CURRENT MICROBIOLOGY Vol. 49 (2004), pp. 145–151 DOI: 10.1007/s00284-004-4303-x
Current Microbiology An International Journal © Springer Science⫹Business Media, Inc. 2004
A Novel Exopolymer-Producing Bacterium, Paracoccus zeaxanthinifaciens subsp. payriae, Isolated from a “Kopara” Mat Located in Rangiroa, an Atoll of French Polynesia Ge´rard Rague´ne`s,1 Xavier Moppert,1 Laurent Richert,2 Jacqueline Ratiskol,1 Claude Payri,3 Bernard Costa,2 Jean Guezennec1 1
Institut Franc¸ais de Recherche pour l’Exploitation de la Mer, Centre de Brest, DRV/VP/BMM, B.P. 70, 29280 Plouzane´, France Cairap, B.P. 14521, 98701 Arue, Tahiti, Polyne´sie Franc¸aise 3 Universite´ de Polyne´sie Franc¸aise, B.P. 6570, 98702 Faaa, Tahiti, Polyne´sie Franc¸aise 2
Received: 9 January 2004 / Accepted: 18 February 2004
Abstract. An aerobic, mesophilic and heterotrophic marine bacterium designated RA19, able to produce two different exocellular polymers and zeaxanthin, was isolated from a French polynesian bacterial mat (localy named “kopara”) situated in the atoll of Rangiroa. This microorganism, on the basis of its phenotypical features and the genotypic investigations, can be clearly assigned to the Parococcus zeaxanthinifaciens species and the name Parococcus zeaxanthinifaciens subsp. payriae is proposed. Optimal growth occurs between 30°C and 35°C, at pH between 6.5 and 7.5 and at ionic strength between 20 and 40 g/L of NaCl. The guanine-plus-cytosine content of DNA was 65.6%. This bacterium excreted, under laboratory conditions, two different polymers: a water-soluble exopolysaccharide (EPSI) consisting of 5 different sugars and a non-water-soluble macromolecule assumed to be of a glycoproteinic nature. The high sulfate content of the EPS1 and preliminary biological tests clearly showed that applications could be found in the very near future for both polymers in the cosmetic area. Their contribution to the viscous laminated microbial mat locally called “kopara” can be also mentioned.
As previously described by Mao Che et al. [14], marine microbial mats are encountered in a large variety of sites. They can also be found in some atolls of French Polynesia [1], where they are called “kopara”. These environments are known to produce a large number of viscous laminated layers due to the exopolysaccharides (EPS) secreted by the microbial communities [14, 22]. So far, most studies have focused on the cyanobacterial diversity present in these mats and, conversely, little is known about the bacterial diversity in such environments. In the course of the discovery of novel metabolites of biotechnological interest, it is now widely accepted that extremophilic microorganisms will provide a valuable resource, not only for exploitation in biotechnological processes but also as models for investigating how biomolecules are stabilized when subjected to unusual conditions. The aim of this study was to describe a new EPSCorrespondence to: G. Rague´ne`s; email:
[email protected]
producing strain designated as RA19 which was isolated from a “kopara” mat of the Rangiroa atoll. In a medium supplemented with a suitable carbon substrate, this bacterium was shown to produce two different extracellular polymers of biotechnological interest. Materials and Methods Bacterial strain. In November 2001, many samples of “kopara” were collected from the different microbial mats located on the Rangiroa atoll [2]. Enrichment cultures were purified on Marine Agar 2216E (MA, Difco Laboratories, Detroit, MI). Strain RA19 was selected because of its ability to exhibit a swarming mucoid phenotype on Marine Agar 2216E supplemented with 30 g/L of glucose. Reference strains. Paracoccus zeaxanthinifaciens type strain [4] (ATCC 21588T) was purchased from ATCC collection (Manassas, VA). Characterization of the bacterium. Isolation procedures, cytological and physiological characterizations and DNA base composition were performed as described by Rague´ne`s et al. [18, 19]. For phylogenetic characterization, DNA preparations and 16S rDNA sequence analysis
