Staphylococcal virolysin,a phage-induced lysin; its differentiation from the autolysis of normal cells

Virolysin is a lysin which appears in Staphylococcus aureus K₁ cells infected with phage P₁₄; together with phage, virolysin is released from phage-infected cells at the time of lysis. Autolysin is a lysin formed by uninfected cells of the K₁ strain; autolysin is released from uninfected cells by autolysis. They show the following similarities: Both agents act within the genus Micrococcus. They lyse cells only after the cell has been subjected to a damaging or "sensitizing" treatment, such as heat, bacteriophage, acetone, or ultraviolet irradiation. The course of lysis of heated cells by both lysins has been found to proceed in a similar manner. A constant percentage of cells is lysed, independent of the concentration of lysin; the residual cells remain resistant to either lysin. Lysis proceeds logarithmically with time, and the velocity constants K are proportional to the lysin concentration. K increases with increasing temperature. Both lysins are unaffected by antiserum to the phage. They are inhibited alike by a number of chemicals, including known enzyme inhibitors. Both agents are destroyed by proteolytic enzymes and are precipitated by 40 per cent saturation with (NH₄)₂SO₄. Both lysins are very thermolabile. The two lysins differ with respect to their pH optimum, antigenic relationship and specificity for Micrococcus lysodeikticus. These results suggest that (1) both lysins have many properties associated with enzymes, (2) the lysis of heated cells, which they produce, has some of the characteristics of a chemical reaction, (3) the lysin from the phage-infected cell is clearly different from the lysin of the uninfected cell.     

Interaction of Bacteriophage Infection and Low Penicillin Concentrations on the Performance of Yogurt Cultures

Bacteriophage infection of a mixed-strain Streptococcus thermophilus culture, one strain of which is phage sensitive and the other phage resistant, may induce lysis of both strains. Experiments were carried out with three different phage-resistant strains. One such strain lysed in penicillin-free growth medium and another needed penicillin G (0.005 IU/ml) for lysis, while the third strain continued to grow in the presence of this concentration of antibiotic. Growth of the latter strain was inhibited when the medium contained a relatively high concentration of phage lysin. The different penicillin concentrations required to induce “lysis from without” of these phage-resistant strains correlated with their individual sensitivities to the antibiotic. The apparent relationship between the sensitivities of these strains to penicillin and to phage lysin could be explained by a difference in the degree of polymerization of the cell wall peptidoglycan.     

Export of the pneumococcal phage SV1 lysin requires choline‐containing teichoic acids and is holin‐independent

Streptococcus pneumoniae bacteriophages (phages) rely on a holin–lysin system to accomplish host lysis. Due to the lack of lysin export signals, it is assumed that holin disruption of the cytoplasmic membrane allows endolysin access to the peptidoglycan. We investigated the lysis mechanism of pneumococcal phage SV1, by using lysogens without holin activity. Upon phage induction in a holin deficient background, phage lysin was gradually targeted to the cell wall, in spite of lacking any obvious signal sequence. Our data indicate that export of the phage lysin requires the presence of choline in the teichoic acids, an unusual characteristic of pneumococci. At the bacterial surface, the exolysin remains bound to choline residues without inducing lysis, but is readily activated by the collapse of the membrane potential. Additionally, the activation of the major autolysin LytA, which also participates in phage‐mediated lysis, is equally related to perturbations of the membrane proton motive force. These results indicate that collapse of the membrane potential by holins is sufficient to trigger bacterial lysis. We found that the lysin of phage SV1 reaches the peptidoglycan through a novel holin‐independent pathway and propose that the same mechanism could be used by other pneumococcal phages.     

Mechanism of phage-induced lysis in pneumococci

Earlier studies have suggested the possible role of host autolytic enzyme in the release of progeny phage from Dp-1 infected pneumococci. Several new experiments described here reinforce this notion. Specifically, the resistance of an autolysis-defective mutant to infection at low phage to cell ratios could be eliminated by prior 'coating' of the host bacteria with pneumococcal autolysin isolated from wild-type cells. Similar, productive infection was also possible by lowering the temperature of incubation to 30 degrees C, a condition that leads to a partial activation of the thermosensitive residual autolysin in the mutant cells. Other experiments, however, clearly indicate the role of the newly discovered phage-associated lysin (PAL), reported in the accompanying communication, in bacteriophage release and culture lysis; specifically, lysis was stimulated by reducing agents and inhibited by cardiolipin. It seems that both the host-related and the PAL activities are involved with Dp-1 induced lysis of pneumococci.     

