Continuous culture studies on the production of staphylocoagulase by Staphylococcus aureus

The production of staphylocoagulase was studied with continuous cultures of various S. aureus strains in a simple salts medium supplemented with mannitol, casein hydrolysate and three vitamins. Conditions of low oxygen availability and magnesium-limitation were required for optimal steady-state staphylocoagulase production. It was demonstrated that the specific rate of staphylocoagulase production was dependent on the growth rate.In two bovine strains, the production rate pattern was similar to that of an inducible enzyme sensitive to catabolite repression, although no specific inductor or repressor could be demonstrated. The human strain, on the other hand, produced staphylocoagulase constitutively. In all strains the specific rate of production of total extracellular protein was strictly proportional to the growth rate. The bovine strains produced 6 times more staphylocoagulase in chemostat culture as compared with batch cultures of the same organisms.It is likely that mannitol functioned as an energy source rather than as a carbon source because it was converted for a major part to acetate and for a minor part to lactate and not to new cell material. Repression of staphylocoagulase production by mannitol, acetate or lactate was not observed. The probable nature of the regulating mechanism(s) underlying staphylocoagulase production is discussed.     

Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Characteristics of Penicillinase Secretion by Growing Cells and Protoplasts of Bacillus licheniformis

Cultures of the inducible penicillinase-producing strain 749 of Bacillus licheniformis, induced with small amounts of benzylpenicillin, synthesized penicillinase at a high rate for a short period, after which the rate of synthesis slowly declined. During the period of active synthesis, the rate of secretion, as a fraction of the level of cell-bound penicillinase (which is originally high), gradually decreased to a constant level. Chloramphenicol, at a concentration (40 mug/ml) which completely inhibited synthesis of penicillinase, partially inhibited secretion if added during the period of active synthesis. During the phase of reduced synthesis, chloramphenicol was without effect on secretion. Penicillinase secretion, by actively growing cultures of the constitutive penicillinase-producing mutant 749/C, was inhibited by 75% immediately after addition of chloramphenicol. The secretion of part of the penicillinase released during active growth is probably dependent on synthesis of penicillinase, but part of the secreted penicillinase can be released in the absence of synthesis. Protoplasts were obtained from which periplasmic penicillinase has been removed, and these protoplasts were capable of substantial growth and penicillinase synthesis without lysis. At pH 7.5, there was no net incorporation of penicillinase into the cell membrane; the enzyme released was almost entirely of the exo form and was roughly equivalent to the amount of new enzyme formed. At pH 6.0, there was some incorporation of penicillinase into the plasma membrane, and approximately half of the extracellular penicillinase was in the exo form; the remainder perhaps represented membrane fragments. In the presence of chloramphenicol, a small amount of penicillinase was released at pH 7.5 as the exo form; at pH 6.0, practically none was released. We suggest that, with the removal from protoplasts of the periplasmic penicillinase-containing particles, a restriction on secretion has been lifted.     

Isolation and characterization of staphylocoagulase chymotryptic fragment. Localization of the procoagulant- and prothrombin-binding domain of this protein

Several strains of Staphylococcus aureus secrete a protein, staphylocoagulase, that binds stoichiometrically to human prothrombin, resulting in a coagulant complex designated staphylothrombin. In the present study, staphylocoagulase was digested with alpha-chymotrypsin and the resulting fragments were isolated by gel filtration. One fragment (Mr 43,000) exhibited a high affinity for human prothrombin (Kd = 1.7 X 10(-9) M), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 4.6 X 10(-10) M). A complex of the Mr 43,000 fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. A second fragment (Mr 30,000) exhibited weaker affinity for prothrombin (Kd = 1.2 X 10(-7) M). While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. A third fragment (Mr 20,000) was found to bind to prothrombin, but the resultant complex did not exhibit clotting or amidase activity. Amino-terminal sequence analyses of these staphylocoagulase fragments revealed that the Mr 43,000 fragment constitutes the amino-terminal portion of staphylocoagulase and also contains the Mr 30,000 and 20,000 fragments. Moreover, the amino-terminal sequence of the Mr 20,000 fragment was identical to that observed for the Mr 30,000 fragment. From these results, we conclude that the functional region of staphylocoagulase for binding and activation of human prothrombin is localized in the amino-terminal region of the intact bacterial protein.     