146 were done according to Wery et al. [30]. A total of 1372 bp was sequenced and then a total of 1335 bp was compared with others sequences avalaible in GenBank (www.ncbi.nlm.nih.gov.). The quantitative DNA reassociations between Paracoccus zeaxanthinifaciens type strain and strain RA19 were determined by spectrophotometry as reported elsewhere [18]. Production, isolation and purification of RA19 exopolymers. Exopolymer production was performed at 30°C in a 2 L fermenter (New Brunswick, Toulouse, France) containing 1 L of 2216E-glucose broth. A batch of culture medium was inoculated at 10% (v/v) with a suspension of cells in exponential phase. The pH was adjusted and maintained at 7.6 by automatic addition of KOH 0.25 mol/L. Foaming was avoided by the addition of Pluronic-PE6100 oil (BASF, Levallois/ Perret, France) at 0.1% (v/v). The air flow was fixed at 30 L/h and the agitation rate at 200 – 400 rpm to maintain the level of dissolved O2 around 25%. A water-soluble exopolysaccharide (EPS1) was recovered from the culture medium by high-speed centrifugation (20,000 g for 2 h) after 4 days. The residual gelatinous pellet contained bacterial cells along with a second polymer refered as EPS2. This latter polymer was recovered from the pellet as suggested by Samain et al. [23]. EPS1 and EPS2 were then treated separately by ultracentrifugation using a Pellicon-2 Mini Holder equipped with a Biomax 100K filter (Millipore Corporation, Bedford, MA) and lyophilized prior to further analysis. Chemical analysis. The total neutral carbohydrate content and hexuronic acids were determined using the orcinol-sulfuric method [21, 26] and the meta-hydroxydiphenyl method [6] respectively. The molar ratios of monosaccharides were determined according to Kamerling et al. [10] and Montreuil et al. [15]. The monosaccharides were analyzed after either aqueous hydrolysis or acidic methanolysis of the polymers and subsequent GC analyses as peracetylated derivatives or trimethylsilyl derivatives respectively. Protein and sulfate contents were determined by the method of Lowry et al. [13] with bovine serum albumin as standard and by Fourier transform infrared spectroscopy [12] and elemental analysis respectively. Methylation analysis of the polysaccharides were carried out using a modification of the Hakomori procedure [9] using lithium dimethyl sulfinyl anion [11] and MeI in Me2SO. The methylated compounds were recovered by the use of Sep-Pak C18 cartridges (Millipore, Bedford, MA) [27]. Reduction of ester groups with “Superdeuteride” LiB(C2H5)3D was carried out according to York et al. [31]. The methylated product was then hydrolyzed in 2 M trifluoroacetic acid (TFA) for 2 h at 120°C, reduced, acetylated and analyzed by gas-liquid chromatography–Mass spectrometry (GLC/MS). Pyruvate was detected by gas chromatography analysis as OTMS derivative after polysaccharide hydrolysis by a solution of 0.5% (w/v) of 2,4-dinitrophenylhydrazine in 2 M HCl [24]. Acetate content was determined by high-performance liquid chromatography analysis after hydrolysis of the polymer with TFA (4 M for 4 h at 100°C). Glycosyl composition of the EPS was determined by methanolysis using methanolic HCl. The methyl glycosides were converted to the corresponding trimethylsilyl derivatives as described by Montreuil et al. [15]. The percentage and identification of the different monosaccharides were determined by GLC using erythritol as the internal reference. The absolute configuration of the sugars was determined by GLC analysis of their corresponding trimethylsilylated R(⫺)-2-butyl derivatives by comparison with reference compounds [7]. Analytical GLC was performed on a Fisons instruments GC 8000 series fitted with a WCOT fused silica CP-SIL 5CB (60 m ⫻ 0.25 mm) with a temperature program of 50 –120°C at 20°C/min, then 120 – 240°C at 2°C/min and hydrogen as carrier gas. GLC-MS analysis was performed on a Carlo Elba HRGC 5160 gas chromatograph, fitted with
