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A Thermophilic Bacillus Isolated From an Eolian Shallow Hydrothermal Vent Able to Produce Exopolysaccharides
System. Appl. Microbiol. 23, 426-432 (2000) © Urban & Fischer Verlag _htt-,-p:_Ilw_w_w_.ur_ba_nf_isc_h_er_.de--.:./jo_u_rn_als_/s_am_ _ _ _ _ _ _ _ _ _ _ _
SYSTErvL4TIC AND APPLIED MICROBIOLOGY
A Thermophilic Bacillus Isolated From an Eolian Shallow Hydrothermal Vent, Able to Produce Exopolysaccharides BARBARA NICOLAUS!, ADRIANA PANICO I , MARIA CRISTINA MANCAt, LICIA LAMAI, AGATA GAMBACORTAt, TERESA MAUGERI2, CETTINA GUGLIANDOLQ2, and DANIELA CACCAM0 2
lIstituto per la Chimica di Molecole di Interesse Biologica, CNR, Arco Felice (Na), Italy 2Dipartimento di Biologia Animale ed Ecologia Marina, Messina, Italy Received August 2, 2000
Summary A thermophilic aerobic microorganism, able to produce two exocellular polysaccharides (EPS1 and EPS2), was isolated from a shallow hydrothermal vent at Vulcano island (Eolian Islands, Italy). EPS1 and EPS2 were based on mannose and glucose although in a different ratio. EPS2 possessed a trisaccharide repeating unit with a manno-pyranoside configuration. New isolate phenotype was studied by physiological and morphological observations, including biochemical and antimicrobial susceptibility tests (134). Previous analyses carried out on 87 field isolates and 8 thermophilic reference bacilli displayed low phenotypic similarity level (SSM = 65%) with Bacillus thermodenitrificans DSM 465. Optimal growth occurs at 65 DC and pH 7.0. Oxidase and catalase are negative. The guanine-plus-cytosine (G+C) content of DNA is 52.7%. Genotypic investigations demonstrated the diversity of the isolate with fifteen selected thermophilic Bacillus spp. when we compared the restriction patterns of the amplified 16S rDNA. The membrane lipids are based on fatty acids mainly belonging to the iso-family. Key words: Thermophilic Bacillus - Hydrothermal vent - Exopolysaccharides - Lipids - Numerical taxonomy
Introduction The isolation of thermophilic bacteria from hydrothermal sites is of great interest due to its potential for biotechnology (KRISTjANSSON, 1991). It is widely accepted that thermophiles offer important insights into the biology and evolution of many organisms. They provide also a valuable resource for exploitation in novel biotechnological processes (MANCA et al., 1996; NICOLAUS et al., 1999). The use of marine prokaryotic biopolymers in the biotechnology and biopharmaceutical industries is more and more increasing. Marine shallow hydrothermal vents around volcanic Eolian islands, close to Sicily coasts (Italy), represent accessible fields for isolation of thermophilic bacteria. Previous studies described diversity and distribution of bacterial communities within deep and shallow hydrothermal systems at Porto di Levante, Vulcano, revealing the presence of chemosynthetic, thermophilic, archaeal and bacterial strains (GUGLIANDOLO and MAUGERI, 1993; 1998; GUGLIANDOLO et al., 1999; HUBER et al., 1986; HUBER and STETTER, 1989; STETTER et al., 1983; ZILLIG et al., 1983). The most abundant bacterial population is represented by aerobic 0723-2020/00/23103-426 $ 15.00/0
spore-forming bacteria easy to be isolated (MAUGERI et al., 1998). In this paper we describe the composition and partial characterisation of two exopolysaccharides produced by a thermophilic Bacillus isolated from Porto Levante (Vulcano, Italy). Phenotypic and genotypic characterisation of this Bacillus are also reported.
Materials and Methods Study site A seawater sample was collected from a shallow hydrothermal vent of Vulcano Island (Eolian Islands, Italy) at Porto di Levante. Sampling was made at a 0.7 m depth. Recorded water temperature and pH were 70 DC and 5.2, respectively. Enrichment and isolation
Water sample was inoculated into Bacto Marine Broth 2216 (MB) (Difco) and incubated at 55 DC and 70 DC for three days in aerobic conditions. Sub-cultures were made on the same media supplemented with agar (2%). All colonies obtained on
Exopolysaccharide-Producing Thermophilic Bacillus plates were picked and purified by streaking onto Bacto Marine Agar 2216 (MA) (Difco) at least three times. The isolate B3-72 was routinely maintained at room temperature onto MA slopes.
Morphological and cultural characteristics Cells of the isolate were Gram stained and end os pores presence was observed either by phase contrast microscopy or in stained specimens. All growth tests were performed at 60°C in liquid medium. Growth optimum was measured by the increase in turbidity at 600 nm with a spectrophotometer (Ultraspec 3000, Pharmacia). Temperature and pH range for growth was determined after incubation in MB at 37 0, 55 0, 60 0, 65 0, 70 ° and 75°C and pH 5.5, 6.0, 7.0, 8.0 and 9.0. Halotolerance was tested in Nutrient Broth (Oxoid) supplemented with 0%, 2%, 5%, 7% and 10% (w/v) NaCi. Biochemical properties and antibiotic sensitivity Biochemical characteristics were screened by API 20 E, API 20 NE, API 50 CHB, API ZYM (bioMerieux) according to SHARP et ai. (1980) and LOGAN and BERKELEY (1984). Gelatin, starch and casein hydrolysis was tested according to MARTEINSSON et ai. (1996); xylan and dextran hydrolysis was tested according to WHITE et al. (1993). Lipolytic activity on Tween 20 and Tween 80 was tested on Sierra agar modified (DEGRYSE et ai., 1978). Sensitivity to the following ten antibiotics: nalidixic acid (30 rg), polymyxin B (300 u), chloramphenicol (3 rg), bacitracin (10 U), kanamycin (30 rg), tetracycline (30 rg), novobiocin (30 rg), penicillin G (10 U), streptomycin (10 rg) and erythromycin (15 rg) was tested using paper discs (Oxoid). The morphological and physiological characteristics of the isolate were compared with those of the following reference strains: B. stearothermophilus DSM 22 T , "B. thermodenitrificans" DSM 465, B. thermoleovorans DSM 5366T, "B. caldolyticus" DSM 405, "B. caldotenax" DSM 406, "B. caldovelox" DSM 411, B. kaustophilus DSM 7263, B. thermocatenulatus DSM 730, B. pallidus DSM 3670. DNA base composition The DNA was isolated according to CASHION et ai. (1977). The guanine-plus-cytosine (G+C) content of the DNA was determined as described by MESBAH et al. (1989). Extraction and amplification of 165 rONA The isolate was grown overnight onto MA at 20°C. Genomic DNA extraction and 16S rDNA amplification were performed according to the method of RAINEY et ai. (1994). Restriction analysis of PCR products Approximately 1.5 mg of amplified 16S rDNA was cleaved with 3 units of the restriction enzyme AluI (Boehringer Mannheim) in a total volume of 20 ml at 37°C for 3 hours. The enzyme was then inactivated by heating the reaction mixtures at 65 °C for 15 minutes. The reaction products were analysed by agarose gel (2.5% w/v) electrophoresis in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA) IX with 1 mg/ml ethidiurn bromide. The restriction pattern of 16S rDNA of the isolate was compared with that of the following reference strains: B. stearothermophilus DSM 22 T , "B. thermodenitrificans" DSM 465, B. thermoleovorans DSM 5366 T , "B . caldolyticus" DSM 405, "B. caldotenax" DSM 406, "B. caldovelox" DSM 411, B. kaustophilus DSM 7263, B. thermocatenulatus DSM 730, B. pallidus DSM 3670. Six thermophilic Bacillus spp., isolated from Eolian Islands and previously analysed at genetic level (MAUGERI et ai., submitted), were also used for comparison.
