Partial Purification and Characterization of a Bacteriolytic Enzyme Secreted by Tetrahymena*

Synopsis. Tetrahymena pyriformis strain HSM secretes large quantities of acid hydrolases into the culture medium. An enzyme secreted by the ciliate and capable of degrading walls of streptococci was identified and purified to a considerable degree. The pH optimum of this enzyme was 3–4, and it was eluted after cytochrome c from Sephadex G-75 columns. Unlike lysozyme, the enzyme was thermolabile at pH 2.9, but relatively thermostable at pH 8.1. It degraded “C-labeled cell walls of streptococci releasing reducing groups. Cell walls prepared from different strains of streptococci differed in susceptibility to this enzyme, the most sensitive strain tested being of group A, type T12. It was shown in immunologic studies that this hydrolase released the group-specific carbohydrate from the walls. Secretions of Tetrahymena from early stationary-phase cultures had more bacterio-lytic activity than those from cells from late stationary-phase cultures. Further, cells from cultures grown in glucose-supplemented medium secreted less of the enzyme than ciliates of comparable age grown in unsupplemented proteose-peptone. The newly isolated bacteriolytic enzyme, presumably of lysosomal origin, may be helpful in characterizing streptococcal cell walls.     

Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100.

We have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving [14C]taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three- to fivefold higher than in cells from cultures grown in medium free of bile salts. In cell-free extracts, however, the activity was about equal, 1.41 and 1.53 mumol/min per mg of protein, respectively, whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two- to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Both had similar substrate specificities, highest on taurodeoxycholic and glycocholic acid, and pH optima between 3.8 and 4.5. The kinetic properties were also similar, with Vmaxs of 17 and 53 micromoles/min per mg of protein and Kms of 0.76 and 0.95 mM taurocholic acid for A and B, respectively. Therefore, whether the enzyme exists in two forms in the cells remains to be determined.     

A bacteriolytic muramidase from the basidiomycete Schizophyllum commune

The basidiomycete Schizophyllum commune produces an extracellular bacteriolytic enzyme when grown on heat-killed cells of Bacillus subtilis as sole C, N and P source. The enzyme catalyses the dissolution of isolated B. subtilis cell walls at an optimum pH of 3.2-3.4, releasing muramyl reducing groups, which indicates that it is a muramidase. Although low levels of enzyme activity are present when the fungus is grown in the absence of bacteria, full enzyme production appears to be induced by bacterial cells and repressed by glucose. Whole bacteria are not lysed by the enzyme at pH 3.3, but are rendered osmotically fragile, and lyse when the pH is raised to 7 or higher. The muramidase is effective against several Gram-positive bacteria but did not lyse any of the Gram-negative species tested.     

Formation of Bacteriolytic Enzymes in Batch and Continuous Culture of Staphylococcus aureus

The formation of bacteriolytic enzymes of Staphylococcus aureus, with special reference to strain M18, was investigated under a variety of conditions. The bacteriolytic activity was tested by using whole cells of Micrococcus lysodeikticus as a substrate. Complex media were required for production, and a Casein Hydrolysate-Yeast Extract medium (CCY(I)) was superior to Brain Heart Infusion and Trypticase Soy Broth. The optimal pH level for production was 7.0. Effective oxygenation and exchange of the beta-glycerophosphate of the CCY(I) medium for glucose increased the rates of growth and autolysis and the rate of appearance of extracellular bacteriolytic enzymes. However, the extracellular lytic activity decreased more rapidly at the end of the growth period than under the standard culture conditions. The appearance of inhibitor(s), probably derived from autolysis, might be responsible for this rapid decrease. The highest yields were obtained in a continuous process in which the activity was almost twice that of batch cultures grown under the same conditions. The bacteriolytic activity produced in continuous culture had a considerably increased stability in the purification process. The advantage of producing unstable bacterial proteins in continuous culture under controlled growth conditions is discussed.     

