Vol. 130, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Apr. 1977, p. 455-463 Copyright © 1977 American Society for Microbiology
Regulation of Galactose Oxidase Synthesis and Secretion in Dactylium dendroides: Effects of pH and Culture Density ALLAN R. SHATZMAN AND DANIEL J. KOSMAN* Bioinorganic Graduate Research Group, Departments of Biochemistry and Chemistry, State University of New York at Buffalo, Buffalo, New York 14214
Received for publication 13 October 1976
The effects of pH and growth density on the amount of an extracellular enzyme, galactose oxidase, synthesized by the fungus Dactylium dendroides were studied. Growth at a pH below 6.7 caused a decrease in the ability of the organism to release galactose oxidase. The enzyme retained by these fungal cells was liberated whenever the pH was raised to 7.0. Cycloheximide addition failed to inhibit the appearance of this protein; [3H]leucine added prior to pH adjustment was not incorporated into the released protein. These observations indicate the released protein is not newly synthesized protein. The retained enzyme would be secreted slowly over a 2-day period if the pH was not increased. In addition to regulating protein retention, pH was also shown to be associated with vacuolization, cell volume, culture density, and inhibition of protein synthesis. Cultures maintained at low pH were characterized by a dense growth consisting of highly vacuolated, buoyant, fungal hyphae. Increasing the pH from 6 to 7 caused a decrease in vacuole size. Cells grown at neutral pH maintained a lower density of growth and, based on activity measurements, synthesized 33% more galactose oxidase. Furthermore, cultures grown at pH 6.0 and maintained at a lower cell density produced galactose oxidase at a level similar to that of cells grown at neutral pH. Thus, the elevated density of the cell culture was inhibitory to galactose oxidase synthesis. The observed effects on protein synthesis and release were rather specific for galactose oxidase, since other extracellular proteins appeared in the earliest stages of growth. Many fungi secrete extracellular enzymes into their growth medium (1, 16). Although these enzymes have been studied extensively with respect to their isolation and characterization, the regulation of their synthesis and release has received far less consideration. However, the mechanisms of intracellular and extracellular protein syntheses per se are known to be identical (5), although the two protein classes are undoubtedly subjected to different post-translational processing events. Galactose oxidase (EC 1.3.3.9; GOase) is an extracellular copper-containing enzyme elaborated by several fungal genera (9). Although some difficulty has been encountered in the identification of specific species that produce this enzyme (19), it has been purified from shake flask cultures of Dactylium dendroides (Bulliard) Fries (2, 16). Except for that of Markus and co-workers (18) and Gancedo et al. (8), little effort has been made to detail either the biological function of this enzyme or the factors mediating its synthesis and release. Within the context of our work on the structure/function relationship in 455
copper-containing proteins, we initiated a study of the events leading up to the appearance of GOase in the growth medium of D. dendroides. In this report we describe experiments that focus on the control of GOase synthesis and the effectors of protein release. Our results are of practical use in isolating large amounts of GOase and may aid in isolating extracellular enzymes from other organisms. MATERIALS AND METHODS Terminology. "Release" refers to the appearance of GOase, quantitated by enzyme activity assay, in the extracellular environment. "Synthesis" refers to both intra- and extracellular GOase and indicates that cycloheximide and/or [3H]leucine was used to prove that the released protein was synthesized prior to the perturbation that stimulated the release. "Culture density" refers to the volume occupied by cells in 100 ml of fungal culture; i.e., 100 ml of culture may contain 10 ml of cells and 90 ml of growth medium. "Cell volume" refers to the actual size of the mycelium as seen by phase-contrast microscopy. Volume increases and decreases are associated with
456
SHATZMAN AND KOSMAN
changes in vacuole size and are also related to the ratio wet cell weight/dry cell weight. Growth of organism. The organism used in this work, D. dendroides (Bulliard) Fries (NRRL 2903), was provided by J. J. Ellis of the Northern Regional Research Lab, Peoria, Ill. This is the original Bacila (2) isolate formerly misidentified as Polyporus circinatus (19). Isolates were maintained on Sabouraud dextrose agar (Difco Laboratories) slants at 4°C. A starter flask containing a culture medium, similar to that described by Markus et al. (18), with dextrose (Fisher Scientific Co.) as the carbon source was inoculated from a stock slant and shaken at room temperature for 48 h. At this point, 12 2-liter shaker flasks, each containing 1.25 liters of medium with L(-)-sorbose (1%) (Sigma Chemical Co.) as the carbon source, were inoculated from the starter flask with a 1.5% (vol/vol) inoculum. The flasks were than shaken for 108 to 144 h at 180 rpm at 22°C. The pH was monitored with a Radiometer pH meter and adjusted to the desired pH by the addition of either concentrated NH4OH (Fisher) or concentrated H3PO4 (Baker). Ionic strength was varied by adding NaNO3 (Fisher). Thiamine hydrochloride (General Biochemicals) was substituted for yeast extract (Difco) in the preparation of an artificial medium. Preparation of mycelia. Cells were removed from shaker flasks to thin the growth as well as for examination and experimentation. Cells were filtered through four layers of cheesecloth and suspended in 0.1 M sodium phosphate (monobasic) adjusted to various pH values with NaOH. Mycelial weight determination. Wet weights were obtained by removing 100-ml samples from shaker flasks and centrifuging at 12,000 x g for 15 min. The resulting pellet, after removal of occluded medium by passage through cheesecloth, was weighed on a Mettler single-pan balance. To obtain dry weights, the mycelia were placed on a filter disk that had been dried previously to a constant weight. The mycelia were placed in a dryheat oven at 75°C until a constant weight was reached. Protein determination. Extracellular protein concentrations were determined by the biuret method (10) with bovine serum albumin (Sigma) as the standard. To measure only fungal extracellular protein, an artificial medium that had no endogenous protein was used. Inhibition of protein synthesis. Cycloheximide (Sigma, lot 54C-0332), at a concentration of 10 ,uM, was used to inhibit synthesis of fungal proteins. Protein labeling. [4,5-3H]leucine incorporation was measured on a Packard liquid scintillation spectrometer (model 3200). [3H]leucine was purchased from New England Nuclear Corp. (specific activity, 50 mCi/mmol). Labeling was attempted by adding 25 ,uCi of [3H]leucine to 500 to 1,250 ml of culture 5, 30, and 60 min before pH increase and subsequent precipitation of extracellular protein. The times were chosen to allow for transport of [3H]leucine into the cells without, however, substantial utilization in normal protein synthesis. Protein was precipitated with 7.5% ice-cold trichloroacetic acid, and after 30
