Enzymic Lysis Research Articles

Restriction endonuclease analysis using Hhal and Hpall to discriminate among group B Pasteurella multocida associated with haemorrhagic septicaemia.

The purpose of this study was to improve and standardise restriction endonuclease analysis (REA) for discriminating isolates of serogroup B Pasteurella multocida associated with haemorrhagic septicaemia in wild and domestic animals and to create a reference database that can be used for epidemiological studies. Two techniques for extraction and isolation of chromosomal DNA were compared, a DNAzol method and an enzymic lysis followed by a two-phase partition method. No differences were observed between DNA fingerprint profiles with either technique; however, the former technique was faster and easier to perform. P. multocida isolated from different animals in different countries representing serotypes B:2, B:3, B:3,4 and B:4 were subjected to REA with HhaI and HpaII endonucleases. Forty-eight fingerprint profiles were distinguished among 222 isolates when only HhaI was used. By combining the data from REA with HhaI and HpaII used separately, 88 different groups could be distinguished among the same isolates. Following digestion with HhaI and electrophoresis, the DNA of all serotype B:2 isolates produced fingerprint profiles characterised by two trailing bands at approximately 8.4-7.1 kb which have not been observed in any other serotypes of P. multocida. Passage of three serotype B:2 isolates on laboratory media or two serotype B:2 isolates through mice did not result in a change of DNA fingerprint profile detectable by REA. The findings with 59 isolates from Sri Lanka showed that REA was highly discriminative in determining the genetic diversity of serotype B:2 P. multocida in an area where haemorrhagic septicaemia is endemic.

Cell wall degradation of Fusarium oxysporum f.sp. lycopersici race 2 by lytic enzymes from different Fusarium species for its biocontrol

The lysis of Fusarium oxysporum f.sp. lycopersici cell wall and cell wall fractions F1S (glucogalactomannan) and F4 (β-1,3-glucan chitin) was studied with lytic enzymes from 13 Fusarium species and four forms. The fungi were classified into three groups: A, fungi whose lytic enzymes do not degrade either Fol2 cell walls or F1S and F4 fractions (F. culmorum and F. solani); B, fungi whose enzymes degrade them to a very low extent after an incubation period of 72 h (F. acuminatum, F. decemcellulare, F. oxysporum, F. reticulatum, F. semitectum var. majus and F. tricinctum); C, fungi that produce enzymes that degrade Fol2 cell wall and fractions (F. equiseti, F. graminearum, F. moniliforme, F. oxysporum f.sp. gladioli, F. oxysporum f.sp lini, F. oxysporum f.sp. lycopersici, F. sambucinum and F. subglutinans). Lysis of the cell walls and fractions was followed by the release of reducing substances and by the decrease in optical density of insoluble samples (lysis degree) with the incubation time. The neutral sugars released were quantified and characterized by GLC and N-acetylglucosamine by a spectrophotometric procedure. Fusarium graminearum, F. moniliforme, F. oxysporum f.sp lini, F. oxysporum f.sp. lycopersici and F. subglutinans were selected as possible mycoparasites of Fol2 because they exhibited high hydrolytic activity on F1S fraction known to be very resistant to the enzymic lysis by other fungi, as well as on F4 fraction and isolated cell walls.

The lysis of Gram-negative Alcaligenes eutrophus by enzymes from Cytophaga

The use of Cytophaga lysing enzymes was investigated for the liberation of poly-β-hydroxybutyrate (PHB) granules from the Gram-negative bacterium Alcaligenes eutrophus. Complete cell lysis was approached within a 60 minute period. Contrary to previous findings for the lysis of Gram-negative bacteria, prior removal of the outer membrane was not essential for enzymic lysis. The destabilisation of the outer membrane by the removal of divalent cations resulted in no significant improvement in the disruption process.

Structural Aspects of the Parasitism of Sclerotia of Sclerotinia sclerotiorum (Lib.) de Bary by Coniothyrium minitans Campb.

AbstractSclerotia of Sclerotinia sclerotiorum inoculated with pycnidiospores of Coniothyrium minitans were studied by means of light microscopy and transmission and scanning electron microscopy. The hyperparasite penetrated the walls of the rind cells by means of physical pressure and destroyed the cell contents. Penetration of medullary hyphae was by enzymic lysis and physical pressure; there was evidence to suggest that the hyperparasite may coil around the host cells before inserting infection hyphae.