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Lipid and fatty acid analysis Lipid analysis was performed according to MANCA et ai. (1992). Lipid hydrolysis was performed by acid methanolysis. Fatty acid methyl esters were analysed by separation on TLC and on GC-MS as reported in Nicolaus et al. (1995). Identification of compounds was obtained with standards and by interpretation of mass spectra. Production of EP5 Microbial growth and EPS production were monitored quantitatively in batch culture by sampling 10 ml of culture broth at 0, 8, 16, 24, and 48 h; biomass production was followed turbidimetrically as described before. EPS production was tested on cell free cultural broth with phenol-sulphuric acid method using glucose as standard (DUBOIS et ai., 1956). Isolation and purification of EP5 Cells were harvested by cultural broth centrifugation in a stationary phase of growth (1 litre, 9800 g, 20 min). The liquid phase was treated with 1 volume of cold ethanol added drop by drop under stirring. Alcoholic solution was kept at -18°C overnight and then centrifuged at 15000 g for 30 min. The pellet was dissolved in hot water. The same procedure was repeated again. The final water solution was dialysed against tap water (48 h) and distilled water (20 h), then freeze-dried and weighted. The sample was tested for carbohydrate, protein, and nucleic acid contents. Polysaccharide fractions were purified by Gel Chromatography (Sephadex G-50; 2.5 x 50 cm) using H 2 0/Pyridine/AcOH (50015/2, by vol) as eluant, fractions were collected with a flux of 6 ml/h (5 ml each fraction), followed by anion exchange chromatography (Sepharose DEAE CI-6B; 1.5x40 cm) eluted with 0.1 I of H 2 0 and 1 I of NaCI gradient from 0 to 1 M with a flux of 12 mllh. The volume of each fraction was 10 ml. Fractions were tested for carbohydrate qualitatively by spot test on thin layer chromatography (TLC) sprayed with a-naphthol (STAHL, 1965) and quantitatively by Dubois method (DUBOIS et aI., 1956). The a-naphthol positive fractions were pulled. Each step of purification was estimated for protein content using Bradford reagent (Bio-Rad), for nucleic acid content reading the absorbance at 260 nm. Pulled fractions were exhaustively dialysed against water, freeze-dried and weighted. This material was used for all analytic works. Chemical characterization of EP5s Pyruvate was detected after polysaccharide hydrolysis using a solution of 0.5% (w/v) of 2,4-dinitrophenylhydrazine in 2 M HCl (DUCKWORTH et ai., 1993). Molecular weight was estimated by: Gel filtration on Sepharose GL-6B column (lx80 cm) using H 20/Pyridine/AcOH (500/5/2, by vol) as eluant, with a flux of 3.7 mllh, and density gradient centrifugation method, using a sucrose gradient from 0 to 50% w/v at 13000 g for 16 h (PAZUR et aI., 1994). In both methods 10 mg of EPS and a mixture of dextran for calibration curves (10 mg of T-700, MW 670000; T-400, MW 410000; T150, MW 154000) were used. Sugar analysis was performed by hydrolysis of EPS with 2 M trifluoroacetic acid (TFA) at 120°C for 2 h. Sugar mixture was identified by TLC and HPAE-PAD using standards for identification and calibration curves. TLC was developed with the following solvent system: a) acetone/ButOH/H 20 (8/2/2, by vol) for neutral sugars; b) ButOHJH 2 0/AcOH (31111, by vol) for acidic sugars; HPAE-PAD Dionex equipped with Carbopac PA 1 column was eluted isocratically with a) 15 mM NaOH for neutral sugars; b) buffer 100 mM NaOH and 150 mM NaOAc for acidic sugars (CLARKE et aI., 1991). Methylation analysis of the polysaccharides was carried out according to the methods described by JANSSON et ai. (1976)
428
B. NICOLAUS et al.
and WAEGHE et al. (1983). The methylated material (0.5 mg) was hydrolysed with 2 M TFA at 120°C for 2 h and then transformed in partially methylated alditol acetates. Identification of sugars was obtained by GLC and GC-MS using standards. GLC runs were performed on a Hewlett-Packard 5890A instrument, fitted with a FID detector and equipped with a HP-5-V column and N2 flux of 100 ml/min. The temperature program used was: 170°C 1 min from 170 to 180 °C 1 °C/min, 180°C 2 min, from 180 to 210 °C at 4 °Clmin. GC-MS was performed on a Hewlett-Packard 5890-5970 instrument, equipped with a HP5-MS column and with a N2 flux of 50 mllmin; 170°C 1 min, from 170 to 250 °C at 3 °C/min was used as temperature program. The absolute configuration of the sugars was performed as described by LEONTEIN et al. (1978) using optically active (+)-2butanol by GLC of their acetylated-( +)-2-butyl glycosides. For GLC runs the same instrument and conditions described III methylation analysis were used.
Spectroscopic analysis Infrared spectrum of polymer (KBr tablet, 10 mg) was recorded at room temperature using a FT-IR BIO-RAD spectrometer. Ultraviolet spectra of EPS were obtained reading the absorbance of aqueous solutions (3 mg/ml) from 350 to 210 nm on a Varian DMS-90 instrument. Optical rotation value was obtained on a Perkin-Elmer 243 B polarimeter at 25°C in water. NMR spectra were obtained on a Bruker AMX-500 (500.13 MHz for lH and 125.75 MHz for 13C) at 70°C. EPS samples were prepared for NMR spectra as reported in MANCA et al. (1996). Chemical shifts were reported in parts per million relative to sodium 2,2,3,3-d4 - (trimethylsilyl)propanoate for 'H NMR (PERLIN and CASU, 1982) and CDCl] for BC NMR (MANCA et al., 1996).
