Thermomonas hydrothermalis sp. nov., A New Slightly Thermophilic γ-Proteobacterium Isolated from a Hot Spring in Central Portugal

System. Appl. Microbiol. 26, 70–75 (2003) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam

Thermomonas hydrothermalis sp. nov., A New Slightly Thermophilic γ-Proteobacterium Isolated from a Hot Spring in Central Portugal Marta P. Alves1, Fred A. Rainey2, M. Fernanda Nobre3, and Milton S. da Costa1 1

Departamento de Bioquímica, Universidade de Coimbra, Coimbra, Portugal Department of Biological Sciences, Louisiana State University, La, USA 3 Departamento de Zoologia, Universidade de Coimbra, Coimbra, Portugal 2

Received: November 21, 2002

Summary Several non-pigmented bacterial isolates, with an optimum growth temperature of about 50 °C, were recovered from the hot spring at São Gemil in Central Portugal. Phylogenetic analyses of the 16S rRNA gene sequence of strain SGM-6T indicated that this organism represents a new species of the γ-subclass of the Proteobacteria that is closely related to the newly described slightly thermophilic species Thermomonas haemolytica. The major fatty acids of strains SGM-6T and SGM-7 are C15:0 iso, C16:0 iso, C11:0 iso and C11:0 iso 3OH. Ubiquinone 8 is the major respiratory quinone. The new isolates are strictly organotrophic and aerobic. Strain SGM-6 T only assimilated D-glucose, D-maltose, D-cellobiose, D-furanose, L-glutamate, L-glutamine, L-lysine, L-proline, L-ornithine, acetate, L-glutamic acid and pyruvate of sixty-five carbon sources tested. Strain SGM-7 also assimilates L-serine, but does not assimilate L-ornithine. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we propose that strains SGM-6T and SGM-7 represent a new species most closely related to Thermomonas haemolytica for which we propose the name Thermomonas hydrothermalis. Key words: Thermomonas hydrothermalis – γ-subclass – Proteobacteria – thermophilic

Introduction The vast majority of the species within the Proteobacteria grow at temperatures that rarely exceed 45 °C. However, a few species within this phylum grow at higher temperatures and are slightly or moderately thermophilic. Some of the species that grow at elevated temperatures belong to the same genera that include mesophilic organisms. Species such as Porphyrobacter tepidarius [9], P. cryptus [27] and Albidovulum inexpectatum [2] with optimum growth temperatures in the neighborhood of 50 °C are members of the α-subclass. Within the β-subclass, Tepidimonas ignava and Thiomonas thermosulfata have optimum growth temperatures around 50 °C [22, 23, 29], while Hydrogenophilus spp. have optimum growth temperatures of about 60 °C [10, 33]. The species Thermothrix azorensis which also belongs to the β-subclass may be the most thermophilic species known within the Proteobacteria with a reported optimum growth temperature of 76 °C and a maximum growth temperature of 86 °C [24]. Unfortunately, the strain is no longer available from public culture col-

lections for confirmation of these extraordinary growth temperatures. The species Desulfacium hydrothermalis and Desulfurella kamchatkensis with optimum growth temperatures of about 60 °C are members of the δ-subclass [20, 30]. Nautilia lithotrophica and Caminibacter hydrogenophilus, belong to the ε-subclass of the Proteobacteria and have optimum growth temperatures in the vicinity of 50 and 60 °C, respectively [1, 21]. Despite the large number of species known to belong to the γ-subclass, only two slightly thermophilic species have been recently described; Thermomonas haemolytica, was isolated from kaolin slurry used in paper manufacture [3]. This organism has an optimum growth temperature between about 37 and 50 °C and is distantly related

Nucleotide sequence accession numbers. The GenBank accession number for the 16S rRNA gene sequence for strain SGM-6T is AF 542054. 0723-2020/03/26/01-070 $ 15.00/0

Thermomonas hydrothermalis sp. nov.

to the species of the genera Xanthomonas, Pseudoxanthomonas and Stenotrophomonas. The species Pseudomonas thermotolerans has an optimum growth temperature of about 50 °C and is at present the most thermophilic species within this subclass [17]. We recently isolated an organism from the hot spring at São Gemil in Central Portugal that, based on phylogenetic analysis, was closely related to Tm. haemolytica, but has a higher growth temperature range than this species. On the basis of the phylogenetic analysis, physiological and biochemical characteristics, we propose that strains SGM-6T and SGM-7 represent a new species for which we propose the name Thermomonas hydrothermalis.

