Nov 24, 1993 - qualitative modifications of P3-D-glucosidase in marine snow. ... of horizontal gene transfer for such enzymes in natural eco- systems.
APPLIED AND ENVIRONMENTAI MICROBIOLOGY, Mar. 1994, 0099-2240/94/$04.00 +() Copyright © 1994, American Society for Microbiology
p.
Vol. 60, No. 3
807-813
Characteristics and Diversity of -D-Glucosidase (EC 3.2.1.21) Activity in Marine Snow JOHANNES RATH AND GERHARD J. HERNDL* Department of Marine Biology, Institute of Zoology, University of Vienna, Althanstrasse 14, 1090 Vielnna, Aulstria Received 12 July 1993/Accepted 24 November 1993
Large amorphous aggregates commonly described as marine snow were sampled in the water column of the northern Adriatic Sea in August 1991. P-D-Glucosidase activity associated with these aggregates was characterized. Enzymatic activity was measured with the fluorogenic substrate analog 4-methylumbelliferylP-D-glucoside and found to be mainly associated with particles; on average, only 24% of the whole ,-D-glucosidase activity remained in the supernatant after centrifugation. Although the temperature optimum for P-D-glucosidase activity was -40°C, incubation of the previously liberated particle-attached enzyme at 500C for 20 min caused a >90% reduction of enzymatic activity relative to the activity at 400C. The level of inactivation of P-D-glucosidase was much lower, however, when whole marine snow was incubated, indicating qualitative modifications of P3-D-glucosidase in marine snow. Separation of P-D-glucosidase by different approaches indicated that the diversity of isoenzymes is restricted. In samples taken from the pycnocline, only one major isoenzyme was present in noticeable amounts. This isoenzyme contributed up to 70% of the whole P3-D-glucosidase activity detectable by two different chromatographic separations (anion-exchange chromatography and size exclusion chromatography). Although the same isoenzyme was dominant in marine snow taken from surface waters (0.5-m depth), we found a second peak of activity which eluted at lower NaCl concentrations from the anion-exchange column. Generally, the diversity of isoenzymes exhibiting P-Dglucosidase activity seems to be surprisingly small in amorphous pelagic aggregates.
bacteria has frequently been cited (38). The occurrence in natural ecosystems and the potential role of 3-D-glucosidaseone component of this complex enzyme system-have been shown in a number of investigations (10, 14, 19, 23, 36, 39). Chrost (10) pointed out that f-D-glucosidase is predominantly produced by bacteria because its activity is associated mainly with the bacterium-sized fraction. The expression of 1-Dglucosidase in limnetic ecosystems was positively correlated with the expression of cellobiohydrolase-another enzyme of the cellulolytic system (26). On the regulatory level, r-Dglucosidase activity was induced by the presence of dissolved polymeric glucose (26). Competitive inhibition of P-D-glucosidase activity has been documented by the addition of glucose and cellobiose (10). On the genetic level, recombination and different alignments of the functional domains have played a central role in the evolution of the cellulolytic enzyme system (6). 1-D-Glucosidase can be divided into two molecular subfamilies on the basis of similarities in their catalytic domains (6). Members of very distinct phylogenetic groups are found within the same subfamily, raising questions about the importance and frequency of horizontal gene transfer for such enzymes in natural ecosystems. In the present study, we aimed to address the question of isoenzyme diversity in marine snow. In particular, we wanted to determine the variety of isoenzymes present in a distinct natural environment, such as marine snow, and whether the expression of isoenzymes changes under different ecological conditions. Furthermore, interactions between the enzyme and the polymeric marine snow matrix as well as possible interferences of this matrix with the enzyme assay itself have been addressed. (This work was done in partial fulfillment of the requirements for a Ph.D. degree at the University of Vienna by J.R.)