Heterologous expression of a thermophilic esterase in <Emphasis Type="Italic">Kluyveromyces</Emphasis> yeasts

In the present work, a thermophilic esterase from Thermus thermophilus HB27 was cloned into Kluyveromyces marxianus and into Kluyveromyces lactis using two different expression systems, yielding four recombinant strains. K. lactis showed the highest esterase expression levels (294 units per gram dry cell weight, with 65% of cell-bound enzyme) using an episomal system with the PGK promoter and terminator from Saccharomyces cerevisiae combined with the K. lactis k1 secretion signal. K. marxianus showed higher secretion efficiency of the heterologous esterase (56.9 units per gram dry cell weight, with 34% of cell-bound enzyme) than K. lactis. Hydrolytic activities for the heterologous esterases were maximum at pH values between 8.0 and 9.0 for both yeast species and at temperatures of 50 °C and 45 °C for K. marxianus and K. lactis, respectively. When compared to previously published data on this same esterase produced in the original host or in S. cerevisiae, our results indicate that Kluyveromyces yeasts can be considered good hosts for the heterologous secretion of thermophilic esterases, which have a potential application in biodiesel production or in resolving racemates.     

Production,characteristics and applications of the cell-bound phytase of <Emphasis Type="Italic">Pichia anomala</Emphasis>

Among several yeasts isolated from dried flowers of Woodfordia fruticosa, Pichia anomala produced a high titre of cell-bound phytase. The optimization of fermentation variables led to formulation of media and selection of cultural variables that supported enhanced phytase production. The enzyme productivity was very high in fed batch fermentation in air-lift fermentor as compared to that in stirred tank fermentor. Amelioration in the cell-bound phytase activity was observed when yeast cells were permeabilized with Triton-X-100. The enzyme is thermostable and acid stable with broad substrate specificity, the characteristics that are desirable for enzymes to be used in the animal feed industry. The phytase-encoding gene was cloned and sequenced. The 3D structure of the enzyme was proposed by comparative modeling using phytase of Debaryomyces occidentalis (50% sequence identity) as template. When broiler chicks, and fresh water and marine fishes were fed with the feed supplemented with yeast biomass containing phytase, improvement in growth and phosphorus retention, and decrease in the excretion of phosphorus in the faeces were recorded. The cell-bound phytase of P. anomala could effectively dephytinize wheat flour and soymilk.     

β-Galactosidase production by the psychrotolerant yeast Guehomyces pullulans 17-1 isolated from sea sediment in Antarctica and lactose hydrolysis

A psychrotolerant yeast Guehomyces pullulans 17-1 was isolated from sea sediment in Antarctica. It was found that it could yield both extracellular and cell-bound β-galactosidase. After optimization of the medium and cultivation conditions, it was found that the yeast strain produced over 17.2 U mL⁻¹ of β-galactosidase activity within 120 h at the flask level while the yeast strain produced over 25.3 U mL⁻¹ of β-galactosidase activity within 144 h during the 2-L fermentation. This is the highest β-galactosidase activity produced by the wild type yeast strains reported so far. However, the optimal pH and temperature for the crude β-galactosidase were 4.0 and 50 °C, respectively. Lactose could be converted into glucose and galactose and a large amount of reducing sugar could be released from milk under catalysis of the yeast culture.     

Incorporation of fucose in the intact heart and dissociated heart cells of the chick embryo

The incorporation of [³H]fucose into cell-bound and medium-released TCA-precipitable fractions was determined in intact hearts and dissociated heart cells of the 4-day chick embryo. The amount of released label was found to be much greater in the dissociated cells than in intact hearts both in absolute quantities and in proportion to cell-bound label.     

Surface-Bound Nuclease of Staphylococcus aureus: Localization of the Enzyme

The cellular localization of staphylococcus nuclease, previously known as an exoenzyme, was investigated, and the following results were obtained. (i) When Staphylococcus aureus cells were converted to protoplasts by cell wall lytic enzyme L-11 (a bacteriolytic enzyme purified from Flavobacterium sp. which specifically hydrolyzes amide and peptide linkages of murein layers), over 80% of the cell-bound nuclease was released into the surrounding sucrose medium. (ii) The cell-bound nuclease was associated with the cell-wall membrane fraction of mechanically disrupted cells. (iii) The nuclease activity of cell-wall membrane fractions from cells during early and late stages of protoplast formation were compared. Less activity was found in the late stage. These results suggest that nuclease may be located at or near the surface of the cells. The distribution of cell-bound nuclease in the cell-wall membrane fraction varied with the growth conditions of S. aureus. The activity of alkaline phosphatase, another surface enzyme, was also investigated. Less of this enzyme than nuclease was released when the cells were converted to protoplasts.     