Results and Discussion Morphological and physiological characteristics Strain B3-72 cells were Gram-positive rods with oval endospores, catalase and oxidase negative. It grew aerobically, with the lower limit temperature of 45°C and the upper limit of 70 0c. Optimal temperature was 65°C. The pH range for growth was 6.0-9.0 with optimal pH 7.0. Strain B3-72 grew in a range 0%-2% NaCl with optimal growth without salt. Positive tests for the strain were: hydrolysis of gelatin, casein, esculin and Tween 20; reduction of nitrate; production of acetoin; fermentation of ribose, D-xylose, fructose, mannose, sorbose, esculin, N-acetyl-glucosammine, arbutin, salicin, cellobiose, maltose, trehalose, melibiose, sucrose, raffinose, turanose, 2-K-gluconate and 5-k-gluconate; resistance to nalidixic acid; activity of phosphatase alcaline esterase (C4), esterase lipase (C8), lipase C14, valine arylamidase, trypsin, ~-galactosidase, and a-naphthol-A5-BI-phosphohydrolase. Negative tests were: ONPG, arginine dehydrolase; lysine and ornithine decarboxylase; urease; tryptophan deaminase; production of indole and hydrogen sulphide; assimilation of arabinose, adipate and citrate; fermentation of glycerol, erythritol, D- and L-arabinose, L-xylose, adonitol, ~-methyl-D-glucoside, galactose, glucose,
Table 1. Differential phenotypic and chemo-taxonomic properties of Bacillus isolate B3-72 and Bacillus thermodenitrificans DSM 465.
Bacillus strain B3 -72
Bacillus thermodenitrificans DSM465
G+Cmol%DNA 62.7 Catalase Oxidase 45-70°C Temperature range 65°C Optimal temperature 6-9 pH range Growth with 5% NaCI ONPG Nitrate reduction + Denitrification Hydrolysis of starch Tween 20 + Acid from: glucose fructose + sucrose + melibiose + raffinose + salicine + L-xylose trehalose + Assimilation of arabinose Valine arylamidase + Phosphatase acid
52.9 + + 37-70°C 65 °C 6-9 + + + + +
+ + +
rhamnose, dulcitol, inositol, mannitol, sorbitol, amethyl-D-mannoside, a-methyl-D-glycoside, amygdalin, lactose, inuline, melezitose, starch, glycogen, xylitol, gentiobiose, D-lyxose, D-tagatose, D- and L-fucose, D- and L-arabitol and gluconate, hydrolysis of starch; activity of leucine and cystine arylamidase, chymotrypsin, a-galactosidase, ~-glucuronidase, a-glucosidase, phosphatase acid, ~-glucosidase, N-acetyl-~-glucosaminidase, a-mannosidase, a-fucosidase; resistance to streptomycin, tetracycline, novobiocin, penicillin G, chloramphenicol, erythromycin, kanamycin, bacitracin and polymyxin B. In a previous numerical analysis study, the isolate clustered at 65% similarity level with Bacillus thermodenitri{icans DSM 465 (MAUGERI et al., submitted). Differential phenotypic and chemotaxonomic characters of strain B3-72 in comparison with B. thermodenitri{icans DSM 465 are shown in Table 1. DNA base composition The G+C content of DNA determined in this study, 52.7 mol%, was in the range of thermophilic bacilli of the Bacillus rRNA Group 5 (ASH et al., 1991). DNA amplification and restriction analysis The 165 rDNA amplified restriction patterns generated by AluI showed that strain B3-72 was different from reference and field strains used for comparison. Since the enzyme AluI often generates species-specific restriction
Exopolysaccharide-Producing Thermophilic Bacillus
patterns, enabling bacterial strains to be divided into groups corresponding to a given species (MARTINEZMURCIA et aI., 1995), we could exclude that strain B3-72 represents one of the selected reference species. Further molecular investigations are now in progress in order to obtain more firm conclusions about the identity of this strain.
Polar lipids and fatty acid content The total lipid content in this strain constituted about 4-5% of the total dry weight of cells. The polar lipid pattern on TLC showed 7 spots phosphorus positive; the first one at higher Rf (0.68) was also positive to the test for amino lipid, and on the basis of TLC comparison with authentic standard could be identified as phosphatidylethanolamine (PEA). This component is one of the major polar lipids present in the mixture. Other abundant components were three phospholipids in the Rf range 0.56-0.48. The other phospholipids were in a trace amount as well as minor phosphoglycolipid. Glycolipids were not visualised, and this was probably due to their absence or occurrence in very low amounts. The phospholipids in our isolate represented the main polar lipids found, consistent with literature data for Bacillus species; on the contrary, the absence of glycolipids was reported only for few species (O'LEARY and WILKINSON, 1988; NICOLAUS et aI., 1995). Fatty acid composition after methanolysis of polar lipids was identified by GLC and mass spectrometry. The branched-iso-family was the most abundant component of the fatty acid mixture (FAMEs). These acids were a combination of iC15:0 (21 %), iC16:0 (24%), iC17:0 (18%); and accounted 63% of total FAMEs; the other major component was nC16:0 (14%) . Unsaturated acids -
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were absent. Each other component was less than 7% of total FAMEs: iC14 (1.2%), nC14 (1.2%), aiC15 (1.5%), aiC17 (6 .5%), nC17 (1.4%), iC18 (1.8%), nC18 (5.5%), iC19 and aiC19, lesser than 0.5%. Although the fatty acid profile found in this isolate, (e.g. the preponderance of branched fatty acids) was consistent with that reported for the Bacillus species (O'LEARY and WILKINSON, 1988) it should be noted that in this isolate the nC18 present in a relative high amount was unusually for the Bacillus species analysed so far (KAMPFER, 1994).