Relationship of Cellular Components to the Stability of Concentrated Lactic Streptococcus Cultures at -17 C

Concentrated cultures of lactic streptococci varied with respect to survival at -17 C. Cells of each strain grown at pH 6.0 were more stable to freezing than were those grown statically. The lipid fraction of the cells from static cultures was important in preventing death during freezing. As the percentage of octadecenoic acid in the cellular lipids from different cultures increased, the percentage of survivors decreased. Capsular material associated with cells from cultures grown both statically and at pH 6.0 was also important in protecting the cells at -17 C. The amount of capsular material, measured as percentage of cellular glucose, varied among the cultures tested. Cultures containing larger amounts of the capsular material were more resistant to the stress of freezing than those containing low levels.     

Autolysis of Lactococci: Detection of Lytic Enzymes by Polyacrylamide Gel Electrophoresis and Characterization in Buffer Systems

Lactococcal strains were screened for bacteriolytic activity against Micrococcus luteus cells, lactococcal cells, and cell walls. Thirty strains were screened for bacteriolytic activity against cells and cell walls incorporated into agar medium. Enzymes from all strains hydrolyzed the substrates; however, the activity against Micrococcus cells was much higher than against Lactococcus cells or cell walls. Electrophoretic profiles of bacteriolytic activities of culture supernatants, sodium dodecyl sulfate-treated cell extracts, cell wall fractions, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing M. luteus cells or lactococcal cell walls as the substrate. The 22 strains tested contained two to five lytic bands in the culture supernatant, ranging in size between 32 and 53 kDa. The cell extracts, the sodium dodecyl sulfate-treated cell extracts, and the cell wall fractions revealed two lytic bands of 47 and 53 kDa. Effects of external factors on autolysis of some strains were determined in buffer systems. Optimal autolysis was observed in the exponential growth phase at pH 6.0 to 7.5 and at a temperature of 30(deg)C. Two of three strains tested seemed to contain a glycosidase, and all three strains contained an N-acetylmuramyl-l-alanine amidase or an endopeptidase.     

Fratricide is essential for efficient gene transfer between pneumococci in biofilms

Streptococcus pneumoniae and a number of commensal streptococcal species are competent for natural genetic transformation. The natural habitat of these bacteria is multispecies biofilms in the human oral cavity and nasopharynx. Studies investigating lateral transfer of virulence and antibiotic resistance determinants among streptococci have shown that interspecies as well as intraspecies gene exchange takes place in these environments. We have previously shown that the action of a competence-specific murein hydrolase termed CbpD strongly increases the rate of gene transfer between pneumococci grown in liquid cultures. CbpD is the key component of a bacteriolytic mechanism termed the fratricide mechanism. It is secreted by competent pneumococci and mediates the release of donor DNA from sensitive streptococci present in the same environment. However, in nature, gene exchange between streptococci takes place in biofilms and not in liquid cultures. In the present study, we therefore investigated whether CbpD affects the rate of gene transfer in laboratory-grown biofilms. Our results show that the fratricide mechanism has a strong positive impact on intrabiofilm gene exchange, indicating that it is important for active acquisition of homologous donor DNA under natural conditions. Furthermore, we found that competent biofilm cells of S. pneumoniae acquire a Nov(r) marker much more efficiently from neighboring cells than from the growth medium. Efficient lysis of target cells requires that CbpD act in conjunction with the murein hydrolase LytC. In contrast, the major autolysin LytA does not seem to be important for fratricide-mediated gene exchange in a biofilm environment.     

High performance liquid chromatographic analysis of catecholamines in growing and non-growing Tetrahymena pyriformis

Tetrahymena pyriformis, strains NT-1 and W, harvested in logarithmic (growing) and stationary (non-growing) phases, were found by high-performance liquid chromatography to contain considerable quantities of dopamine. In addition, small amounts of epinephrine and norepinephrine were detected. Logarithmic-phase strain NT-1 cells contained 249±44 pg dopamine/10⁶ cells compared to 477±42 pg/10⁶ cells for logarithmic-phase strain W cells for logarithmic-phase strain W cells. The dopamine content of stationary-phase cells was approximately half the value of the logarithmic-phase cells. There was a significant amount of dopamine in the growth medium from stationary-phase cultures and, to a lesser extent, logarithmic-phase cells.     

