Purification and properties of staphylococcal beta hemolysin. II. Purification of beta hemolysin

Staphylococcal beta hemolysin from the 681 strain of Staphylococcus aureus grown in a Heart Infusion dialysate semisolid medium under 10% carbon dioxide was obtained in an immunoelectrophoretically pure form by a combination of procedures of precipitation with 2 volumes of acetone followed by chromatography on diethylaminoethyl cellulose at pH 6.0. The acetone precipitation procedure did not show any deleterious effect on the hemolytic activity of the beta hemolysin unless the precipitate was left in contact with the acetone for at least 4 hr. The crude preparations contained two types of beta hemolysin. One of these represented the major portion of the total activity of beta hemolysin and behaved as a cation. The other represented a minor (1/5,000) portion of the total beta hemolysin activity and behaved as an anion. These active principles were designated as cationic and anionic beta hemolysins, respectively. An unexpected increase in the total beta hemolysin activity of the crude preparations was noted when these were concentrated by dialysis against polyethylene glycol (20 m). This effect was probably due to polyethylene glycol. A further unexpected increase in the titer of the acetone-precipitated preparations occurred when these were lyophilized. The reason for this incremental increase is not known. It may be due to fragmentation of the beta hemolysin.     

Isolation and characterization of hemolysin activated by reductant from Prevotella intermedia

The hemolysin from Prevotella intermedia was partially purified from culture supernatant and then characterized. The hemolysin produced a clear beta-hemolytic zone on a blood agar plate. Hemolytic activity was 2.5-fold greater in culture supernatant compared to that cell-associated. The isolation and purification procedure involved ammonium sulfate and polyethylene glycol precipitations and ion-exchange chromatographies on DEAE-Sephacel and CM-Sepharose. The activity of this hemolysin was stimulated by reductants such as cysteine, dithiothreitol, glutathione etc., and was lost upon oxidation. Trypsin or heat treatment resulted in complete inhibition of hemolytic activity. Ca(2+), Mg(2+) and EDTA did not affect the activity. The optimal pH of this hemolysin was 7.5.     

Purification and Some Properties of Crystalline Acid Protease from Acrocylindrium sp.

A crystalline acid protease produced by a strain of Acrocylindrium in a submerged culture was prepared by treatment with acetone (60%), salting out with ammonium sulfate (saturated) and, after chromatography on Duolite GS-101 column, dialysis against distilled water. This preparation was homogeneous on sedimentation analysis, starch-gel electrophoresis and gel filtration with Sephadex G-75. The optimum pH was 2.0 for milk casein digestion and the pH stability was for 2.0~5.0 at 30°C for one day. The crystalline enzyme was completely stable below 50°C, but lost the activity at 70°G in ten minutes. The acid protease was almost equal to pepsin on specific activity when milk casein solution (pH 2.0) was used as substrate.     

A hemolysin from the mushroom Pleurotus eryngii

A monomeric 17-kDa hemolysin designated as eryngeolysin was isolated from fresh fruiting bodies of the mushroom Pleurotus eryngii, using a protocol that involved gel filtration on Superdex 75, ion exchange chromatography on Mono Q and gel filtration on Superdex 75. Its N-terminal sequence demonstrated striking homology to that of its counterparts ostreolysin from the oyster mushroom Pleurotus ostreatus and aegerolysin from the mushroom Agrocybe cylindracea. Its hemolytic activity was unaffected over the pH range 4.0–12.0, but no activity was observed at pH 13 and at and below pH 2. The hemolysin was stable between 0 and 30 °C. At 40 °C, only residual activity was detectable. At and above 50 °C, activity was indiscernible. Eryngeolysin exhibited cytotoxicity toward leukemia (L1210) cells but not toward fungi. The hemolysin was inactivated by treatment with trypsin. It exhibited antibacterial activity against Bacillus sp. but not against other species. It inhibited basal as well as ConA-stimulated mitogenic response of murine splenocytes. N-Glycolyneuraminic acid was the only sugar capable of inhibiting the hemolytic activity. Eryngeolysin-induced hemolysis was osmotically protected by polyethylene glycol (PEG) 10000 with a mean hydrated diameter dose to 9.3 nm. However, no protection was offered by PEG 10000 to the anti-mitogenic and antiproliferative activities of eryngeolysin. The susceptibility of erythrocytes from different classes of vertebrates to eryngeolysin was mammalian > avian > reptilian > piscine.     