Mutations in coliphage p1 affecting host cell lysis

A total of 103 amber mutants of coliphage P1 were tested for lysis of nonpermissive cells. Of these, 83 caused cell lysis at the normal lysis time and have defects in particle morphogenesis. Five amber mutants, with mutations in the same gene (gene 2), caused premature lysis and may have a defect in a lysis regulator. Fifteen amber mutants were unable to cause cell lysis. Artificially lysed cells infected with five of these mutants produced viable phage particles, and phage particles were seen in thin sections of unlysed, infected cells. However, phage production by these mutants was not continued after the normal lysis time. We conclude that the defect of these five mutants is in a lysis function. The five mutations were found to be in the same gene (designated gene 17). The remaining 10 amber mutants, whose mutations were found to be in the same gene (gene 10), were also unable to cause cell lysis. They differed from those in gene 17 in that no viable phage particles were produced from artificially lysed cells, and no phage particles were seen in thin sections of unlysed, infected cells. We conclude that the gene 10 mutants cannot synthesize late proteins, and it is possible that gene 10 may code for a regulator of late gene expression for P1.     

The effect of dexamethoxin on the integrity of cytoplasmic membrane in gram-positive and gram-negative microorganisms

Decamethoxin is shown to be able to increase membrane permeability of Pseudomonas aeruginosa, Escherichia coli and Micrococcus lysodeikticus, that is confirmed by a loss of compounds with the absorption maximum at 260 nm by cells. Parallel with this the number of viable individuals has fallen and activity of dehydrogenases has been inhibited. The aspartate and alanine aminotransferase activity was not inhibited by decamethoxin and even increased. Decamethoxin lysed the protoplasts of the tested microorganisms. At high decamethoxin concentrations (over 500 micrograms/ml for P. aeruginosa and over 200 mu/ml--for E. coli) the outflow of components from the cells of gram-negative bacteria ceased, that may be associated with the coagulation changes in the cytoplasm. A loss of the low-molecular components by M. lysodeikticus cells and lysis of protoplasts proceeded less intensely than the same processes in the gram-negative microorganisms, that is explained by a less resistance of M. lysodeikticus to decamethoxin and earlier coagulation of the cytoplasm preventing lysis.     

Identification of a lysin associated with a bacteriophage (A25) virulent for group A streptococci.

A phage-associated lysin was found in culture lysates resulting from the propagation of virulent bacteriophage A25 on the group A streptococcal strain designated K56. In contrast to the previously described group C streptococcal phage-associated lysins, A25 phage-associated lysin was more active on chloroform-treated cells, was not phage bound, and was active on some group G and H strains, as well as on group A and C strains. A25 phage-associated lysin had an optimum pH of 6.7 and was inactivated by 10(-3) M p-hydroxymercuribenzoate. Group A cells exposed to penicillin were more susceptible to A25 phage-associated lysin, whereas chloramphenicol-treated cells became resistant to lysis. Release of lipoteichoic acid appeared to precede lysis, and cardiolipin treatment of cells reversed the effects of chloroform and penicillin treatments. These results suggest the possibility that A25 phage-associated lysin may have a mechanism similar to the mechanism of an autolysin or that cell lysis may be due to the activation of an autolysin.     

Lysis from without of S. aureus K1 by the combined action of phage and virolysin

Lysis from without (LFW) occurs in two steps: (1) sensitization of cells by phage, which renders the cells susceptible to (2) destruction of an essential cell structure by an extracellular lytic enzyme. Virolysin, from phage-infected cells, was used in these studies. Normal cell autolysin is also effective. Evidence is presented that: 1. Neither phage nor lysin alone causes LFW. 2. Sensitization requires phage adsorption. 3. It can be caused by non-infectious particles. This establishes a new biological activity of the particle. 4. Heat, U.V., detergents, penicillin, and other damaging agents also sensitize cells. 5. Sensitization involves a non-lethal, reversible reaction. 6. Sensitization by phage prevents virus synthesis. Following adsorption, a cell can undergo sensitization or infection but not simultaneously. When only a few particles are adsorbed, infection can occur; when sufficient particles are adsorbed, sensitization takes place. 7. Quantitative aspects of LFW are described. Lysis proceeds logarithmically. The lysis end-point depends upon the phage concentration but is independent of the enzyme concentration.     

