Effects of Detergents on Lysis of Micrococcus lysodeikticus by Lysozyme

The effects of detergents on the lysozyme-catalyzed hydrolysis of Micrococcus lysodeikticus cells were investigated by changing the concentration of Na-phosphate buffer and pH in the presence or absence of sucrose. Also, a parallel study of the hydrolysis of glycolchitin by lysozyme was conducted and compared to the lytic reaction. Electron microscopy was utilized to follow the changes in cell morphology during the various treatments.

None of the detergents changed turbidity of the cell suspension. However, they did affect the change in turbidity during lysis in unique ways. SDS, which is an anionic detergent, inhibited lysozyme activity and its addition to the reaction mixture caused a rapid and large decrease in the turbidity. Brij 35 and Triton X-100, which are non-ionic detergents, did not inhibit lysozyme activity, but their presence in the reaction mixture changed the rate of turbidity change. Apparently non-ionic detergents disrupt only the protoplast, while anionic detergents disrupt both the protoplast and the damaged cell. The lytic mechanism of M. lysodeikticus by lysozyme was discussed in detail.     

Serratia marcescens-Lytic Enzyme Produced by Micromonospora sp. Strain No. 152

A strain of Micromonospora sp. producing a lytic enzyme toward Serratia marcescens was isolated from soil. The lytic enzyme, called 152-enzyme, was purified from the culture filtrate by salting-out with ammonium sulfate, DEAE-cellulose column chromatography, and gel filtration on Sephadex G-75. The molecular weight of 152-enzyme was 17,000 and the isoelectric point was pH 7.3. The 152-enzyme showed lytic activity toward S. marcescens, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Bacillus subtilis, but was completely intert toward Staphylococcus aureus. The enzyme also showed caseinolytic activity. The lytic and caseinolytic activities of 152-enzyme were maximum around pH 11.0 and at 60°C. Both activities were inhibited by DFP and API-2c. Liberation of amino groups from cell walls of P. aeruginosa by incubation with 152-enzyme suggested that the enzyme was a kind of cell wall-lytic peptidase.     

Autolytic Enzyme System of Clostridium botulinum

Isolated cell walls of Clostridium botulinum type A strain 190L released an autolysin during autolysis of the cell walls. The autolysin was isolated from the cell walls, and partially purified 18.6-fold by ammonium sulfate precipitation, chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The purified preparation of the autolysin showed 2 major and 2 minor protein bands on Polyacrylamide gel electrophoresis. Some properties of the autolysin were examined using SDS-treated cell walls of the organisms as a substrate. The autolysin was active over a pH range of 6 to 8, with a maximum near pH 6.8. The lytic activity was stimulated by 10^?4 M each of Co⁺⁺, Mg⁺⁺ and Ca⁺⁺ in the order, whereas it was inhibited markedly by Cu⁺⁺. Mercaptoethanol (10^?4–10^?3 M) significantly activated the lytic action. Trypsin and nagarse (10 μg/ml) also stimulated the lytic activity. The lytic spectrum of the autolysin toward the SDS-treated cell walls obtained from various types of C. botulinum and C. perfringens indicated a relatively high specificity. After treatment with hot formamide the cell walls of C. botulinum increased in susceptibility to the autolysin.     

Purification and Properties of Phage HM 7-Induced Lytic Enzyme of Clostridium saccharoperbutylacetonicum

A lytic enzyme was isolated from phage HM 7-induced lysate of Clostridium saccharoperbutylacetonicum, and purified about 200-fold by precipitation with ammonium sulfate, gel filtration with Sephadex G–75 and ampholine isoelectric focusing. The purified lytic enzyme had an apparent homogeneity on disc-electrophoresis, and the character of acidic protein showing isoelectric point at pH 4.0. The molecular weight of lytic enzyme was estimated to be about 100,000 from the result of SDS-polyacrylamide gel electrophoresis. The optimum pH for the lytic enzyme activity was 6.5. Maximum activity occurred at 30 to 35°C, and at the ionic strength of 0.04 m or above. The lytic enzyme activity was stimulated about 140% by 10?³ m EDTA. The lytic enzyme lysed the living cells, but it had a narrow specificity which was restricted to a certain species of Clostridium such as Cl. saccharoperbutylacetonicum, Cl. butyricum, Cl. botulinum, Cl. sporogenes, and Cl. thiaminolyticum.     