J. BACTERIOL.
min the mixture was centrifuged at 10,000 x g for 10 min. The resulting pellet was dissolved in 0.1 ml of 0.1 M NaOH. This procedure was repeated two times after which the basic protein solution was neutralized by the addition of 0.9 ml of 0.2 M citric acid. This was then added to 10 ml of scintillation fluid (three parts xylene, one part Triton X-114,4 g of 2,5diphenyloxazole per liter) and stored in the dark for 30 min before counting. Assay for GOase activity. Enzyme activity was monitored by the method of Kosman et al. (16). One unit of activity is defined as the amount of GOase in 1 ml needed to bring about a change of 1 absorbance unit per min at 460 nm in a peroxidase-coupled oxidation of o-dianisidine. At 12-h periods, 0.2-ml samples were withdrawn from several flasks, pooled, and assayed to monitor the appearance of GOase in the growth medium. Determination of total extracellular GOase. To detect all GOase present extracellularly (inactive as well as active), immunoelectrophoretic studies were carried out on samples of the growth medium concentrated by ultrafiltration with an Amicon PM10 membrane. Agar-coated microscope slides were used by the method of Scheidegger (24). Antisera to GOase has been obtained from a goat that had been inoculated twice with purified GOase. Each subcutaneous inoculation consisted of 1 mg of enzyme in 1 ml of 0.1 M phosphate buffer (pH 7.0). One microliter of antiserum is equivalent to 3 U of precipitable GOase.
RESULTS
Quantitation of extracellular GOase. To quantitate the amount of GOase present in the extracellular fluid, immunoelectrophoresis was carried out on samples of media, and the results were quantitated by comparison to standard GOase solutions ranging in concentration from 0.25 to 2 mg/ml. Differences in precipitant arc area were sought since with this method the area of the precipitant arc is directly related to the concentration of the antigen. Any inactive GOase present, in addition to active GOase, would appear as an abnormally large precipitant arc (compared with that of the standard). At all concentrations tested, precipitant arcs of similar sizes were obtained for standards and medium samples. There was no evidence that extracellular GOase was present in any form other than the holoenzyme. This work was done assuming that all GOase forms present would be cross-reacting, since in a separate experiment apo- and holo-GOases were shown to cross-react equally with the antibody. Occurrence of GOase extracellularly and the effect of pH. GOase was released extracellularly throughout the entire 5- to 6-day growth period rather than at one specific phase of growth (Fig. 1). Although the amount of GOase release with respect to the entire culture in-
VOL. 130, 1977
C-)
#r
0
0 0
GFWTH TIME (days) FIG. 1. Influence of pH on the synthesis and release of GOase followed by enzymic activity in the growth medium. Arrow length indicates the amount of GOase released on pH increase. Cultures were grown at pH 7.0 (a), 6.7 (A), and 6.0 (0). Cultures grown at pH 6.0 were also treated with cycloheximide after 2 days of growth (a).
creased for the first 5 days of growth (Fig. 1), the rate (change in units per day) of release per milligram of mycelium actually decreased after its peak at 24 h of growth. These data parallel the results of Markus et al. (18). Extracellular GOase is stable in the growth medium over the growth period, since shaker flasks supplemented with GOase showed a surplus of activity at the end of the growth phase equal to the amount of GOase supplemented at time zero. The growth medium at the time of inoculation has a pH of 6.75. As noted by Markus et al. (18), during the first 48 h of growth, the pH decreased to 6.4 at which point it leveled off. To ascertain the effect of pH on GOase synthesis and mycelial growth, the organism was cultured in shaker flasks at pH 6.0 and 7.0, and the appearance of extracellular GOase was followed. Although GOase appeared extracellularly throughout growth at both pH values, the actual amount of enzyme released during each 24-h time period varied with the pH of the growth medium (Fig. 1). Release of GOase. Decreased levels of GOase were found in the extracellular medium of cells cultured below pH 7.0 compared with that of
457 cells grown at pH 7.0. To determine whether
GALACTOSE OXIDASE BIOSYNTHESIS
the pH was affecting synthesis or release of the enzyme, the pH of cells grown for at least 2.5 days at low pH was raised to 7.0 by dropwise addition of NH4OH. Within minutes of the increase in pH, there was a substantial increase in the extracellular activity of GOase. The enzyme activity in cell-free media was not changed by an increase in pH. On the other hand, phosphate buffer (pH 7.0) with no initial GOase activity showed 2 to 3 units of activity per ml soon after cells alone had been added. The protein assay showed that only GOase was being released, based on the specific activity of the pure enzyme (16). Cells had been washed previously in pH 6.0 phosphate buffer to remove any occluded GOase. Addition of cycloheximide (10 uM) did not inhibit the appearance of this additional activity. Increasing the pH above 7.0 gave no further enzyme release, nor did dropping the pH to 6.0 cause a decrease in activity at any time. Cells grown at pH 7.0 released no GOase upon an increase of pH. Sonic treatment at pH 7.0 of cells grown at all pH values showed that little GOase remained as intracellular protein after the spontaneous release. If the pH of the growth medium is not increased from 6.0 to 7.0 at the end of the growth cycle, the activity one sees immediately after increasing the pH appears slowly within the next 24 to 48 h. The cells grown at pH 6.0 for 96 h ceased release of GOase whether raised to pH 7.0 at that point or not. In fact, synthesis stopped at 72 h (Fig. 1). The earlier in the growth cycle that the pH was increased to 7.0, the greater the amount of overall synthesis and release (Fig. 2). Cells grown at pH 7.0 and then decreased to pH 6.0 before 72 h of growth showed a corresponding decrease in GOase levels. Accompanying the decreased time span of GOase synthesis in cells grown at low pH was the retention of the enzyme by the fungal cells. To examine the level of unreleased GOase and its release without change in pH, cycloheximide was added to inhibit further protein synthesis. At 48 h of growth, cycloheximide was added to cultures grown at pH 6.0 while other flasks were raised to pH 7.0. The culture raised to pH 7.0 increased in activity to 8.1 U/ml within 5 min, whereas those to which cycloheximide was added increased in activity to 8.1 U/ ml within 24 h (Fig. 1). These experiments were repeated at 24-h intervals and again showed that the fungal cells retain 1.5 to 2 U of GOase per ml from 48 through 108 h of growth after which time GOase synthesis comes to a halt and retained enzyme is gradually released.