CURRENT MICROBIOLOGY Vol. 49 (2004) a WCOT fused silica CP-SIL 5CB (60 m ⫻ 0.32 mm) connected to a Nermag R10-10 mass spectrometer. The temperature program used was 50 –170°C at 20°C/min, then 170 –240°C at 4°C/min followed by 10 min at 240°C. In the electron impact mode, an ionization potential of 70 eV was used. NMR analysis. 1H-NMR and 13C-NMR spectra of solutions in D2O were recorded on a Bruker AMX 500 at 25°C after removal of the acyl groups by treatment in aqueous 2% acetic acid (AcOH) for 5 h at 100°C. The chemical shifts were expressed in parts per million relative to acetone as internal reference (␦H 2.22 ppm, ␦C 31.07 ppm). Heteronuclear single quantum coherence (HSQC) were performed according to standard pulse sequences. Size exclusion chromatography. SEC was performed using H2O, NaNO3 0.1 M as eluant protected by 200 ppm sodium azide at room temperature. Flow rate was 1 cm3/min using an isocratic pump Spectra Physics P100. A set of two columns was used: a TSK gel PW5000 and a TSK gel PW6000. For detection, a differential refractometer (Shodex R171) was employed and a home-made multiangle light-scattering detector described elsewhere [5]. Lipid analysis. Lipids were extracted from lyophilized cells (50 mg) using a modified Bligh and Dyer method. The extracted lipids were fractionated into neutral lipids, glycolipids and polar lipids by silicic column chromatography using a series of solvents of increasing polarity, dichloromethane, acetone and methanol. Fatty acid methyl esters were prepared from the esterified lipids in the polar (methanol) fraction by mild alkaline methanolic transesterification and analyzed by gas chromatography and gas chromatography-mass spectrometry (GC/MS) [8]. GC/MS analyses were carried out using a Carlo Erba (Rodano, Italy) model 5160 HRGC chromatograph coupled to a quadrupole Nermag R10-10H mass spectrometer. The GC/MS analyses were performed under the same conditions as those described above, but helium was used instead as the carrier gas (1.5 bar). Identification of fatty acid methyl esters was based on comparison of relative retention times against internal standard and GC spectra. Electron energy in electron impact mode was 70 eV. Double bond position and geometry of monounsaturated FAME were determined after the formation of dimethyl-disulfide (DMDS) adducts prepared according to methods described previously [16]. Fatty acids are designated by the total number of carbon atoms: number of double bonds, followed by the position of the double bond from the terminal () end of the molecule. The suffixes c and t indicate cis and trans geometry. Pigment analysis. For analysis of the pigment by HPLC, a 250 mL shake flask with four baffles (Wheaton, Millville, NJ) containing 100 mL of Marine Broth was inoculated with 10 mL of a liquid culture in exponantial phase (12 h) and incubated at 30°C for 24 h with shaking. Cells were collected by centrifugation. Pigments were extracted from the cells by suspending the pellet in 9 mL of acetone/methanol (7:2). Solids were removed from the extracts by centrifugation. The supernatant was analyzed by HPLC equipped with a diode array detector and using a Kromasil C18 column along with a guard column.
Results Morphology. Strain RA19 was a non-motile, strictly aerobic, pigmented, nonfermentative, Gram-negative short rod, 0.9 ⫻ 1.3 m in size. Five-day-old colonies on MA 2216E-glucose (30 g/L) medium appeared orange
G. Rague´ ne`s et al.: A Novel Exopolymer-Producing Bacterium, Paracoccus zeaxanthinifaciens
147
Fig. 1. Phylogenetic position of the new bacterium (RA19) within the genus Paracoccus. Rhodobacter capsulatus was used as the outgroup. The topology shown is an unrooted tree obtained using a neighbor-joining algorithm. A bootstrap analysis using parsimony is shown as percentages of replications at the branches.