In marine pelagic systems, macroscopic mucoid aggregations have attracted considerable attention in recent years (2, 9, 18, 34). Now special emphasis is being placed on particle formation and the role of these aggregations in the transport of organic matter into deeper layers of the ocean (3, 4). Transformation of particles by bacterial activity associated with these aggregations leads to qualitative changes in the composition of the particles (21, 33). Recently, the general characteristics of marine snow in the northern Adriatic Sea were summarized in a series of papers (7, 8, 17, 18, 20, 22, 25). These papers clearly demonstrated that there is a characteristic sequence in the development of biotic and chemical parameters during the formation and appearance of marine snow in these waters. Investigations on the role of heterotrophic bacteria in the sea have demonstrated their importance in the cycling of energy and nutrients in the pelagic ecosystem (5, 32). A particularly important parameter in this respect is the activity of extracellular enzymes (13, 19). Although a large variety of organisms excrete enzymes into the extracellular space, bacteria are believed to be one of the major sources of ectoenzymes (l1) which may be involved in the degradation of organic matter (14). The importance of enzymatic activity in degradation processes and in the generation of nutrients by transformation of nonutilizable substances into substances suitable to be taken up by microbes has been shown (12). Beyond the general agreement on the role of enzymes, the quantification of real substrate turnover driven by enzymatic activity is hampered by a variety of methodological problems. As an example of complex degradation mechanisms, the degradation of cellulosic material in which several endo- and exoglucohydrolases act synergistically in fungi as well as in *
Corresponding author.
Phone:
43-1-31336-1351. Fax: 43-1-31336-
70)0. 807
808
RATH AND HERNDL
MATERLALS AND METHODS
Study site and sampling. Samples were obtained from the Gulf of Trieste (northern Adriatic Sea), about 1 km off the Istituto di Biologia Marina at Trieste-Aurisina, Italy. During the period of sampling in August 1991, phytoplankton-derived marine snow was abundant and present in single flocs (up to 3 cm in diameter) and in large clouds (up to 3 m in diameter; for terminology, see reference 37). Samples taken from a 0.5-m water depth represented mainly the single-floc type of marine snow, whereas samples taken from the pycnocline were predominantly the cloud type. Samples were taken by scuba divers from 0.5- to 15-m depths by use of 0.1 N HCl-rinsed 600-ml syringes as described elsewhere (18). Determination of enzyme solubilization efficiency with Triton X-100 (Sigma Chemical Co., St. Louis, Mo.) and ammonium sulfate precipitation were carried out immediately after sampling. For chromatographic separation of the enzymes, marine snow samples were frozen and stored at - 20°C until further processing. Solubilization of the enzyme. Samples of 250 ml of the cloud type of marine snow and 600 ml of the single-floc type of marine snow were centrifuged (15,000 x g at 4°C for 15 min). Enzymatic activity was measured in the supernatant as described below and regarded as free enzymatic activity. The marine snow pellet was resuspended in 10 ml of artificial seawater (31). Solubilization of particle-attached enzymatic activity was performed by adding Triton X-100 to a final concentration of 0.1% (wt/vol) (12). After resuspension of the pellet with a Pasteur pipette, the sample was centrifuged again (15,000 x g at 4°C for 15 min). For determination of particleattached enzymatic activity, 0.3 ml of the supernatant was diluted with artificial seawater to yield a final volume of 3 ml. Enzymatic activity was measured as described below. For chromatographic techniques, 7 ml of the supernatant was desalted with PD 10 columns (Pharmacia LKB Biotechnology, Uppsala, Sweden) and transferred to Tris-HCl buffer (25 mM, pH 7.5) by use of commercially available size exclusion chromatography columns (PD 10 columns); dilution increased the volume to about 10 ml. Protein measurement. The protein concentration was determined spectrophotometrically with bicinchoninic acid protein assay reagent (Pierce Chemicals, Rockford, Ill.) following the recommendations of the manufacturer. Incubations were done in a water bath at 60°C for 30 min. For calibration, a commercial albumin standard (Pierce) was used. Ammonium sulfate precipitation. Precipitation was performed by adding ammonium sulfate to 6 ml of the Triton X-100-solubilized enzyme (see above). Ammonium sulfate was added stepwise (20% intervals of ammonium sulfate saturation) up to a final concentration of 100% ammonium sulfate saturation. After each step, precipitated protein was centrifuged (15,000 x g at 4°C for 15 min) and the resulting pellet was resuspended in 2 ml of artificial seawater. To avoid possible interference of different ammonium sulfate concentrations with enzymatic activity measurements, the resuspended precipitate was desalted with PD 10 columns. The columns had been equilibrated previously with artificial seawater. Enzymatic activity measurements were performed as described below. Separation of enzymes. Separation of enzymes was carried out with a fast protein liquid chromatography system (Pharmacia) connected to a Mono-Q HR 5/5 anion exchanger (5 by 50 mm; Pharmacia). The desalted samples (see above) were sterilely filtered (0.2-jim-pore-size filters; Filtron Kapadisc) and transferred to the anion-exchange column. The proteins were eluted with 25 mM Tris-HCl buffer (pH 7.5) by applying
APPL. ENVIRON. MICROBIOL.
