Addition Of Lysozyme Research Articles

Lysis of Grouped and Ungrouped Streptococci by Lysozyme

Thirty strains of streptococci were tested for lysis with lysozyme, and 29 of these could be lysed by the following method: (i) suspension of the cells to a Klett reading of 200 units (no. 42 filter) in 0.01 m tris(hydroxymethyl)aminomethane buffer, pH 8.2, after washing twice with the buffer; (ii) addition of lysozyme to a final concentration of 250 mug/ml with incubation for 60 min at 37 C; (iii) addition of sodium lauryl sulfate (SLS) to a final concentration of 0.2% and incubation up to an additional 15 min at 37 C. Significant lysis was obtained only after the addition of SLS. (Strains of groups A, E, and G were treated with trypsin at a concentration of 200 mug/ml for 2 hr at 37 C before exposure to lysozyme.) These parameters for optimal lysis of streptococci by lysozyme were established by testing the group D Streptococcus faecalis strain 31 which lyses readily with lysozyme and the group H strain Challis which is less susceptible to the action of the enzyme. Viability of S. faecalis decreased 96% after 3 min of exposure to 250 mug of lysozyme per ml, whereas the more resistant strain Challis retained 27% of the initial viability after the same period. After 60 min, there was almost total loss of viability in each case. Variations of three methods of lysing streptococci with lysozyme were compared with respect to the decrease in turbidity and the release of protein and deoxyribonucleic acid (DNA) effected by each variation. The method presented in this paper allowed the greatest release of these cytoplasmic constituents from S. faecalis and strain Challis. Transformation experiments using DNA obtained from strain Challis (streptomycinresistant) by this method showed that the DNA released is biologically active.

Calcium phosphate formation in vitro: I. Factors affecting initial phase separation

The major events comprising the de novo formation of calcium phosphate salts from buffered solutions were: (1) the initial deposition of amorphous calcium phosphate; (2) its subsequent transformation into small crystals of apatite; and (3) the ultimate growth or ripening of those crystals. The degree of metastability for any given calcifying solution was shown to be an exact function of pH, ionic strength, temperature, sol Ca P molar mixing ratio, and initial Ca × P millimolar product. Initial mineral-phase formation lag time was also sensitive to changes in solution viscosity and dielectric constant. The presence of pyrophosphate, fluoride, magnesium, carbonate, collagen, or gelatin always enhanced amorphous calcium phosphate formation, while addition of lysozyme, casein, phosvitin, poly- l-lysine, or protein-polysaccharides enhanced initial mineral-phase formation only at relatively low initial Ca × P millimolar products. Citrate, poly- l-glutamate, and polyacrylate always inhibited amorphous calcium phosphate formation, whereas chondroitin sulfate and protein-polysaccharides were inhibitory only at relatively high initial Ca × P millimolar products.

Stability and comparative transport capacity of cells, mureinoplasts, and true protoplasts of a gram-negative bacterium.

The outer layers of the cell envelope of a pseudomonad of marine origin were removed by washing the cells in 0.5 m NaCl followed by suspension in 0.5 m sucrose. The term mureinoplast has been suggested for the rod-shaped forms which resulted from this treatment. As previously established, these forms lacked the outer cell wall layers but still retained a rigid peptidoglycan structure. Mureinoplasts remained stable if suspended in a balanced salt solution containing 0.3 m NaCl, 0.05 m MgSO(4), and 0.01 m KCl but, unlike whole cells, lost ultraviolet (UV)-absorbing material if suspended in 0.5 m NaCl or 0.05 m MgCl(2). Sucrose added to the balanced salt solution also enhanced the loss of UV-absorbing material. Addition of lysozyme to suspensions of mureinoplasts in the balanced salt solution produced spherical forms which, by electron microscopy and the analysis of residual cell wall material, appeared to be true protoplasts. Only undamaged mureinoplasts, as judged by their capacity to fully retain alpha-aminoisobutyric acid, were capable of being converted to protoplasts. Protoplasts and undamaged mureinoplasts retained 100% transport capacity when compared to an equal number of whole cells. The Na(+) requirement for transport of alpha-aminoisobutyric acid and the sparing action of Li(+) on this Na(+) requirement were the same for both protoplasts and whole cells. These observations indicate that, in this gram-negative bacterium, the cell wall does not participate in the transport process though it does stabilize the cytoplasmic membrane against changes in porosity produced by unbalanced salt solutions. The results also indicate that the requirements for Na(+) for transport and for the retention of intracellular solutes are manifested at the level of the cytoplasmic membrane.