due to the accumulation of zeaxanthin, smooth, gummy, and about 0.7 cm in diameter (0.4 cm without glucose). Under the same conditions, the reference strain P. zeaxanthinifaciens exhibits no difference between medium with or without glucose. As a result, we can conclude that the type strain is not an EPS-producing bacterium. Culture conditions. The optimal temperature for growth was between 30°C and 35°C, the optimal pH was between 6.5 and 7.5 and the optimal ionic strength was between 20 and 40 g/L of NaCl. The doubling time, under optimal conditions, was 35 min. Metabolic properties. For standing comparison between strain RA19 and the P. zeaxanthinifaciens type strain, tests were performed using API20NE, APIZYM, ATB7 strips and Biolog GN microplates. No difference was observed except on Biolog GN microplates, where the strain RA19 differs from the type strain by its assimilation of ␣-ketobutyric acid, L-ornithine and inosine. The other chemical and nutritional characteristics showed wide utilization of carbohydrate substrates as the sole source of carbon. Antibiotic susceptibility tests showed that strain RA19 is bacitracin (2 mg/L) resistant. Computer-based analysis of the results of Biolog GN microplates indicated clearly that RA19 was a P. zeaxanthinifaciens-close bacterium. DNA base composition. The G⫹C content of strain RA19 was 65.6 mol%. This value is close to that of the type strain of Paracoccus zeaxanthinifaciens (66.9 – 67.7 mol%) [4]. Phylogenetic analyses. The sequence of the 16S rRNAencoding gene of strain RA19 was determined (1372 bp) and deposited in the EMBL sequence database under
accession number AJ507806. Phylogenetic analyses using the BLAST program showed that strain RA19 belonged to the alpha subdivision of the phylum Proteobacteria and that it was closely related to Paracoccus zeaxanthinifaciens. More analyses were performed using the sequences of the type strain of the genus Paracoccus. Results are shown in Fig. 1. All three methods used (neighbor joining, maximum parsimony, maximum likelihood) placed RA19 in a monophyletic group with P. zeaxanthinifaciens. The percentage sequence similarity between RA19 and P. zeaxanthinifaciens was 99%, clearly above the limit of intraspecies variability (97%) as proposed by Stackebrandt and Goebel [25]. Nevertheless, we conducted quantitative DNA/DNA hybridization to confirm (or not) RA19 as a subspecies of P. zeaxanthinifaciens. DNA reassociation. The measurement of DNA–DNA homology between strain RA19 and the most closely related Paracoccus as deduced from the phylogenetic analysis showed a homology value of 87.3% with P. zeaxanthinifaciens. Taking into consideration the criteria recommended by Wayne et al. [28], this result confirmed strain RA19 as a subspecies of P. zeaxanthinifaciens type strain. Lipid analysis. Phospholipid ester linked fatty acids are listed in Table 1. The fatty acid profile is characterized by the predominance of C16:17, accounting from 82% of the total acids. Saturated fatty acids were present as lower concentration, with C16:0 accounting for 15% while C18:0 was present only as trace. This is consistent with other fatty acid profiles reported in other Paracoccus strains [3].
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Table 1. Cellular fatty acid composition of strain RA19 FAME
C14:0
C14:17
C16:0
C16:17
C18:0
C18:17
EPS1
0.5
0.4
14.9
82.1
0.3
1.4
Production of exopolymers. During the batch fermentation of strain RA19, the production of the two exoplymers began at the end of the exponential phase and continued during the entire stationary phase, reaching a value of 3.7 g/L (dry weight) for EPS1 and 21 g/L (dry weight) for EPS2 at the end of the fermentation (4 days). Characterization of EPS. Fourier transform infrared spectroscopy (FTIR) was performed on both polymers. The water-soluble EPS (EPS1) exhibited a broad band beyond 3000 cm⫺1, resulting from O-H and C-H stretching bands at 3420 cm⫺1 and 2900 cm⫺1 respectively, and an intense absorption band at 1630 –1650 cm⫺1 with a shoulder at 1730 cm⫺1 due to the presence of carboxylic groups of the uronic acids. This spectrum is also characterized by an intense doublet at 1250 and 1230 cm⫺1 indicative of the presence of ester sulfate groups associated with the polysaccharide (Fig. 2). The second polymer exhibited a more complex spectrum characterized by bands characteristic of proteins and a carbohydrate moiety (data not shown). The low amount of protein in EPS1 (⬍3%) indicated a good efficiency of the purification protocol applied to this polymer. This water-soluble polysaccharide is characterized by a high content of neutral sugars while uronic acids were present at low concentration (Table 2). No traces of amino-sugars were detected for this polymer. Very interestingly, sulfate as determined by FTIR, a colorimetric method and CHNS elemental analysis accounted for up to 29% of the polysaccharide. GC analysis of monosaccharides as per-O-trimethylsilyl methylglycosides is listed in Table 3. Fucose, galactose and rhamnose predominated as neutral sugars in equal amounts while galacturonic acid was the only uronic acid identified in these fractions. The 1H NMR spectrum of the water-soluble polysaccharide (EPS1) also indicated the presence of sugar substituants such as succinate and acetate in the alkylic region (up 1.5 to 2.5 ppm). The second polymer was characterized by a very high concentration of proteins, indicating the occurrence of a glycoproteinic polymer. The carbohydrate moiety of this glycoprotein consisted of glucose, galactose, rhamnose and fucose in equal relative proportions. Molecular weight. This water-soluble polymer consisted of a high-molecular-weight chain estimated at
3.2 ⫻ 106 Da with a low polydispersity (Ip) ⬍ 1.4 along with with a second one with a molecular weight of 3 ⫻ 10 5 Da (Ip ⫽ 1.3) (Fig. 3). Due to its low water solubility, no experiment was performed on the second polymer. Pigment. HPLC analysis clearly indicated zeaxanthin as the major carotenoid produced by the strain RA19. Other isomers of zeaxanthin were also present in low concentrations.
