Cell wall degradation ofStaphylococcus aureus by lysozyme

In contrast to former findings lysozyme was able to attack the cell walls ofStaphylococcus aureus under acid conditions. However, experiments with¹⁴C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. attack from the inside). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but stabilized protoplasts were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.     

Surface charge measurements on Micrococcus lysodeikticus and the catalytic implications for lysozyme

Electrophoresis measurements on Micrococcus lysodeikticus have shown that the net surface charge density on the cell wall is constant at around -1.5 microC/cm2 for the pH range 4-8. This result has enabled a quantitative analysis to be made of how the electrostatic field associated with the negatively charged cell wall influences the ionic strength and pH dependency of the lytic activity of lysozyme towards M. lysodeikticus. A dominant effect is the creation of a local pH gradient at the cell wall, and at high ionic strengths the lytic activity is found to be controlled by an electrostatic force of attraction between the lysozyme molecule and the cell wall. As the ionic strength of the supporting electrolyte is decreased, however, an electrostatic force of repulsion becomes dominant and is associated with a negative charge carried by the lysozyme molecule, which could possibly be the ionized Asp-52 residue at the active site. This is considered to arise from the fact that at low ionic strengths the fine details of the heterogeneous charge distribution on the cell wall and lysozyme molecule are only partially screened by counter ions.     

l-Glutamic Acid Fermentation with Molasses

Conditions suitable for the cell wall lysis of a l-glutamate-producing bacterium, Microbacterium ammoniaphilum, by egg white lysozyme were studied, in order to make clear the correlation of the fatty acid composition of the cellular fractions and the extracellular accumulation of l-glutamate,

The cell wall of a phage-resistant strain was recognized to be almost completely lyzed by the lysozyme.

Using this result, the relationship between the fatty acid composition of each fraction and extracellular accumulation of l-glutamate was investigated, and the following thesis was proposed: The extracellular accumulation of l-glutamate in large quantity took place when the molar ratio of saturated/unsaturated fatty acid in the cell membrane fraction was above 1.     

A turbidimetric and electron microscopic study of the effects of several parameters on the lysis of Streptococcus faecalis by lysozyme

The lytic effect of lysozyme on Streptococcus faecalis ATCC 9790 was studied by spectrophotometry and electron microscopy and it was found to be highly dependent on the ionic strength of the suspending media and on the ratio lysozyme to bacterial cell mass. When 7.2 X 10(8) bacteria/mL are exposed to 0.4 mg/mL of lysozyme in media with low ionic strength, the enzyme is bound in great amounts, as deduced from protein determinations and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS--PAGE); the binding prevents bacteriolysis in spite of the removal of the cell wall. Extensive lysis of S. faecalis could be obtained by reducing the ratio of lysozyme to bacterial cell mass. Stabilization of S. faecalis by lysozyme was also observed when exponential phase cells incubated under conditions that promote spontaneous autolysis (incubation in 0.05 M tris(hydroxymethyl)aminomethane buffer, pH 8.0, ionic strength = 0.01675) do not lyse and do not leak material which absorbs at 260 nm when lysozyme was present at the highest concentration.     

Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge

The surface positive charges of human lysozyme were either increased or decreased to alter the electrostatic interaction between enzyme and substrate in the lytic action of human lysozyme using site-directed mutagenesis. The amino acid substitutions accompanying either the addition or the removal of two units of positive charge have shifted the optimal ionic strength (NaCl concentration in 10 mM Mes buffer, pH 6.2) for the lysis of Micrococcus lysodeikticus cell from 0.04 M to 0.1 M and from 0.04 M to 0.02 M respectively. In addition to the change in ionic strength-activity profile, the pH-activity profile and the effect of a polycationic electrolyte, poly-L-Lys-HCl, on the lytic activity were significantly changed. Owing to the shifts in both ionic strength profiles and pH profiles the Arg74/Arg126 mutant has become a better catalyst than wild-type enzyme under the conditions of high ionic strength and high pH, and the Gln41/Ser101 mutant has become a better catalyst under the conditions of low ionic strength and low pH.     