Immunoregulatory role of Ig isotypes. I. Induction of contrasuppressor T cells for contact sensitivity responses by antibodies of the IgM, IgG1, and IgG3 isotypes

A profound state of specific tolerance for the contact sensitivity reaction can be produced by i.v. exposure to hapten on the surface of syngeneic macrophages. When the same haptenated cells are incubated with specific antibody to form cell-bound Ag-antibody complexes, i.v. injection induces immunity rather than tolerance. We observe that such cell-bound Ag-antibody complexes induce not only effector cells for contact sensitivity but also hapten-specific contrasuppressor T (Tcs) cells, which are capable of rendering effector cells resistant to the inhibitory effects of Ts cells. Whereas the induction of the effector cells of contact sensitivity by cell-bound complexes required I region compatibility between the injected cells and the recipient, the induction of Tcs cells showed no genetic restriction. On the other hand, induction of contrasuppression required intact Fc on the complexed antibody, inasmuch as F(ab')2 fragments of specific antibody did not induce immunity. In addition, Tcs cells could also be induced by Ag-antibody complexes on opsonized TNP-mouse RBC treated with anti-TNP antibody. Immunity induced by cell-bound Ag-antibody complexes was observed only when antibodies of the IgM, IgG3, or IgG1 isotypes are used to generate the complexes. Further studies demonstrated that the Tcs cells induced in this way displayed the phenotype of Tcs cells described in other systems (Lyt-1+,2- I-J+, Vicia villosa lectin-adherent) and released a hapten-specific contrasuppressor factor. These studies indicate that Tcs cells can be induced independently of other T cells (such as the effector cells of contact sensitivity) and are likely to be responsible for some of the immunoregulatory effects of cell-bound Ag-antibody complexes. The role of antibody isotype in the induction of Tcs cells is discussed.     

Characteristics of penicillinase release by washed cells of Bacillus licheniformis

Saline-washed cells of Bacillus licheniformis strain 749/C (constitutive for penicillinase) were able to release exopenicillinase in the presence of concentrations of chloramphenicol that prevented protein synthesis completely. The release reaction was strongly pH-dependent, occurring at a faster rate at alkaline pH in anionic or cationic buffers than at neutral pH. A strongly pH-dependent release reaction was noted in growing cells also. The reaction in washed cells can be stopped completely by changing the pH to 6.0. Within 30 min at pH 9.0, about 55% of the cell-bound penicillinase was released; thereafter, release continued at a greatly reduced rate. Suspensions of washed cells retained their capacity to release penicillinase at pH 9.0 for 90 min. Penicillinase released at pH 9.0 from either cells or protoplasts was not readsorbed over a 60-min period after changing the pH to 6.0. The release reaction was strongly temperature-dependent. We examined the effect of a large number of metabolic inhibitors and other compounds on the pH-dependent release phenomenon. Quinacrine hydrochloride, chloroquine diphosphate, and chlorpromazine hydrochloride reduced secretion substantially at 10(-4)m. Deoxycholate and Triton X-100 were active at 10(-3)m, but tungstate, arsenate, and molybdate had small effects at 10(-1)m. The rate of exopenicillinase release at pH 9.0 from fully stabilized protoplasts was one-half that of intact cells. Protoplasts lysed in hypotonic media or detergents showed even greater reduction in releasing activity. Penicillinase released from washed cells at pH 7.5 or 9.0 appeared to be derived from the periplasmic tubule and vesicle fraction that was released by protoplast formation.     

Extracellular Peptidases in Group N Streptococci used as Cheese Starters

Strains of Streptococcus cremoris which utilized peptides had a mercaptoethanol-sensitive, Mg²⁺-activated dipeptidase which could be released from the cells by lysozyme. A similar enzyme was inferred to be present in Strep. lactis. An apparently cell wall-bound peptidase was only found in Strep. lactis ; this was also Mg²⁺-activated but was insensitive to mercaptoethanol. The peptidase recovered from culture-supernatants of Strep. cremoris and Strep. lactis was similar to one of the two EDTA-sensitive intracellular dipeptidases present in both organisms, but was distinct from the lysozyme-released enzymes. The significance of these cell-bound extracellular peptidases in relation to peptide utilization is discussed.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Occurrence of a thermoacidophilic cell-bound exo-pectinase inAlicyclobacillus acidocaldarius

Alicyclobacillus acidocaldarius was able to degrade pectin under thermoacidophilic conditions of high temperature and acidity. Both extracellular and cell-bound pectolytic activities were found (28 and 72% of total activity, respectively). WhenA. acidocaldarius was subjected to lysozyme or sonication, more than 50% of the activity was found to be bound with the cell debris. The cell-bound enzyme presented principally exopectolytic activity. SDS-PAGE and zymogram showed that the estimated molar mass of the crude enzyme was 52 kDa. pH optimum was between 1.5 and 2.0 and the enzyme was thermostable at 70°C for 1 h at pH 2.0.     

