Mailing address: CNRS. .... Mass.). Samples were maintained at the appropriate temper- ature for 5 min prior to measurements of ...... Company, Inc., New York.
Vol. 60, No. 11
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1994, p. 4134-4141
0099-2240/94/$04.00+0 Copyright ©D 1994, American Society for Microbiology
Production and Characterization of an Exopolysaccharide Excreted by a Deep-Sea Hydrothermal Vent Bacterium Isolated from the Polychaete Annelid Alvinella pompejanat P. VINCENT,1 P. PIGNET,2 F. TALMONT,3 L. BOZZI,4 B. FOURNET,3 J. GUEZENNEC, C. JEANTHON,1 AND D. PRIEURl* LP 4601, Station Biologique, Centre National de la Recherche Scientifique (CNRS), 29682 Roscoff Cedex,1 IFREMER, DERO/EP, Centre de Brest, 29280 Plouzane,2 UMR 111, Laboratoire de Chimie Biologique, UST Lille, CNRS, 59650 Villeneuve d Ascq,3 and CERMAV, CNRS, 38041 Grenoble,4 France Received 9 March 1994/Accepted 30 August 1994
The heterotrophic and mesophilic marine bacterium HYD-1545 was isolated on a metal-amended medium from the dorsal integument of the hydrothermal vent polychaete Alvinella pompejana. This strain, which can be assigned to the genus Alteromonas on the basis of its G+C content and phenotypical features, produced large amounts of an acidic polysaccharide in batch cultures. The polysaccharide was excreted during the stationary phase of growth and contained glucose, galactose, glucuronic acid, galacturonic acid, and 4,6-O-(1-carboxyethilidene)-galactose as major components. This polysaccharide was a polyelectrolyte, and the viscosity of its solutions depended on the ionic strength. The decrease in viscosity with increasing NaCl concentrations and the effect of Ca21 in decreasing the viscosity at low Ca2+ concentrations support a model in which the polysaccharide carries anionic groups. However, an unusual behavior was observed at higher concentrations and could be related to intermolecular interactions involving Ca2+ ions.
The adhesion of bacteria to solid surfaces in aquatic environments involving exocellular polysaccharides has been investigated from an ecological and structural standpoint by using light and electron microscopy (10, 16, 35). Many authors have discussed the role and properties of these polymers in the attachment and survival of the cells and their interactions with ions, such as heavy metals (30, 49). More recently, considerable attention has been focused on the chemical characterization of novel exopolysaccharides (EPS) excreted by marine bacteria (8, 33) in association with the study of their rheological properties (6) or antitumoral activities (32). Only a few interesting molecules, e.g., Marinactan (45), have yet been found, purified, and totally characterized. In the course of searches for new marine biopolymers of potential use to industry, 479 heterotrophic bacteria isolated from various deep-sea hydrothermal vent samples were screened for their ability to produce EPS (46). These ecosystems are characterized by the existence of rich animal communities explained by the abundance of local chemosynthetic bacterial primary production (23), and several associations between bacteria and invertebrates have been described. The strain HYD-1545 examined in this study was isolated from the dorsal integument of the marine polychaete Alvinella pompejana (12), which builds mineralized organic tubes on the walls of sulfide diffusers. This worm lives in areas where hot, acidic, and metal-rich hydrothermal fluids are mixing with cold and well-oxygenated seawater (11).