458
J. BACTERIOL.
SHATZMAN AND KOSMAN
8
i6 E 4
d, 10) 0
6
23
6
4
4 2 0
~~2
GROWTH
4 3 TIME (days)
5
2 3 4 5 6 GROWTH TIME (dWyO FIG. 3. Growth patterns of D. dendroides expressed as a function of the wet weight of cells contained in a 100-ml sample from cultures grown at pH 7.0 (a) and 6.0 (U).
6
FIG. 2. Effect of pH increase at different time periods on GOase synthesis and secretion as followed by activity measurements. The pH of cultures was raised from 6.0 to 7.0 at time zero (0), 1 day (0), 2 days (0), 3 days (a), 4 days (A), and 5 days (A); pH 6.0 control (A).
That this released protein, after cycloheximide addition, pH increase, or termination of growth, is actually retained and not newly synthesized protein was indicated further by pulse labeling with [3H]leucine. Isotope added before pH increase did not appear in precipitable protein. Although 1 to 2 U of activity per ml appeared after 108 h of growth, addition of [3H]leucine at this time gave no isotope in the protein associated with this activity. Effect of pH on fungal cell morphology. Cells grown at pH 6.0 achieved greater cell volume and culture density than those grown at pH 7.0. Those grown at pH 6.0 were flocculant, buoyant, and leafy in appearance, whereas those grown at pH 7.0 had more of a ricelike appearance and tended to sink. Furthermore, cells grown at the lower pH were found to have a wet weight almost three times that of the pH 7.0 cells (Fig. 3). However, when dry weights were recorded, it was found that they were approximately the same for all cells (Fig. 4). Thus, the difference in culture density
O
1 2 3 4 5 6 GROWTH TIME (days)
FIG. 4. Growth patterns of D. dendroides expressed as a function of the dry weight of cells contained in a 100-ml sample from cultures grown at pH 7.0 (a) and 6.0 (A) in a normal yeast extract-containing medium, as well as from cultures grown in an artificial, thiamine-containing medium (U).
VOL. 130, 1977
is due to a greater cell volume for cells grown at pH 6.0. Microscopic examination of the fungi revealed that those grown at pH 6.0 had numerous large vacuoles, whereas those grown at pH 7.0 had comparatively tiny vacuoles, although slightly greater in number (Fig. 5). The release of enzyme upon raising the pH from 6 to 7 was accompanied by a distinct morphological change. Mycelia adjusted to pH 7.0 an hour before examination showed a closer resemblance to cells grown at pH 7.0 than those grown at 6.0 with respect to vacuolization. Efficiency of GOase synthesis. A correlation was made between the extracellular concentration of GOase and the dry weight of mycelium (efficiency of synthesis). Five-day cultures grown at pH 7.0 secreted about 30% more GOase per mg of mycelium than cells grown at pH 6.0 (Fig. 6). When cells were removed from pH 6.0 shaker flasks to maintain culture density at a level equal to that found in cultures grown at pH 7.0, the extracellular concentration of GOase per mg of mycelium was ultimately equal in both cases (Fig. 6). The depressed efficiency in the first 3 days of growth
GALACTOSE OXIDASE BIOSYNTHESIS
459
in the pH 6.0 cultures was thus due to increased retention of GOase rather than to decreased synthesis. When the density of pH 7.0 cultures was increased by removing the growth medium, GOase synthesis and secretion ceased earlier in growth, and the efficiency of synthesis decreased. The same occurred when cells were grown in flasks of smaller volumes. Total extracellular protein studies. In the first 24 h of growth, less than 0.5% of all protein appearing extracellularly was GOase. This represents about 50 ng of GOase per ml compared with 13.5 ,ug of other extracellular proteins per ml. After the initial 24-h period, the secretion of extracellular proteins decreased greatly, except for GOase, whose level in the culture increased sharply (Fig. 7). Over the 6-day growth period, the concentration of GOase in the extracellular medium became appreciable, accounting for over 11% of all extracellular protein (Fig. 8). An effort was made to determine what some of these other extracellular proteins might have been. Enzyme assays excluded f8-mannosidase, ,3-N-acetylglucosaminidase, 8-xylosidase, ,3glucosidase, glucose oxidase, laccase, and tyrosinase. The absence of the latter two enzymes is
FIG. 5. Phase-contrast micrographs of cells cultured at pH 7.0 (A) and 6.0 (B), accentuating differences in vacuolization. Mycelia magnified x300. Micrographs courtesy of James LaFountain.