a linear gradient of NaCl (0 to 1,000 mM) from fraction 3 to fraction 24. The fraction size was 2 ml, and the flow velocity was 1 ml min- . Fractionation was done with Frac 100 (Pharmacia). Because of the low enzyme concentration, the Mono-Q anion-exchange column was cleaned carefully by running the same elution program as that described above but without applying any sample to the column. This procedure was repeated until there was no further elution of proteins detectable while the NaCl gradient was being run. Another separation was performed by size exclusion chromatography with 3 ml of the Triton X-100-solubilized enzyme. The gel material was Sephadex G-150 (Pharmacia), the column size was 17 by 450 mm, and artificial seawater was used as the elution buffer. The fraction size was 2 ml, the flow velocity was adjusted to 1 ml min- ', and fractionation was done with Frac 100. The voided volume of the column was determined with blue dextran (average Mr, 2,000,000; Sigma); fractionation started 4 ml before elution of blue dextran occurred. Determination of enzymatic activity. The substrate used for the determination of P-D-glucosidase activity was 4-methylumbelliferyl-3-D-glucoside (Sigma), a structural analog of the natural substrate cellobiose. Fluorescence caused by the enzymatic cleavage of 4-methylumbelliferone (Sigma) was detected with a spectrofluorometer (820-FP; JASCO, Tokyo, Japan). The excitation wavelength was 360 nm, and the emission wavelength was 444 nm. Calibration of the fluorescence yield was performed for each measurement with different concentrations. The substrate was added to a final concentration of 2.5 p.M. Incubation time varied between 30 and 45 min, and incubation temperature was 20°C. For standardization, all enzymatic activity measurements were carried out with artificial seawater, except for the determination of free enzymatic activity, for which the assay was performed by direct addition of the substrate to the supernatant (see above). To avoid interference of high NaCl concentrations in the later fractions of the anion-exchange chromatography with enzymatic activity measurements, all fractions were desalted with PD 10 columns prior to enzymatic activity measurements. The PD 10 columns had been equilibrated previously with artificial seawater. Temperature profile. The temperature profile ranged from 5 to 50°C, with 5°C intervals. For each temperature interval, 0.3 ml of the enzyme solution obtained after Triton X-100 addition (see above) was diluted with artificial seawater to obtain a final volume of 3 ml. All samples were adjusted to the appropriate incubation temperature for 4 min in a water bath before the substrate was added. Enzymatic activity was assayed by adding 2.5 p.M 4-methylumbelliferyl-p-D-glucoside and incubating the samples at the appropriate temperature for 20 min. Enzymatic activity was measured as described above. Temperature stability. To test the influence of marine snow on the stability of 3-D-glucosidase, three different types of samples were used: (i) Triton X-100-solubilized 3-D-glucosidase, (ii) natural marine snow samples to which 1 % (wt/vol; final concentrations) Triton X-100 had been added before incubation at the appropriate temperature, and (iii) natural marine snow samples to which 1% (wt/vol; final concentration) Triton X-100 was added after incubation at the appropriate temperature. Duplicates were incubated at 20 or 50°C for 20 min. To avoid interference of particles with the enzyme assays (see Discussion), marine snow was centrifuged as described above and only the supernatant was used for the determination of enzymatic activity. Enzyme assays were carried out at 30°C for 20 min. The fluorescence yield was determined as described above.