Inhibition by Liposomal Cholesterol of Streptococcus Pneumoniae Induced Lung Epithelial Cell Damage

Abstract

Streptococcus pneumoniae was shown to be capable of lysing A549 cells in culture. Membrane damage to cells as assessed by trypan blue exclusion increased with increasing concentration of bacteria. After 45 min of incubation with 7.5 × 10⁸ bacteria/ml less than 20% of A549 cells excluded trypan blue. The lytic activity of S. pneumoniae was inhibited by phosphatidylcholine liposomes containing cholesterol. Using an haemolysis assay and S. pneumoniae's culture filtrates, the efficiency of the anti-lytic activity of liposomes was found to be distearoylphosphatidylcholine (DSPC) > dipalmitoylphosphatidylcho-line (DPPC) > dimyristoylphosphatidylcholine (DMPC). Furthermore, the anti-lytic activity also depended on the cholesterol content in a non-trivial manner. There was no protection against haemolytic activity at cholesterol content of less than 20% for DSPC and 35 mole% for DPPC and DMPC liposomes respectively. Above these threshold values inhibition of lytic activity increased sharply. In agreement with the haemolysis results, A549 cells were protected by liposomes against the lytic activity of S. pneumoniae with the efficiency also being DSPC > DPPC > DMPC. Clearly the efficiency of liposomal cholesterol is increased with increasing gel to liquid crystalline phase transition temperature of the lipid matrix. The results suggest that liposomal cholesterol may be used to protect the host against cell damage caused by S. pneumoniae.     

Synthesis of (±)-Xanthoxins

The enzymatic properties of P2-2 enzyme were determined by using cells of M. radiodurans. The enzyme was: most active at 60°C incubation temperature, stable at 40°C in neutral buffer, and inactivated by heating at 80°C for 15min. Maximal lytic activity occurred at pH 8.5 in Tris-HCl buffer. The range of enzyme stability was between pH 5.5 and 8. Bivalent metal ions, p-chloromercuribenzoate and monoiodo acetate inhibited lytic activity. The molecular weight was estimated to be 16,000 daltons by gel filtration on Sephadex G-75. The enzymatic digestion of peptidoglycans from the cell walls of M. radiodurans and M. lysodeikticus liberated free amino groups, but neither reducing groups nor N-acetylhexosamine, indicating that the enzyme was an endopeptidase. From analysis of the N-terminal amino acids of the digests, it is suggested that the P2-2 enzyme cleaves the peptide bond at the carboxyl group of D-alanine in peptidoglycan.     

Taxonomic Characters and Culture Conditions of a Bacterium which Produces a Lytic Enzyme on Rhizopus Cell Wall

A bacterium R–4 which produces a novel type of lytic enzyme which lyses fungal and yeast cell walls was isolated from the air and was identified to belong to the genus Bacillus.

Production of the enzyme appeared to require a high concentration of nitrogen source in medium. No inducing substance was needed for the enzyme production.

A crude preparation of the enzyme was used to characterize the lytic activity. From the lytic spectrum, the enzyme seemed to have the highest activity toward the cell walls of species in the genus Rhizopus among various fungi and yeasts tested, A proteolytic activity was shown to be parallel with the lytic activity. The lytic activity was also accompanied with the liberation of reducing sugars from Rhizopus cell wall, but no activity on some known carbohydrates tested was detected in the preparation.     

Fungus fruit body lytic enzyme from a myxomycete,Badhamia utricularis

Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and 8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH 5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations (Driselase and Usukizyme).     

Biomphalaria glabrata: lysozyme activities in the hemolymph, digestive gland, and headfoot of the intermediate host of Schistosoma mansoni.

Lysozyme (mucopeptide N-acetylmuramylhydrolase EC 3.2.1.17) activity has been found in the hemolymph, digestive gland, and headfoot extracts of Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. Partial purification of the bacteriolytic enzyme was attained by gel chromatography on Sephacryl S-200 and active lytic fractions were concentrated by Amicon filtration. The properties of the lytic enzymes from the three tissue extracts were identical. Enzyme activity was determined by the rate of lysis of cell wall suspension of Micrococcus lysodeikticus. Lysis of the cell walls was accompanied by a release of reducing sugar groups and N-acetylhexosamines. The enzyme was stable to heating at 100 C for 2 min and had an optimum activity at pH 4.5 to 5.0 in 0.066 M glycylglycine buffer. Low concentrations (5 mM) of NaCl, KCl, and LiCl increased the activity of the enzyme, whereas high concentrations (25 mM) of the same ions caused about 50% inhibition of the enzyme activity. MgCl₂ and CaCl₂ also inhibited the enzyme activity. Addition of 1 mM EDTA or EGTA resulted in about a twofold increase in enzyme activity. Double reciprocal plots of enzyme velocities and substrate concentrations yielded an apparent Michaelis-Menten constant (K_m) of 0.05 ± 0.01 mg/ml of M. lysodeikticus.     