Identification and characterization of novel cell wall hydrolase CwlT: a two-domain autolysin exhibiting n-acetylmuramidase and DL-endopeptidase activities

A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to dl-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a dl-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents "a new category of cell wall hydrolases."     

Mode of Action of Clostridium Phage HM 7-Induced Lytic Enzyme on Clostridium saccharoperbutylacetonicum Cell Wall Peptidoglycan

The action of Clostridium phage HM 7-induced lytic enzyme on the cell wall peptidoglycan of Clostridium saccharoperbutylacetonicum was investigated. The cell wall peptidoglycan of this strain contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00: 2.08: 0.97; 0.92: 0.68. It was strongly digested when incubated with the lytic enzyme. This digestion was accompanied by the release of NH₂-terminal l-alanine without a concomitant release of COOH-terminal amino acids and reducing groups. Chromatography of the lytic enzyme digest resulted in only two fractions, each of which was chromatographically homogeneous. One was a polysaccharide consisting of glucosamine and muramic acid in molar ratios 1.00: 0.78, and other was a peptide composed of glutamic acid, alanine and diaminopimelic acid in molar ratios of 1.00: 2.09: 1.05. These results indicate that phage HM 7-induced lytic enzyme is N-acetylmuramyl-l-alanine amidase, which cleaves the linkage between N-acetylmuramic acid and l-alanine.

A possible structure for the cell wall peptidoglycan was also proposed.     

Electron microscopy of Staphylococcus aureus cell wall lysis

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Mu?oz, 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.     

A special morphogenetic wall defect and the subsequent activity of “murosomes” as the very reason for penicillin-induced bacteriolysis in staphylococci

The actual reason for the penicillin-induced bacteriolysis of staphylococci was shown to be the punching of one or a few minute holes into the peripheral cell wall at predictable sites. These perforations were the result of the lytic activity of novel, extraplasmatic vesicular structures, located exclusively within the bacterial wall material, which we have named murosomes.In untreated staphylococci the punching of holes into the peripheral wall is a normal process which follows cross wall completion and represents the first visible step of cell separation. Under penicillin, however, analogous holes are punched by the murosomes at sites of presumptive cell separation even if no sufficient cross wall material had been assembled before at this site (but had rather been deposited at other sites). Consequently, because of the internal pressure of the protoplast, lytic death is the inevitable result of this perforation of the protective peripheral wall.Hence, the real mechanism of penicillin-induced bacteriolysis in staphylococci is considered to be mainly the result of a special morphogenetic wall defect: bacteriolysis is taking place regularly when a cell separation process is no longer preceeded by sufficient cross wall assembly at the correct place. However, hypotheses which are based purely on some variations of overall biochemical processes like total wall enzyme activities or total wall synthesis are not regarded to be sufficient to explain this type of lytic death.Dedicated to Prof. Dr. Gerhart Drews on the occasion of his 60th birthday     

The Lysozyme-Induced Peptidoglycan N-Acetylglucosamine Deacetylase PgdA (EF1843) Is Required for Enterococcus faecalis Virulence

Lysozyme is a key component of the innate immune response in humans that provides a first line of defense against microbes. The bactericidal effect of lysozyme relies both on the cell wall lytic activity of this enzyme and on a cationic antimicrobial peptide activity that leads to membrane permeabilization. Among Gram-positive bacteria, the opportunistic pathogen Enterococcus faecalis has been shown to be extremely resistant to lysozyme. This unusual resistance is explained partly by peptidoglycan O-acetylation, which inhibits the enzymatic activity of lysozyme, and partly by d-alanylation of teichoic acids, which is likely to inhibit binding of lysozyme to the bacterial cell wall. Surprisingly, combined mutations abolishing both peptidoglycan O-acetylation and teichoic acid alanylation are not sufficient to confer lysozyme susceptibility. In this work, we identify another mechanism involved in E. faecalis lysozyme resistance. We show that exposure to lysozyme triggers the expression of EF1843, a protein that is not detected under normal growth conditions. Analysis of peptidoglycan structure from strains with EF1843 loss- and gain-of-function mutations, together with in vitro assays using recombinant protein, showed that EF1843 is a peptidoglycan N-acetylglucosamine deacetylase. EF1843-mediated peptidoglycan deacetylation was shown to contribute to lysozyme resistance by inhibiting both lysozyme enzymatic activity and, to a lesser extent, lysozyme cationic antimicrobial activity. Finally, EF1843 mutation was shown to reduce the ability of E. faecalis to cause lethality in the Galleria mellonella infection model. Taken together, our results reveal that peptidoglycan deacetylation is a component of the arsenal that enables E. faecalis to thrive inside mammalian hosts, as both a commensal and a pathogen.     