Differential lytic response of enterococci associated with addition order of lysozyme and anions.

Suspensions of Streptococcus faecium, prepared by washing with and resuspending in water, were lysed slowly if sodium chloride was added prior to lysozyme; however, if brief incubation with lysozyme was followed by addition of sodium chloride, lysis was immediate and extensive. Relatively lysozyme-resistant strains of S. faecalis could be lysed readily by adding lysozyme first. The primary addition of lysozyme apparently resulted in a "sensitized" cell with a damaged wall, as evidenced by N-acetylhexosamine release. Anionic detergents could replace sodium chloride in lysing these sensitized cells. The difference in activity associated with the order of addition probably involved a competition for reactive sites on the cell surface.

Differential Leakage of Enzymes from Escherichia coli During Immune Bacteriolysis

Abstract Escherichia coli B and its mutants were converted to BAC′ cells by lysozyme-free antiserum and lysozyme-free complement. BAC′ cells had normal morphologic rod shape, and lost little β-d-galactosidase, an intracellular enzyme. With lysozyme BAC′ cells were converted to serum spheroplasts and lost β-d-galactosidase. The liberation of the enzyme was inhibited in hypertonic medium. In contrast, plakin, a phospholipase A from rabbit blood platelets, converted BAC′ cells to non-stainable rod-shaped ghosts and liberated β-d-galactosidase even in hypertonic sucrose medium. Alkaline phosphatase, a surface-bound enzyme, was released from bacteria during the formation of BAC′. The release was accelerated by addition of either lysozyme or plakin. It was also demonstrated that lysozyme could act on its substrate to form serum spheroplasts only after BAC′1a42a356789 had formed.

クックドソーセージに添加された卵白リゾチームの保存効果について

The effect of lysozyme in the presence or absence of NaCl and NaNO2 as a preservative agent for cooked sausage was studied. The addition of lysozyme at levels of 50 and 200ppm in combination with 3% NaCl and 12.5ppm NaNO2 plus heat was more effective in preserving sausage for four weeks at 10°C than additions of either lysozyme or a mixture of NaCl and NaNO2 plus heat. Omitting the effect of heat, lysozyme retarded microbial growth to greater extent than did the standard preservative mixture of NaCl and NaNO2.

The influence of lysozyme on the appearance of epiphyseal cartilage in organ culture.

1. A new method is described for preparing the osteocartilaginous junction of growing dog scapular cartilage free of periosteum. This preparation is suitable for organ culture. 2. Addition of lysozyme to organ cultures of such material results in an increase in opacity of the tissue. This “darkening” is induced in viable epiphyseal cartilage, but not in non-viable epiphyseal cartilage or in cartilage which does not ossify (nasal septum). 3. Protamine, another basic protein, also induces “darkening” which, in contrast to lysozyme, is persistent after withdrawal of the protein. 4. The “darkening” induced by lysozyme, but not protamine, spares the zone of hypertrophic cartilage cells, the zone where lysozyme is present in highest concentrationin vivo. 5. Histologically, both lysozyme and protamine induce changes in the staining reactions of the cartilage matrix. The distribution of these changes appears to be the same as that of the gross darkening effect. 6. Microradiography indicates that these changes are not a consequence of calcification. 7. These data support the concept that lysozyme may have a functional role in either osteogenesis, chondrogenesis, or calcification.