Variation in the growth medium during the culture cycle of Tetrahymena: with special reference to ammonia (NH3), ammonium (NH4+), and pH1

The ciliate Tetrahymena pyriformis was grown in a peptone medium without added glucose. The interrelationship between increasing cell density and pH of the growth medium was studied from mid-log to the stationary phase, i.e. from 50,000 to 1,000,000 cells/ml, by continuous registration of the pH of the growth medium. The present findings correlate with the known physiological, biochemical, and structural changes occurring in Tetrahymena as it passes through the culture cycle. The ammonia production of the cells and the buffer capacity of the growth medium were determined throughout the growth cycle. The results revealed that the ammonia excreted by the cells can explain the increase in pH of the medium from 6.8 to about 8.3 normally seen during the culture cycle. Moreover, neither the increased pH nor the raised level of ammonia were found to be the responsible factor for cessation of cell proliferation in the stationary growth phase although these factors may affect cell proliferation in concentrations well beyond the range found in normal cultures.     

Spontaneous mutagenesis in exponentially growing and stationary-phase, umuDC-proficient and -deficient, Escherichia coli dnaQ49

Spontaneous mutations arise not only in exponentially growing bacteria but also in non-dividing or slowly dividing stationary-phase cells. In the latter case mutations are called adaptive or stationary-phase mutations. High spontaneous mutability has been observed in temperature sensitive Escherichia coli dnaQ49 strain deficient in 3'-->5' proofreading activity assured by the e subunit of the main replicative polymerase, Pol III. The aim of this study was to evaluate the effects of the dnaQ49 mutation and deletion of the umuDC operon encoding polymerase V (Pol V) on spontaneous mutagenesis in growing and stationary-phase E. coli cells. Using the argE3(OC) -->Arg+ reversion system in the AB1157 strain, we found that the level of growth-dependent and stationary-phase Arg+ revertants was significantly increased in the dnaQ49 mutant at the non-permissive temperature of 37 degrees C. At this temperature, in contrast to cultures grown at 28 degrees C, SOS functions were dramatically increased. Deletion of the umuDC operon in the dnaQ49 strain led to a 10-fold decrease in the level of Arg+ revertants in cultures grown at 37 degrees C and only to a 2-fold decrease in cultures grown at 28 degrees C. Furthermore, in stationary-phase cultures Pol V influenced spontaneous mutagenesis to a much lesser extent than in growing cultures. Our results indicate that the level of Pol III desintegration, dependent on the temperature of incubation, is more critical for spontaneous mutagenesis in stationary-phase dnaQ49 cells than the presence or absence of Pol V.     

Production and detection of muramidase and acetylglucosaminidase from Agaricus bisporus

The production and regulation of extracellular bacteriolytic enzymes of Agaricus bisporus are being studied to understand better the nutrition of this fungus and to identify factors that regulate the selectivity of mushroom compost as a growth medium. Both muramidase (EC.3.2.1.17) and N -acetyl-β- D -glucosaminidase (β-GlcNAcase, EC.3.2.1.30) have been detected in liquid cultures of A. bisporus , and in cultures fruiting in sterile and non-sterile compost. A turbidometric assay, based on the decrease in optical density of suspended Bacillus subtilis bacterial cell walls, was used to measure muramidase production by A. bisporus . A colorimetric assay was used to measure β-GlcNAcase. Both bacteriolytic enzyme activities were produced on a range of sole carbon sources, including killed freeze-dried B. subtilis cells. Muramidase activity was highest in axenic compost cultures. Bacteriolytic enzyme activity peaked as the first group of fruit bodies was harvested in both sterile and non-sterile compost.     