Comparative study of the effects of salinity and UV radiation on metabolism and morphology of the red macroalga <Emphasis Type="Italic">Acanthophora spicifera</Emphasis> (Rhodophyta,Ceramiales)

Increase of harmful radiation to the Earth’s surface due to ozone depletion results in higher exposure to harmful ultraviolet- B radiation (UV), while fluctuations in seawater salinity may alter water density, ionic concentration, nutrient uptake, and osmotic pressure. This study evaluated the effects of salinity and UV on metabolism and morphology of Acanthophora spicifera (M.Vahl) Børgesen. Water with 30 and 37 psu [g(salt) kg^–1(sea water)] was used for experiments during 7 d of exposure to UV (3 h per day). We demonstrated that UV treatment predisposed, irrespective of salinity, A. spicifera to a decrease in its growth rate and cell viability, as well as affected its morphological parameters. After exposure to PAR + UVA + UVB (PAB), samples showed structural changes and damage, such as increasing cell wall thickness and chloroplast disruption. Our results indicate that UV led to dramatic metabolic changes and cellular imbalances, but more remarkable changes were seen in samples exposed to high salinity.     

A fluorescent assay for bacterial cell wall lytic enzymes

A sensitive fluorimetric assay is described for the measurement of N-acetylmuramic acid l-alanine amidase as well as lysozyme. The method uses Bacillus subtilis cell walls labeled with fluorescamine on the free amino group of diaminopimelic acid. The method can easily detect the lytic activity of 0.02 μg of pure N-acetylmuramic acid l-alanine amidase in 30 min and of 1 μg of hen egg-white lysozyme in the same period. The method is particularly suitable for measurement of competition between various cell wall preparations for the same enzyme.     

Degradation and turnover of bacterial cell wall mucopeptides in growing bacteria

The mucopeptide layer of the cell wall ofBacillus megaterium is broken down into separate components during growth of the cells. The released diaminopimelic acid is partly decarboxylated to lysine, which is incorporated in the proteins and partly used for cell wall resynthesis. The smaller portion of the degraded mucopeptide is released into the medium in the form of non-utilized fragments. The rate of the mucopeptide turnover is a function of the rate of growth of the culture. About 15–20% of the rigid layer of the cell wall is degraded during on cell division. The sensitivity ofBacillus megaterium to lysozyme and the rate of its conversion to protoplasts is also proportionate to the rate of growth of the culture. There is no measurable mucopeptide turnover in non-growing cells, either in the stationary phase of the culture or in starvation in nitrogen-free medium. The resistance of the cell wall to lysozyme also increases during the stationary phase. The rigid component of the cell wall is probably also broken down during growth ofBacillus cereus andEscherichia coli cultures.     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

Lytic agents, cell permeability, and monolayer penetrability

Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20–30 % lipid and 50–75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2–3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.     