the submersible Nautile at 13°N on the East Pacific Rise (2,600 m depth, 12°48.56'N, 103°56.72'W). While we were searching for marine bacteria producing EPS, this strain was selected for its ability to show a mucoid phenotype on solid medium and to produce viscous broth. Vibrio natriegens (DMS 759) used as a reference for polyhydroxybutyrate synthesis was obtained from the Deutsche Sammlung von mikroorganismen und Zellkulturen collection (RFA). Alteromonas haloplanktis used as a reference for the G+C content measurements was a gift of M. J. Gauthier Institut National de la Sante et de la Recherche Medicale, Unite 303, Nice, France). Media. The strain HYD-1545 was isolated on a metalamended medium (24) prepared by adding to the 2216E medium (34) 1 mg of AsO43- ml-' and was tested for polysaccharide production in shake flasks by using a sugar-containing medium composed of the following: 5 g of peptone, 1 g of yeast extract, 250 ml of distilled water, 750 ml of filtered seawater, and 30 g of glucose (31). The medium was supplemented with agar (20 g/liter) for culture on solid medium. The strain isolated in pure culture was preserved by being frozen in glycerol (20% [vol/vol]) at -20°C, and from these organisms precultures for each experiment were prepared. Medium 2216E was used for the determination of the optimal temperature for growth. Optimal pH was then determined by using the same medium supplemented with Tris-HCl (50 mM) in a pH range of 7.5 to 8.0, MOPS (morpholinepropanesulfonic acid; 50 mM) in a pH range of 7.0 to 7.5, and PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid); 20 mM] in a pH range of 5.5 to 7.0. The optimal ionic strength was determined by using a modification of a medium described by Boyle and Reade (6) containing the following: MgCl2 6H20, 5.9 g; Na2SO4, 3.24 g; CaCl2 * 2H20, 1.8 g; KCl, 0.55 g; NaHCO3, 0.16 g; ferrous citrate, 0.1 g; tryptone, 0.7 g; yeast extract, 1 g; and distilled water, 1 liter. NaCl in a range of S to 70% was added. Culture conditions. In all experiments concerning EPS production, plates and flasks were incubated for 5 days at 23°C
MATERIALS AND METHODS Microorganisms. The bacterium HYD-1545 was isolated from the epidermis of the hydrothermal vent polychaete A. pompejana, collected in 1987 during the "Hydronaut" cruise by * Corresponding author. Mailing address: CNRS. LP 4601, Station Biologique, B.P. 74, 29682 Roscoff Cedex, France. t This article is dedicated to B. Fournet.
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EPS-PRODUCING BACTERIUM FROM VENT POLYCHAETE
andEPS were obtained during the stationary phase of growth. Flasks (500 ml) containing 100 ml of glucose-containing medium were inoculated with a loopful of the primary bacterial culture and incubated at 150 rpm on a shaker. Optimal growth conditions were determined in 1-liter flasks containing 200 ml of the appropriate medium. Cultures were incubated for 36 h, and cell growth was monitored spectrophotometrically (A520). The media were inoculated with 4 ml of a bacterial suspension spectrophotometrically standardized at an optical density at 520 nm of 0.1 which was prepared from cells cultured overnight on the same medium. The doubling times were calculated from the slopes of the growth curves. The optimal temperature for growth was determined for cultures incubated at 16, 22, 25, 27, 29, 31, 33, 35, and 39°C, at pH 7.2. The effect of pH on growth was tested at pH 5.7, 6.0, 6.5, 7.0, 7.3, 7.5, and 7.8, at the optimal temperature, which was 27°C. The optimal ionic strength for growth was determined at 27°C and pH 7.3 by using 11 different NaCl concentrations, 0, 5, 10, 20, 25, 30, 40, 50, 60, and 70 g/liter. Morphological, biochemical, and nutritional tests. The presence of a capsule was determined by using wet mounts with India ink and the staining technique of Allison and Sutherland (1). Flagellation was characterized by the staining method of Rhodes described by Conn et al. (9). The occurrence of an oxidase reaction was determined by using one-day cultures according to the procedure of Kovacs (26). H2S production, glucose fermentation, and Tween degradation were recorded after 7 days by the methods described by Harrigan and McCance (22). Proteolytic activity on gelatin was tested as described by Kohn (25). The presence of a constitutive arginine dihydrolase system was determined by the method of Baumann and Baumann (2). Amounts of indole production, nitrate and nitrite reduction, starch hydrolysis, and urease, catalase, and 3-galactosidase activities were determined by standard methods (27). Polyhydroxybutyrate accumulation was tested according to the method of Baumann and Baumann (2) for 4 days, with V natriegens (DSM 759) serving as a control strain. Chitinase activity was tested according to the method of Reichenbach and Dworkin (38). The utilization of 19 sugars and 16 other carbohydrate substrates as the sole source of carbon was tested according the method of Baumann and Baumann (2) by using a basal medium (BM) supplemented with the substrate at a final concentration of 0.2% (wt/vol for solids; vol/vol for liquids) for sugars and 