Oxidation of n-alkanes: Isolation of alkane hydroxylase from Pseudomonas putida

The disruption of Pseudomonas putida cells capable of n-alkane assimilation was investigated by enzymic lysis and mechanical disruption in a high pressure-homogeniser, with a view to the isolation of alkane hydroxylase activity. Examination of the conditions for enzymic lysis showed that disruption with lysozyme/EDTA could be replaced effectively with lysozyme alone in phosphate buffer, pH 8.0 (I=0.05). This allowed inclusion of DNase during the lysis procedure for high bacterial concentrations and gave improved cell disruption. Mechanical disruption resulted in the solubilisation of alkane hydroxylase activity. In contrast enzymic lysis allowed the isolation of an insoluble fraction containing alkane hydroxylase activity, and although some solubilisation of the enzyme system did occur much of the activity was retained in the insoluble fraction. This fraction also contained a high level of n-alkane or diethoxymethane inducible, NAD-independent alcohol dehydrogenase activity.

Effect of growth conditions on production of rhamnose-containing cell wall and capsular polysaccharides by strains of Lactobacillus casei subsp. rhamnosus.

Strains of Lactobacillus casei subsp. rhamnosus possessing two cell wall polysaccharides, a hexosamine-containing H-polysaccharide and a rhamnose-containing R-polysaccharide, were examined for the effect of growth conditions on the production of these two components. In strain NCTC 6375, R- and H-polysaccharides accounted for an estimated 44 and 20%, respectively, of the cell wall for organisms grown in batch culture with glucose as the carbohydrate source. Growth on fructose-containing media reduced the amount of R-polysaccharide by approximately 50% without affecting the amount of H-polysaccharide. Subculture of fructose-grown organisms in glucose restored the original proportions of the two polysaccharides. Galactose- and sucrose-grown cells behaved similarly to glucose-grown cells with respect to polysaccharide production, whereas growth in rhamnose or ribose showed values close to those for fructose-grown cells. Continuous culture of strain NCTC 6375 for more than 100 generations showed a gradual and irreversible reduction of the R-polysaccharide to less than 5% of the cell wall and an increase of the H-polysaccharide to 40% of the cell wall. Other type culture strains of L. casei subsp. rhamnosus, NCIB 7473 and ATCC 7469, behaved similarly in batch and continuous culture. In contrast, strains of L. casei subsp. rhamnosus isolated at the Institute of Dental Research showed phenotypic stability with respect to the relative proportions of R- and H-polysaccharides in both batch and continuous culture. Changes in polysaccharide composition of type culture strains were also mirrored in changes in the immunogenicity of the two components and resistance to the rate of enzymic lysis of whole organisms. For L. casei subsp. rhamnosus strain NCTC 10302 the R-polysaccharide is present entirely as capsular material. The amount of R-polysaccharide produced was also markedly dependent on the carbohydrate component of the medium in batch culture and both dilution rate and nature of the limiting carbohydrate in continuous culture, varying over a 10-fold range, whereas the cell wall H-polysaccharide remained constant.

Enzymic lysis and structure of the cell walls of the oral Streptococcus mutans BHT

Solubilization of purified Streptococcus mutans BHT cell walls by Flavobacterium L-11 cell-wall lytic enzyme preparation was accompanied by the exposure of 0.92 mole of N-terminal l-alanine and 0.31 mole of C-terminal d-alanine per mole of glutamic acid residues. No other terminal amino acids and soluble reducing groups were exposed during cell-wall lysis. A small but significant amount (0.23 mole) of free l-alanine was also liberated by aminopeptidase activity of this enzyme preparation. These results suggest that about 60 per cent of N-acetylmuramyl- l-alanine linkages and 30 per cent of d-alanyl- l-alanine linkages of cell-wall peptidoglycan were cleaved by amidase and endopeptidase activities of the cell-wall lytic enzyme preparation, respectively, a view supported by the chromatographic isolation of penta-and hexa-peptide fractions consisting of glutamic acid, alanine, and lysine in molar ratio of 1.0:3.0–4.0:1.0 from the cell-wall digests. Analyses of N- and C-terminal amino acids suggested that the major peptides isolated have the chemical structures of N α -( l-alanyl- d-iso-glutaminyl)- N ϵ -( l-alanyl)1–2- l-lysyl- d-alanine or N α -( l-alanyl- d-isoglutaminyl)- N ϵ -( l-alanyl)2–3- l-lysine. A possible structure of peptidoglycan of the cell walls was proposed.