Amino acid compostion of walls from single and filamentous cells of Clostridium acidiurici

Gaffar, Abdul (Brigham Young University, Provo, Utah), David R. Terry, and Richard D. Sagers. Amino acid composition of walls from single and filamentous cells of Clostridium acidiurici. J. Bacteriol. 91:1618-1624. 1966.-The walls from single and filamentous cells of Clostridium acidiurici were shown to contain 11 amino acids: aspartic acid, serine, glutamic acid, proline, d-alanine, glycine, valine, methionine, valine, leucine, phenylalanine, and lysine. In the walls from cells grown at 37 C, d-alanine was the amino acid present in largest quantity, but in the walls from cells grown at 44 C there was a 50% reduction in the d-alanine content while the levels of the other amino acids were unchanged. Filamentous cells grown at 44 C, then brought to 37 C and transferred to fresh medium, fragmented into short cells within 30 min. Alanine racemase activity was the same in extracts from cells grown at both 37 and 44 C, suggesting that this enzyme was not the major controlling factor in the low content of d-alanine in filaments grown at 44 C. Spent medium from cultures grown at 44 C contained a significant amount of d-alanine, whereas there was no evidence of this amino acid in the spent medium from cultures grown at 37 C.     

Differential increase in activity of acid phosphatase induced by phosphate starvation in Tetrahymena.

We have studied the effects of phosphate starvation on the levels and distributions of activities of acid phosphatase and beta-hexosaminidase in cultures of Tetrahymena thermophila. The cells were grown in synthetic nutrient medium and refed every day with fresh medium. After 4 days of growth in the complete medium, the cultures were divided into two portions. One received complete medium and the other phosphate-free, but otherwise complete, medium. Population densities and activities of acid phosphatase and beta-hexosaminidase in cells plus medium and in cell-free samples were determined in aliquots removed every day before medium replacement. In cultures having complete medium the enzyme levels remained fairly constant; in the phosphate-starved cultures both total and extracellular activities of acid phosphatase increased sixfold. beta-Hexosaminidase levels remained essentially unaltered in both cases. These results indicate that phosphate starvation can induce differential increase in acid phosphatase activity in cultures of Tetrahymena. Somewhat less than 50% of the total activities of both enzymes are found in the cell-free extracellular fluid at any time.     

Ammonium uptake in carrot cell structures is influenced by pH-dependent cell aggregation

Semicontinuously grown wild carrot ( Daucus carota L.) cells were used in an investigation of the effect of culture medium pH on ammonium uptake in suspension cultures as a first step in exploring the relationship between pH and anthocyanin biosynthesis. In contrast to published data showing decreasing uptake rates with decreasing culture medium pH, ammonium-limited, semicontinuous carrot cell cultures showed a 25% greater ammonium uptake rate at pH 4.5 than at pH 5.5. When cells that had been grown semicontinuously in medium with a pH of 4.5 or 5.5 were grown in batch cultures at pH 4.5, 5.5 or 6.5 the ammonium uptake rates were those of the semicontinuous cultures, indicating that the pH of the batch culture medium had no effect on ammonium uptake rates over 7 days. The cell culture was composed of very small aggregates when it was grown semicontinuously in medium at pH 4.5, but was composed of large aggregates when it was grown semicontinuously in medium at pH 5.5. The aggregation/disaggregation of the cells was pH dependent, as changing the pH of the semicontinuous culture medium altered the extent of the aggregation. We conclude that the change in culture medium pH caused the cells to aggregate or disaggregate which in turn decreased or increased the rate of ammonium uptake from the medium.     

