coefficients were used as measures of hydrophobicity of the surface of the cells and correlated with the susceptibility to an antimicrobial agent (dodecylpyridinium iodide). This agent is lethal to the cells and the effect of pressure on its concentration for a lethal effect on E. coli was determined. A good correlation was found between the hydrophobicity of the cells and their death rate on treatment with this reagent.     

Termination of Deoxyribonucleic Acid in Escherichia coli by 2′,3′-Dideoxyadenosine

2′,3′-Dideoxyadenosine was previously shown to be lethal to Escherichia coli and to inhibit deoxyribonucleic acid (DNA) synthesis irreversibly in this organism. It was also shown that triphosphate of this analogue terminates DNA chains in an in vitro system. Data presented here show that the nucleoside is relatively insensitive to E. coli adenosine deaminase and is converted intracellularly into the dideoxynucleotide, including the triphosphate. Thymine nucleotide pools were not reduced in inhibited bacteria, nor did preformed DNA break down. Some adenine was liberated from the dideoxyadenosine on incubation, and the latter was incorporated into ribonucleic acid. Nevertheless, about 4,000 molecules of the dideoxynucleoside were incorporated into DNA per cell. The dideoxynucleotide occurred in DNA chains in a terminal position, liberated selectively by venom phosphodiesterase. The possible nature of the lethal event is discussed.     

The effect of hyperbaric oxygenation on thein vitro growth ofEscherichia coli in environments with and without blood cells

The aim of this study was to investigate whether thein vitro presence of blood cells influences the anti-microbial activity of hyperbaric oxygen (HBO) againstEscherichia coli. FiftyE. coli isolates from clinical samples were used in the study. A small number of colonies belonging to each isolate from the nutrient media were transferred into two K₃EDTA tubes (the blood group) and two Mueller-Hinton broth tubes (the broth group). Then, both groups were divided into subgroups according to whether HBO was administered (HBO subgroup) or not (non-HBO subgroup). HBO treatment was applied for one hour at 2.5 absolute atmospheres. The tubes in the non-HBO subgroup were left at room temperature during this period. Subsequently, all the tubes were cultured on Mueller-Hinton and Eosin Methylene Blue agar using the quantitative counting technique. After 18 to 24 h incubation at 37 °C, the colonies formed in the plates were counted. In the blood group, compared with non-HBO subgroup samples, the number of colonies decreased in 56% of samples, increased in 32% of samples and did not change in 12% of samples in the HBO subgroup. Whereas, in the broth group the number of colonies decreased in only 32% of samples increased in 38% of samples and did not change in 30% of samples in the HBO subgroup compared with the non-HBO subgroup. The difference between the blood and the broth groups revealed a statistical significance using Pearson’s Chi-square test (P=0.025). We concluded that the antibacterial effect of HBO onE. coli increases in the cellular environment belonging to the host organism.     

Intercellular Transfer by Phage Receptor Site Lipopolysaccharide

TREATMENT of Escherichia coli cells with lysozyme and EDTA partially removes the outer layer of the cell wall containing lipopolysaccharide (LPS), leaving osmotically unstable spheroplasts¹. These can be infected with phage nucleic acid² and can produce viable phage particles. Removal of LPS-containing phage receptor sites^3–5, however, leaves spheroplasts resistant to infection by intact phages¹. We now show that LPS, obtained from phage-sensitive cells by aqueous phenol extraction, can provide functional phage receptor sites to spheroplasts prepared from cells lacking receptor sites.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

Differences in chromatin proteins of resting and growing human lymphocytes.

Two-dimensional polyacrylamide gel electrophoresis comparisons were made for the non-histone “Chromatin fraction II” proteins of normal, phytohemagglutinin-stimulated and acute leukemic lymphocytes. The “Chromatin fraction II” proteins were extracted from the nuclear residue fraction after initial treatment with (a) 0.075 M NaCl containing 0.025 M EDTA, pH 8; (b) 0.01 M Tris-HCl, pH 8; and (c) 0.4 N H₂SO₄. Most of the proteins found earlier in the “Chromatin fraction II” of rodent liver and hepatomas were also found in the human cells. Some changes such as the decrease in amount of protein BA of normal rodent cells were found in the comparison of normal and stimulated human cells. By comparison with normal lymphocytes, the phytohemagglutinin-treated cells had decreased spot densities and sizes for proteins BA and Bv and an increase in densities and sizes of proteins CB, C25, CS and CT. In the acute lymphocytic leukemic cells there was a decrease in spots A24, BA, Bv, CD and CD′ by comparison with the normal lymphocytes. Protein CG′ which was found earlier in the hepatomas was found in acute lymphocytic leukemic cells but not in the control or phytohemagglutinin-treated cells. These studies show that there is a loss in specific Chromatin proteins BA and Bv from the Chromatin of rapidly turning over cells. Concomitantly, increases are observed for the amounts of protein spots CB, C25, CS and CT in the actively growing cell samples.