Expression of an iron-regulated hemolysin by Edwardsiella tarda

Abstract The ability of Edwardsiella tarda to hemolyse red blood cells was investigated. Most E. tarda strains (> 80%) produced a hemolysin when assayed by either an agar overlay or contact-dependent hemolysis technique. This activity was cell-associated (CAH) and not released into the culture supernatant under routine conditions. When quantified, E. tarda strains significantly produced 30–40-fold higher levels of hemolytic activity against guinea pig, sheep, or rabbit erythrocytes than either E. hoshinae or E. ictaluri . When grown under iron restricted-conditions in the presence of ethylenediamine di( o -hydroxyphenylacetic acid), hemoglubin, hematin and hemin were found to stimulate growth in both liquid and agar bioassays. Hemolysin activity could be released from selected E. tarda strains when grown in L broth supplemented with EDDA; hemolytic activity was 3- to > 40-fold under these conditions when compared to L broth alone. Preliminary characterization of the hemolysin of strain ET-13 indicates that it is a heat-labile protein with active sulphydryl and thiol groups. These results indicate that, in addition to its invasive capabilities, E. tarda produces a hemolysin which is at least partially regulated by the relative availability of iron and may play a role in human disease.     

Purification and characterization of cloacin DF13 receptor from Enterobacter cloacae and its interaction with cloacin DF13 in vitro.

Extraction of the crude cell envelope fraction of cloacin DF13-susceptible Enterobacter cloacae strain 02 with Triton X-100 and ethylenediaminetetraacetate solubilized an outer membrane fraction which neutralized the lethal activity of cloacin DF13. A similar fraction could not be isolated from strains known to be lacking functional cloacin DF13 receptors. On this basis the isolated outer membrane fraction was assumed to contain the specific cloacin DF13 receptor. The receptor was purified to homogeneity by acetone precipitation and affinity chromatography, using cloacin DF13 as a ligand. The purified receptor was identified as a protein which consisted of a single polypeptide chain with an apparent molecular weight of 90,000 and a preponderance of acidic amino acids (pI = 5.0). The interaction of equimolar amounts of purified receptor and cloacin DF13 in vitro resulted in a complete, irreversible neutralization of the lethal activity of the bacteriocin. This interaction showed a temperature optimum at 43 degrees C but was only slightly affected by variation of the pH between 5.0 and 8.5 or by increasing the ionic strength of the incubation buffer. The receptor had no neutralizing activity towards other bacteriocins, such as colicin E1 or colicin E3.     

Characterization of rhizosphere colonization by luminescent Enterobacter cloacae at the population and single-cell levels.

A bioluminescence marker system was used to characterized colonization of the rhizosphere by a bacterial inoculum, both in terms of population activity and at the single-cell level. Plasmid pQF70/44, which contains luxAB genes under the control of a strong constitutive phage promoter, was introduced into the rhizobacterium and model biocontrol agent Enterobacter cloacae. Light output from the lux-modified strain was detected by luminometry of samples from growing cultures of E. cloacae and from inoculated soil and wheat root samples. The minimum detection limits for fully active cells under optimum conditions were 90 and 445 cells g-1 for liquid culture and soil, respectively. The metabolic activities of the lux-marked population of E. cloacae, characterized by luminometry, contrasted in rhizosphere and nonrhizosphere soil. Cells in the rhizosphere were active, and there was a linear relationship between light output and cell concentration. The activity of cells in nonrhizosphere coil could not be detected unless the soil was supplied with substrate. Novel use of a charge-coupled device is reported for the spatial characterization of rhizosphere colonization by E. cloacae (pQF70/44) at the single-cell and population levels. Used macroscopically, the charge-coupled device identified differences in colonization due to competition from indigenous soil organisms. The lux-marked bacterium was able to colonize all depths of roots in the absence of competition but was restricted tot he spermosphere in the presence of competition (nonsterile soil).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of termolabile enterotoxin of Enterobacter cloacae on phagocytic activity of mice peritoneal macrophages

Influence of termolabile enterotoxin (LT-enterotoxin) of Enterobacter cloacae on functional activity of mice peritoneal macrophages was studied and following combinations were used: LT-enterotoxin-producing E. cloacae, its isogenic pair--LT-enterotoxin non-producing E. cloacae, supernatantof broth culture containing LT-enterotoxin, and physiological salt solution (in control group). Data showing decrease in phagocytic and lysosomal activity, disorder in functions of hexosemonophosphate shunt enzymes in peritoneal phagocytes were obtained.     