Purification and characterization of a Streptomyces albus endo-N-acetylmuramidase lytic for group A and other beta haemolytic streptococci.

The purification and characterization of the streptolytic exo-enzyme from the Maxted-McCarty strain of Streptomyces albus is described. This enzyme was shown to be an endo-N-acetylmuramidase with a molecular weight of 10 to 12,000 and optimal activity at pH 8 and 45 degrees C. The enzyme is lytic for streptococci of various groups, Micrococcus lysodeikticus, Staphylococcus aureus, as well as Escherichia coli. It closely resembles the F1 endo-N-acetylmuramidase described by Ghuysen et al. (1966) except for small differences in the products of lysis of streptococcal cell walls and the resistance of Escherichia coli to lysis by the F1 enzyme. Lysates of group A and A variant streptococcal cell walls prepared with purified Streptomyces albus muramidase contained serologically active M protein and C carbohydrate-peptidoglycan complexes. The chemical and immunological characteristics of these enzymmatic products of streptococcal cell walls are reported and their utility as immunologic reagents is described.     

Enhanced lysis of herpes simplex virus type 1-infected mouse cell lines by NC and NK effectors

Spontaneously cytotoxic murine lymphocytes lysed certain cell types infected by herpes simplex virus type 1 (HSV-1) better than uninfected cells. The levels of virus-directed lysis varied widely from target to target, and we found that differences in virus-directed lytic efficiency could be attributed both to the characteristics of HSV-1 replication in the different targets and to the subgroup of natural effector cells which mediated lysis. Although HSV-1 adsorbed to the surface of all the target cells, those in which the virus replicated more efficiently were lysed to a greater extent. As targets, we used cell lines that, when uninfected, were spontaneously lysed by NK cells (YAC-1) or by NC cells (WEHI-164). We also used a fibroblastoid cell line (M50) and a monocytic tumor line (PU51R), which were not spontaneously killed. Using complement-mediated elimination of Qa-5-positive or asialo-GM1-positive NK cells to distinguish NK from NC activity, we found that NK cells lysed HSV-1-infected YAC cells better than uninfected cells, and an NC-like activity selectively lysed HSV-1-infected WEHI cells. In addition, we showed that both NK and NC cytotoxicities contributed to the lysis against the HSV-1-infected fibroblastoid line, M50, but the infected PU51R cells were killed by only NK effectors. These findings were consistent with the results of experiments performed to define the role of interferon in induction of virus-augmented cytolysis. Increased lysis of YAC-HSV and PU51R-HSV was entirely due to interferon activation and was completely abolished by performing the 51Cr-release assay in the presence of anti-interferon serum. Because NC activity was not augmented by interferon, virus-enhanced NC lysis of M50-HSV and WEHI-HSV was not due to this nonspecific mechanism. Together, our data show that HSV-1 infection of NK/NC targets induces increased cytotoxicity, but the effector cell responsible for lysis is determined by the uninfected target, or by an interaction between the virus and target cell, rather than by a viral determinant alone.     

Factors affecting the hemolytic action of "lysolecithin" upon rabbit erythrocytes

1. Lysolipid was prepared by the action of snake venom on egg yolk, and a study was made of the factors affecting its hemolytic action upon rabbit erythrocytes. 2. Lysis proceeded very rapidly at first, then ceased within a few minutes at room temperature. A given amount of lysin appeared to hemolyze a fixed number of cells, under specified conditions. 3. The more dilute erythrocyte suspensions required relatively more lysin per cell, for 50 per cent hemolysis of the suspension. There may be an equilibrium between the lysin dissolved in the medium and that adsorbed on the cells. 4. The degree of hemolysis for varying lysin concentrations was measured, and the cells showed a typical distribution of resistance to hemolysis. 5. As the temperature was lowered lysis was more extensive. Adsorption of the lysin on the cell surface was apparently increased. 6. The resistance of the erythrocytes to lysis increased slightly as the pH was raised from 5.5 to 7.8. 7. Resistance to lysis was independent of the tonicity of the medium and of initial cell volume. The magnitude of the cell surface was probably the determining factor. 8. A marked shrinkage of the erythrocytes was observed in the presence of calcium ions and lysin, but not in the absence of the lysin. 9. Hemolytic resistance curves obtained by the Wilbrandt technique were of the "colloid-osmotic" type. However, there was no evidence of prolytic loss of potassium ions. 10. Hypotonic fragility of the cells was slightly increased in the presence of the lysin. The rate of penetration of thiourea was greatly increased.     