Materials and Methods Isolation and bacterial strains Strains SGM-6T (T = type strain), SGM-2, SGM-3, SGM-4 and SGM-7 were isolated from the hot spring at São Gemil in Central Portugal. Water samples were transported at ambient temperature and filtered through membrane filters (Gelman type GN-6; pore size 0.45 µm, diameter 47 mm) within 12 hours, which were placed on the surface of Thermus agar plates [35]. These preparations were wrapped in plastic bags and incubated at 50 °C for up 7 days. Cultures were purified by sub-culturing and were preserved at –80 °C in Thermus medium containing 15% glycerol. The type strain of Thermomonas haemolytica A50-7-3 (= DSM 13605) was kindly donated by Mirja SalkinojaSalonen (Helsinki, Finland) and the type strain of Pseudomonas thermotolerans CM3 (= DSM 14292) was kindly donated by Célia Manaia (Porto, Portugal) for comparative purposes. Growth and morphology Growth of the organisms was examined in several media that included Nutrient Agar (Difco), R2A agar (Difco) and R3A agar [28] without sodium pyruvate, Luria-Bertani broth (LB) plus 2.0% agar, MacConkey agar (Difco), Cetrimide agar (Difco), Tryptic Soy Agar (Difco), Brain Heart Infusion (Difco), medium 162 [6] and Bosea thiooxidans medium (Deutsche Sammlung von Mikroorganismen und Zellkulturen, www.dsmz.de/media/med763.htm) containing 1.0 g · l–1 yeast extract instead of 0.1 g · l–1. Growth was consistently better with Thermus medium, which was adopted to grow the organisms, unless otherwise stated. Cell morphology, dimensions and motility were examined by phasecontrast microscopy during exponential growth in liquid medium. The number and position of flagella were determined by light microscopy after staining of the cells with the Ryu stain [11]. Physiological and biochemical characteristics Growth at different temperatures, pH and NaCl concentrations was determined in Thermus medium as described previously [2]. Anaerobic growth was assessed in Thermus medium 162 with 1.0 g · l–1 of KNO3, dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMA), incubated in anaerobic chambers with CO2 atmosphere (BioMérieux) in the dark. Fermentation of glucose was assessed with OF Basal Medium (Difco). The IMViC test was performed as described by Smibert and Krieg [31]. The reduction of nitrate, nitrite and tellurite, the presence of catalase and cytochrome oxidase, the hydrolysis of urea, elastin, fibrin, gelatin, casein, starch, xylan, Tween 20, 40, 60 and 80, hippurate, arbutin and esculin and activity of DNAse, αgalactosidase, β-galactosidase, α-glucosidase and β-glucosidase and lipase were determined as described previously by Hudson et