Purification and Genetic Determination of Bacteriocin Production in Enterobacter cloacae

Enterobacter cloacae (strain DF13) was found to produce a bacteriocin which could be induced by mitomycin C. In the supernatant fluid of the induced culture phagelike particles were found. The bacteriocin was partially purified from induced cultures by ammonium sulfate precipitation and gel-filtration on Sephadex G-150. Ultraviolet-absorbing material was eluted from the Sephadex column in three fractions. The biological activity was mainly present in the second fraction and is associated with a protein with a molecular weight of about 61,000. The phagelike particles were found in the first fraction and show no biological activity. Upon conjugation of E. cloacae strain DF13 with another strain of the same species and with Escherichia coli K-12S, the ability to produce bacteriocin was transferred. The new bacteriocinogenic strain produced bacteriocin, which could not be distinguished from that produced by E. cloacae strain DF13. Although transfer of the bacteriocinogenic factor often occurred together with transfer of the ability to produce phagelike particles, it was shown that these two factors are two separate genetic entities. In addition to a bacteriocinogenic factor, E. cloacae strain DF13 was found to carry two other transferable plasmids: one determining resistance against streptomycin and sulfanilamide and another determining resistance against penicillin.     

alpha-Hemolysin of E. coli]

The activity of alpha-hemolysin increased at the log growth phase in the culture of E. coli P678 Hly+ hemolytic strain; this activity diminished with the change into the stationary phase, and then fell sharply. Replacement of the culture medium in the stationary growth phase by fresh one led to restoration of the hemolytic activity of the culture. The culture fluid separated from the cells at the stationary growth phase produced an inhibitory action on the alpha-hemolysin Ca ions activated and stabilized the alpha-hemolysin. Sodium citrate and sucrose served as hemolysis inhibitors. The action of alpha-hemolysin was maximal against human erythrocytes at pH 6.5. Hemolytic activity was characterized in time by a distinct lag-phase and the phase of the greatest rate of reaction. The duration of the lag-phase and also the rate of hemolysis depended on the concentration of alpha-hemolysin (with the increase of the hemolysin concentration lag-phase was shortened and the reaction was accelerated). There proved to be a linear relationship between the amount of erythrocytes taken into the reaction and the rate of hemoglobin release, and also there was noted a temperature activation of the hemolytic reaction.     

Evidence for the production of a proteinaceous hemolytic exotoxin by wild-type strain of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 (Cyanobacteria)

A proteinaceous hemolysin produced by a wild-type strain of Synechocystis sp. PCC 6803 was purified from cell-free culture supernatants by successive column chromatography on DEAE-Sepharose Fast Flow and Sephacryl S-300 High Resolution. The molecular mass of the hemolysin, determined by SDS-PAGE, was approximately 81 kDa. The hemolysin was heat labile and showed potent hemolytic activity against rabbit and sheep erythrocytes. The hemolysin started to be secreted during the exponential growth phase and accumulated maximally at the stationary phase. The production of hemolysin varied with the amount of calcium present in modified BG-11 culture medium. Hemolysin production decreased in calcium-free medium, whereas it increased in medium containing 0.48 mM calcium. In contrast, the potency of hemolysin, as shown by hemolysis assay, was enhanced by deprivation of calcium (EDTA treatment) but decreased in the presence of calcium. Our results show that calcium stimulated production and secretion of hemolysin, but inhibited hemolytic potency.     