460
J. BACTERIOL.
SHATZMAN AND KOSMAN
buffers (I = 0.32 and 0.62 M). In no instance did an increase in ionic strength alter the characteristics of the pH 6.0 cells. Enzyme retention and vacuolization were not affected. Thus, pH and not ionic strength modulates the properties of the cell culture.
z
EJ2 C-,
cm
_E
I
2 3 4 5 GROWTH TIME (days) FIG. 6. Efficiency of GOase secretion. The extracellular enzyme was quantitated in relation to the dry weight of the mycelium from cultures maintained at pH 7.0 (a) and 6.0 (A), as well as from thinned cultures grown at pH 6.0 (A).
not surprising inasmuch as they are associated with the sexual phase of this fungal genus (17). The percentage of new extracellular protein appearing each day, which was GOase, increased gradually through the first 4 days (Fig. 8). The rate at that time (33%) was maintained for the next 24-h period. Between days 5 and 6 of growth, all new extracellular protein was GOase, based on the known specific activity of the pure enzyme (16). Cycloheximide did not inhibit the appearance of the protein, and pulse labeling with [3H]leucine gave no 3H incorporation into precipitable protein. This indicates that the protein appearing extracellularly in this time period was synthesized prior to this time but remained cell associated. Ionic strength effects. Phosphate (Na) is the ion that makes the predominant contribution to the ionic strength of both the media and buffers used. The total phosphate concentration is ca. 0.1 M in both cases; thus the total molar ionic strength varied between 0.12 M (pH 6.0) and 0.34 M (pH 7.0). To ascertain whether ionic strength differences were responsible for the effects noted, cells were grown at pH 6.0 in the presence of NaNO3 (I = 0.32 M) and washed with high-ionic-strength (NaNO3) pH 6.0
FIG. 7. Extracellular protein production by D. dendroides. The secretion of GOase (a) and all other extracellular proteins (a) was followed by activity measurements and biuret protein determinations. These cultures were grown in artificial medium containing 10 pg of thiamine per ml and no endogenous amino acids or proteins.
I
Io0
0
0
0
x
c
a.
c,
0~
a-
C.)
4) U) a
0
0
H-
0
0
0D
a
a.
x
E w
'I
4-a 0
-
2 3 4 5 ( Growth Time (days) FIG. 8. Extracellular concentration of GOase in relation to other extracellular proteins. The percentages of all extracellular protein, which is GOase, at any point in the growth period (a) and the percentage of new extracellular protein, which is GOase, within each 24-h period (-) are shown.
461 shaker flasks whose pH 7.0 medium volume was reduced to 700 ml from the normal 1,250 ml between 24 and 48 h of growth ceased GOase synthesis between days 3 and 4 of growth. The efficiency of GOase synthesis decreased as well. When transferred to 700 ml of fresh medium these mycelia did not resume synthesis, indicating that nutrient depletion was not responsible for the observed effect, although a change in nutrient utilization cannot be ruled out. These results indicate that GOase synthesis is responsive to culture density. The conclusion that pH adjustment before 72 h is critical for the maintenance of GOase synthesis agrees with the finding that cell cultures grown at pH 6.0 reach an inhibitory level of cell density between 48 and 72 h of growth. Note that the culture density at pH 7.0 is never limiting with respect to GOase synthesis and that the inoculum size has a distinct effect on GOase synthesis (18). The greater the inoculum size, the smaller the amount of GOase produced. This decreased synthesis again may be attributed to the more rapid growth associated with the large inoculum, which naturally leads to a cell culture of greater density. The decrease in GOase synthesis with increasing culture density appears to be a relatively specific phenomenon, inasmuch as almost all of the extracellular proteins except for GOase are secreted prior to the achievement of inhibitory levels of culture density. The reason why GOase synthesis is inhibited remains a mystery, but deprivation of nutrients and oxygen is not responsible since the mycelia continue to grow for 24 to 48 h after GOase synthe-
GALACTOSE OXIDASE BIOSYNTHESIS
VOL. 130, 1977
DISCUSSION
The appearance of extracellular GOase accompanies the growth of D. dendroides at all stages, with the major portion being synthesized and secreted during the logarithmic phase of growth. The appearance of an extracellular enzyme in large quantity throughout the growth cycle is somewhat unusual, since many enzymes of this class, such as the bullulanases of Klebsiella aerogenes (3), catalase of Salmonella typhimurium (7), superoxide dismutase of Escherichia coli (13), and several proteases from Neurospora crassa (4), appear almost entirely during the stationary phase of the life cycle. Thus, GOase secretion by D. dendroides appears to be a normal cell process rather than a result of cell lysis. Other extracellular enzymes that do appear throughout the growth cycle, such as the invertase and aryl a-glucosidase of N. crassa (26), comprise a small percentage of the organisms' total extracellular protein complement in contrast to the relatively large amount of GOase synthesized by D. dendroides. One must show caution when comparing GOase with other "extracellular" proteins. Many enzymes are considered extracellular even though they are not secreted. Rather, they are located between the cell membrane and the cell wall. Such enzymes, considered extracellular because of their localization outside the cell membrane, are released only as a result of cell lysis or extraction with organic solvents (11). Most proteins found associated with the cell wall are glycoproteins containing at least 3 to 5% carbohydrate (5, 6). GOase, on the other hand, contains -
JOURNAL OF BACTERIOLOGY, Apr. 1977, p. 455-463 Copyright © 1977 American Society for Microbiology
Regulation of Galactose Oxidase Synthesis and Secretion in Dactylium dendroides: Effects of pH and Culture Density ALLAN R. SHATZMAN AND DANIEL J. KOSMAN* Bioinorganic Graduate Research Group, Departments of Biochemistry and Chemistry, State University of New York at Buffalo, Buffalo, New York 14214
Received for publication 13 October 1976