Discussion On the basis of morphological and biochemical data and G⫹C content, the strictly aerobic, mesophilic, heterotrophic new bacterium isolated from a Polynesian “kopara” mat clearly belongs to the genus Paracoccus and more precisely to the species P. zeaxanthinifaciens. Phylogenetic analyses of 16S rDNA clearly demonstrated that this bacterium forms, with P. zeaxanthinifaciens, a welldefined taxon that deserves the rank of genus. Since the measurement of genomic DNA-DNA homology revealed a relatedness of 87.3% between the two bacteria, it is appropriate to conclude that they belong to the same species. In fact, this homology is less than 90% and owing to the facts that (i) the two bacteria have some different metabolic patterns, (ii) the P. zeaxanthinifaciens type strain does not produce EPS under the same conditions and (iii) the two bacteria have distant origins (African Red Sea and a Pacific Polynesian atoll), we propose that they should be dissociated, and the name P. zeaxanthinifaciens subsp. payriae is proposed for the bacterium RA19. The chemical composition of the water-soluble exopolysaccharide excreted by RA19 strain appeared to be different from those produced by other bacteria from extreme environments. The most important feature of the water-soluble exopolysaccharide is its high sulfate content (up to 29%) as native state. Introducing sulfate groups through enzymatic and/or chemical means can improve the biological activity of polysaccharides. But to the best of our knowledge the biosynthesis of a highly sulfated polysaccharide by a marine bacterium has not been described in the literature. Polysaccharides from different origins with antiviral activity, including human immunodeficiency virus activity, and physiological functions such as blood anticoagulants or regulators of growth factors amongst other biological activities, have been shown to have activity related to both the sulfate content and location of the sulfate group on the repeating unit of the polymers [29]. Dextran sulfate has been stated to have both anticoagulant and antilipemic properties useful in the treatment of lipemia and arterioscleosis. The
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G. Rague´ ne`s et al.: A Novel Exopolymer-Producing Bacterium, Paracoccus zeaxanthinifaciens
Fig. 2. FTIR spectrum of the water-soluble exopolysaccharide (RA19-1). Table 2. Chemical composition of the two different EPSs produced by strain RA19
RA19
Neutral sugars (%)
Uronic acids (%)
Proteins (%)
Sulfates (%)
Osamines (%)
EPS1 EPS2
48 12
6 4
3 33
29 9
– 3
Table 3. Monosaccharide ratios of the two polymers produced by strain RA19 RA19
Gal
Glc
Rha
Fuc
GalUA
EPS1 EPS2
11.7 2.0
4.4 2.1
12.4 1.7
14.7 2.2
2.9 –
Gal, galactose; Glc, glucose; Rha, rhamnose; Fuc, fucose; GalUA, galacturonic acid.