0.1% (wt/vol for solids; vol/vol for liquids) for the other carbohydrate substrates. Most carbohydrate solutions were sterilized separately by being autoclaved at 120°C for 20 min, except xylose, fructose, and maltose solutions, which were sterilized by filtration with 0.2-,um-pore-size filter units (Merck). DNA was prepared as described by Beji et al. (4), and the GC content was determined by both high-performance liquid chromatography (HPLC) (36) and the method of Ulitzur (44). Viscosimetry. Solution viscosity was measured with a Brookfield viscosimeter model DV-II supplied with a small sample adapter (SC4-18/13R) at 25°C and 30 rpm unless otherwise indicated (Brookfield Engineering Laboratories, Stoughton, Mass.). Samples were maintained at the appropriate temperature for 5 min prior to measurements of viscosity. Polysaccharide production. The EPS production by strain HYD-1545 was studied by using a 2-liter fermentor (LSL Biolafitte, St-Germain-en-Laye, France) initially containing 1.4 liters of the appropriate medium. Batch fermentation was started by inoculating a suspension of cells (2% [vol/vol]) grown overnight in 2216E medium into the fermentor. The temperature was maintained at 25°C. Air was supplied at 40 liters per volume of medium per h, and the culture was mixed
4135
at an impeller speed within a range of 350 to 800 rpm. The pH was adjusted to 7.2 at the beginning of the culture and measured but not controlled during the experiment. Samples (40 ml) were removed at regular intervals for growth and viscosity measurements, EPS isolation, and residual glucose analysis. The viscosity of the culture broth was measured as described above, and the residual concentration of glucose in the medium was determined enzymatically by using a reagent kit purchased from Boehringer Mannheim Biochemicals (Mannheim, Germany). Growth was measured by CFU counts and turbidimetrically at 520 nm, sterile medium being used as a blank. EPS that were analyzed with respect to composition were obtained during the stationary phase of growth, after 5 days of culture. Polysaccharide isolation. (i) Procedure 1. Culture broths were diluted when necessary and centrifuged at 4°C and 4,000 x g for 15 min in a Sorvall RC-5B centrifuge (Rotor GSA). When EPS was firmly bound to the cells as insoluble material, a loosely packed layer of cells covering a compact cell pellet was observed. Both the layer of cells and the pellet were then recovered, and cell dry weight was determined. Cold ethanol (3 volumes/1 volume) was added to the supernatant, and the solution was maintained overnight at 4°C. The precipitated polymer was redissolved in a small volume of distilled water, and the precipitation procedure was repeated. The EPS was then redissolved in distilled water and dialyzed (molecular weight cutoff, 6,000 to 8,000) against distilled water for 3 days at 4°C. The polysaccharide preparation was then dried at 40°C, freeze-dried, and stored at 4°C until required. (ii) Procedure 2. The culture broth was diluted with water to yield a polymer concentration of about 1 g/liter. The solution was centrifuged for 2 h at 24,000 x g in order to remove insoluble material and cell debris, and the supernatant was heated for 5 min at 90°C to denature the proteins and improve the filtration. Finally, the solution was filtered through Sartorius membranes (3- to 0.45-,um pore diameters) and NaCl was added to a final concentration of 20 g/liter. To the solution was added ethanol (50% [vol/vol]), and the precipitate was washed with 70 to 100% (vol/vol) ethyl alcohol (EtOH)-H20 mixtures to eliminate excess sodium chloride. The polysaccharide was dried under vacuum at 30°C for 48 h. Chemical analysis. Protein content was determined by both the method of Lowry et al. (28) with bovine serum albumin as the standard (Sigma Chemical Co., St. Louis, Mo.) and by the method of Bradford (7) by using a protein assay (Laboratories Bio-Rad S.A., Paris, France). Uronic acid levels were determined by both the carbazole-sulfuric acid reaction (13) and the m-phenylphenol method (5), with glucuronic acid as the standard. Hexosamine levels were determined by the Elson-Morgan reaction (15) with glucosamine as the standard. Neutral sugar levels were determined by the orcinol-sulfuric acid reaction of Tillmans and Philippi (42), as modified by Rimington (39), with 1 mannose molecule-1 galactose molecule as the standard. Polysaccharide hydrolysis. A freeze-dried polysaccharide sample (500 jig) was placed in a screw-cap glass tube, 4 N trifluoroacetic acid (0.5 ml) was added, and the tube was tightly capped (Teflon-lined cap) and heated for 4 h at 100°C. The hydrolyzed sample was then cooled to 25°C, uncapped, and dried under reduced pressure for 24 h. The residue was resuspended in water, and the pH was adjusted to 9 with 0.1 M ammonia. Reduction was performed with sodium borohydride, and the mixture was left overnight at room temperature. Excess borohydride was removed by addition of acetic acid (pH 4). Borate ions were removed by codistillation with methanol. The alditol acetates were prepared by adding pyri-
4136
APPL. ENVIRON. MICROBIOL.