The effects of pH and growth density on the amount of an extracellular enzyme, galactose oxidase, synthesized by the fungus Dactylium dendroides were studied. Growth at a pH below 6.7 caused a decrease in the ability of the organism to release galactose oxidase. The enzyme retained by these fungal cells was liberated whenever the pH was raised to 7.0. Cycloheximide addition failed to inhibit the appearance of this protein; [3H]leucine added prior to pH adjustment was not incorporated into the released protein. These observations indicate the released protein is not newly synthesized protein. The retained enzyme would be secreted slowly over a 2-day period if the pH was not increased. In addition to regulating protein retention, pH was also shown to be associated with vacuolization, cell volume, culture density, and inhibition of protein synthesis. Cultures maintained at low pH were characterized by a dense growth consisting of highly vacuolated, buoyant, fungal hyphae. Increasing the pH from 6 to 7 caused a decrease in vacuole size. Cells grown at neutral pH maintained a lower density of growth and, based on activity measurements, synthesized 33% more galactose oxidase. Furthermore, cultures grown at pH 6.0 and maintained at a lower cell density produced galactose oxidase at a level similar to that of cells grown at neutral pH. Thus, the elevated density of the cell culture was inhibitory to galactose oxidase synthesis. The observed effects on protein synthesis and release were rather specific for galactose oxidase, since other extracellular proteins appeared in the earliest stages of growth. Many fungi secrete extracellular enzymes into their growth medium (1, 16). Although these enzymes have been studied extensively with respect to their isolation and characterization, the regulation of their synthesis and release has received far less consideration. However, the mechanisms of intracellular and extracellular protein syntheses per se are known to be identical (5), although the two protein classes are undoubtedly subjected to different post-translational processing events. Galactose oxidase (EC 1.3.3.9; GOase) is an extracellular copper-containing enzyme elaborated by several fungal genera (9). Although some difficulty has been encountered in the identification of specific species that produce this enzyme (19), it has been purified from shake flask cultures of Dactylium dendroides (Bulliard) Fries (2, 16). Except for that of Markus and co-workers (18) and Gancedo et al. (8), little effort has been made to detail either the biological function of this enzyme or the factors mediating its synthesis and release. Within the context of our work on the structure/function relationship in 455
copper-containing proteins, we initiated a study of the events leading up to the appearance of GOase in the growth medium of D. dendroides. In this report we describe experiments that focus on the control of GOase synthesis and the effectors of protein release. Our results are of practical use in isolating large amounts of GOase and may aid in isolating extracellular enzymes from other organisms. MATERIALS AND METHODS Terminology. "Release" refers to the appearance of GOase, quantitated by enzyme activity assay, in the extracellular environment. "Synthesis" refers to both intra- and extracellular GOase and indicates that cycloheximide and/or [3H]leucine was used to prove that the released protein was synthesized prior to the perturbation that stimulated the release. "Culture density" refers to the volume occupied by cells in 100 ml of fungal culture; i.e., 100 ml of culture may contain 10 ml of cells and 90 ml of growth medium. "Cell volume" refers to the actual size of the mycelium as seen by phase-contrast microscopy. Volume increases and decreases are associated with
456
SHATZMAN AND KOSMAN
changes in vacuole size and are also related to the ratio wet cell weight/dry cell weight. Growth of organism. The organism used in this work, D. dendroides (Bulliard) Fries (NRRL 2903), was provided by J. J. Ellis of the Northern Regional Research Lab, Peoria, Ill. This is the original Bacila (2) isolate formerly misidentified as Polyporus circinatus (19). Isolates were maintained on Sabouraud dextrose agar (Difco Laboratories) slants at 4°C. A starter flask containing a culture medium, similar to that described by Markus et al. (18), with dextrose (Fisher Scientific Co.) as the carbon source was inoculated from a stock slant and shaken at room temperature for 48 h. At this point, 12 2-liter shaker flasks, each containing 1.25 liters of medium with L(-)-sorbose (1%) (Sigma Chemical Co.) as the carbon source, were inoculated from the starter flask with a 1.5% (vol/vol) inoculum. The flasks were than shaken for 108 to 144 h at 180 rpm at 22°C. The pH was monitored with a Radiometer pH meter and adjusted to the desired pH by the addition of either concentrated NH4OH (Fisher) or concentrated H3PO4 (Baker). Ionic strength was varied by adding NaNO3 (Fisher). Thiamine hydrochloride (General Biochemicals) was substituted for yeast extract (Difco) in the preparation of an artificial medium. Preparation of mycelia. Cells were removed from shaker flasks to thin the growth as well as for examination and experimentation. Cells were filtered through four layers of cheesecloth and suspended in 0.1 M sodium phosphate (monobasic) adjusted to various pH values with NaOH. Mycelial weight determination. Wet weights were obtained by removing 100-ml samples from shaker flasks and centrifuging at 12,000 x g for 15 min. The resulting pellet, after removal of occluded medium by passage through cheesecloth, was weighed on a Mettler single-pan balance. To obtain dry weights, the mycelia were placed on a filter disk that had been dried previously to a constant weight. The mycelia were placed in a dryheat oven at 75°C until a constant weight was reached. Protein determination. Extracellular protein concentrations were determined by the biuret method (10) with bovine serum albumin (Sigma) as the standard. To measure only fungal extracellular protein, an artificial medium that had no endogenous protein was used. Inhibition of protein synthesis. Cycloheximide (Sigma, lot 54C-0332), at a concentration of 10 ,uM, was used to inhibit synthesis of fungal proteins. Protein labeling. [4,5-3H]leucine incorporation was measured on a Packard liquid scintillation spectrometer (model 3200). [3H]leucine was purchased from New England Nuclear Corp. (specific activity, 50 mCi/mmol). Labeling was attempted by adding 25 ,uCi of [3H]leucine to 500 to 1,250 ml of culture 5, 30, and 60 min before pH increase and subsequent precipitation of extracellular protein. The times were chosen to allow for transport of [3H]leucine into the cells without, however, substantial utilization in normal protein synthesis. Protein was precipitated with 7.5% ice-cold trichloroacetic acid, and after 30