Enzymic lysis of three species of yeasts

The cell wall structures of three species of yeasts — Torulopsis aeris Marcilla & Feduchy, Geotrichum lactis Fres. and Oospora suaaeolens (Linder) Lindau — have been studied using lytic enzymes. Extensive lysis was only achieved by mixtures of enzymes or by complex lytic systems derived from soil organisms. The ‘structural’ components of the walls, mainly chitin and glucan, seem to be protected from the lytic action of the enzymes by other ‘cementing’ polysaccharides. The glucan is sensitive to β(1–3) and β(1–6) glucanases. Those polysaccharides containing sugars other than glucose are released from the wall after lysis without complete degradation to monomers.

Effects of the interaction of penicillins with other antimicrobial agents of the enzymic lysis of Staphylococcus aureus.

SummaryThe penicillin G- and nafcillin-mediated lysis of growing cells of S. aureus by lysozyme and trypsin was used as a test to determine the influence of sublethal concentrations of other antibiotics on this response. Seventeen antibiotics were tested with two strains of S. aureus (G50 and CHP). Rifamycin, novobiocin, tetracycline HCl, actinomycin D, erythromycin, and streptomycin interfered with or blocked lysis whereas methicillin, cephalothin, cloxacillin, dicloxacillin, cycloserine, and vancomycin either enhanced it or had no effect depending on the concentrations of these antibiotics. Rifamycin, chloramphenicol, and novobiocin were found to be the most active antagonists of penicillin G and nafcillin. Under the experimental conditions, none of the bactericidal antibiotics acted synergistically with either penicillin G or nafcillin. The results suggest that the change in lytic activity when certain antibiotics are combined with penicillin depends on some specific biologic activity against S. aureus...

Effect of Antimicrobial Drug Combinations on Enzymic Lysis of Staphylococcus aureus

SummarySublethal concentrations of nafcillin, ampicillin, cloxacillin and cephalothin react with growing cells of S. aureus CHP and bring about a characteristic lytic response when these cells are subsequently incubated with lysozyme and trypsin. The same lytic effect is apparent to a much lesser degree with novobiocin but not with inhibitors of protein synthesis, e.g., chloramphenicol, tetracycline, erythromycin or actinomycin D, which in fact antagonize this effect. In combination with nafcillin, novobiocin was shown to antagonize the nafcillin-promoted enzymic lysis, while ampicillin, cloxacillin and cephalothin exerted neither antagonism or significant synergism.

Enzymic lysis of plasma clots: The influence of fibrin stabilization on lysis rates

Fibrin clots, freshly formed from recalcified plasma, displayed high susceptibility to lysis by the plasminogen activator, urokinase, or the proteolytic enzymes, plasmin or trypsin; later these clots became insoluble in 5 m urea or 1% monochloroacetic acid and developed increased resistance to enzymic lysis (5- to 10-fold that of the unstabilized clot). In contrast, clots formed from EDTA plasma and clotted with thrombin usually remained soluble in solvents capable of rupturing hydrogen bonds, and did not develop resistance to enzymic lysis. Transamidation inhibitors (glycine amide, methylester, and glycyl derivative) and sulfhydryl inhibitors (iodoacetamide, p-chloromercuribenzoic acid, etc.) inhibited fibrin stabilization and the development of clot resistance to enzymic lysis. Only limited correlation existed between conventional solubility measures of clot stabilization and clot resistance to enzymic lysis, the latter increasing after solubility criteria demonstrated fibrin stabilization. It is suggested that clot resistance to enzymic lysis may constitute an additional valuable measure and criterion of fibrin stabilization.

INFLUENCE OF NAFCILLIN ON THE ENZYMIC LYSIS OF STAPHYLOCOCCUS AUREUS

During growth of Staphylococcus aureus cells, sublethal concentrations of nafcillin induce changes in the cell wall resulting in a pronounced increase in susceptibility to lysis by lysozyme and proteolytic enzymes. Data presented demonstrate that this property of nafcillin is dose dependent, and the relatively low concentration needed to influence lysis significantly, approximately 1/80th the minimum inhibiting concentration, suggests that we are dealing with a specific and primary action on cell wall synthesis. The enhancement in lytic response observed during the logarithmic phase of growth is consistent with the hypothesis that an impairment of cross-linking in mucopeptide synthesis occurs following exposure to nafcillin. Since heat treatment (60 °C, 20 min) of nafcillin-treated cells considerably reduces lysis, the rate and extent must depend in part on the activity of one or more autolytic factors. Enzymic lysis is also enhanced by addition of sodium bicarbonate to control and nafcillin-treated suspensions, a phenomenon interpreted as a reversible bicarbonate-induced effect on cell permeability.