Stability and activity of an<Emphasis Type="Italic"> Enterobacter aerogenes</Emphasis>-specific bacteriophage under simulated gastro-intestinal conditions

A bacteriophage, designated UZ1 and showing lytic activity against a clinically important strain (BE1) of Enterobacter aerogenes was isolated from hospital sewage. The stability and lytic activity against this strain under simulated gastro-intestinal conditions was evaluated. After addition of bacteriophage UZ1 to a liquid feed at gastric pH 2, the phage was immediately inactivated and could not be recovered. However, by use of an antacid to neutralize stomach acidity, no significant changes in phage titer were observed after 2 h incubation at 37 °C. After supplementing pancreatic juice and further incubation for 4 h, the phage titer remained stable. The persistence of UZ1 in a mixed microbial ecosystem that was representative for the large intestine was monitored using an in vitro simulation of the human intestinal microbial ecosystem. A pulse administration of bacteriophage UZ1 at a concentration of 10⁵ plaque-forming units (PFU)/ml to reactor 3 (which simulates the ascending colon) showed that, in the absence of the host, bacteriophage UZ1 persisted for 13 days in the simulated colon, while the theoretical washout was calculated at 16 days. To assess its lytic activity in an intestinal microbial ecosystem, a green fluorescent protein (gfp)-labeled E. aerogenes BE1 strain was constructed and gfp-specific primers were designed in order to quantify the host strain using real-time PCR. It was observed that bacteriophage UZ1 was able to replicate and showed lytic activity against E. aerogenes BE1/gfp in an intestinal microbial ecosystem. Indeed, after 17 h a 2 log unit reduction of E. aerogenes BE1/gfp was measured as compared with the assay without bacteriophage UZ1, while the phage titer increased by 2 log units at an initial multiplicity of infection of 0.07 PFU/colony-forming unit. This is the first report of an in vitro model to study bacteriophage activity in the complex intestinal microbial community.     

Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito,Anopheles gambiae

Abstract Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N‐acetyl‐d ‐glucosamine (UDP‐GlcNAc) and Mg⁺⁺. The optimal pH was about 6.5–7.0, and the highest activity was detected at temperatures between 37°C and 44°C. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GlcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500 ×g supernatant and the 40 000 ×g pellet. Our study revealed only slight in vitro inhibition of A. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5 μmol/L) examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.     

Ecto-alkaline phosphatase considered as levamisole-sensitive phosphohydrolase at physiological pH range during mineralization in cultured fetal calvaria cells

Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p-nitrophenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6–8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP were close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole was uncompetitive (K_i = 45 μM). Phosphatidylinositol-specific phospholipase C (PI-PLC) produced the release into the medium of a p-nitrophenyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The released activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto-pNPPase activity. After 75 min of PI-PLC treatment the ecto-pNPPase activity remained unchanged despite the 30% decrease in Nonidet P-40-extractable ALP activity. High levels of ⁴⁵Ca incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in accordance with ecto-pNPPase activity variations. These results evidenced the levamisole-sensitive activity of a glycosylphosphatidylinositol-anchored pNPPase consistent with ALP acting as an ecto-enzyme whose functioning under physiological conditions was correlated to ⁴⁵Ca incorporation and permit the prediction of the physiological importance of the enzyme dynamic equilibrium at the cell surface in cultured fetal calvaria cells. © 1996 Wiley-Liss, Inc.     