Purification and characterization of lysozyme produced by Streptomyces erythraeus.

A species of lysozyme (SE lysozyme) was purified from culture filtrate of Streptomyces erythraeus. The enzyme has a molecular weight of 18,500 as determined by ultracentrifugation. Its isoelectric point is 9.5, and it shows optimal activity at pH 4.0 with an optimal ionic strength of 0.1. Investigation of the substrate specificity showed SE lysozyme to be an N-acetyl-muramidase. The simplest product in the digest of cell walls of Micrococcus lysodeikticus was identified as a disaccharide, [GlcNAcbeta(1 leads to 4) MurNAc]. While S. aureus as well as M. lysodeikticus was lysed by this lysozyme, chitin and its derivatives were not.     

Staphylococcal Bacteriophage-Associated Lysin: a Lytic Agent Active Against Staphylococcus aureus

A lytic enzyme active against viable, intact staphylococci is released into culture fluids upon lysis of bacteriophage-infected Staphylococcus aureus PS53 cells. This enzyme, staphylococcal phage-associated lysin (PAL), was partially purified by ammonium sulfate precipitation and gel filtration through Sephadex G-200. PAL is optimally active at pH 6.5 and 30 C, and lytic activity is greatly enhanced by the addition of reducing agents. Lytic activity was observed against all strains of staphylococci tested and against purified staphylococcal cell walls, but no activity was noted against other bacterial species. PAL possesses peptidase activity and results in the production of spheroplasts which can be osmotically stabilized for extended periods by the addition of 7.5% polyethylene glycol 4000.     

Characteristics of Bacteriophage N1 and Its Attachment to Cells of Micrococcus lysodeikticus

Bacteriophage N1 was purified by differential and equilibrium gradient centrifugation and characterized with respect to bouyant density in CsCl, one-step growth properties, host range, and morphology by electron microscopy. In a tris (hydroxymethyl) aminomethane-magnesium buffer (pH 7.15), the irreversible adsorption of N1 to cells of Micrococcus lysodeikticus strain 1 (ML-1) followed first-order reaction kinetics with an adsorption-velocity constant of 1.6 x 10(-9)/min at 32 C. The rate of phage attachment was not significantly altered when adsorption mixtures contained 0.01 m KCN or 1% casein hydrolysate, 0.01 m CaCl(2), and 0.001 m tryptophan. The activation energy for the irreversible adsorption reaction was 8.6 kcal. Treatment of ML-1 cells by any of the following procedures reduced the irreversible phage receptor activity over 90%: (i) mechanical disruption, (ii) lysozyme digestion, (iii) incubation in 1% cetyltrimethylammonium bromide, or (iv) incubation of heated cells (100 C, 15 min) with trypsin, Pronase, or lysozyme. The sensitivity of the phage receptor activity of ML-1 cells to lysozyme suggests that the bacterial cell wall is involved in the receptor site for the virus. Destruction of receptor activity by the other treatments cited above implies that, in addition to the cell wall, other cellular components may participate in the irreversible attachment of N1 phage to cells.     

An assay system for factors involved in mammalian DNA replication

An assay for cellular factors stimulating DNA synthesis by partially lysed CHO cells is presented. The assay is based on the observation that in highly lysed cells, DNA synthesis, as determined by [3H]dTTP incorporation, was only 2-5% of that in gently lysed cells, and that this low level of DNA synthesis could be increased by a factor of approx. 50 by the addition of CHO cell extract (i.e. supernatant of a cell homogenate subjected to high-speed centrifugation). Highly lysed cells were obtained by treatment with 0.1% Brij-58 and 240 mM KCl, while for the preparation of gently lysed cells, 0.01% Brij-58 and 80 mM KCl were used. Incorporation of [3H]dTTP reflected DNA synthesis qualitatively similar to that in intact cells. It was semiconservative, and no repair synthesis was detected unless cells were irradiated with ultraviolet light prior to parital lysis. DNA molecules of 4 S were synthesized and converted to DNA of more than 25 S via 6-12-S intermediates. DNA synthesis was restricted to nuclei from cells in S phase, and cell extract did not induce DNA synthesis in nuclei from cells in G1 phase. Stimulation of DNA synthesis by cell extract was concentration-dependent. Cell extract activity was recovered to more than 50% after (NH4)2SO4 precipitation. Heat-inactivation experiments suggested that cell extract contained at least tow factors timulating DNA replication. This system may, therefore, be used for the purification and characterization of factors participating in DNA replication of mammalian cells.     