Two novel bacterial biosensors for detection of nitrate availability in the rhizosphere

The nitrate-regulated promoter of narG in Escherichia coli was fused to promoterless ice nucleation (inaZ) and green fluorescent protein (GFP) reporter genes to yield the nitrate-responsive gene fusions in plasmids pNice and pNgfp, respectively. While the promoter of narG is normally nitrate responsive only under anaerobic conditions, the L28H-fnr gene was provided in trans to enable nitrate-dependent expression of these reporter gene fusions even under aerobic conditions in both E. coli DH5alpha and Enterobacter cloacae EcCT501R. E. cloacae and E. coli cells containing the fusion plasmid pNice exhibited more than 100-fold-higher ice nucleation activity in cultures amended with 10 mM sodium nitrate than in nitrate-free media. The GFP fluorescence of E. cloacae cells harboring pNgfp was uniform at a given concentration of nitrate and increased about 1,000-fold when nitrate increased from 0 to 1 mM. Measurable induction of ice nucleation in E. cloacae EcCT501R harboring pNice occurred at nitrate concentrations of as low as 0.1 microM, while GFP fluorescence was detected in cells harboring pNgfp at about 10 microM. In the rhizosphere of wild oat (Avena fatua), the whole-cell bioreporter E.cloacae(pNgfp) or E. cloacae(pNice) expressed significantly higher GFP fluorescence or ice nucleation activity when the plants were grown in natural soils amended with nitrate than in unamended natural soils. Significantly lower nitrate abundance was detected by the E. cloacae(pNgfp) reporter in the A. fatua rhizosphere compared to in bulk soil, indicating plant competition for nitrate. Ice- and GFP-based bacterial sensors thus are useful for estimating nitrate availability in relevant microbial niches in natural environments.     

Purification and characterization of a hemolysin of Vibrio mimicus that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.     

A Salmonella typhimurium strain genetically engineered to secrete effectively a bioactive human interleukin (hIL)-6 via the Escherichia coli hemolysin secretion apparatus

Human interleukin-6 (hIL-6) cDNA was genetically fused with the Escherichia coli hemolysin secretorial signal ( hlyA_S ) sequence in a plasmid vector. Recombinant E. coli XL-1 Blue and attenuated Salmonella typhimurium secreted a 30 kDa hIL-6-HlyA_S fusion protein, with an additional form of higher apparent molecular mass produced by S. typhimurium . In S. typhimurium cultures hIL-6-HlyA_S concentrations entered a plateau at 500 to 600 ng ml⁻¹ culture supernatant. In contrast to E. coli XL-1 Blue, in S. typhimurium culture supernatants hIL-6-HlyA_S was accumulated faster reaching three-fold higher maximal concentrations. The cell proliferating activity of hIL-6-HlyA_S fusion protein(s) was equivalent to that of mature recombinant hIL-6. Furthermore, hIL-6-secreting S. typhimurium were less invasive than the attenuated control strain. Therefore, the bulky hemolysin secretorial peptide at the C-terminus of the fusion protein does not markably affect hIL-6 activity, suggesting that the hemolysin secretion apparatus provides an excellent system to study immunomodulatory effects of in situ synthesized IL-6 in Salmonella vaccine strains.     

An improved medium for preparation of filterable Escherichia coli hemolysin

We found that Tween 80 has potent activity in enhancing passage of E. coli hemolysin through a membrane filter and, on the basis of this finding, we devised a medium, heart infusion broth supplemented with 0.5% Tween 80 and 0.1% glucose (HI-TG) for preparation of culture filtrates containing fairly large amounts of hemolysin. When 27 strains of hemolytic E. coli were cultivated to late logarithmic growth phase in the HI-TG broth at 37°C, the culture filtrates of most strains contained 64 to 128 HU₅₀ (50% hemolytic unit) per ml. From these and other results, we established a routine method for partially purifying E. coli hemolysin.     