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al. [12] and Smibert and Krieg [31]. Hemolysis was examined on Blood Agar plates (Difco) incubated at 37 and 40 °C. Other enzyme activities were determined with the API ZYM test system (BioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions at 37 °C and 50 °C. Antibiotic susceptibility of strains SGM-6T and SGM-7 to disks (BioMérieux) containing ampicillin (10 µg), carbenicillin (100 µg), cephalothin (30 µg), cefazolin (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), doxycyclin (30 µg), erythromycin (15 µg), gentamycin (10 µg), lincomycin (2 µg), neomycin (30 µg), ofloxacin (5 µg), penicillin G (10 U/IE), rifampicin (30 µg), streptomycin (10 µg), sulphamethoxazole/trimethoprim (25 µg), tetracyclin (30 µg) and vancomycin (30 µg) was examined on half-strength Mueller Hinton Agar (diluted 1:1; with the addition of agar to 1.5%), since the new organisms did not grow on Mueller-Hinton agar (Difco) at 37 °C and 50 °C for 48 hours, while the antibiotic sensitivity of the type strain of Tm. haemolytica was examined on Mueller Hinton Agar (Difco). Single carbon source assimilation tests were determined in a defined medium composed of Thermus basal salts containing 0.2 g of yeast extract per liter to which filter-sterilized carbon sources (2.0 g · l–1) and ammonium sulfate (0.5 g · l–1) were added. Growth was examined by measuring the turbidity of unshaken cultures daily, for 5 days, incubated at 50 °C in 20-ml screw-cap tubes containing 10 ml of medium. Carbon source assimilation tests on Tm. haemolytica were performed in the same medium at 37 °C. Positive and negative control cultures were grown in Thermus medium and in defined medium without a carbon source. The oxidation of sodium sulfite (0.5 and 1.0 g · l–1), sodium sulfide (0.5 and 1.0 g · l–1), sodium thiosulfate (0.5, 1.0 and 2.0 g · l–1) and potassium tetrathionate (1.0 g · l–1) was examined in Thermus medium without sulfate at the optimum growth temperature. The production of sulfate was determined as described by Sörbo [32]. Polar lipid, lipoquinone and fatty acid composition The cultures used for polar lipid analysis were grown in 1l Erlenmeyer flasks containing 200 ml of Thermus medium at 50 °C in a reciprocal water-bath shaker for 24 hours. Harvesting of the cultures and extraction of lipids was performed as described previously [7, 25]. The individual polar lipids were separated by mono-dimensional thin-layer chromatography (TLC) on silica gel G plates (Merck; 0.25 mm thick) with a solvent system consisting of chloroform/methanol/acetic acid/water (80:12:15:4, v/v). Lipoquinones were extracted from freeze dried cells, purified by thin layer chromatography and separated with a Gilson high-performance liquid chromatography apparatus by using a reverse phase column (RP18, Spherisorb, S5 ODS2) with methanol/heptane (10:2, v/v) as the mobile phase and were detected at 269 nm [34]. Cultures for fatty acid analysis were grown on half-strength TSA plates incubated in sealed plastic bags submerged in a water-bath at the optimum growth temperature for 24 h. Fatty acid methyl esters (FAMEs) were obtained from fresh wet biomass by saponification, methylation and extraction as described previously by Kuykendall et al. [15] and separated, identified and quantified as described previously [22]. Determination of G+C content of DNA and 16S rRNA gene sequence determination and phylogenetic analyses The DNA for the determination of the G+C content of the DNA was isolated as described by Cashion et al. [5]. The G+C content of DNA was determined by high-performance liquid chromatography as described by Mesbah et al. [19]. The extraction of genomic DNA for 16S rRNA gene sequence determination, PCR amplification of the 16S rRNA gene

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and sequencing of the purified PCR products were carried out as described previously [26]. Purified reactions were electrophoresed using a model 310 Genetic Analyzer (Applied Biosystems, Foster City, Ca.). The 16S rRNA gene sequences determined in this study were aligned against representative reference sequences of members of the Proteobacteria using the ae2 editor [16]. The method of Jukes and Cantor [14] was used to calculate evolutionary distances. Phylogenetic dendrograms were generated using various treeing algorithms contained in the PHYLIP package [8].