Characterization of the staphylococcal bacteriophage lysin CHAP(K)

Aims: To develop an efficient purification strategy for the bacteriophage lysin CHAP_K. To evaluate its antibacterial spectrum_, enzymatic properties, optimal reaction conditions and lytic activity against live Staphlyococcus aureus. Methods and Results: Recombinant CHAP_K was purified to homogeneity by cation exchange chromatography, with yields of up to 10 mg from 1 l of Escherichia coli culture. The lytic spectrum of CHAP_K includes all staphylococcal species and also members of the genera Micrcococcus, Streptococcus, Nesterenkonia, Arthrobacter, Leuconostoc and Carnobacterium. The enzyme was active from pH 6 to 11 with an optimum activity at pH 9, from 5 to 40°C, with an optimum activity at 15°C. When cell lysis by CHAP_K and lysostaphin was compared over a concentration range of 2·5–10 μg ml^?1 using live Staph. aureus for 5 min at 37°C, CHAP_K gave rise to greater turbidity reduction indicating that it works more rapidly than lysostaphin. Conclusions: This study describes in detail the purification and characteristics of the novel phage‐derived enzyme CHAP_K demonstrating that it has excellent biochemical properties as an anti‐staphylococcal agent. Significance and Impact of the Study: Currently, there is a need for new antimicrobial agents due to the increasing worldwide prevalence of antibiotic resistance. Our findings demonstrate the potential for development of CHAP_K as an alternative therapeutic against pathogenic staphylococci including MRSA.     

Kinetic study on pH dependence of growth and death of Streptococcus faecalis

A chemostat culture was used for lactic acid fermentation with Streptococcus faecalis at various pH values (8.0, 7.0, 6.0, 5.5, 5.0) and glucose concentrations (10, 20, 30 g/l). At every pH value, the reciprocals of the specific consumption rate of glucose and the specific production rate of lactic acid were linearly correlated to the reciprocal of the specific growth rate. The product, lactic acid, caused non-competitive inhibition of the specific growth rate at every pH value. Moreover, it was found that the cell death rate was dependent on pH and lactic acid. The death rate was smallest at pH 7.0 and increased with increasing lactic acid concentration. The kinetic equations of growth and death are proposed in a broader pH range. Correspondence to: H. Ohara     

Detection, cellular localization and antibacterial activity of two lytic enzymes of Pediococcus acidilactici ATCC 8042

Aim: In Pediococcus acidilactici ATCC 8042, two activities of peptidoglycan hydrolase (PGH) with lytic effect against Micrococcus lysodeikticus and Staphylococcus aureus have been detected. This work intends to elucidate the growth phase of maximum lytic activity, the localization and the effectiveness of the activity against pathogenic Gram‐negative and Gram‐positive bacteria. Methods and Results: Cells were grown in MRS medium and collected at different growth stages, and the proteins were extracted. The highest PGH activity was found during the logarithmic growth phase in the protein fraction bound to the cell membrane. From this fraction, two distinct proteins bands (110‐ and 99‐kDa) in SDS–PAGE were partially purified with a three‐step procedure. Both bands showed lytic activity against M. lysodeikticus. Mass spectrometry analysis (LC/ESI‐MS/MS) indicated that the 110‐kDa band corresponded to a protein of unknown function. The 99‐kDa band corresponded to a N‐acetylmuramidase that harboured catalytic sites with N‐acetylmuramoyl‐l ‐alanine amidase and N‐acetylglucosaminidase activities. Both proteins are reported in the Ped. acidilactici 7_4 genome. The fraction containing the concentrated proteins (110 and 99 kDa) inhibited the growth of several pathogenic strains as: Bacillus cereus, Listeria monocytogenes and Salmonella typhimurium. The growth of S. aureus was diminished by 3 logarithmic units as early as 0·5 h of growth, while inhibition of Escherichia coli and Ped. acidilactici was observed after 18 and 8 h, respectively (both in one logarithmic unit). The minimum inhibitory concentration against S. aureus was 10 μg ml^?1. Conclusion: Pediococcus acidilactici harbours at least two lytic enzymes, one of them recognized as PGH for the first time, which exert antibacterial activity against several bacterial strains. Significance and Impact of the Study: Both PGH activities have a broad growth inhibition spectrum and could be used to control pathogenic bacteria. Because this activity comes from a lactic acid bacterium, it could be safely used in manufacturing processes of fermented foods.     

Lethal effect of protamine and histone on competent Bacillus subtilis cells. Inhibition of genetic transformation by protamine in sublethal concentration.

Summary Under experimental conditions of genetic transformation, protamine and total histone were bactericidal for Bacillus subtilis cells. The abilities to cause lethality were very similar for both, either protamine or histone, with no antagonistic effects amongst these natural polycations. With both basic proteins acting simultaneously the enhancement was higher than a summation of the separate lethal effects. Sublethal concentration of protamine added at the beginning of transformation time, produced a strong inhibition of transforming efficiency. The same concentration added later than 10 min from the start of transformation had no inhibitory effect. These facts together with the absence of inhibition by simple pretreatment of DNA alone as well as the cell protection by protamine against lytic activity of lysozyme, suggest a protamine-cell surface interaction which impedes DNA uptake events.     

Effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes in continuous culture

Summary The effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes from glucose was investigated in a microaerobic continuous culture. At a dilution rate of 0.20 h^–1 and a fixed oxygen uptake rate (OUR) of 31.5 mmol l^–1 h^–1 the biomass concentration increased with pH ranging from 5.0 to 7.0, while the specific ATP requirement of the cells decreased. In the pH range 5.5–6.5 the product concentration (butanediol + acetoin) was maximal and nearly constant. However, the specific production continuously declined with increasing pH. Experiments with addition of acetic acid showed that the various effects of pH are due to inhibition of the by-product acetic acid on cell growth. The strength of the acetic and inhibition depended only on the concentration of its undissociated form [HAc]. The biomass concentration and the specific OUR were also only functions of [HAc], irrespective of the pH. Although the specific ATP requirement (q ATP) strongly depended on the pH, [HAc] at constant pH. Offprint requests to: W.-D. Deckwer     

Red Yeast Cell Lysis by Red Yeast Cell Wall Lytic Enzyme and Protease

The purified red yeast cell wall lytic enzyme of Penicillium lilacinum No. 2093 has a potent saccharifying activity against cell walls, but the living cell lytic activity of it is considerably lower than that of the culture filtrate. Therefore, the living cell lytic factors in the culture filtrate were examined. The alkaline protease of Pen. lilacinum played an important role for living cell lysis. The synergistic effect on living cell lysis was also detected, when acid proteases from various origins were combined with the cell wall lytic enzyme. These results indicated that the protein layers of red yeast cell surface inhibited the action of a glycanase,cell wall lytic enzyme, and the protein molecule contributed to retain the rigid structure of the wall.     

Purification and Properties of Lytic Enzymes from Streptomyces globisporus 1829

Two lytic enzymes capable of lysing Streptococcus mutans have been purified to give a single band on disc-gel electrophoresis, respectively. The M–1 and M–2 enzymes were both proved to be N-acetylmuramidases. However, these enzymes were entirely different on their enzymatic properties. The molecular weights were about 20,000 and 11,000 for M–1 and M–2 enzymes, respectively, The maximal lytic activity of M–1 enzyme was obtained at ionic strength 0.05, while lytic activity of M–2 enzyme did not change within the ionic strength range of 0 to 0.05. The M–1 enzyme constituted the majority of the total lytic activity against the cell walls of Streptococcus mutans BHT of cultured filtrate. The M–2 enzyme showed less specific lytic activity on the cell walls of Streptococcus mutans BHT than M–1 enzyme.     

Fermenting Escherichia coli Is Able to Grow in Media of High Osmolarity, But Is Sensitive to the Presence of Sodium Ion

Escherichia coli is able to grow at increased NaCl concentrations that provides an increase in medium osmolarity and cellular Na⁺ content. The addition of 0.5 M NaCl to the growth medium led to a substantial decrease in growth rate during anaerobic fermentation on glucose at pH of 7.3 or 9.0. This inhibitory effect of 0.5 M NaCl was at least threefold stronger than that seen under aerobic conditions, and stronger than equivalent concentrations of sucrose, KCl, or potassium glutamate under anaerobic conditions. Further, proline was found to stimulate the growth rate at high NaCl concentration under anaerobic and to a lesser extent, under aerobic conditions. Wild-type cells and mutants having a functional NhaA or ChaA alone grown under anaerobic conditions at pH 9.0 and subsequently loaded with Na⁺ were shown to extrude Na⁺ at a rate that were lower than the extrusion rate reported for appropriate aerobically grown bacteria (Sakuma et al. [1998] Biochim Biophys Acta 1363:231–237). The growth rate and Na⁺ extrusion activity of a mutant having a functional NhaA were similar to that of the wild type and higher than that of a mutant with an active ChaA. A mutant defective for both NhaA and ChaA was unable to grow under anaerobic conditions at pH 9.0 in the presence of 0.15 M Na⁺. It is suggested that the observed strong inhibition in the growth of E. coli during fermentation under anaerobic conditions in the presence of increased NaCl concentration could be due to a decrease in Na⁺ extrusion activity. Received: 18 September 1998 / Accepted: 2 April 1999