Cytochrome P-450 and the activation of promutagens in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the cellular content of cytochrome P-450 was investigated and shown to be related to the growth phase of aerobic cultures when glucose was the carbon source. When grown on glucose medium the log-phase cells of the diploid strain D5 contained about 9× more cytochrome P-450 than log-phase cells of the diploid strain D4. The D4 cells grown on medium containing glucose contained about 10× more cytochrome P-450 than D4 cell grown on medium containing galactose as carbon source. Cells of strain D4, harvested from log-phase cultures grown on glucose, were capable of metabolizing aflatoxin B₁, dimethylnitrosamine, β-naphthylamine, ethyl carbamate, cyclophosphamide and dimethylsulphoxide to products active genetically in the same cells. The metabolism of the compounds tested was attributed to cyctochrome P-450-dependent mixed-function oxidation since genetic activity was high in log cells grown on medium containing glucose but negligible in log cells grown on medium containing galactose. However, aflatoxin B₁ differed from the other promutagens tested since the genetic activity of this compound in cells grown on galactose medium was similar to the activity in cells grown on glucose medium. This result is discussed in relation to enzyme systems which could metabolize aflatoxin B₁. The results of treating log-phase cells of the strain D5, grown on medium containing glucose, with aflatoxin B₁ and dimethylnitrosamine are presented and compared with the results from the strain D4.     

Bacteriolytic enzymes from Staphylococcus aureus. Specificity of action of endo-β-N-acetylglucosaminidase

The bacteriolytic enzyme with an isoelectric point of 9.5 that is produced by all strains of Staphylococcus aureus investigated was purified from strain M18 (Wadström & Hisatsune, 1970). This enzyme released reducing groups from cell walls of Micrococcus lysodeikticus and was thus shown to be a bacteriolytic hexosaminidase. Although dinitrophenylation and acid hydrolysis of cell walls hydrolysed by a partially purified enzyme gave DNP-alanine and DNP-glycine from staphylococcal peptidoglycan, which indicated the presence of a peptidase and probably also an N-acetylmuramyl-l-alanine amidase, hydrolysis of cell walls by the extensively purified enzyme did not give any DNP-amino acids. The enzyme digest was purified by Amberlite CG-120 and Sephadex G-10 chromatography. Reduction by sodium borohydride of the disaccharide obtained was followed by acid hydrolysis and paper chromatography. Glucosamine completely disappeared after this treatment and a new spot identical with glucosaminitol appeared. The muramic acid spot remained unchanged. The purified enzyme was found to be devoid of exo-β-N-acetylglucosaminidase activity. These results are compatible with the action of a bacteriolytic endo-β-N-acetylglucosaminidase. It is also proposed that this enzyme is probably identical with the staphylococcal lysozyme. The mode of action of this has not previously been investigated.     

Host-Pathogen Interactions: II. Parameters Affecting Polysaccharide-degrading Enzyme Secretion by Colletotrichum lindemuthianum Grown in Culture

The effect of a number of physiological variables on the secretion of polysaccharide-degrading enzymes by culture-grown Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner was determined. The number of spores used to inoculate cultures grown on isolated bean hypocotyl cell walls affects the time after inoculation at which enzyme secretion occurs, but has no significant effect on the maximal amount of enzyme ultimately secreted. Cell walls isolated from bean leaves, first internodes, or hypocotyls (susceptible to C. lindemuthianum infection), when used as carbon source for C. lindemuthianum growth, stimulate the fungus to secrete more α-galactosidase than do cell walls isolated from roots (resistant to infection). The concentration of carbon source used for fungal growth determines the final level of enzyme activity in the culture fluid. The level of enzyme secretion is not proportional to fungal growth; rather, enzyme secretion is induced. Maximal α-galactosidase activity in the culture medium is found when the concentration of cell walls used as carbon source is 1% or greater. A higher concentration of cell walls is necessary for maximal α-arabinosidase activity. Galactose, when used as the carbon source, stimulates α-galactosidase secretion but, at comparable concentrations, is less effective in doing so than are cell walls. Polysaccharide-degrading enzymes are secreted by C. lindemuthianum at different times during growth of the pathogen on isolated cell walls. Pectinase and α-arabinosidase are secreted first, followed by β-xylosidase and cellulase, then β-glucosidase, and, finally, α-galactosidase.     