EPS purification Mucous colonies indicative of polymer production were observed on agar plates incubated aerobically at 60°C. Strain B3-72 produced highly viscous flasks cultures at concentration of glucose or sucrose of 0.6 gil. The production started at the end of the exponential phase of growth and continued during the stationary phase. The highest concentrations of polysaccharide harvested after 3 days of culture was approximately of 70 mgll. After centrifugation the supernatant formed precipitate with ethanol. The first ethanol precipitate could not be completely dissolved in distilled water even after heating to 100°C for 30 min. This pellet was collected and called "insoluble fraction" which accounts for 10% of the total carbohydrate fraction. Soluble fraction was tested for carbohydrate 75%, protein 5%, and nucleic acid 3%. The polymeric fraction was desalted on Sephadex G-50 with a yield of 70% and then chromatographed on DEAE-Sepharose CL-6B with a yield of 94%. The elution profile was shown in Fig. 1. Two fractions were obtained: the first (EPS1) was eluted only in water, the second (EPS2) eluted at 20% NaCI. On weight basis, EPSI
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Table 2. Partial characterisation of EPS2. Properties
Results
Carbohydrate content Protein content Molecular weight Optical rotation Sugar analysis Repeating Unit Configuration
80% 3% 400.000 D -14 Man:Glc 1:0.2 Trisaccharide
and sulfate group (S=O stretching 1240 em-I) were not observable suggesting the absence of uronic acid and sulfate residues. The UV spectra of EPSs did not indicate any strong absorption peaks in the range of 350 to 210 nm. The optical rotation of EPS2 was [al~O-14°, concentration of 1 mg/ml of H 2 0. Hydrolysis of EPS1 and EPS2 with 2 M trifluoroacetic acid yielded mannose and glucose, as principal constituents, in a relative ratio of 0.3:1 and 1:0.2, respectively. The 13e and IH NMR spectra of EPS2 were performed (Fig. 2). From lH-NMR we can observe the presence of three an orne ric signals at 8 5.00 (s), 8 5.05 (s) and 8 5.22 (s), all of them have a little coupling constant (ca. 1 Hz) probably due to a manno configuration. This observation seems to be confirmed by 13C-NMR spectrum. In fact the anomeric carbon region contains three signals at 8 101.2, 8 103.4 and 8 104.8. Remaining signals (lHNMR and 13C-NMR) are attributable to ring protons and carbons and confirm the presence of a pyranosidic hexose. From the comparison of the chemical shift values in IH and 13e spectra and little values of coupling constant of H1-H2 (less than 1 Hz) we could conclude that EPS2 of the strain B3-72 was a trisaccharide repeating unit essentially constituted by sugars having a mannopyranosidic configuration (Table 2). Studies of the chemical structure of these molecules, substituent identification and physical properties are essential for understanding their possible application. Increasing attention is being paid to biopolymers because of
Manno-pyranosidic
and EPS2 represented about 20 and 80% respectively. The two fractions were assayed for carbohydrate and protein content; EPS1 and EPS2 contained at least 80% sugars and less than 3% of proteins (Table 2).
EPS characterisation The molecular weight of EPS2 was estimated from a calibration curve of standard dextrans obtained by gel filtration on Sepharose CL-6B and also by density gradient centrifugation. In both cases, the molecular weight was approximately 4.0x10 s (Table 2). The IR spectrum of EPS2 was similar to those of other bacterial exopolysaccharides. In fact, it showed strong signal at 3420 cm-1 attributable to OH stretching, CH stretching signal at 2929 em-I; signal at 1420 cm- 1 an intense signal attributable to CH deformation and at 1055 cm- 1 attributable to OH deformation. Signals characteristics of uronic acid (C=O stretching 1730-1660 em-I)
Fig. 2.
145
140
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35 PPP
Exopolysaccharide-Producing Thermophilic Bacillus
their bioactive role and their wide range of commercial applications, which include uses as food additives (xanthan, alginate, dextran, glucomannan) and in non food applications such as viscosity control, gelation and flocculation (agar, glucan, mannan) (NICOLAUS et aI., 1996). It has been hypothesised that the synthesis of exo-cellular polysaccharides in microorganisms plays a major role in protecting cells from stress in extreme habitats (DE PHILIPPIS and VINCENZINI 1998). Therefore the production of exopolysaccharides by Bacillus, isolated from Vulcano, could serve as a boundary between the bacterial cell and its immediate environment.
Acknowledgements
This work was supported by the Mast project: MAS3CT9595-0034 "Microorganisms in deep sea vents and marine hot springs as sources of potentially valuable chemicals" coordinated by Prof. DANIEL PRIEUR. The author thanks EDUARDO PAGNOTTA and VINCENZO SCHIANO MORIELLO for assistance in some experiments and VINCENZO MIRRA for NMR service.
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HUBER, R., STETTER, K. 0.: Thiobacillus prosperus sp. nov. Represents a new group of halotolerant metal-mobilizing bacteria isolated from a marine geothermal field . Arch. Microbiol. 151,479-485 (1989). JANSSON, P. E., LENNART, K., LIEDGREN, H., LINDBERG, B., LONNGREN, J.: A practical guide to methylation analysis of carbohydrates. Chern. Commun. Univ. Stockholm 8, 1-76 (1976). KAMPFER, P.: Limits and possibilities of total fatty acid analysis for classification and identification of Bacillus species. System. Appl. Microbio!. 17,86-98 (1994). KRISTJANSSON, J. K.: Thermophilic bacteria. Boca Raton, CRC Press Inc., 1991. LEONTEIN, K., LINDBERG, B., LONNGREN, J.: Assignment of absolute configuration of sugar by G.L.c. of their acetylated glycosides formed from chiral alcohols. Carbohydr. Res. 62, 359-362 (1978). LOGAN, N. A., BERKELEY, C. W.: Identification of Bacillus strains using the API system. j. Gen. Microbio!. 130, 1871-1882 (1984). MANCA, M. c., NICOLAUS, B., LANZOTTI, V., TRINCONE, A., GAMBACORTA, A., PETER-KATALINIC, J., EGGE, H., HUBER, R., STETTER, K. 0.: Glycolipids from Thermotoga maritima, a hyperthermophilic microorganism belonging to Bacteria domain. Biochem. Biophys. Acta 1124,249-252 (1992). MANCA, M. c., LAMA, L., IMPROTA, R., ESPOSITO, E., GAMBACORTA, A., NICOLAUS, B.: Chemical composition of two exopolysaccharides from Bacillus thermoantarcticus. Appl. Environ. Microbiol. 62, 3265-3269 (1996). MARTEINSSON, V. T., BIRRIEN, J. L., jEANTHON, C., PRIEUR, D.: Numerical taxonomic study of thermophilic Bacillus isolated from three geographically separated deep-sea hydrothermal vents. FEMS Microbio!. Ecol. 21 , 255-266 (1996). MARTINEZ-MuRCIA, A. j ., ACINAS, S. G., RODRIGUEZ-VALERA, E: Evaluation of prokaryotic diversity by restrictase digestion of 16S rDNA directly amplified from hypersaline environments. FEMS Microbio!. Eco!. 17,247-256 (1995). MAUGERI, T. L., GUGLIANDOLO, c., CACCAMO, D.: Bacterial communities from marine, shallow hydrothermal vents off the Eolian Islands (Italy). 