sulfate groups in heparin play a critical role in its antithrombotic activity [17]. Thus the high sulfate content of this particular bacterial polysaccharide needs special consideration. While the water solubility of the polysaccharide was enhanced by its high degree of sulfation the presence of acetate and succinate groups is responsible for the interesting rheological properties of the polysaccharide, including its gel-like behavior [20]. Carotenoids are natural pigments synthesized by plants and microorganisms. Zeaxanthin is known to have applications in poultry pigmentation and in the prevention of human macular degeneration [4]. But one of the natural function of the carotenoids is to absorb sunlight during photosynthesis and to protect cells from photosensitization. Because of this property, its evaluation in
the cosmetic area and especially in solar protection is under way. Moreover, these exopolysaccharides may contribute to the viscous laminated microbial mat locally called “kopara” and pigment to the orange coloration of the mats. The fact that bacterium RA19 produced exopolymers, but not its type strain, is probably connected to the “extreme” conditions of “kopara” mats. Description of P. zeaxanthinifaciens subsp. payriae subsp. nov. Paracoccus zeaxanthinifaciens subsp. payriae (pay.ri.ae. N.L. gen. n. payriae in honour of Claude Payri, a great figure of Polynesian atoll ecology). Cells are Gram-negative, non-motile, short rods 0.9 ⫻ 1.3 m. Colonies on Marine Agar are circular, convex, smooth and pigmented deep yellow. Strictly aerobic. Catalase,
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Fig. 3. 1H NMR spectrum of the water-soluble polysaccharide (RA19-1).
oxidase, -galactosidase and -glucosidase are positive. Growth occurs at 20 – 40°C but not at 4°C and weakly at 10°C. Seawater base obligate for growth. The G⫹C content of the DNA is 65.6%. DNA- DNA homology with P. zeaxanthinifaciens type strain is 87.3%. Produces zeaxanthin. Isolated from a “kopara” mat on the atoll of Rangiroa (French Polynesia). The type strain P. zeaxanthinifaciens subsp. payriae has been deposited in the Collection Nationale de Culture de Microorganismes (Institut Pasteur, Paris, France) as strain CNCM I-2926.
6.
7.
8.
9.
ACKNOWLEDGMENTS 10. The authors would like to thank the Cairap SA (Arue, Tahiti) for financial support, Serge Andrefouet (University of South Florida, St Petersburg, FL) for his great contribution to the ground localization of the mats, and Bernard Rousseau and Jean Claude Thomas (ENS, Paris, France) for the pigment determination.
Literature Cited 1. Andre´ foue¨ t S, Claereboudt M, Matsakis P, Page`s J, Dufour P (2001) Typology of atoll rims at landscape scale in Tuamotu Archipelago (French Polynesia) using SPOT-HRV images. Int J Remote Sens 22:987–1004 2. Andre´ foue¨ t S, Hochberg EJ, Payri C, Atkinson MJ, Muller-Karger FE, Ripley H (2003) Multi- scale remote sensing of microbial mats in atoll environment. Int J Remote Sens 24:2661–2682 3. Baj J (2000) Taxonomy of the genus Parococcus. Acta Microbiol Pol 49:185–200 4. Berry A, Janssens D, Hu¨ mbelin M, Jore JPM, Hoste B, Cleenwerck I, et al. (2003) Paracoccus zeaxanthinifaciens sp. nov., a zeaxanthin-producing bacterium. Int J Syst Evol Microbiol 53: 231–238 5. Busnel JP, Degoulet C, Nicolai T, Woodley W, Patin P (1985)
11.
12.
13.
14.
15.
Multiangle light scattering angle and viscosimetric detector for size-exclusion chromatography. J Phys III France 5:1501–1512 Filisetti-Cozzi TMCC, Carpita NC (1991) Measurement of uronic acids without interference from neutral sugars. Anal Biochem 197:157–162 Gerwig GJ, Kamerling JP, Vliegenthart JFG (1978) Determination of the D and L configuration of neutral monosaccharides by highresolution capillary GLC. Carbohydr Res 62:349 –357 Guezennec J, Ortega-Morales O, Rague´ ne`s G, Geesey G (1998) Bacterial colonization of artificial substrate in the vicinity of deepsea hydrothermal vents. FEMS Microbiol Ecol 26:89 –99 Hakomori S (1964) A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinil carbanion in dimethyl sulfoxide. J Biochem 55:205–208 Kamerling JP, Gerwig GJ, Vliegenhart JFG, Clamp JR (1975) Characterization