VINCENT ET AL.
dine (300 ,ul) and acetic anhydride (300 ,ul) and incubating the solution overnight at room temperature. The reaction mixtures were evaporated to dryness, and the alditol acetate derivatives were dissolved in dichloromethane for gas chromatographic analysis. Methanolysis, N acetylation, and trimethylsilylation. Freezedried sugar (500 pg) was dissolved in 0.5 ml of 0.5 M HCl in methanol. After methanolysis for 24 h at 800, the acid solution was neutralized by the addition of silver carbonate. Re-N acetylation was carried out by the addition of 20 pl. of acetic anhydride. This mixture was kept at room temperature for 24 h in the dark. The precipitate was centrifuged (3000 x g for 5 min), and the supernatant was collected. Lipids were removed with heptane, and methanol was evaporated under a stream of nitrogen. Finally the sample was trimethylsilylated with silylating agent (40 p.l of pyridine plus 40 p.1 of BSTFA plus 1% TMCS) for 2 h at room temperature. The sample was dried under a stream of nitrogen and dissolved in dichloromethane for analysis. Gas chromatography. Gas chromatography was performed on a Carlo Erba gas chromatograph equipped with a flame ionization detector and an automatic injector "on column." The carrier gas was hydrogen, and the flow rate was 2 ml min-'. A fused silica capillary column (CP-Sil 5 CB; 25 m; inside diameter, 0.25 mm) was operated with temperature gradients (50 to 120°C, 20°C/min; and 120 to 250°C, 2°C/min). The initial temperature was maintained for 1 min at 50°C, and the final temperature was maintained for 5 min at 250°C. Rheological analysis. The polysaccharide 1545 was purified as described above (procedure 2) but without heating. The average molecular weight was determined by using a Chromatix KMX6 light scattering detector. The measurements were carried out in 0.1 M NaCl with different polymer concentrations (0.05 to 0.4 g liter-'). The solutions were filtered through 0.2-p.m-pore-size Sartorius membranes. The dn/dc value was determined on the same solutions at 25°C by using a BricePhoenix differential refractometer. The polymer characterization, e.g., determination of the molecular weight distribution, was carried out by size exclusion chromatography (columns, Shodex OH-Pak 804 and 805) by using a 1545 solution (0.625 g/liter) in 0.1 M NH4NO3. Three detectors were used on-line, a low angle light scattering detector (Chromatix CMX100), a capillary viscometer, and a refractometer, as previously described (43). Viscosity measurements were performed at 25 ± 0.01°C by using a Contraves Low Shear 30 viscometer in a range of shear rate from 10-2 to 128 s- . For high viscosities, a Carrimed CS-50 rheometer, equipped with a Rheo 1000 C system and 5.0 software which allows direct viscosity shear rate determination by angular speed control, was used. For these measurements a cone with a 6-cm diameter and a 10 angle was used. Polymer solutions were obtained by dissolving the polysaccharide in distilled water and adding 1 M NaCl solution in order to reach the desired NaCl concentration. A potentiometric titration was performed by using a Tacussel (Minisis 6000) potentiometer. The protonated form of the polymer was obtained by passage of a sodium salt solution through an ion-exchange column (Amberlite IR120-H+ form). The titration was carried out at 25 ± 0.1°C by using a 24 mM NaOH solution. RESULTS Strain HYD-1545 appeared as a motile, nonfermentative, gram-negative rod (0.5 by 1.3 to 1.5 p.m) with a single polar flagellum when cultured on 2216E medium for 36 h. Five-dayold colonies on 2216E medium supplemented with glucose
160 140
120 100
80 60 40
20 10
15 20 25 30 35 40 45 50
a - Temperatue (°C) 70
65 60
55
b4) 50
,0
45
0
n
40
35c
5.5
6.5
6
7.5
7
8
b -pH 7S 70
65 60 55
SO
45 40
0
10
20
30
40
50
60
70
80
c - NaCI concentration (gA) FIG. 1. Influence of incubation temperature, pH (0, initial pH; El, final pH), and NaCl concentration on growth of strain HYD-1545. The doubling times were calculated from the slopes of the growth curves
(not shown).
viscous, glistening, and irregular shaped, with a lobate edge, and they were about 1.0 to 1.2 cm in diameter. The optimal temperature for growth was between 25 and 29°C, with a doubling time of 38 min at 27°C (Fig. la). The optimal pH was between 7.0 and 7.3. For initial pHs of 6.5, 7.0, and 7.3, the final values after 36 h of culture were, respectively, pH 7.2, 7.3, and 7.5 (Fig. lb). The optimal ionic strength was 30 g of NaCl per liter (Fig. lc), with a longer doubling time (45
were
EPS-PRODUCING BACTERIUM FROM VENT POLYCHAETE
VOL. 60, 1994
100
250
90
225
80
200
j
70
175
._
O
60
15
.