J. BACTERIOL.
min the mixture was centrifuged at 10,000 x g for 10 min. The resulting pellet was dissolved in 0.1 ml of 0.1 M NaOH. This procedure was repeated two times after which the basic protein solution was neutralized by the addition of 0.9 ml of 0.2 M citric acid. This was then added to 10 ml of scintillation fluid (three parts xylene, one part Triton X-114,4 g of 2,5diphenyloxazole per liter) and stored in the dark for 30 min before counting. Assay for GOase activity. Enzyme activity was monitored by the method of Kosman et al. (16). One unit of activity is defined as the amount of GOase in 1 ml needed to bring about a change of 1 absorbance unit per min at 460 nm in a peroxidase-coupled oxidation of o-dianisidine. At 12-h periods, 0.2-ml samples were withdrawn from several flasks, pooled, and assayed to monitor the appearance of GOase in the growth medium. Determination of total extracellular GOase. To detect all GOase present extracellularly (inactive as well as active), immunoelectrophoretic studies were carried out on samples of the growth medium concentrated by ultrafiltration with an Amicon PM10 membrane. Agar-coated microscope slides were used by the method of Scheidegger (24). Antisera to GOase has been obtained from a goat that had been inoculated twice with purified GOase. Each subcutaneous inoculation consisted of 1 mg of enzyme in 1 ml of 0.1 M phosphate buffer (pH 7.0). One microliter of antiserum is equivalent to 3 U of precipitable GOase.
RESULTS
Quantitation of extracellular GOase. To quantitate the amount of GOase present in the extracellular fluid, immunoelectrophoresis was carried out on samples of media, and the results were quantitated by comparison to standard GOase solutions ranging in concentration from 0.25 to 2 mg/ml. Differences in precipitant arc area were sought since with this method the area of the precipitant arc is directly related to the concentration of the antigen. Any inactive GOase present, in addition to active GOase, would appear as an abnormally large precipitant arc (compared with that of the standard). At all concentrations tested, precipitant arcs of similar sizes were obtained for standards and medium samples. There was no evidence that extracellular GOase was present in any form other than the holoenzyme. This work was done assuming that all GOase forms present would be cross-reacting, since in a separate experiment apo- and holo-GOases were shown to cross-react equally with the antibody. Occurrence of GOase extracellularly and the effect of pH. GOase was released extracellularly throughout the entire 5- to 6-day growth period rather than at one specific phase of growth (Fig. 1). Although the amount of GOase release with respect to the entire culture in-
VOL. 130, 1977
C-)
#r
0
0 0
GFWTH TIME (days) FIG. 1. Influence of pH on the synthesis and release of GOase followed by enzymic activity in the growth medium. Arrow length indicates the amount of GOase released on pH increase. Cultures were grown at pH 7.0 (a), 6.7 (A), and 6.0 (0). Cultures grown at pH 6.0 were also treated with cycloheximide after 2 days of growth (a).
creased for the first 5 days of growth (Fig. 1), the rate (change in units per day) of release per milligram of mycelium actually decreased after its peak at 24 h of growth. These data parallel the results of Markus et al. (18). Extracellular GOase is stable in the growth medium over the growth period, since shaker flasks supplemented with GOase showed a surplus of activity at the end of the growth phase equal to the amount of GOase supplemented at time zero. The growth medium at the time of inoculation has a pH of 6.75. As noted by Markus et al. (18), during the first 48 h of growth, the pH decreased to 6.4 at which point it leveled off. To ascertain the effect of pH on GOase synthesis and mycelial growth, the organism was cultured in shaker flasks at pH 6.0 and 7.0, and the appearance of extracellular GOase was followed. Although GOase appeared extracellularly throughout growth at both pH values, the actual amount of enzyme released during each 24-h time period varied with the pH of the growth medium (Fig. 1). Release of GOase. Decreased levels of GOase were found in the extracellular medium of cells cultured below pH 7.0 compared with that of
457 cells grown at pH 7.0. To determine whether
GALACTOSE OXIDASE BIOSYNTHESIS
the pH was affecting synthesis or release of the enzyme, the pH of cells grown for at least 2.5 days at low pH was raised to 7.0 by dropwise addition of NH4OH. Within minutes of the increase in pH, there was a substantial increase in the extracellular activity of GOase. The enzyme activity in cell-free media was not changed by an increase in pH. On the other hand, phosphate buffer (pH 7.0) with no initial GOase activity showed 2 to 3 units of activity per ml soon after cells alone had been added. The protein assay showed that only GOase was being released, based on the specific activity of the pure enzyme (16). Cells had been washed previously in pH 6.0 phosphate buffer to remove any occluded GOase. Addition of cycloheximide (10 uM) did not inhibit the appearance of this additional activity. Increasing the pH above 7.0 gave no further enzyme release, nor did dropping the pH to 6.0 cause a decrease in activity at any time. Cells grown at pH 7.0 released no GOase upon an increase of pH. Sonic treatment at pH 7.0 of cells grown at all pH values showed that little GOase remained as intracellular protein after the spontaneous release. If the pH of the growth medium is not increased from 6.0 to 7.0 at the end of the growth cycle, the activity one sees immediately after increasing the pH appears slowly within the next 24 to 48 h. The cells grown at pH 6.0 for 96 h ceased release of GOase whether raised to pH 7.0 at that point or not. In fact, synthesis stopped at 72 h (Fig. 1). The earlier in the growth cycle that the pH was increased to 7.0, the greater the amount of overall synthesis and release (Fig. 2). Cells grown at pH 7.0 and then decreased to pH 6.0 before 72 h of growth showed a corresponding decrease in GOase levels. Accompanying the decreased time span of GOase synthesis in cells grown at low pH was the retention of the enzyme by the fungal cells. To examine the level of unreleased GOase and its release without change in pH, cycloheximide was added to inhibit further protein synthesis. At 48 h of growth, cycloheximide was added to cultures grown at pH 6.0 while other flasks were raised to pH 7.0. The culture raised to pH 7.0 increased in activity to 8.1 U/ml within 5 min, whereas those to which cycloheximide was added increased in activity to 8.1 U/ ml within 24 h (Fig. 1). These experiments were repeated at 24-h intervals and again showed that the fungal cells retain 1.5 to 2 U of GOase per ml from 48 through 108 h of growth after which time GOase synthesis comes to a halt and retained enzyme is gradually released.