VOL. IN MARINE SNOW V-D-GLUCOSIDASE 6(), 19'94
TABLE 1. Distribution of free- and particle-attached enzymatic activities compared with enzymatic activity measured after direct addition of the enzyme substrate to marine snow collected at the pycnocline"
809
A 80 70
Enzymatic activity (% of total)
Triton X-t100 and centrifugation
Sampling date
With
(day in August 1991)
After direct addition of the substrate to marine snow
9
71.4 55.6 85.5
10 11
Frec
Particle attached
27.1 16.7
72.9 83.3 71.1
28.9
Samplcs were collected on three consecutive days in August 1991 from the northern Adriatic Sea. Enzymatic activities are expressed as the percentage of total (free plus particle-attached) P-D-glucosidase aictivity in marine snow before and after treatment with t).1 Triton X-100 and suhscquent centrifugation; the supernataint was used for the measurement of enzymattic activitics. For further details, sce Materials and Methods.
60
5040 30
20 10 -
tn -4
0-20
RESULTS Free and attached enzymatic activities. On average, free 3-D-glucosidase activity accounted for 27% (17 to 29%; n = 3) of the total f-D-glucosidase activity in marine snow (Table 1). The glucosidase activity obtained after direct addition of the substrate to marine snow was always lower than the total glucosidase activity (free plus particle-attached enzymatic activities) obtained after enzyme solubilization (Table 1). Temperature profile. The increase in temperature was accompanied by an increase in ,-D-glucosidase activity up to a temperature of 40°C. At temperatures above 40°C, enzymatic activity declined rapidly. 1-D-Glucosidase activity at 50°C was only 8% of the activity at 40°C (Fig. 1). Ammonium sulfate precipitation. Stepwise precipitation of proteins by the addition of ammonium sulfate was used in an attempt to separate f-D-glucosidase activity from the protein pool. Precipitation of P-D-glucosidase obtained from the cloud type of marine snow yielded only one major peak of 13-Dglucosidase activity. More than 70% of the residual activity was found in the precipitate of the fraction at 40 to 60% ammonium sulfate saturation (Fig. 2A). The recovery of P-D-glucosidase activity isolated from clouds after ammonium sulfate precipitation was 20%. In marine snow from a 0.5-m depth, noticeable amounts of residual activity were found in two fractions. The addition of ammonium sulfate up to 20% of the saturation concentration resulted in the precipitation of 13-D120I--
80 4.
40-60
60-80
+80
ammoniumsulfate saturation (%)
0-20
20-40
40-60
60-80
+80
ammoniumsulfate saturation (%) FIG. 2. Distribution of P-D-glucosidase activity in the ammonium sulfate precipitate at different levels of ammonium sulfate saturation. One example is given for a total of five determinations for each type of marine snow. Activities are expressed as percentages of the total enzymatic activity remaining after ammonium sulfate precipitation. (A) Cloud type of marine snow (100% is 3.4 pmol h-'). (B) Single-floc type of marine snow (1 00% is 2.8 pmol h -').
100-
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-
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I
.-
10 15 20 25 30 35 40 45 50 55 temperature (0 C) FIG. 1. Temperature profile for 3-o-glucosidase activity liberated from the cloud type of marine snow, collected at the pycnocline. Activity is expressed as a percentage of the activity at the temperature optimum of 40°C; 1(00% was 3.0 pmol h-' mg of protein'.
0
5
glucosidase activity contributing 35% of the total residual activity; 55% of the total residual activity, however, was found in the precipitate of the fraction at 40 to 60%c ammonium sulfate saturation (Fig. 2B). The recovery of f-D-glucosidase activity after ammonium sulfate precipitation was 18% and therefore was close to the recovery efficiency for marine snow collected at the pycnocline. Column chromatography. To avoid losses of enzymatic activity, solubilized enzymes from marine snow were immediately transferred to Tris-HCI buffer (25 mM, pH 7.5) and introduced to an anion-exchange chromatography column. Nearly all of the P-D-glucosidase activity bound to the column and could be eluted by applying an NaCl gradient of 0 to 1,000 mM. Separation of the proteins along the NaCl gradient
Al'I'L. ENVIIZON. MICROBIOL.