Effect of pH oscillations on a competing mixed culture

Two microorganisms, E. coli and S. cerevisiae, competing for glucose were maintained in a stable cycle of coexistence by alternating the growth advantage between the two organisms by oscillating the pH in a Chemostat. Pure culture experiments found S. cerevisiae to be insensitive to pH between 5 and 4.3 with a maximum specific growth rate (mu(max)) of 0.4/hr; while mu(max) of E. coli decreased from 0.6 h(-1) at pH 5 to 0.1 h(-1) at pH 4.3. Steady-state and cross-inoculation chemostat runs at a dilution rate of 0.17 h(-1) confirmed the expectation that the mixed culture system is unstable at constant pH with E. coli dominating at pH 5 and S. cerevisiae dominating at pH 4.3. Three pH oscillation experiments were performed at D =0.17 h(-1) with 1 g per liter glucose feed. The 16 h/16 h cycle was stable for six periods with a stable alternating cycle of E. coli and S. cerevisiae being quickly established. A 18 h pH 5/14 h pH 4.3 cycle was found to be stable with smaller yeast concentrations. A 6 h/6 h cycle was found unstable with yeast washout. Simulation results were compared with these runs and were used to predict the onset of instability. Oscillations of pH can force stable persistence of a competing mixed culture that is otherwise unstable. Thus, varying conditions are experimentally demonstrated to be one explanation for competitive coexistence.     

Purification and characterization of hemolysin from Porphyromonas gingivalis A7436

Porphyromonas gingivalis, a periodontal pathogen, has the ability to lyse erythrocytes. The hemolytic activity of P. gingivalis A7436 was purified as a 45-kDa protein from the culture supernatant of a 3-days old culture using nickel-nitrilotriacetic acid chromatography. Erythrocytes treated with purified P. gingivalis hemolysin showed the presence of pores and extracellular debris by scanning electron microscopy. Active immunization of mice with 15 micrograms hemolysin induced neutralizing antibodies to hemolysin. Heating at 60 degrees C and treatment with trypsin and dithiothreitol abolished hemolytic activity, while incubation with the protease inhibitor Na-p-tosyl-L-lysine chloromethyl ketone caused no effect. We report here for the first time purification of a hemolysin from P. gingivalis A7436. The amino acid sequence of an internal peptide of hemolysin showed sequence similarity with fimbrillin from P. gingivalis HG564. However, the amino acid composition of purified hemolysin was different from that of P. gingivalis fimbrillin. Also, the ability to lyse but not agglutinate erythrocytes and to bind to nickel-nitrilotriacetic acid differentiates P. gingivalis hemolysin from fimbrillin.     

Host-dependent, thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae.

The thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae, was studied. pJY5 consisted of 61 million daltons of covalently closed circular (CCC) deoxyribonucleic acid (DNA) with a buoyant density of 1.714 g/cm3 (55 mol % guanine plus cytosine). In Escherichia coli, this plasmid replicated stringently at 32 degrees C, but ceased its CCC DNA replication after a short incubation at 42 degrees C, resulting in production of R- segregants. The thermosensitive replication of pJY5 was not overcome by the coexistence of non-thermosensitive R plasmids. The plasmid manifested an inhibitory effect on host bacterial cell growth at 42 degrees C, although the effect was less prominent than that of R plasmids belonging to the T-incompatibility group, Rts1, R401, and R402. When the pJY5 plasmid was transferred into E. cloacae, however, no R- segregants were detected at any culture temperature, even 42 degrees C. Alkaline sucrose gradient analysis revealed that a significant amount of pJY5 CCC DNA was synthesized in E. cloacae at the high temperature but not in E. coli. Furthermore, the growth-inhibitory effect of pJY5 on hosts at 42 degrees C was not observed in E. cloacae. On the other hand, Rts1 and R401 were found to be thermosensitive in E. cloacae as well as in E. coli.     

Production and characteristics of hemolysins of Escherichia coli

Snyder, Irvin S. (University of Iowa, Iowa City), and Nancy A. Koch. Production and characteristics of hemolysins of Escherichia coli. J. Bacteriol. 91:763-767. 1996.-Filterable and nonfilterable hemolysins were produced by Escherichia coli in beef-heart infusion, casein hydrolysate, and chemically defined media. Differences were found among the three media in the time of appearance and in the relationship between production of these two hemolysins. The nonfilterable hemolysin produced in the three media, as well as the filterable hemolysin produced in the beef-heart infusion medium, were destroyed in 1 hr at 56 C. The filterable hemolysin produced in the casein hydrolysate and chemically defined media was unaffected by exposure to 56 C for 1 hr. Formalin inactivated the hemolysins produced in all three media. The optimal pH for activity of the nonfilterable hemolysin varied with time of production, whereas the optimal pH for the filterable hemolysin was constant. Certain carbohydrates were required for the production of filterable hemolysin by E. coli grown in casein hydrolysate and chemically defined media.