458
J. BACTERIOL.
SHATZMAN AND KOSMAN
8
i6 E 4
d, 10) 0
6
23
6
4
4 2 0
~~2
GROWTH
4 3 TIME (days)
5
2 3 4 5 6 GROWTH TIME (dWyO FIG. 3. Growth patterns of D. dendroides expressed as a function of the wet weight of cells contained in a 100-ml sample from cultures grown at pH 7.0 (a) and 6.0 (U).
6
FIG. 2. Effect of pH increase at different time periods on GOase synthesis and secretion as followed by activity measurements. The pH of cultures was raised from 6.0 to 7.0 at time zero (0), 1 day (0), 2 days (0), 3 days (a), 4 days (A), and 5 days (A); pH 6.0 control (A).
That this released protein, after cycloheximide addition, pH increase, or termination of growth, is actually retained and not newly synthesized protein was indicated further by pulse labeling with [3H]leucine. Isotope added before pH increase did not appear in precipitable protein. Although 1 to 2 U of activity per ml appeared after 108 h of growth, addition of [3H]leucine at this time gave no isotope in the protein associated with this activity. Effect of pH on fungal cell morphology. Cells grown at pH 6.0 achieved greater cell volume and culture density than those grown at pH 7.0. Those grown at pH 6.0 were flocculant, buoyant, and leafy in appearance, whereas those grown at pH 7.0 had more of a ricelike appearance and tended to sink. Furthermore, cells grown at the lower pH were found to have a wet weight almost three times that of the pH 7.0 cells (Fig. 3). However, when dry weights were recorded, it was found that they were approximately the same for all cells (Fig. 4). Thus, the difference in culture density
O
1 2 3 4 5 6 GROWTH TIME (days)
FIG. 4. Growth patterns of D. dendroides expressed as a function of the dry weight of cells contained in a 100-ml sample from cultures grown at pH 7.0 (a) and 6.0 (A) in a normal yeast extract-containing medium, as well as from cultures grown in an artificial, thiamine-containing medium (U).
VOL. 130, 1977
is due to a greater cell volume for cells grown at pH 6.0. Microscopic examination of the fungi revealed that those grown at pH 6.0 had numerous large vacuoles, whereas those grown at pH 7.0 had comparatively tiny vacuoles, although slightly greater in number (Fig. 5). The release of enzyme upon raising the pH from 6 to 7 was accompanied by a distinct morphological change. Mycelia adjusted to pH 7.0 an hour before examination showed a closer resemblance to cells grown at pH 7.0 than those grown at 6.0 with respect to vacuolization. Efficiency of GOase synthesis. A correlation was made between the extracellular concentration of GOase and the dry weight of mycelium (efficiency of synthesis). Five-day cultures grown at pH 7.0 secreted about 30% more GOase per mg of mycelium than cells grown at pH 6.0 (Fig. 6). When cells were removed from pH 6.0 shaker flasks to maintain culture density at a level equal to that found in cultures grown at pH 7.0, the extracellular concentration of GOase per mg of mycelium was ultimately equal in both cases (Fig. 6). The depressed efficiency in the first 3 days of growth
GALACTOSE OXIDASE BIOSYNTHESIS
459
in the pH 6.0 cultures was thus due to increased retention of GOase rather than to decreased synthesis. When the density of pH 7.0 cultures was increased by removing the growth medium, GOase synthesis and secretion ceased earlier in growth, and the efficiency of synthesis decreased. The same occurred when cells were grown in flasks of smaller volumes. Total extracellular protein studies. In the first 24 h of growth, less than 0.5% of all protein appearing extracellularly was GOase. This represents about 50 ng of GOase per ml compared with 13.5 ,ug of other extracellular proteins per ml. After the initial 24-h period, the secretion of extracellular proteins decreased greatly, except for GOase, whose level in the culture increased sharply (Fig. 7). Over the 6-day growth period, the concentration of GOase in the extracellular medium became appreciable, accounting for over 11% of all extracellular protein (Fig. 8). An effort was made to determine what some of these other extracellular proteins might have been. Enzyme assays excluded f8-mannosidase, ,3-N-acetylglucosaminidase, 8-xylosidase, ,3glucosidase, glucose oxidase, laccase, and tyrosinase. The absence of the latter two enzymes is
FIG. 5. Phase-contrast micrographs of cells cultured at pH 7.0 (A) and 6.0 (B), accentuating differences in vacuolization. Mycelia magnified x300. Micrographs courtesy of James LaFountain.