RATH AND HERNDL
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FIG. 4. Size exclusion chromatography of the enzyme solubilized from the cloud type of marine snow, collected at the pycnocline. One example is given for a total of four detcrminations. Activities are expressed as percentagcs of the total activity obtained after size exclusion chromatography (100% is 6.7 pmol h- '). The arrow indicates the elution of blue dextran.
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FIG. 3. Anion-exchange chromatography of solubilized enzymes. One example is given for a total of seven determinations for each type of marine snow. Activities are expressed as percentages of the total 3-D-glucosidase activity obtained after anion-exchange chromatography. (A) Cloud type of marine snow (100% is 2.7 pmol h-'). (B) Single-floc type of marine snow (100% is 5.9 pmol h-').
resulted in different elution patterns for P-D-glucosidase activity (Fig. 3). For all samples, 3-D-glucosidase activity was eluted at an NaCl concentration of 750 mM. This was the only major activity detectable for 3-D-glucosidase liberated from the cloud type of marine snow (Fig. 3A). For the single-floc type of marine snow, taken in surface waters, however, we found a second activity which eluted at an NaCl concentration of 400 mM (Fig. 3B). The relative proportions of this enzyme differed from sample to sample (data not shown). The recovery of enzymatic activity after this separation process ranged from 36 to 51 %. Size exclusion chromatography of the solubilized enzyme from the cloud type of marine snow supported the results obtained by anion-exchange chromatography. Only one major peak of ,B-D-glucosidase activity was detectable when the solubilized enzyme was introduced to a column filled with Sephadex G-150 (Fig. 4). Elution of this dominant portion of 3-D-glucosidase activity was close to the voided volume of the column. Temperature stability. Pretreatment of the samples at 50(C
for 20 min resulted in a rapid loss of 3-D-glucosidase activity compared with the activity obtained at 20°C (Fig. 5). However, when beta-D-glucosidase was not isolated from marine snow, its activity remained at a higher level. Thus, it seems that marine snow has a stabilizing effect on enzymatic activity. This stabilizing effect was detectable both before and after incubation of the enzyme with Triton X-100. DISCUSSION Mucoid aggregates in the northern Adriatic Sea occur in a wide range of morphologically diverse stages (37). The biological and chemical characteristics of the two different stages of marine snow investigated in this study have been described previously (20, 25). Degradation of these aggregates is mediated by the combined action of physical disruption and extracellular enzymes (35). Enzymatic activity per volume of marine snow is among the highest measured in pelagic ecosystems (25). The measurement of enzymatic activity associated with large particles is accompanied by a variety of methodological problems. For example, sedimentation of the particles leads to differences in the shielding of excitation light, consequently leading to an underestimation of the actual enzymatic activity. Furthermore, these measurements are influenced by effects similar to those occurring after the immobilization of enzymes (16), such as partitioning effects between the polymeric matrix and the bulk solution, caused by electrostatic and hydrophobic interactions, and diffusional effects, both of which may reduce the actual substrate concentration within the particles. As shown in Table 1, total 3-D-glucosidase activity measured after liberation of the enzyme by Triton X-100 and removal of the particles by centrifugation was always higher than enzymatic activity measured after the direct addition of the substrate to marine snow. Furthermore, a massive reduction of diffusion within the particles may also lead to an underestimation of potential enzymatic activity. As shown previously (1, 29, 30), limitation of diffusion within pelagic particles is strong enough to establish stable gradients for pH and oxygen and allows the development of an anaerobic community within the polymeric matrix. We are aware, however, that not all of the enzyme was
IN MARINE SNOW VOL. V-D-GLUCOSIDASE 6(), 1994
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marine snow enzyme attached to marine snow +0.1% Triton X 100 marine snow FIG. 5. Temperature stability of 3-[)-glucosidase; residual enzymatic activities after incubation at 50°C are expressed as percentages of the activity detectable at 20(C (100% is 0.8 pmol h '). The enzyme was isolated from the cloud type of marine snow, collected at the pycnocline. enzyme without