Purification and characterization of three separate bacteriolytic enzymes excreted by Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus saprophyticus.

As a further development of previous investigations showing that different staphylococcal species display different bacteriolytic activity patterns (lyogroups), the bacteriolytic enzymes excreted by three different Staphylococcus species, Staphylococcus aureus (lyogroup I), S. simulans (lyogroup II), and S. saprophyticus (lyogroup IV); have been purified and characterized. A representative strain from each species was grown in a preselected medium made of fully dialyzable products. Culture supernatants were collected in the appropriate growth phase. Two different affinity adsorbents were used for enzyme purification. One was obtained by coupling lysozyme-digested pure peptidoglycan from Micrococcus luteus to cyanogen bromide-activated Sepharose 4B. The second affinity adsorbent used was chitin. The S. aureus bacteriolytic enzyme bound to the solubilized peptidoglycan but not to chitin, whereas the opposite was true for the S. simulans enzyme. The bacteriolytic enzyme from S. saprophyticus did not bind to either the Sepharose 4B-peptidoglycan resin or to chitin, and its purification was achieved by two ion-exchange chromatography steps combined with gel filtration. All three enzymes were purified to apparent homogeneity. Their subsequent characterization indicated that all acted as endo-beta-N-acetylglucosaminidases. However, the three glucosaminidases differed significantly in their kinetics of activity and bacteriolytic spectrum against heat-killed cells of a variety of microorganisms. Very different values also resulted from molecular weight determinations: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme, and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their stability, optimal pH and ionic strength for their activity, and their responses to temperature and divalent cations. These results confirmed the previous proposal that different staphylococcal species excrete different lytic enzymes.     

Nitrogen fixation associated with the rice plant grown in water culture

Two bacteriolytic enzymes were produced when Hartmanella glebae was grown in the presence of both Enterobacter aerogenes and Alcaligenes faecalis. The identification of enzyme I as N-acetylmuramidase was reported earlier. Enzyme II was purified by gel filtration on a Bio-Gel A column. A recovery of 68.76% with 72.3-fold purification was obtained. It was found that 5 and 10 mM MgCl2 significantly increased the bacteriolytic activity. It is a basic protein. The cell walls of Micrococcus lysodeikticus were lysed by the enzyme, and the products of digestion were purified by Amberlite CG-120 and Sephadex G-15 chromatography to facilitate the detection of amino sugars. After reduction of the oligosaccharides with sodium borohydride and acid hydrolysis, the amino sugars were identified by paper chromatography. It was found that enzyme II cleaved the glycosidic bond between N-acetylmuramic and and N-acetylglucosamine of the peptidoglycan moiety of the cell walls. Thus, the enzyme was identified as endo-beta-N-acetylmuramidase.     

Extracellular Lytic Enzymes of Myxococcus virescens

An easy and rapid method for the purification of a bacteriolytic endopeptidase produced by Myxococcus virescens is described. The bacteria were grown in casitone media and the cells were sedimented by centrifugation. About 1.2 g of montmorillonite were added per liter of cell-free culture solution. The clay was sedimented by centrifugation and the enzyme was then eluted by 0.05 M Na-phosphate buffer pH 6.0, containing 0.4 M NaCl. The enzyme was diluted with water and chromatographed on carboxymethyl-cellulose columns. The purified enzyme liberated free amino groups but no reducing sugars or N-acetylhexosamines when acting on purified N-acetylated cell walls of Micrococcus lysodeikticus. Analysis of N- and C-terminal amino acids in the digestion products showed that the enzyme had liberated about 110 nmoles of lysine ε-amino groups and 60 nmoles of alanine carboxyl groups per mg of cell wall. When it acted on a bisdisaccharide pentapeptide dimer isolated from M. lysodeikticus cell walls, it cleaved about 30% of the alanyl-lysine linkages. Consequently the enzyme was an alanyl-lysine endopeptidase. It had no muramyl-alanine amidase activity.