to
50
125
10
lo
0-%
_ W4) oo
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5
0 0
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4137
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40
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75
30
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U 2
-
20
50
25
10
0
0
0
5
10
15
20
25
50
30
70
Glucose (g.1-1) FIG. 2. Effect of the initial glucose concentration on EPS production by strain HYD-1545.
min) when the modified medium of Boyle and Reade (6) was used, at 27°C and pH 7.3. Strain HYD-1545 produced H2S from cysteine, reduced nitrates, and degraded Tween 80, starch, and gelatin. Positive responses were obtained for catalase, oxidase, P-galactosidase, and arginine dihydrolase tests. Glucose was not fermented, and the strain did not accumulate polyhydroxybutyrate or reduce nitrite. Negative responses were obtained for urease, citrate indole, and chitinase tests. The other biochemical and nutritional characteristics indicated below showed a large amount of utilization of carbohydrate substrates. Use of the following sugar substrates as the sole source of carbon produced significant growth of strain HYD-1545: D-glucose, D-fructose, maltose, D-trehalose, D-melibiose, sucrose, lactose, D-cellobiose, D-melezitose, and D-raffinose. Use of D-xylose and D-galactose produced slight growth, and D-mannose, D-arabinose, L-arabinose, L-rhamnose, L-sorbose, and D-ribose were not utilized by the strain. Among nonsugar carbohydrate substrates, glycerate and ethanol produced significant growth and succinate and hexadecane produced slight growth, whereas malate, aconitate, butyrate, mannitol, sorbitol, N-acetylglucosamine, fumarate, gluconate, glycolate, erythritol, and sarcosine were not utilized. The G+C content of the DNA of strain HYD-1545 was 42% as determined by HPLC (36) and 45.1% + 2.3% as determined spectroscopically by the method of Ulitzur (44). Strain HYD-1545 produced highly viscous shake flask cultures at concentrations of glucose ranging from 5 to 70 g/liter (Fig. 2). After centrifugation the supernatants formed stringy precipitates with ethanol. The highest concentrations of polysaccharide harvested after 5 days of culture were approximately 1.8 to 2.0 g/liter for an initial glucose concentration in the medium of 20 to 70 g/liter. In the absence of glucose in the medium, the strain did not liberate any material in the culture broth. However, capsules and filamentous structures between the cells were observed. The maximum viscosity (166 cP at 12 rpm) was reached with an initial glucose concentration of 70 g/liter, but the increase in the viscosity above that obtained with a glucose level of 30
g/liter may have been the result of a cooperative effect between the remaining glucose and the EPS. Polysaccharide was firmly bound to the cells, and after centrifugation a loosely packed layer of cells was found to cover a compact cell pellet. This layer was more important, as the glucose concentration was higher. Dry cell weight corresponding to both fractions of cells was then higher, in contrast to the soluble EPS concentration, which remained almost constant between 30 and 70 g of glucose per liter. The optimal initial glucose concentration for the formation of the polymer was between 20 and 30 g/liter, with a yield coefficient of polysaccharide determined on the basis of consumed glucose (Yp/s) ranging from 7 to 9%. Over this value the polymer remained firmly bound to the cells and residual glucose in the medium increased. Batch culture of strain HYD-1545 performed by using a 2-liter fermentor and the 2216E medium supplemented with glucose (30 g/liter) allowed an optimization of EPS production (Fig. 3). The production started at the end of the exponential phase of growth and continued during the stationary phase, reaching a value of 11.0 g/liter at harvest (120 h). At the same time, the broth viscosity increased to 510 cP at 3 rpm (Fig. 3b). All the substrate was utilized after 4 days of culture, and the Yp/s was 37%. The pH of the culture decreased to 6.5 as glucose was consumed and then increased to 8.3 with the utilization of residual peptones (Fig. 3a). The cell dry weight, composed of the cells and firmly bound EPS, was maximal after 3 days of culture and then decreased as EPS were released into the medium. EPS-1545 is characterized by a high uronic acid content, between 32.5 and 39% for the sample prepared by procedure 1 and between 33 and 36% for the sample prepared by the second procedure (Table 1). Differences between the two samples with respect to amino sugar and protein content were detected, although both were minor components. Amino sugars found in sample 1 represented 2.7%. Protein content determined by the method of Lowry et al. (28) was also higher when the first procedure was used (3% instead of 0.8%). The protein content was lower when the Bradford (Bio-Rad) protein assay was used.
APPL. ENVIRON. MICROBIOL.
VINCENT ET AL.
4138
a
b -8
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60
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VL)
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4-
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cc
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--
:0
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=
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Co CL)
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0: D--_
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12-
-0
100 120
Time (hours)
Time (hours)
FIG. 3. EPS production by strain HYD-1545 during
an
aerobic batch culture in
a
1.4-liter-working-volume reactor.