Effect of sublethal concentrations of penicillins on the lysis of bacteria by lysozyme and trypsin.

Resistance of Staphylococcus aureus and other bacteria to lysis by lysozyme and trypsin is abolished in cultures grown in sublethal concentrations of antibiotics. Antibiotics which inhibit synthesis of the mucopeptide polymer of bacterial cell walls differ widely in their ability to influence the lysis of cell suspensions. Such differences are revealed by both the speed of reduction of turbidity and final level of turbidity reduction. Under the influence of nafcillin, cells of Staphylococcus aureus, Streptococcus faecalis and Diplococcus pneumoniae are readily lysed but Neisseria catarrhalis, Sarcina lutea and Gaffkya tetragena are resistant, suggesting that bacterial cells differ in resistance or susceptibility to enzymic lysis. Lysis is not conditioned by the in vitro antibacterial activity of the antibiotics; nafcillin, which has the same activity against Staphylococcus aureus as oxacillin, cloxacillin, cephalothin, novobiocin and vancomycin, gives a greater rate and extent of lysis than the other antibiotics when used in comparable concentrations. Of the antibiotics interfering with protein synthesis, only erythromycin gives a significant lytic response. The results suggest that sublethal levels of antibiotics may mediate a favorable interplay between the host tissue environment and the parasite.

PRODUCTION OF A POLYSACCHARIDE BY STAPHYLOCOCCUS AUREUS. 3. ACTION OF PENICILLINS AND POLYSACCHARIDES ON ENZYMIC LYSIS.

Summary1. The resistance of S. aureus to lysis by lysozome and trypsin was abolished by exposing growing cells to subinhibitory concentrations of nafcillin. This observation, together with the results of electron microscopic studies of nafcillin-treated cells exposed to maximal enzymatic action, reflect an actual interference with cell wall synthesis. 2. Although the precise cytological action of the subinhibitory levels of the penicillins on the integrity of the cell wall is not known, the limited lytic action of lysozyme on cells treated with oxacillin and cloxacillin, as well as the resistance of these cells to further lysis by trypsin, suggest at least a quantitative difference between nafcillin and the other penicillins in the disorganization of the cell wall structure. 3. On the basis of the experiments described, evidence is presented which indicates that subinhibitory levels of nafcillin produce an alteration of cell wall structure, thereby increasing permeability and rendering the cell wall and p...

酵素による細菌細胞の融解

酵素による細菌細胞の融解

Cell structure and the enzymic lysis of bacteria.

SUMMARY: Untreated cells of three Gram-negative and four Gram-positive bacteria were resistant to lysis by crude and crystallbe trypsin. The resistance of the Gram negative organisms to lysis by trypsin was abolished by heating suspensions for 5 min. at 100° the rates and extent of lysis of the three organisms by crude trypsin were comparable. After maximal lysis of heated suspensions of Bacterium coil and Pseudomonas fluorescens, by crude trypsin, examination of the residual material in the electron microscope showed that cel walls were the principal components. When heated suspensions of Micrococcus lysodeikticus, Bacillus megaterium and Staphylococcus aurm were incubated with crude or crystalline trypsin, slow lysis occurred and the extent of lysis was less than that observed for the Gram-negative bacteria. Electron microscopic examination of trypsin-lysed cells of B. megMum showed incomplete digestion of cytoplasmic contents. Heated and autochved cells of Streptococcus faecalis were resistant to digestion with trypsin. Cell-wall suspensions of B. meguterium were rapidly lysed on incubation with crystalline lysozyme. Heated cells of M. lysodeikticus were only partially lysed by lysozyme, but the subsequent addition of trypsin resulted in a rapid dissolution of the cell bodies. Although treatment of heated B. megaterium cells with lysozyme resulted in turbidity increases, the enzyme completely digested the cell wall leaving coagulated protoplasmic bodies. The protoplasmic bodies were rapidly lysed by crude trypsin.