35Th CIESM Congress Procs. 35, 354-355 (1998). MAUGERI, T. L., GUGLIANDOLO, c., CACCAMO, D. (submitted): Phenetic diversity of thermophilic bacilli isolated from shallow, marine vents of the Eolian Islands. MESBAH, M., PREMACHANDRAN, U., WHITMAN, W. B.: Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int. ]. Syst. Bacteriol. 39,159-167 (1989). NICOLAUS, B., MANCA, M. c., LAMA, L., ESPOSITO, E., GAMBACORTA, A.: Effects of growth temperature on the polar lipid pattern and fatty acid composition of seven thermophilic isolates from the Antarctic continent. System. App!. Microbiol. 18,32-36 (1995). NICOLAUS, B., LAMA, L., MANCA, M. c., GAMBACORTA, A.: Extremophiles: polysaccharides and enzymes degrading polysaccharides. Recent Res. Devel. Biotech. Bioeng. 2, 37-64 (1999). O'LEARY, W. M., WILKINSON, S. j.: Gram-positive bacteria, pp. 117-185. In: Microbial lipids, Vo!' I (C. RATLEDGE, S. J. WILKINSON, eds.), New York, Academic Press 1988. PAZUR, J. K.: Neutral polysaccharides, pp. 73-124. In: Carbohydrate analysis (M. E CHAPLIN, E KENNEDY, eds.) 2nd ed., Oxford, IRL press 1994. PERLIN, A. S., CASU, B.: Spectroscopic methods, pp. 133-193. In: The polysaccharides, Vo!' I (G. O. ASPINAL, ed.), London, Academic Press Ltd. 1982. RAINEY, E A., FRITZE, D., STACKEBRANDT, E.: The phylogenetic diversity of thermophilic members of the genus Bacillus as
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revealed by 16S rDNA analysis. FEMS Microbiol. Lett. 115, 205-212 (1994). SHARP, R. ]., BOWN, K. J., ATKINSON, A.: Phenotypic and genotypic characterization of some thermophilic species of BacilIus.]. Gen. Microbiol. 117,201-210 (1980). STAHL, E.: Thin layer chromatography: a laboratory handbook. Berlin, Springer-Verlag KG 1965. STETTER, K. 0., KONIG, H ., STACKEBRANDT, E. : Pyrodictium gen. nov., a new genus of submarine disc-shaped sulphur-reducing Archaebacteria growing optimally at 105°C. System. Appl. Microbiol. 4, 535-551 (1983). WAEGHE, T. J., DARVILL, A. G., McNEIL, M., ALBERTSHEIM, P.: Determination, by methylation analysis, of the glycosyl-linkage composition of microgram quantities of complex carbohydrate. Carbohydr. Res. 123,281-304 (1983).
WHITE, D., SHARP, R. J., PRIEST, F. G.: A polyphasic taxonomic study of thermophilic bacilli from a wide geographical area. Antonie van Leeuwenhoek 64, 357-386 (1993). ZILLIG, W., HOLZ, I., jANEKovIC, D., SCHAFER, W., REITER, W. D.: The archaebacterium Thermococcus celer represents a novel genus within the thermophilic branch of the archaebacteria. System. Appl. Microbiol. 4,88-94 (1983).
Corresponding author BARBARA NICOLAUS, Istituto per la Chi mica di Molecole di Interesse Biologico, CNR, via Toiano 6, 80072 Arco Felice (Na), Italy Tel: 0818534179, Fax: 0818041770 e-mail: [email protected]
SYSTErvL4TIC AND APPLIED MICROBIOLOGY
A Thermophilic Bacillus Isolated From an Eolian Shallow Hydrothermal Vent, Able to Produce Exopolysaccharides BARBARA NICOLAUS!, ADRIANA PANICO I , MARIA CRISTINA MANCAt, LICIA LAMAI, AGATA GAMBACORTAt, TERESA MAUGERI2, CETTINA GUGLIANDOLQ2, and DANIELA CACCAM0 2
lIstituto per la Chimica di Molecole di Interesse Biologica, CNR, Arco Felice (Na), Italy 2Dipartimento di Biologia Animale ed Ecologia Marina, Messina, Italy Received August 2, 2000
Summary A thermophilic aerobic microorganism, able to produce two exocellular polysaccharides (EPS1 and EPS2), was isolated from a shallow hydrothermal vent at Vulcano island (Eolian Islands, Italy). EPS1 and EPS2 were based on mannose and glucose although in a different ratio. EPS2 possessed a trisaccharide repeating unit with a manno-pyranoside configuration. New isolate phenotype was studied by physiological and morphological observations, including biochemical and antimicrobial susceptibility tests (134). Previous analyses carried out on 87 field isolates and 8 thermophilic reference bacilli displayed low phenotypic similarity level (SSM = 65%) with Bacillus thermodenitrificans DSM 465. Optimal growth occurs at 65 DC and pH 7.0. Oxidase and catalase are negative. The guanine-plus-cytosine (G+C) content of DNA is 52.7%. Genotypic investigations demonstrated the diversity of the isolate with fifteen selected thermophilic Bacillus spp. when we compared the restriction patterns of the amplified 16S rDNA. The membrane lipids are based on fatty acids mainly belonging to the iso-family. Key words: Thermophilic Bacillus - Hydrothermal vent - Exopolysaccharides - Lipids - Numerical taxonomy
Introduction The isolation of thermophilic bacteria from hydrothermal sites is of great interest due to its potential for biotechnology (KRISTjANSSON, 1991). It is widely accepted that thermophiles offer important insights into the biology and evolution of many organisms. They provide also a valuable resource for exploitation in novel biotechnological processes (MANCA et al., 1996; NICOLAUS et al., 1999). The use of marine prokaryotic biopolymers in the biotechnology and biopharmaceutical industries is more and more increasing. Marine shallow hydrothermal vents around volcanic Eolian islands, close to Sicily coasts (Italy), represent accessible fields for isolation of thermophilic bacteria. Previous studies described diversity and distribution of bacterial communities within deep and shallow hydrothermal systems at Porto di Levante, Vulcano, revealing the presence of chemosynthetic, thermophilic, archaeal and bacterial strains (GUGLIANDOLO and MAUGERI, 1993; 1998; GUGLIANDOLO et al., 1999; HUBER et al., 1986; HUBER and STETTER, 1989; STETTER et al., 1983; ZILLIG et al., 1983). The most abundant bacterial population is represented by aerobic 0723-2020/00/23103-426 $ 15.00/0
spore-forming bacteria easy to be isolated (MAUGERI et al., 1998). In this paper we describe the composition and partial characterisation of two exopolysaccharides produced by a thermophilic Bacillus isolated from Porto Levante (Vulcano, Italy). Phenotypic and genotypic characterisation of this Bacillus are also reported.
Materials and Methods Study site A seawater sample was collected from a shallow hydrothermal vent of Vulcano Island (Eolian Islands, Italy) at Porto di Levante. Sampling was made at a 0.7 m depth. Recorded water temperature and pH were 70 DC and 5.2, respectively. Enrichment and isolation
Water sample was inoculated into Bacto Marine Broth 2216 (MB) (Difco) and incubated at 55 DC and 70 DC for three days in aerobic conditions. Sub-cultures were made on the same media supplemented with agar (2%). All colonies obtained on
Exopolysaccharide-Producing Thermophilic Bacillus plates were picked and purified by streaking onto Bacto Marine Agar 2216 (MA) (Difco) at least three times. The isolate B3-72 was routinely maintained at room temperature onto MA slopes.