Purified Recombinant Phage Lysin LySMP: An Extensive Spectrum of Lytic Activity for Swine Streptococci

Bacteriophage lysin has attracted considerable attentions as possible antimicrobial agents for solution of antibiotic resistance. SMP was a Streptococcus suis serotype 2 bacteriophage isolated from nasal swabs of healthy Bama minipigs. The putative SMP bacteriophage lysin, designated LySMP, was recombinantly expressed in Escherichia coli BL21, and chromatographically purified. Treated with 0.8% of β-mercaptoethanol, LySMP exhibited an extensive lysin spectrum than those of whole phage against bacteria investigated. S. suis serotype 2, S. suis serotype 7 and S. suis serotype 9 strains were recovered from diseased pigs between 1998 and 2005 in China. Fifteen of seventeen strains of S. suis serotype 2 could be lysed, as well as S. suis serotype 7 and 9, Streptococcus equi ssp. zooepidemicus and Staphylococcus aureus. But E. coli and Salmonella enterica were not affected. Purified LySMP showed high degrading efficiency against PMSF or lysozyme treated cells comparing to PBS washed cells. Optimum pH and temperature conditions for the lysin were investigated by turbidity reduction assay. The lysin exerted efficient lysis activity at 37°C, pH 5.2. The turbidity of bacterium investigated was observed to decrease by 1.2–68% in 30 min. Result indicated that putative LySMP could be a candidate antimicrobial agent in controlling S. suis infection.     

Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin.

LL-H, a virulent phage of Lactobacillus delbrueckii subsp. lactis, produces a peptidoglycan-degrading enzyme, Mur, that is effective on L. delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, and Pediococcus damnosus cell walls. In this study, the LL-H gene mur was cloned into Escherichia coli, its nucleotide sequence was determined, and the enzyme produced in E. coli was purified and biochemically characterized. Mur was purified 112-fold by means of ammonium sulfate precipitation and cation-exchange chromatography. The cell wall-hydrolyzing activity was found to be associated with a 34-kDa protein. The C-terminal domain of Mur is not essential for catalytic activity since it can be removed without destroying the lytic activity. The N-terminal sequence of the purified lysin was identical to that deduced from the nucleotide sequence, but the first methionine is absent from the mature protein. The N-terminal part of this 297-amino-acid protein had homology with several Chalaropsis-type lysozymes. Reduction of purified and Mur-digested L. delbrueckii cell wall material with labeled NaB3H4 indicated that the enzyme is a muramidase. The temperature optimum of purified Mur is between 30 and 40 degrees C, and the pH optimum is around 5.0. The LL-H lysin Mur is stable at temperatures below 60 degrees C.     

The N-terminal region of the Oenococcus oeni bacteriophage fOg44 lysin behaves as a bona fide signal peptide in Escherichia coli and as a cis-inhibitory element, preventing lytic activity on oenococcal cells

The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed in E. coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O. oeni-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known O. oeni and Lactococcus lactis phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.     

Isolation and characterization of three autolytic enzymes associated with sporulation of Bacillus thuringiensis var. thuringiensis

Cells of Bacillus thuringiensis containing refractile spores autolyzed readily when suspended in buffer. The autolysate contained enzymes which lysed vegetative cell walls of the organism. Three enzymes were isolated from the autolysate, and each was purified approximately 30-fold. One enzyme, most active near pH 4.0, was found to be an N-acetylmuramidase. The other two enzymes exhibited pH optima at 8.5. One was stimulated by cobalt ions and the other was not. The cobalt-stimulated enzyme was shown to be an N-acetylmuramyl-l-alanine amidase. The cobalt insensitive enzyme exhibited both N-acetylmuramyl-l-alanine amidase and endopeptidase activity. The amidase activity may reflect incomplete separation of the cobalt-stimulated enzyme. The endopeptidase cleaved the peptide bond between l-alanine d-glutamic acid. A cell wall lytic endopeptidase with this specificity has not been previously reported. All three enzymes were extremely limited in the range of bacterial cell walls which they attacked. Except for cell walls of Micrococcus lysodeikticus, which were lysed by the muramidase, only cell walls of members of the genus Bacillus were attacked.