liberated by Triton X-100 treatment. Thus, the portion of the particle-attached enzymatic activity is probably larger. In summary, our findings suggest that current techniques applied to the measurement of enzymatic activity in marine snow underestimate actual rates. Although the optimal temperature for ,B-D-glucosidase activity was -40°C, a temperature much higher than the ambient water temperature in the Adriatic Sea, activity declined rapidly at temperatures above 40°C (Fig. 1). However, the temperature stability of P-D-glucosidase activity present at natural concentrations in marine snow increased fourfold (Fig. 5). This result indicates that the presence of the polysaccharide fibrils forming the matrix of marine snow and not the binding of the enzyme to a special (cellular) binding structure is crucial for the increase in the thermostability of the enzyme when associated with marine snow. The stabilizing effects of different chemical and physical factors on enzymatic activity have been shown (24). Recently, Nagata and Kirchman (27) showed the importance of one of these interactions for natural environments. Acid phosphatase derived from flagellates was protected against proteolytic digestion by association with liposomes. Molecular interactions with other macromolecules seem to be of crucial importance for in situ enzymatic activity. Enzymes work within a certain "molecular environment," which alters enzymatic activity on qualitative as well as quantitative levels. The highly polymeric matrix of marine snow seems to modify the temperature stability of ,B-D-glucosidase activity. An unsolved question in considering extracellular enzymatic activity is how many isoenzymes are expressed in a specific natural environment. Olsson (28) showed that free and particle-attached (phytoplankton-attached) acid phosphatases had slightly different elution profiles during size exclusion chromatography. He speculated that this result might have been caused by the production of two different isoenzymes, one for excretion and one for attachment on the cell surface. Cotner and Wetzel (15) isolated bacteria from different natural habitats and determined alkaline phosphatase activity. They found that alkaline phosphatase was expressed from different bacterial species. These enzymes, however, differed enormously in their characteristics, although the molecular mass of each of the different enzymes was about 40,000 Da. Fluorogenic enzyme assays are extremely sensitive and are therefore used
to estimate extracellular enzymatic activity under in situ conditions. The major problem, however, in investigating enzyme diversity is the low concentration of an enzyme found in a natural environment. r-D-Glucosidase activity was highly enriched in our marine snow (the level was up to 3 orders of magnitude higher than in the ambient water; see reference 25). This high level of activity allowed further processing of an enzyme isolated directly from a natural sample. We chose the ectoenzyme 3-D-glucosidase for further characterization because of its participation in the degradation of a homopolymeric substance and its relatively high level of activity compared with that of ct-D-glucosidase found in our samples. The occurrence of 3-D-glucosidase activity in marine snow seems to be restricted to only a few isoenzymes. Marine snow samples collected from surface waters showed two distinct peaks of 3-D-glucosidase activity when separation was performed by anion-exchange chromatography (Table 2 and Fig. 3B). The occurrence of at least two distinct isoenzymes was also supported by ammonium sulfate precipitation (Fig. 2B). Although we have no data on substrate turnover for the two isoenzymes, different properties of different isoenzymes seem to be quite common (15). Therefore, the occurrence of more than one isoenzyme in a distinct sample may limit the conclusions that can be drawn from enzyme characteristics such as Km and Vmax. For the cloud type of marine snow, obtained, from the pycnocline, however, only one of these isoenzymes contributed up to 80% of the total residual ,B-D-glucosidase activity isolated from these particles (Table 2 and Fig. 3A). Precipitation of the solubilized enzyme with ammonium sulfate supported these results (Fig. 2A). More than 70% of the residual ,B-D-glucosidase activity was found in the precipitate at 40 to 60% ammonium sulfate saturation. An additional column chromatographic separation revealed a very similar pattern. Separation of the proteins by size exclusion chromatography showed the dominance of one major peak for P-D-glucosidase activity close to the voided volume of a column filled with Sephadex G-150 resin, a result which may indicate a high molecular mass for the enzyme (> 100,000 daltons; Fig. 4). The small degree of diversity for ,-D-glucosidase within natural samples is remarkable. Explaining this phenomenon is difficult. One potential explanation is that a highly specialized group of bacteria produces large amounts of a specific P3-D-glucosidase isoenzyme and thereby dominates the degradation of cellulosic
812
RATH AND HERNDL
APPL. ENVIRON. MIC'ROBIOL.