The average molecular mass of the polymer was 1.8 x 106 g mol-', and the dn/dc value was 0.146 ml g-. Potentiometry. The mass per anionic equivalent found by pH measurements was 542 ± 50 g mol-'. The pKo value
The percentage of monomers recovered after hydrolysis or methanolysis was low, between 20 and 34% (Table 2), probably because of the stability of osidic linkage in this acidic polymer. Rhamnose, glucose, galactose, and mannose were found in all the samples as glucuronic acid, galacturonic acid, and 4,6-0(1-carboxyethylidene)-galactose after methanolysis (Table 2). However, the values obtained for mannose and rhamnose were very low. The percentage of recovery for monosaccharides was lower when the sample was prepared by procedure 1 (20.4 to 30.8%), and the highest value (33.9%) was obtained for sample 2, after methanolysis. After hydrolysis, molar glucose/galactose ratios were, respectively, 3:5.1 and 3:3 for samples 1 and 2. After methanolysis, molar glucose/galactose/glucuronic acid/ galacturonic acid ratios were, respectively, 3:1.2:0.9:0.5 and 3:1.7:0.7:0.4 for samples 1 and 2. These molar ratios are only indicative, because of the low degree of hydrolysis of the polymer.
extrapolated for
a
null net charge,
aT = 0
(LT
=
tN +
cxH,
is the degree of neutralization and aH = [H+]I/cP) where was 3.2 ± 0.2. Viscosimetry. Figure 4 shows the influence of ionic strength on the viscosities of EPS solutions prepared from EPS-1545. A salt excess screens the electrostatic repulsions between the charges and leads to a decrease in the rigidity of the polymeric chain and consequently in viscosity. However, in the presence of CaCl2, a stabilization and even an increase in viscosity are observed. The influence of the polymer concentration on the solution viscosities was also tested in O.1M NaCl. When -q (specific viscosity) is plotted against C[-q] (overlap parameters ([rj], intrinsic viscosity), the curve shows an abnormal increase aN
TABLE 1. Fractional analysis (grams per 100 g [dry weight]) of crude EPS-1545 % of total composition EPS-1545
sample'
Neutral sugars
Carbazol-sulfuric acidb
1 2* 2** 2***
51 49 51 55
39 35 33 33
Hexuronic acidsm-Phenylphenol' 32.5 36 33 33
Total
Hexosamines
carbohydrates
Lowryd
2.7 0.2 0.2 0.2
927, 86.2 84.2, 85.2 84.2 88.2
3.0 0.8 0.8 0.8
ProteinsBradforde NDf 0.2 0.2 0.2
a The polysaccharide was obtained during the stationary phase of growth, after 5 days of culture. Samples 2*, 2**, and 2*** were prepared by procedure 2 with 1 Gal molecule, 1 Glc molecule and 1 Gal molecule, and 2 Glc molecules and 1 Gal molecule, respectively, as the reference for neutral sugar determination. b Determined by the carbazol-sulfuric acid reaction. 'Determined by the m-phenylphenol method. d Determined by the method of Lowry et al. (28). e Determined by the method of Bradford (7). f ND, not determined.
EPS-PRODUCING BACTERIUM FROM VENT POLYCHAETE
VOL. 60, 1994
4139
TABLE 2. Fractional composition (grams per 100 g [dry weight]) of monosaccharides released from EPS-1545 by acici hydrolysis % of total composition GalX 6-4 GlcUA
EPS-1545 samplea
Method of
releaseb
Rha
Glc
Gal
Man
1
Acid hydrolysis Methanolysis
1.4 0.3
6.3 13.0
10.7 5.3
2.0 1.0
4.3
Acid hydrolysis Methanolysis
0.6 0.5
15.6 17.0
15.7 9.4
0.5 0.6
NQc
2
GalUA
GlcNAc
Total
4.0
2.5
0.4
20.4 30.8
4.0
2.4
NDd
32.4 33.9
The EPS was obtained during the stationary phase of growth, after 5 days of culture. Monosaccharides released by acid hydrolysis were measured as polyol acetates; those released by methanolysis were measured as trimethylsilyl glycosides. c NQ, not quantified. d ND, not determined.
a
b
in viscosity for C['r] values of >1 in semidilute solutions compared with that in xanthan solutions (Fig. 5). In the dilute solutions, polysaccharide 1545 exhibits normal behavior with a Huggins constant of about 0.3. Mark-Houwink parameter determination. The steric exclusion chromatography experiments and analysis by light scattering and viscosimetry as a function of elution volume allowed us to obtain the [-q] versus M variation, which is equal to 0.01
WO8.