硫黄細菌の細胞表層の性質について

1. The sulfur oxidation by Thiobacillus thiooxidans was inhibited by long chain fatty acids at neutral and alkaline pH. The rate of inhibition seems to depend on their solubility in water.2. Although DNAase, RNAase, lipase, cellulase and proteases did not inhibit the sulfur oxidation, lysozyme and snake venom enzymes caused inhibition which was enhanced by NaOH treatment.The optical density of cell suspension did not decrease but increased with the addition of lysozyme. Various experiments designed for lysis of the intact cell with lysozyme was not successful. An interpretation of inhibitory action of lysozyme is that lysozyme, a basic protein, acts as a cementing substance to aggregate cells and prevents them from accessing onto solid sulfur.The mode of action of snake venom enzymes was discussed in relation to the inhibition of sulfur oxidation by long chain fatty acids which would be possibly liberated from the cell surface by the action of venom enzymes.3. Isolated cell wall of T. thiooxidans was rapidly disintegrated by the action of trypsin and after the digestion, fibrous structure in the residual material was observed electron-microscopically.

Electron microscopy of Staphylococcus aureus cell wall lysis.

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Muñoz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018-2024. 1966.-A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents.

ELECTRON MICROSCOPE STUDY OF THE RELATIONSHIP BETWEEN MESOSOME LOSS AND THE STABLE L STATE (OR PROTOPLAST STATE) IN BACILLUS SUBTILIS.

Ryter, Antoinette (Institut Pasteur, Paris, France), and Otto E. Landman. An electron microscope study of the relationship between mesosome loss and the stable L-state (or protoplast state) in Bacillus subtilis. J. Bacteriol. 88:457-467. 1964.-In a prior publication, it was postulated that inability of protoplasts to restart cell-wall synthesis and cell division and the inability of stable mass-conversion L forms to return to the bacillary state were both equivalent and both due to the interruption of a membrane-associated reaction sequence. It was further postulated that this reaction sequence might reside in the mesosome. In the present publication, it is shown by means of electron microscopy of thin sections that protoplasts and L forms do not contain mesosomes. The sequence of events leading to loss of the mesosomes during protoplasting is as follows. Soon after lysozyme addition, the mesosomes are extruded from the cell interior into the space between cell wall and cytoplasmic membrane. Mesosome fragments in the form of small vesicles gather at the poles of the cells and are released, along with intact protoplasts, when the wall fragments. (Sudden shift of bacilli to hypertonic environment also causes extrusion and fragmentation of mesosomes, but this damage is later repaired.) In intact bacilli, mesosomes are in contact with both the peripheral membrane and nuclear material. Upon extrusion of the mesosomes, a direct attachment between nuclear material and cytoplasmic membrane is observed. Deoxyribonucleic acid (DNA)-membrane attachment may play a role in the control of DNA replication. Bacillus subtilis L-colonies consist of irregularly shaped bodies of varying sizes, bounded only by a membrane. Many of the smaller bodies do not contain nuclear material, and many of the large ones appear inviable. Division is accomplished by a disorganized-appearing constriction process. There are no septa.

METHICILLIN-INDUCED LYSOZYME-SENSITIVE FORMS OF STAPHYLOCOCCI.

Aldrich, K. M. (The University of Kansas, Lawrence), and C. P. Sword. Methicillin-induced lysozyme-sensitive forms of staphylococci. J. Bacteriol. 87:690-695. 1964.-Staphylococcus aureus and S. epidermidis grown in the presence of sublethal amounts of methicillin were converted to enlarged spheres within 2 to 4 hr, as shown by phase microscopy, Gram stain, and electron microscopy. Addition of lysozyme to cells incubated in the presence of methicillin, and to methicillin-induced spheres suspended in hypotonic saline, caused lysis of methicillin-treated cells but not of untreated cells.

CORRELATION OF SPECIATION WITH LYTIC RESPONSES OF THE ACHROMOBACTER.