TABLE 2. Enrichment and separation characteristics for f3-D-glucosidase isolated from two different stages of marine snow' Walter depth (m)
0.5
10-11 (Pycnocline)
Puritication step
Total protein
Totail
(mg)
activity (pmol h-'
Sp act (pmol h
mg of
Purification
protein ')
(fold)
Crude extract PD 10-desalted extract Mono Q fractions 15-17 9-11
3.2 1.9
9.7 6.7
3.0 3.5
1.0 1.2
0.19 0.27
2.2 1.9
11.2 7.3
3.7 2.4
Crude extract PD 10-desalted extract Mono 0 fractions 16-18
7.1 2.3 0.22
8.1 4.1 1.7
1.2 1.8 8.4
1.( 1.6 7.2
"The siamples were taken on the same day (11 August 1991) at different water depths. For details, see Materials and Methods.
compounds; however, one must keep in mind the fact that 3-D-glucosidase is not a protein that acts in close cooperation with other proteins, such as proteins involved in the ribosomal system, whose genes are therefore much more accessible to horizontal gene transfer. ACKNOWLEDGMENTS We thank C. P. Kubicek and R. Messner for invaluable help. This study was supported by the Austrian Science Foundation (grant 7748 to G.J.H.). REFERENCES 1. Alldredge, A. L., and Y. Cohen. 1987. Can microscale chemical patches persist in the sea? Microelectrode study of marine snow, fecal pellets. Science 235:689-691. 2. Alldredge, A. L., and M. W. Silver. 1988. Characteristics, dynamics and significance of marine snow. Prog. Oceanogr. 20:41-82. 3. Asper, V. L. 1987. Measuring the flux and sinking speed of marine snow aggregates. Deep Sea Res. 34:1-17. 4. Asper, V. L., W. G. Deuser, G. A. Knauer, and S. E. Lohrenz. 1992. Rapid coupling of sinking particle fluxes between surface and deep ocean waters. Nature (London) 357:670-672. 5. Azam, F., T. Fenchel, J. G. Field, J. S. Gray, L. A. Meyer-Reil, and F. Thingstad. 1983. The ecological role of water-column microbes in the sea. Mar. Ecol. Prog. Ser. 10:257-263. 6. Beguin, P. 1990. Molecular biology of cellulose degradation. Annu. Rev. Microbiol. 44:219-248. 7. Bochdansky, A. B., and G. J. Herndl. 1992. Ecology of amorphous aggregations (marine snow) in the Northern Adriatic Sea. III. Zooplankton interactions with marine snow. Mar. Ecol. Prog. Ser. 87:135-146. 8. Bochdansky, A. B., and G. J. Herndl. 1992. Ecology of amorphous aggregations (marine snow) in the Northern Adriatic Sea. V. Role of fecal pellets in marine snow. Mar. Ecol. Prog. Ser. 89:297-303. 9. Caron, D. A., P. G. Davis, L. P. Madin, and J. M. Sieburth. 1986. Enrichment of microbial populations in macroaggregates (marine snow) from surface waters of the North Atlantic. J. Mar. Res. 44:543-565. 10. Chrost, R. J. 1989. Characterization and significance of 3-glucosidase activity in lake water. Limnol. Oceanogr. 34:660-672. 11. Chrost, R. J. 1990. Microbial ectoenzymes in aquatic environments, p. 47-78. In J. Overbeck and R. J. Chrost (ed.), Aquatic microbial ecology. Biochemical and molecular approaches. Springer-Verlag, New York. 12. Chrost, R. J. 1991. Environmental control of the synthesis and activity of aquatic microbial ectoenzymes, p. 29-59. In R. J. Chrost (ed.), Microbial enzymes in aquatic environments. Springer-Verlag, New York. 13. Chrost, R. J. (ed.). 1991. Microbial enzymes in aquatic environments. Springer-Verlag, New York. 14. Chrost, R. J. 1992. Significance of bacterial ectoenzymes in aquatic environments. Hydrobiologia 243/244:61-70. 15. Cotner, J. B., Jr., and R. G. Wetzel. 1991. Bacterial phosphatases from different habitats in a small, hardwater lake, p. 187-205. In
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