DISCUSSION Strain HYD-1545 was isolated from the dorsal integument of A. pompejana, which is characterized by the presence of numerous bacteria covering the posterior part of the animal and forming a felt-like mat (19). The morphological diversity of worm epibacteria has been described, and filamentous, spiral-curved, rod-shaped, and prosthecated bacteria have been observed (18). Rod-shaped bacteria like strain HYD1545 were found on the integument of the worm without any particular location and were often observed to be linked to the cuticle by filamentous structures similar to the polysaccharidic filaments observed in scanning electron microscopic pictures of HYD-1545 (data not shown). The optimal growth temperature of strain HYD-1545 (27°C) corresponds to the thermal gradient measured in the biotope of the annelid (20 to 40°C), and the main metabolic characteristics appeared to be similar to those described for other epibiotic heterotrophic bacteria isolated from A. pompejana in
1984, during the Biocyarise cruise (37). Most of these isolates have been shown to be deficient in fermentative metabolism, to be able to decompose fatty acids, to produce H2S, and to utilize carbohydrate substrates as sole carbon sources. In addition, strain HYD-1545 exhibited unusual proteolytic and amylolitic activities. Tentative identification of strain HYD-1545 by morphological and biochemical tests showed that the organism belonged to the pseudomonad group (2). On the basis of its G+C content and phenotypical features, the strain could be assigned to the genus Alteromonas, and it appeared to be close to Alteromonas macleodii (3, 46). Most of the EPS-producing marine bacteria, isolated from various substrates such as seaweeds (45), sediments (32), artificial immersed surfaces (10), or animal cuticles and shells (48), are gram-negative rods belonging to the genus Vibrio, Flavobacterium, Pseudomonas, or Alteromonas. Alteromonas atlantica and Alteromonas colwelliana are known to produce acidic polysaccharides like that produced by strain HYD-1545 (20, 21). The percentage of recovery of monosaccharides released from EPS-1545 by acid hydrolysis and methanolysis was low because of the stability of osidic linkage in acidic polysaccharides. Glucose, galactose, glucuronic acid, and galacturonic acid, major components of this polymer, are common in marine acidic polysaccharide composition, as described by Christensen et al. (8) for Pseudomonas sp. strain NCMB 2021 and Okutani (33) for a Vibrio sp. Some other marine bacterial EPS, such as Marinactan (45), do not contain any acidic sugars.
/y
2000 -0
\ ~~~~~-
NaCI - Cad2
103
a~~~~~~~~~~~~~~~~cc
100l
_
X
>.
~~~~1545
~
Xi F
500
-
XANTHW
0.1 0.1
10
1
100
0
0.001
0.01
0.1
1
equiv/1 FIG. 4. Effects of NaCl and CaCl2 on viscosity of EPS. Polymer concentration, 0.4 g liter-'; temperature, 25°C. The EPS was obtained during the stationary phase of growth, after 5 days of culture.
[Salt)
c[rI FIG. 5. Comparison of the specific viscosities of polysaccharide EPS-1545 and xanthan in 0.1 M NaCl at 25°C as functions of the overlap parameter (C[-q]). The EPS was obtained during the stationary phase of growth, after 5 days of culture.
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APPL. ENVIRON. MICROBIOL.
VINCENT ET AL.