460
J. BACTERIOL.
SHATZMAN AND KOSMAN
buffers (I = 0.32 and 0.62 M). In no instance did an increase in ionic strength alter the characteristics of the pH 6.0 cells. Enzyme retention and vacuolization were not affected. Thus, pH and not ionic strength modulates the properties of the cell culture.
z
EJ2 C-,
cm
_E
I
2 3 4 5 GROWTH TIME (days) FIG. 6. Efficiency of GOase secretion. The extracellular enzyme was quantitated in relation to the dry weight of the mycelium from cultures maintained at pH 7.0 (a) and 6.0 (A), as well as from thinned cultures grown at pH 6.0 (A).
not surprising inasmuch as they are associated with the sexual phase of this fungal genus (17). The percentage of new extracellular protein appearing each day, which was GOase, increased gradually through the first 4 days (Fig. 8). The rate at that time (33%) was maintained for the next 24-h period. Between days 5 and 6 of growth, all new extracellular protein was GOase, based on the known specific activity of the pure enzyme (16). Cycloheximide did not inhibit the appearance of the protein, and pulse labeling with [3H]leucine gave no 3H incorporation into precipitable protein. This indicates that the protein appearing extracellularly in this time period was synthesized prior to this time but remained cell associated. Ionic strength effects. Phosphate (Na) is the ion that makes the predominant contribution to the ionic strength of both the media and buffers used. The total phosphate concentration is ca. 0.1 M in both cases; thus the total molar ionic strength varied between 0.12 M (pH 6.0) and 0.34 M (pH 7.0). To ascertain whether ionic strength differences were responsible for the effects noted, cells were grown at pH 6.0 in the presence of NaNO3 (I = 0.32 M) and washed with high-ionic-strength (NaNO3) pH 6.0
FIG. 7. Extracellular protein production by D. dendroides. The secretion of GOase (a) and all other extracellular proteins (a) was followed by activity measurements and biuret protein determinations. These cultures were grown in artificial medium containing 10 pg of thiamine per ml and no endogenous amino acids or proteins.
I
Io0
0
0
0
x
c
a.
c,
0~
a-
C.)
4) U) a
0
0
H-
0
0
0D
a
a.
x
E w
'I
4-a 0
-
2 3 4 5 ( Growth Time (days) FIG. 8. Extracellular concentration of GOase in relation to other extracellular proteins. The percentages of all extracellular protein, which is GOase, at any point in the growth period (a) and the percentage of new extracellular protein, which is GOase, within each 24-h period (-) are shown.
461 shaker flasks whose pH 7.0 medium volume was reduced to 700 ml from the normal 1,250 ml between 24 and 48 h of growth ceased GOase synthesis between days 3 and 4 of growth. The efficiency of GOase synthesis decreased as well. When transferred to 700 ml of fresh medium these mycelia did not resume synthesis, indicating that nutrient depletion was not responsible for the observed effect, although a change in nutrient utilization cannot be ruled out. These results indicate that GOase synthesis is responsive to culture density. The conclusion that pH adjustment before 72 h is critical for the maintenance of GOase synthesis agrees with the finding that cell cultures grown at pH 6.0 reach an inhibitory level of cell density between 48 and 72 h of growth. Note that the culture density at pH 7.0 is never limiting with respect to GOase synthesis and that the inoculum size has a distinct effect on GOase synthesis (18). The greater the inoculum size, the smaller the amount of GOase produced. This decreased synthesis again may be attributed to the more rapid growth associated with the large inoculum, which naturally leads to a cell culture of greater density. The decrease in GOase synthesis with increasing culture density appears to be a relatively specific phenomenon, inasmuch as almost all of the extracellular proteins except for GOase are secreted prior to the achievement of inhibitory levels of culture density. The reason why GOase synthesis is inhibited remains a mystery, but deprivation of nutrients and oxygen is not responsible since the mycelia continue to grow for 24 to 48 h after GOase synthe-
GALACTOSE OXIDASE BIOSYNTHESIS
VOL. 130, 1977
DISCUSSION
The appearance of extracellular GOase accompanies the growth of D. dendroides at all stages, with the major portion being synthesized and secreted during the logarithmic phase of growth. The appearance of an extracellular enzyme in large quantity throughout the growth cycle is somewhat unusual, since many enzymes of this class, such as the bullulanases of Klebsiella aerogenes (3), catalase of Salmonella typhimurium (7), superoxide dismutase of Escherichia coli (13), and several proteases from Neurospora crassa (4), appear almost entirely during the stationary phase of the life cycle. Thus, GOase secretion by D. dendroides appears to be a normal cell process rather than a result of cell lysis. Other extracellular enzymes that do appear throughout the growth cycle, such as the invertase and aryl a-glucosidase of N. crassa (26), comprise a small percentage of the organisms' total extracellular protein complement in contrast to the relatively large amount of GOase synthesized by D. dendroides. One must show caution when comparing GOase with other "extracellular" proteins. Many enzymes are considered extracellular even though they are not secreted. Rather, they are located between the cell membrane and the cell wall. Such enzymes, considered extracellular because of their localization outside the cell membrane, are released only as a result of cell lysis or extraction with organic solvents (11). Most proteins found associated with the cell wall are glycoproteins containing at least 3 to 5% carbohydrate (5, 6). GOase, on the other hand, contains -