Morphological and cultural characteristics Cells of the isolate were Gram stained and end os pores presence was observed either by phase contrast microscopy or in stained specimens. All growth tests were performed at 60°C in liquid medium. Growth optimum was measured by the increase in turbidity at 600 nm with a spectrophotometer (Ultraspec 3000, Pharmacia). Temperature and pH range for growth was determined after incubation in MB at 37 0, 55 0, 60 0, 65 0, 70 ° and 75°C and pH 5.5, 6.0, 7.0, 8.0 and 9.0. Halotolerance was tested in Nutrient Broth (Oxoid) supplemented with 0%, 2%, 5%, 7% and 10% (w/v) NaCi. Biochemical properties and antibiotic sensitivity Biochemical characteristics were screened by API 20 E, API 20 NE, API 50 CHB, API ZYM (bioMerieux) according to SHARP et ai. (1980) and LOGAN and BERKELEY (1984). Gelatin, starch and casein hydrolysis was tested according to MARTEINSSON et ai. (1996); xylan and dextran hydrolysis was tested according to WHITE et al. (1993). Lipolytic activity on Tween 20 and Tween 80 was tested on Sierra agar modified (DEGRYSE et ai., 1978). Sensitivity to the following ten antibiotics: nalidixic acid (30 rg), polymyxin B (300 u), chloramphenicol (3 rg), bacitracin (10 U), kanamycin (30 rg), tetracycline (30 rg), novobiocin (30 rg), penicillin G (10 U), streptomycin (10 rg) and erythromycin (15 rg) was tested using paper discs (Oxoid). The morphological and physiological characteristics of the isolate were compared with those of the following reference strains: B. stearothermophilus DSM 22 T , "B. thermodenitrificans" DSM 465, B. thermoleovorans DSM 5366T, "B. caldolyticus" DSM 405, "B. caldotenax" DSM 406, "B. caldovelox" DSM 411, B. kaustophilus DSM 7263, B. thermocatenulatus DSM 730, B. pallidus DSM 3670. DNA base composition The DNA was isolated according to CASHION et ai. (1977). The guanine-plus-cytosine (G+C) content of the DNA was determined as described by MESBAH et al. (1989). Extraction and amplification of 165 rONA The isolate was grown overnight onto MA at 20°C. Genomic DNA extraction and 16S rDNA amplification were performed according to the method of RAINEY et ai. (1994). Restriction analysis of PCR products Approximately 1.5 mg of amplified 16S rDNA was cleaved with 3 units of the restriction enzyme AluI (Boehringer Mannheim) in a total volume of 20 ml at 37°C for 3 hours. The enzyme was then inactivated by heating the reaction mixtures at 65 °C for 15 minutes. The reaction products were analysed by agarose gel (2.5% w/v) electrophoresis in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA) IX with 1 mg/ml ethidiurn bromide. The restriction pattern of 16S rDNA of the isolate was compared with that of the following reference strains: B. stearothermophilus DSM 22 T , "B. thermodenitrificans" DSM 465, B. thermoleovorans DSM 5366 T , "B . caldolyticus" DSM 405, "B. caldotenax" DSM 406, "B. caldovelox" DSM 411, B. kaustophilus DSM 7263, B. thermocatenulatus DSM 730, B. pallidus DSM 3670. Six thermophilic Bacillus spp., isolated from Eolian Islands and previously analysed at genetic level (MAUGERI et ai., submitted), were also used for comparison.
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Lipid and fatty acid analysis Lipid analysis was performed according to MANCA et ai. (1992). Lipid hydrolysis was performed by acid methanolysis. Fatty acid methyl esters were analysed by separation on TLC and on GC-MS as reported in Nicolaus et al. (1995). Identification of compounds was obtained with standards and by interpretation of mass spectra. Production of EP5 Microbial growth and EPS production were monitored quantitatively in batch culture by sampling 10 ml of culture broth at 0, 8, 16, 24, and 48 h; biomass production was followed turbidimetrically as described before. EPS production was tested on cell free cultural broth with phenol-sulphuric acid method using glucose as standard (DUBOIS et ai., 1956). Isolation and purification of EP5 Cells were harvested by cultural broth centrifugation in a stationary phase of growth (1 litre, 9800 g, 20 min). The liquid phase was treated with 1 volume of cold ethanol added drop by drop under stirring. Alcoholic solution was kept at -18°C overnight and then centrifuged at 15000 g for 30 min. The pellet was dissolved in hot water. The same procedure was repeated again. The final water solution was dialysed against tap water (48 h) and distilled water (20 h), then freeze-dried and weighted. The sample was tested for carbohydrate, protein, and nucleic acid contents. Polysaccharide fractions were purified by Gel Chromatography (Sephadex G-50; 2.5 x 50 cm) using H 2 0/Pyridine/AcOH (50015/2, by vol) as eluant, fractions were collected with a flux of 6 ml/h (5 ml each fraction), followed by anion exchange chromatography (Sepharose DEAE CI-6B; 1.5x40 cm) eluted with 0.1 I of H 2 0 and 1 I of NaCI gradient from 0 to 1 M with a flux of 12 mllh. The volume of each fraction was 10 ml. Fractions were tested for carbohydrate qualitatively by spot test on thin layer chromatography (TLC) sprayed with a-naphthol (STAHL, 1965) and quantitatively by Dubois method (DUBOIS et aI., 1956). The a-naphthol positive fractions were pulled. Each step of purification was estimated for protein content using Bradford reagent (Bio-Rad), for nucleic acid content reading the absorbance at 260 nm. Pulled fractions were exhaustively dialysed against water, freeze-dried and weighted. This material was used for all analytic works. Chemical characterization of EP5s Pyruvate was detected after polysaccharide hydrolysis using a solution of 0.5% (w/v) of 2,4-dinitrophenylhydrazine in 2 M HCl (DUCKWORTH et ai., 1993). Molecular weight was estimated by: Gel filtration on Sepharose GL-6B column (lx80 cm) using H 20/Pyridine/AcOH (500/5/2, by vol) as eluant, with a flux of 3.7 mllh, and density gradient centrifugation method, using a sucrose gradient from 0 to 50% w/v at 13000 g for 16 h (PAZUR et aI., 1994). In both methods 10 mg of EPS and a mixture of dextran for calibration curves (10 mg of T-700, MW 670000; T-400, MW 410000; T150, MW 154000) were used. Sugar analysis was performed by hydrolysis of EPS with 2 M trifluoroacetic acid (TFA) at 120°C for 2 h. Sugar mixture was identified by TLC and HPAE-PAD using standards for identification and calibration curves. TLC was developed with the following solvent system: a) acetone/ButOH/H 20 (8/2/2, by vol) for neutral sugars; b) ButOHJH 2 0/AcOH (31111, by vol) for acidic sugars; HPAE-PAD Dionex equipped with Carbopac PA 1 column was eluted isocratically with a) 15 mM NaOH for neutral sugars; b) buffer 100 mM NaOH and 150 mM NaOAc for acidic sugars (CLARKE et aI., 1991). Methylation analysis of the polysaccharides was carried out according to the methods described by JANSSON et ai. (1976)