The role of peptidoglycan structure and structural dynamics during endospore dormancy and germination

Dormant, bacterial endospores are the most resistant living structures known. The spore cell wall (cortex) maintains dormancy, core dehydration, and heat resistance. The cortex peptidoglycan has a unique, spore specific structure that enables it to fulfill its role. The cross-linking index of spore cortex peptidoglycan is very low, occurring at only 2.9% of the muramic acid residues compared to 33% in vegetative cells. The level of cross-linking of the cortex may be important in maintaining spore dormancy and heat resistance. Approximately 50% of the muramic acid residues in spore cortex are substituted with muramic -lactam. This modification is spore specific and is the major characteristic feature of the cortex. The muramic -lactam has no apparent role in establishing core dehydration, maintaining dormancy or heat resistance. However, the muramic -lactam residues are necessary for spore cortex hydrolysis during germination. They constitute part of the substrate recognition profile of the germination specific lytic enzymes (GSLEs) which are responsible for cortex hydrolysis.Germination results in loss of dormant spore properties and hydrolysis of the cortex is essential for later germination events and outgrowth. Application of muropeptide analysis to determine peptidoglycan structural dynamics during germination has revealed an unexpected degree of complexity in peptidoglycan hydrolysis. At least three hydrolytic activities, an N-acetyl glucosaminidase, a lytic transglycosylase and a possible amidase, are involved. A non-hydrolytic acitivity, likely to be an epimerase of muramic acid also occurs early during germination.The lytic transglycosylase generates anhydro-muropeptides which are released during germination and may be recycled during outgrowth to form part of the new vegetative cell wall.     

Serine proteinase from Bacillus brevis: lytic action on intact yeast cells

Summary A serine proteinase which showed lytic acitivity against either intact cell or cell wall preparations of Candida utilis has been isolated from Bacillus brevis culture filtrate by affinity chromatography on bacitracin-silochrome and phenylboronale-Sepharose. Both its proteolytic and lytic activities were completely abolished by inhibitors of serine proteinases, including phenylmethylsul-phonylfluoride, the inhibitor from Actinomyces janthinus, and duck ovomucoid. The optimum pH range for the enzyme is 7.5–9.0, the optimum temperature 40°–50°C, its pI value 8.6 and motecular weight 28000. The amino acid composition of this proteinase is similar to that of serine proteinase from B. amyloliquefaciens (subtilisin BPN), its N-terminal amino acid sequence being identical to that of BPN through 21 residues. The enzyme cleaves chromogenous substrates for subtilisins but shows no activity on a substrate for trypsin. By means of both turbidimetry and electron microscopy the enzyme studied was shown to cause yeast cell lysis.     

Potentialization of antibiotics by lytic enzymes]

Few lytic enzymes, specially papaine and lysozyme, acting on the membrane and cell wall structures facilitate effects of bacitracine, streptomycine and other antibiotics. Streptomycino resistant strains became sensibles to this antibiotic after contact with papaine and lysozyme. The results of tests in physiological suspensions concern only the lytic activity of enzymes. The results on nutrient medium concern together lytic, and antibiotic activities.     

Laser microsurgery: a versatile tool in plant (electro) physiology

Summary In plant cells the cell wall is a formidable obstacle in many physiological studies such as patch-clamp measurements and cell labelling with antibodies. Enzymatic digestion of the cell wall, in order to release a protoplast, has a number of disadvantages; therefore we worked out an alternative method to gain access to the plasma membrane. The wall of specialized cells from three higher plant species and one unicellular alga were perforated using the focussed UV light of a nitrogen laser. In order to enhance the absorption of the UV light by the walls, a dye was used that binds specifically to cell wall components. Extrusion of the protoplast or parts thereof was controlled by a regulated gradual decrease of the osmolarity of the solution surrounding the cells. Cytoplasmic streaming and chloroplast circulation were maintained in the protoplasts, demonstrating their viability after the wall perforation with the laser. Continuous deposition of new cell wall material by the polar tip of pollen tubes after surgical removal of the wall at the tip is another demonstration of the viability of the cells. Formation of high resistance seals between the plasma membrane and a patch pipet was surprisingly difficult. The role of Hechtian strands and continuing synthesis of cell wall material in seal formation is further investigated. Other applications for the surgical laser are: fusion of two cells or vacuoles, analysis of the composition of specific parts of the cell wall, and release of the vacuole from an identified cell type for patchclamp studies.Abbreviations CFW calcofluor white - PM plasma membrane