Mannose and rhamnose were also found in marine EPS excreted by gram-negative rods such as Vibrio spp. and Pseudomonas spp., but these small amounts could be attributed to contamination by components of the culture medium, such as mannose, or cell wall constituents, such as rhamnose (14). Procedure 2 (see Materials and Methods) for isolation of the polysaccharide provides preparations with the lower protein content. The EPS-1545 prepared by procedure 1 was purified by Talmont et al. (41) by using a DEAE Trisacryl ion-exchange column and a Sepharose 4B gel filtration column. The major fraction represented 60% of the crude extract; was made of neutral sugars (50%), uronic acids (40%), and 4,6-O-(1-carboxyethylidene)-galactose (GalX; X as pyruvate) (10%); and was composed of galactose, glucose, glucuronic acid, galacturonic acid, and GalX in a molar ratio of 2.5:3:2:2:1. Pyruvate was found in some marine EPS produced by gram-negative rods, including Pseudomonas atlantica, renamed A. atlantica (6). The presence of acidic sugars in the EPS may be important, considering the heavy-metal-binding properties of this polymer. A. pompejana lives in large colonies on black and white active smokers and diffusers at 13°N hydrothermal vents. The animal is therefore exposed to high concentrations of chemicals (e.g., metallic sulfides), and the polysaccharide coatings of such microorganisms may interact with heavy metals occurring in the environment of the worm. Numerous rod-shaped bacteria which, on metal-amended media, produced EPS were isolated from alvinellids (47). During this screening, about 30% of the isolated strains showed mucoid phenotypes on 2216E plates and 9% produced viscous broth as they were cultured on 2216E medium supplemented with glucose. The susceptibility of those heterotrophic bacteria to heavy metals has been studied by Jeanthon and Prieur (24), who showed an adaptation of alvinellid-associated microflora to this metal-rich environment. Numerous strains (92.3% of the isolates) displayed resistance to cadmium, zinc, arsenate, and/or silver and tolerated large amounts of copper. However, no clear relationship between EPS production and heavy metal resistance was found. Many data about the composition of EPS produced by marine bacteria are available, but little is known about the amounts produced by these organisms. The production and release of EPS during starvation has been studied by Wrangstadh et al. (49). In an attempt to increase the production, strain HYD-1545 was however cultured in a rich nutrient medium. Compared with those of other EPS-producing marine bacteria, the production rate of EPS by strain HYD-1545 following harvest of 11 g/liter during the 2-liter fermentor experiment, with a yield coefficient (Yp/s) of 37%, seems to be particularly significant. The value of the mass per anionic equivalent was in agreement with the presence of one uronic acid for every two neutral sugars in the polymer structure. The pKo value found is typical for carboxyl groups (40), excluding the presence of some strong acidic groups like sulfates or sulfonates on the polymer. The ionic strength had an influence on the viscosities of EPS-1545 solutions, and this behavior is common for polyelectrolyte solutions (17). The stabilization and the increase observed in the presence of CaCl2 may be due to intermolecular bonds or to a polymer conformational change (29). However, it is not certain that all divalent ions were removed during purification of the polysaccharide, and ions may have been responsible for the observed aggregation. Nevertheless, no gelation was produced either by adding an excess of divalent ions or by using acidic medium. The 1545 polymer did not
exhibit any conformational change when monovalent salt concentrations and temperatures were varied. The value found for the Mark-Houwink exponent permits classification of the 1545 polysaccharide among the semirigid polymers. The polysaccharide HYD-1545 is a polyelectrolyte, and the viscosity of its solutions depends strongly on their ionic strength. However, an unusual behavior is observed at higher concentrations, and this behavior could be related to intermolecular interactions in semidilute solutions. In order to better understand these physicochemical properties, more complete studies should be carried out. REFERENCES 1. Allison, D. G., and I. W. Sutherland. 1984. A staining technique for attached bacteria and its correlation to extracellular carbohydrate production. J. Microbiol. Methods 2:93-99. 2. Baumann, P., and L. Baumann. 1981. The marine gram-negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes, p. 1302-1331. In M. P. Starr, H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel (ed.), The prokaryotes. Springer-Verlag KG, Berlin. 3. Baumann, P., M. J. Gauthier, and L. Baumann. 1984. Genus Alteromonas, p. 342-352. In N. R. Krieg, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams and Wilkins Co., Baltimore. 4. Beji, A., D. Izard, F. Gavini, H. Leclerc, M. Leseine-Delstanche, and J. Krembel. 1987. A rapid chemical procedure for isolation and purification of chromosomal DNA from gram-negative bacilli. Anal. Biochem. 162:18-23. 5. Blumenkrantz, N., and G. Asboe-Hansen. 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54: 484-489. 6. Boyle, C. D., and A. E. Reade. 1983. Characterization of two extracellular polysaccharides from marine bacteria. Appl. Environ. Microbiol. 46:392-399. 7. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 8. Christensen, B. E., J. Kjosbakken, and 0. Smidsrod. 1985. Partial chemical and physical characterization of two extracellular polysaccharides produced by marine, periphytic Pseudomonas sp. strain NCMB 2021. Appl. Environ. Microbiol. 50:837-845. 9. Conn, H. J., J. W. Bartholomew, and M. W. Jennison. 1957. Staining methods, p. 10-36. In Society of American Bacteriologists (ed.), Manual of microbiological methods. McGraw-Hill Book Company, Inc., New York. 10. Corpe, W. A. 1970. Attachment of marine bacteria to solid surfaces, p. 73-87. In Adhesion in biological systems. Academic Press, Inc., New York. 11. Desbruyeres, D., F. Gaill, L. Laubier, and Y. Fouquet. 1986. Polychaetous annelids from hydrothermal vent ecosystems: an ecological overview. In M. I. Jones (ed.), The hydrothermal vents
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