428
B. NICOLAUS et al.
and WAEGHE et al. (1983). The methylated material (0.5 mg) was hydrolysed with 2 M TFA at 120°C for 2 h and then transformed in partially methylated alditol acetates. Identification of sugars was obtained by GLC and GC-MS using standards. GLC runs were performed on a Hewlett-Packard 5890A instrument, fitted with a FID detector and equipped with a HP-5-V column and N2 flux of 100 ml/min. The temperature program used was: 170°C 1 min from 170 to 180 °C 1 °C/min, 180°C 2 min, from 180 to 210 °C at 4 °Clmin. GC-MS was performed on a Hewlett-Packard 5890-5970 instrument, equipped with a HP5-MS column and with a N2 flux of 50 mllmin; 170°C 1 min, from 170 to 250 °C at 3 °C/min was used as temperature program. The absolute configuration of the sugars was performed as described by LEONTEIN et al. (1978) using optically active (+)-2butanol by GLC of their acetylated-( +)-2-butyl glycosides. For GLC runs the same instrument and conditions described III methylation analysis were used.
Spectroscopic analysis Infrared spectrum of polymer (KBr tablet, 10 mg) was recorded at room temperature using a FT-IR BIO-RAD spectrometer. Ultraviolet spectra of EPS were obtained reading the absorbance of aqueous solutions (3 mg/ml) from 350 to 210 nm on a Varian DMS-90 instrument. Optical rotation value was obtained on a Perkin-Elmer 243 B polarimeter at 25°C in water. NMR spectra were obtained on a Bruker AMX-500 (500.13 MHz for lH and 125.75 MHz for 13C) at 70°C. EPS samples were prepared for NMR spectra as reported in MANCA et al. (1996). Chemical shifts were reported in parts per million relative to sodium 2,2,3,3-d4 - (trimethylsilyl)propanoate for 'H NMR (PERLIN and CASU, 1982) and CDCl] for BC NMR (MANCA et al., 1996).
Results and Discussion Morphological and physiological characteristics Strain B3-72 cells were Gram-positive rods with oval endospores, catalase and oxidase negative. It grew aerobically, with the lower limit temperature of 45°C and the upper limit of 70 0c. Optimal temperature was 65°C. The pH range for growth was 6.0-9.0 with optimal pH 7.0. Strain B3-72 grew in a range 0%-2% NaCl with optimal growth without salt. Positive tests for the strain were: hydrolysis of gelatin, casein, esculin and Tween 20; reduction of nitrate; production of acetoin; fermentation of ribose, D-xylose, fructose, mannose, sorbose, esculin, N-acetyl-glucosammine, arbutin, salicin, cellobiose, maltose, trehalose, melibiose, sucrose, raffinose, turanose, 2-K-gluconate and 5-k-gluconate; resistance to nalidixic acid; activity of phosphatase alcaline esterase (C4), esterase lipase (C8), lipase C14, valine arylamidase, trypsin, ~-galactosidase, and a-naphthol-A5-BI-phosphohydrolase. Negative tests were: ONPG, arginine dehydrolase; lysine and ornithine decarboxylase; urease; tryptophan deaminase; production of indole and hydrogen sulphide; assimilation of arabinose, adipate and citrate; fermentation of glycerol, erythritol, D- and L-arabinose, L-xylose, adonitol, ~-methyl-D-glucoside, galactose, glucose,
Table 1. Differential phenotypic and chemo-taxonomic properties of Bacillus isolate B3-72 and Bacillus thermodenitrificans DSM 465.
Bacillus strain B3 -72
Bacillus thermodenitrificans DSM465
G+Cmol%DNA 62.7 Catalase Oxidase 45-70°C Temperature range 65°C Optimal temperature 6-9 pH range Growth with 5% NaCI ONPG Nitrate reduction + Denitrification Hydrolysis of starch Tween 20 + Acid from: glucose fructose + sucrose + melibiose + raffinose + salicine + L-xylose trehalose + Assimilation of arabinose Valine arylamidase + Phosphatase acid
52.9 + + 37-70°C 65 °C 6-9 + + + + +
+ + +
rhamnose, dulcitol, inositol, mannitol, sorbitol, amethyl-D-mannoside, a-methyl-D-glycoside, amygdalin, lactose, inuline, melezitose, starch, glycogen, xylitol, gentiobiose, D-lyxose, D-tagatose, D- and L-fucose, D- and L-arabitol and gluconate, hydrolysis of starch; activity of leucine and cystine arylamidase, chymotrypsin, a-galactosidase, ~-glucuronidase, a-glucosidase, phosphatase acid, ~-glucosidase, N-acetyl-~-glucosaminidase, a-mannosidase, a-fucosidase; resistance to streptomycin, tetracycline, novobiocin, penicillin G, chloramphenicol, erythromycin, kanamycin, bacitracin and polymyxin B. In a previous numerical analysis study, the isolate clustered at 65% similarity level with Bacillus thermodenitri{icans DSM 465 (MAUGERI et al., submitted). Differential phenotypic and chemotaxonomic characters of strain B3-72 in comparison with B. thermodenitri{icans DSM 465 are shown in Table 1. DNA base composition The G+C content of DNA determined in this study, 52.7 mol%, was in the range of thermophilic bacilli of the Bacillus rRNA Group 5 (ASH et al., 1991). DNA amplification and restriction analysis The 165 rDNA amplified restriction patterns generated by AluI showed that strain B3-72 was different from reference and field strains used for comparison. Since the enzyme AluI often generates species-specific restriction
Exopolysaccharide-Producing Thermophilic Bacillus
patterns, enabling bacterial strains to be divided into groups corresponding to a given species (MARTINEZMURCIA et aI., 1995), we could exclude that strain B3-72 represents one of the selected reference species. Further molecular investigations are now in progress in order to obtain more firm conclusions about the identity of this strain.