by gas chromatography-mass spectrometry of pertrimethylsilyl glycosides obtained in the methanolysis of glycoproteins and glycolipids. Biochem J 151:491– 495 Kvernhein AL (1987) Methylation analysis of polysaccharides with butyllithium in dimethyl sulfoxide. Acta Chem Scand B41: 150 –152 Lijour Y, Gentric E, Deslandes E, Guezennec J (1994) Estimation of the sulfate content of hydrothermal vent bacterial polysaccharides by Fourier Transform Infrared Spectroscopy. Anal Biochem 220:1–5 Lowry OH, Rosenbrough NJ, Farr A, Randall RJ (1953) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265– 275 Mao Che L, Andre´ foue¨ t S, Bothorel V, Guezennec M, Rougeaux H, Guezennec J, et al. (2001) Physical, chemical and microbiological characteristics of microbial mats (KOPARA) in the South Pacific atolls of French Polynesia. Can J Microbiol 47:994 –1012 Montreuil J, Bouquelet S, Debray H, Fournet B, Spik G, Stecker G (1986) Carbohydrate analysis: A practical approach. In: Chaplin MF, Kennedy JK (eds) Glycoproteins. Washington, DC: IRL Press, pp 143–204
G. Rague´ ne`s et al.: A Novel Exopolymer-Producing Bacterium, Paracoccus zeaxanthinifaciens 16. Nichols PD, Guckert JB, White DC (1986) Determination of monounsaturated fatty acid double bonds and geometry for microbial cultures and complex consortia by capillaty GC-MS and their dimethyl disulphide adducts. J Microbiol Methods 5:49 –55 17. Petitou M (2003) From heparin to synthetic oligosaccharides. Bull Acad Natl Med 187:47–56 18. Rague´ ne`s GHC, Pere`s A, Ruimy R, Pignet P, Christen R, Loaec M, et al. Guezennec JG (1997) Alteromonas infernus sp. nov., a new polysaccharide-producing bacterium isolated from a deep-sea hydrothermal vent. J Appl Microbiol 82:422– 430 19. Rague´ ne`s G, Cambon-Bonavita MA, Lohier JF, Boisset C, Guezennec J (2003) A novel, highly viscous polysaccharide excreted by an Alteromonas isolated from a deep-sea hydrothermal vent shrimp. Curr Microbiol 46:448 – 452 20. Ridoux MJ, Brownsey GJ, York GM, Walker GC, Morris VJ (1997) Effect of o-acyl substituents on the functional behaviour of Rhizobium meliloti succiniglycan. Int J Biol macromols 20:1–7 21. Rimington C (1931) The carbohydrate complex of serum protein. II. Improved method for isolation and redetermination of structure. Isolation of glucosaminodimannose from protein of blood. Biochem J 25:1062–1071 22. Rougeaux H, Guezennec M, Mao Che L, Payri C, Deslandes E, Guezennec J (2001) Microbial communities and exopolysaccharides from polynesian mats. Mar Biotechnol 3:181–187 23. Samain E, Milas M, Bozzi L, Dubreucq G, Rinaudo M (1997) Simultaneous production of two different gel-forming exopolysaccharides by an Alteromonas strain originating from deep sea hydrothermal vents. Carbohydr Polym 34:235–241
151
24. Sloneker JH, Orentas DG (1962) Pyruvic acid, a unique component of an exocellular bacterial polysaccharide. Nature 194:478 – 479 25. Stackebrandt E, Goebel BM (1994) Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846 – 849 ¨ ber den Gehalt der wichtigsten 26. Tillmans J, Philippi J (1929) U Protein der Nahrungsmittel an Kohlehydrat and u¨ ber ein kolorimetrisches Verfahren zur quantitativen Bestimmung von stickstoff-freiem Zucker in Eiweiss. Biochem Z 215:36 – 60 27. Waeghe TJ, Darvill AG, McNeil M, Albertsheim P (1983) Determination, by methylation analysis, of the glycosyl-linkage composition of microgram quantities of complex carbohydrate. Carbohydr Res 123:281–304 28. Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevesky MI, et al. (1987) Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Int J Syst Bacteriol 37:463– 464 29. Weiner R, Langille S, Quintero E (1995) Structure, function and immunochemistry of bacterial exopolysaccharides. J Ind Microbiol 15:339 –346 30. Wery N, Moricet JM, Cueff V, Lesongeur F, Cambon-Bonavita MA, Barbier G (2001) Caloranaerobacter azorensis gen. nov., sp. nov., an anaerobic thermophilic bacterium isolated from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 51:1789 –1796 31. York WS, Darvill AG, McNeil M, Stevenson TT, Albersheim P (1985) Isolation and characterization of plant cell walls and cell wall component. Methods Enzymol 118:3– 40