A novel biocatalytic approach to acetylation of 1-β-<Emphasis Type="SmallCaps">d</Emphasis>-arabinofuranosylcytosine by <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> whole cell in organic solvents

Biocatalytic acylation of 1-β-d-arabinofuranosylcytosine (ara-C) was developed using whole cell of Aspergillus oryzae as a novel catalyst. ¹³C nuclear magnetic resonance (NMR) analysis indicated that the whole-cell biocatalyst had more specific activity toward the 3′-hydroxyl group than 5′-hydroxyl group among the available hydroxyl groups in sugar moiety of ara-C. Except for glucose and maltose, 11 carbon sources supplemented to basal media, including Spans, Tweens, olive oil and oleic acid, exhibited notable enhancement effects on both the cell growth and the acylation reactions. It was suggested that the carbon sources containing controlled-release oleic acid were the important substrates for the production of fungal cell-bound lipase with specific activity, partially due to a gradual induction effect of their released oleic acid on the cell-bound lipase production. Despite the low initial reaction rate and substrate conversion, the addition of 2.0 g/l Span 80 resulted in a higher 3′-regioselectivity of the cells than 81%. By using Tween 85 at its optimum concentration of 5.0 g/l, however, the highest initial rates (3.2 mmol/l h) and substrate conversion (76%) of the whole-cell catalyzed acylation of ara-C can be achieved. It was also found that the 3′-regioselectivity of the cells showed observable increase by extending the culture time. And the activity of cell-bound lipase drastically increased in the early stage of cell growth and then declined in the late culture stage, whatever the culture media used. Our results thus indicated that A. oryzae whole cell was a promising green tool for biosynthesis of nucleoside esters with potential bioactivities.     

The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins. Received: 17 February 1998 / Accepted: 30 June 1998     

In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where, together with fibrin and platelets, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.     

Factors Affecting the Activity and Stability of Alkaline Phosphatase in a Marine Pseudomonad

Conditions optimum for the assay of alkaline phosphatase of marine pseudomonad B-16 (ATCC 19855) and for maintaining the activity of the enzyme have been determined. The pH for optimal activity of the cell-bound enzyme was 9.0, whereas that for the enzyme after its release from the cells exceeded 9.4. Release was effected by first washing the cells in 0.5 M NaCl and then suspending them in 0.5 M sucrose. In the absence of salts, the activity of the cell-bound enzyme decreased rapidly at 25 C and less rapidly at 4 C. This loss of activity could be arrested but not restored by adding Mg(2+). In the presence of Na(+), activity of the cell-bound enzyme dropped to about 50% of that prevailing initially, but in this case adding Mg(2+) restored enzyme activity completely. The activity of the enzyme after its release from the cells into 0.5 M sucrose was approximately 50% of that of the equivalent amount of enzyme in the original cells. This activity was relatively stable at both 25 and 4 C. Adding Mg(2+) to the released enzyme restored its activity to that of the cell-bound form. The synthesis of alkaline phosphatase by the cells was not affected by adding 50 mM inorganic phosphate to the growth medium. The K(m) of the released enzyme for p-nitrophenyl phosphate was found to be 6.1 x 10(-5) M.     

Production of acid and alkaline phosphatases byMyxococcus coralloides

Acid and alkaline phosphatase ofMyxococcus coralloides were examined during vegetative growth in a liquid medium. Two extracellular phosphatases and two cell-bound phosphatases, acid and alkaline in both cases, were produced. The phosphatase production was unaltered by the presence of high concentrations of inorganic phosphate. Both enzymes were produced constitutively. These two hydrolases were released into the growth medium during the exponential growth phase (approximately 10% of total activity). The production of these enzymes was modified by the presence of organic acids and metal ions in the medium.     

Protein secretion in phosphate-limited cultures of Bacillus subtilis 168

The secretion of proteins from Bacillus subtilis was studied under physiologically well-defined conditions in continuous cultures at a range of specific growth rates. The kinetics of secretion was analysed by using pulse-chase and immunoprecipitation techniques that allowed both processing and release to be monitored. Growth conditions were selected that were known to lead to significant changes in the anionic polymer composition of the cell wall. Under magnesium limitation only low levels of native proteins were released into the growth medium. In contrast, much higher amounts of released protein were observed under phosphate limitation. Although synthesis of native secretory proteins appeared to be highly regulated, only minor changes in the secretion of heterologous proteins were detected. Comparable kinetics of protein release of cells grown under different conditions indicated similar cell wall permeabilities. The large changes in the amounts of released proteins were not reflected in the production of chaperones and components required for protein secretion. The data suggest that the capacity of the secretion machinery is not a major limiting step in the export of native secretory proteins. Received: 23 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997