Polar lipids and fatty acid content The total lipid content in this strain constituted about 4-5% of the total dry weight of cells. The polar lipid pattern on TLC showed 7 spots phosphorus positive; the first one at higher Rf (0.68) was also positive to the test for amino lipid, and on the basis of TLC comparison with authentic standard could be identified as phosphatidylethanolamine (PEA). This component is one of the major polar lipids present in the mixture. Other abundant components were three phospholipids in the Rf range 0.56-0.48. The other phospholipids were in a trace amount as well as minor phosphoglycolipid. Glycolipids were not visualised, and this was probably due to their absence or occurrence in very low amounts. The phospholipids in our isolate represented the main polar lipids found, consistent with literature data for Bacillus species; on the contrary, the absence of glycolipids was reported only for few species (O'LEARY and WILKINSON, 1988; NICOLAUS et aI., 1995). Fatty acid composition after methanolysis of polar lipids was identified by GLC and mass spectrometry. The branched-iso-family was the most abundant component of the fatty acid mixture (FAMEs). These acids were a combination of iC15:0 (21 %), iC16:0 (24%), iC17:0 (18%); and accounted 63% of total FAMEs; the other major component was nC16:0 (14%) . Unsaturated acids -
429
were absent. Each other component was less than 7% of total FAMEs: iC14 (1.2%), nC14 (1.2%), aiC15 (1.5%), aiC17 (6 .5%), nC17 (1.4%), iC18 (1.8%), nC18 (5.5%), iC19 and aiC19, lesser than 0.5%. Although the fatty acid profile found in this isolate, (e.g. the preponderance of branched fatty acids) was consistent with that reported for the Bacillus species (O'LEARY and WILKINSON, 1988) it should be noted that in this isolate the nC18 present in a relative high amount was unusually for the Bacillus species analysed so far (KAMPFER, 1994).
EPS purification Mucous colonies indicative of polymer production were observed on agar plates incubated aerobically at 60°C. Strain B3-72 produced highly viscous flasks cultures at concentration of glucose or sucrose of 0.6 gil. The production started at the end of the exponential phase of growth and continued during the stationary phase. The highest concentrations of polysaccharide harvested after 3 days of culture was approximately of 70 mgll. After centrifugation the supernatant formed precipitate with ethanol. The first ethanol precipitate could not be completely dissolved in distilled water even after heating to 100°C for 30 min. This pellet was collected and called "insoluble fraction" which accounts for 10% of the total carbohydrate fraction. Soluble fraction was tested for carbohydrate 75%, protein 5%, and nucleic acid 3%. The polymeric fraction was desalted on Sephadex G-50 with a yield of 70% and then chromatographed on DEAE-Sepharose CL-6B with a yield of 94%. The elution profile was shown in Fig. 1. Two fractions were obtained: the first (EPS1) was eluted only in water, the second (EPS2) eluted at 20% NaCI. On weight basis, EPSI
Dubois
- - 0 - U.V.
.
A 260nm
U.V. A 280nm
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Table 2. Partial characterisation of EPS2. Properties
Results
Carbohydrate content Protein content Molecular weight Optical rotation Sugar analysis Repeating Unit Configuration
80% 3% 400.000 D -14 Man:Glc 1:0.2 Trisaccharide
and sulfate group (S=O stretching 1240 em-I) were not observable suggesting the absence of uronic acid and sulfate residues. The UV spectra of EPSs did not indicate any strong absorption peaks in the range of 350 to 210 nm. The optical rotation of EPS2 was [al~O-14°, concentration of 1 mg/ml of H 2 0. Hydrolysis of EPS1 and EPS2 with 2 M trifluoroacetic acid yielded mannose and glucose, as principal constituents, in a relative ratio of 0.3:1 and 1:0.2, respectively. The 13e and IH NMR spectra of EPS2 were performed (Fig. 2). From lH-NMR we can observe the presence of three an orne ric signals at 8 5.00 (s), 8 5.05 (s) and 8 5.22 (s), all of them have a little coupling constant (ca. 1 Hz) probably due to a manno configuration. This observation seems to be confirmed by 13C-NMR spectrum. In fact the anomeric carbon region contains three signals at 8 101.2, 8 103.4 and 8 104.8. Remaining signals (lHNMR and 13C-NMR) are attributable to ring protons and carbons and confirm the presence of a pyranosidic hexose. From the comparison of the chemical shift values in IH and 13e spectra and little values of coupling constant of H1-H2 (less than 1 Hz) we could conclude that EPS2 of the strain B3-72 was a trisaccharide repeating unit essentially constituted by sugars having a mannopyranosidic configuration (Table 2). Studies of the chemical structure of these molecules, substituent identification and physical properties are essential for understanding their possible application. Increasing attention is being paid to biopolymers because of
Manno-pyranosidic
and EPS2 represented about 20 and 80% respectively. The two fractions were assayed for carbohydrate and protein content; EPS1 and EPS2 contained at least 80% sugars and less than 3% of proteins (Table 2).
EPS characterisation The molecular weight of EPS2 was estimated from a calibration curve of standard dextrans obtained by gel filtration on Sepharose CL-6B and also by density gradient centrifugation. In both cases, the molecular weight was approximately 4.0x10 s (Table 2). The IR spectrum of EPS2 was similar to those of other bacterial exopolysaccharides. In fact, it showed strong signal at 3420 cm-1 attributable to OH stretching, CH stretching signal at 2929 em-I; signal at 1420 cm- 1 an intense signal attributable to CH deformation and at 1055 cm- 1 attributable to OH deformation. Signals characteristics of uronic acid (C=O stretching 1730-1660 em-I)
Fig. 2.
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130
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120
115
110
105
i
I
I
I
I
I
I
I
100
95
90
85
80
75
70
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i
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MR of EP 2.
11
I
45
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35 PPP
Exopolysaccharide-Producing Thermophilic Bacillus
their bioactive role and their wide range of commercial applications, which include uses as food additives (xanthan, alginate, dextran, glucomannan) and in non food applications such as viscosity control, gelation and flocculation (agar, glucan, mannan) (NICOLAUS et aI., 1996). It has been hypothesised that the synthesis of exo-cellular polysaccharides in microorganisms plays a major role in protecting cells from stress in extreme habitats (DE PHILIPPIS and VINCENZINI 1998). Therefore the production of exopolysaccharides by Bacillus, isolated from Vulcano, could serve as a boundary between the bacterial cell and its immediate environment.
Acknowledgements
This work was supported by the Mast project: MAS3CT9595-0034 "Microorganisms in deep sea vents and marine hot springs as sources of potentially valuable chemicals" coordinated by Prof. DANIEL PRIEUR. The author thanks EDUARDO PAGNOTTA and VINCENZO SCHIANO MORIELLO for assistance in some experiments and VINCENZO MIRRA for NMR service.
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Corresponding author BARBARA NICOLAUS, Istituto per la Chi mica di Molecole di Interesse Biologico, CNR, via Toiano 6, 80072 Arco Felice (Na), Italy Tel: 0818534179, Fax: 0818041770 e-mail: [email protected]