Surdy, Theodore E. (Purdue University, Lafayette, Ind.) and S. E. Hartsell. Correlation of speciation with lytic responses of the Achromobacter. J. Bacteriol. 85:1011-1016. 1963.-Lysozymic lysis of six species of Achromobacter was investigated. Three of the six species were lysed with 33, 50, or 100 mug/ml of lysozyme; if higher concentrations of lysozyme were used, precipitation of cells occurred. "Insensitive" cells could be sensitized by the addition of potassium hydroxide, n-butanol, steapsin, or urea, as demonstrated by the subsequent addition of lysozyme. Not all species were sensitive to these agents in the same degree; hence, a spectrum was obtained after the use of the pretreating agents and lysozyme. Optimal clearing of suspensions was observed when cells were suspended in pH 6.6 physiological saline or 0.15 m phosphate buffer and incubated at 45 C. Heat treatment (75 C for 10 min) or freezing (-32 C) and thawing (room temp, 25 C) for one cycle did not increase the sensitivity of the cells to lysozyme. Injury to the cells was evident by the increased amount of lysis noted after pretreatment with potassium hydroxide. When cells were frozen and thawed for three cycles, four of the six species were sensitive to the action of lysozyme. Isolated cell walls elicited a similar lytic pattern to that of whole cells. Individuality of the lytic response of the species (from most sensitive to least sensitive-A. aquamarinus, A. butyri, A. viscosus, A. parvulus, A. guttatus, A. hartlebii) produced a separation scheme. Exhaustive tests proved it to be stable and reliable for these species. The organisms were identified, with the use of the separation scheme, by a person initially unfamiliar with the scheme or the culture.

Formation of Bacterial Protoplasts by Serum Components

Summary Normal mammalian serum plus stabilizing agents effected conversion of the viable bacterial rods of Escherichia coli B and Salmonella typhosa Mrs. S., both rough organisms, into spherical protoplasts. A smooth organism, S. typhosa H901, was refractory, however, to the formation of protoplasts by normal serum, but addition of antiserum, complement, and lysozyme led to the formation of stabilized protoplasts of this organism. These protoplasts may conceivably play a role in the persistence of microbes in the animal body.

Lysozymempfindlichkeit der SpeziesB. megaterium

The 54 strains ofB. megaterium examined could be divided broadly into 2 groups on the basis of their lysozyme sensitivity. In most of the large-celled strains the cell walls were incompletely digested. Loss of turbidity on addition of lysozyme was comparatively slight and few free protoplasts were formed in the presence of saccharose. In contrast, the small-celled strains usually showed a marked drop in turbidity and complete protoplast formation could be obtained. These results suggest that, in some strains ofB. megaterium, particularly those possessing very large cells, certain substances are included in the cell walls which are not depolymerized by lysozyme.

The ultraviolet absorption spectra of desoxypentose nucleic acid

The intensity of the ultraviolet absorption of sodium desoxypentose nucleate (SDN) in aqueous solution is lowered by the addition of salts, such as alkali halides and phosphates. SDN absorbance in sodium chloride solutions has been carefully measured and appears to vary linearly with the log of the sodium chloride concentration over a 10 5 change in salt concentration. In the absence of salt, the ultraviolet absorption of SDN solutions show strong pH dependence between pH 3 and pH 11. Aqueous SDN at pH 6.7 in the absence of added salt has an absorptivity ( k) of 25.5 and A 1cm 1% of 258 at 259 mμ (when measurements are made on concentration less than 0.01%). These values are considerably higher than previously reported. Aqueous SDN solutions in the pH range 2 to 10 have an isosbestic point at 233 m/gm. Addition of lysozyme and bovine plasma albumin to SDN solutions also result in a decrease in the ultraviolet absorption due to SDN. On the other hand, the addition of non-ionic substances such as polyvinyl alcohol, sucrose, or glycerol to aqueous SDN solutions, even in high concentrations, does not decrease its ultraviolet absorption. An explanation of these data in terms of the association of the component purine and pyrimidine groups is suggested. These results indicate that caution must be exercised in using measurements of absorption intensities in the ultraviolet for the determination of the purity of nucleic acid preparations and for the determination of the nucleic acid content of tissues and cells unless the ionic strength, pH, and Beer's Law deviation are known.

The action of egg white lysozyme on ovomucoid and ovomucin

1. 1. No increase in reducing substances, decrease in viscosity or production of amino-sugars was observed when ovomucoid preparations were incubated with egg white lysozyme. 2. 2. Addition of lysozyme to swollen ovomucin resulted in the separation of a compact precipitate of mucin plus lysozyme. 3. 3. The data suggest that the thinning of the thick white gel which occurs during storage of eggs may, in part, be due to the action of lysozyme on mucin.