Results Isolation, morphological and biochemical characteristics of strains SGM-6T and SGM-7 Several non-pigmented organisms, designated SGM-2, SGM-3, SGM-4, SGM-6 T and SGM-7, were isolated from the hot spring, with a vent temperature of 48 °C and a pH of 8.6, at São Gemil in Central Portugal. The fatty acid composition of these organisms were extremely similar and the strains designated SGM-6T and SGM-7 were chosen for in depth characterization. Strains SGM-6T and SGM-7 formed short rod-shaped cells that stained Gramnegative. These organisms, unlike the type strain of Tm. haemolytica, were not motile and did not possess flagella under the conditions examined. Growth was observed on all media examined except TSA, Mueller-Hinton agar, MacConkey agar and Cetrimide agar. However, the SGM strains grew on half-strength TSA and half-strength Mueller-Hinton agar. Strains SGM-6T and SGM-7 had an optimum temperature for growth of about 50 °C, but did not grow at 65 °C. The type strain of Tm. haemolytica, on the other hand, had a lower optimum growth temperature of about 40 to 45 °C. Strain SGM-6T grew at higher temperatures than the type strain of Pseudomonas thermotolerans in Thermus medium and LB without the addition of NaCl (Fig. 1), but the reverse was observed in LB which contains 10.0 g · l–1 of NaCl (w/v) because the organisms from St. Gemil did not grow in media containing more than 0.5% (w/v) NaCl. The optimum pH for growth of strain SGM-6T was between 6.5 and 8.5, but the organism did not grow at pH 4.5 or at pH 9.5. Strains SGM-6T and SGM-7 possessed catalase, cytochrome oxidase, lipase, α-glucosidase and β-glucosidase; other activities determined with the API ZYM are presented in Table 1. These strains hydrolyzed several proteinaceous substrates namely casein, fibrin and gelatin, degraded aesculin but did not degrade urea. Unlike the type strain of Tm. haemolytica, strains SGM-6T and SGM-7 produced a very weak β-hemolysis at 37 and 40 °C that was more reminiscent of the α-type of hemolysis. Growth under anaerobic conditions was not observed. Fermentation was not observed on glucose, but nitrate was reduced to N2. Tm. haemolytica and strains SGM-6T and SGM-7 were sensitive to all the antibiotics examined, except to lincomycin and sulphametoxazole/ trimethoprim. Strain SGM-6T assimilated only a few carbon and energy sources namely D-glucose, D-maltose, Dcellobiose, D-furanose, L-glutamate, L-glutamine, L-lysine, L-proline, L-ornithine, acetate, L-glutamic acid and

Fig. 1. Effect of temperature on the growth rates of strain SGM6T (d) and the type strains of Thermomonas haemolytica (j) and Pseudomonas thermotolerans (s) in Thermus medium.

pyruvate, while strain SGM-7 did not assimilate L-ornithine, but grew on L-serine (Table 1). The type strain of Tm. haemolytica did not assimilate any of the sugars or polyols examined confirming the results of Busse et al. [3]. Yeast extract was required for growth on single carbon sources. Strains SGM-6T and SGM-7 did not oxidize sulfite, sulfide, thiosulfate or tetrathionate. Chemotaxonomic characteristics The polar lipids of Tm. haemolytica and the São Gemil strains consisted primarily of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. Other minor phospholipids were also present in both strains but were not identified. The major respiratory lipoquinone of strain SGM-6T was ubiquinone 8 (87.8%), although Q-7 and Q-9 were also present as minor components (7.8 and 4.4%, respectively). The fatty acid composition of strain SGM-6T and SGM-7 was dominated by C15:0 iso followed by C16:0 iso, C11:0 iso and C11:0 iso 3OH. (Table 2). The fatty acid composition of the type strain of Tm. haemolytica and strains SGM-6T and SGM-7 was very similar. 16S rRNA gene sequence comparison An almost complete 16S rRNA gene sequence (1478 nucleotides) was determined for strain SGM-6T. Comparison of this sequence with representatives of the main lines of descent within the domain Bacteria indicated that strain SGM-6T was a member of the γ-subclass of the Proteobacteria and was most closely related to Thermomonas haemolytica (Fig. 2). The pairwise 16S rRNA gene

Thermomonas hydrothermalis sp. nov. Table 1. Phenotypic and physiological characteristics that distinguish strains SGM-6T and SGM-7 from the type strain of Thermomonas haemolytica.

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sequence similarity between strain SGM-6T and Tm. haemolytica was 97.4%. Other 16S rRNA gene sequences were recovered from the public databases that have been shown to fall within the radiation of Tm. haemolytica and have been included in the phylogenetic analyses (Fig. 2). These strains share 16S rRNA gene sequence similarities in the range 94.8 to 97.1% to strain SGM-6T.

Characteristic

SGM-6 T

SGM-7

Thermomonas haemolytica

Nitrate reduction Hydrolysis of: Aesculin Presence of: β-Glucosidase Urease Utilization of: D-Cellobiose D-Glucose D-Maltose L-Alanine L-Aspartate L-Phenylalanine L-Leucine L-Proline L-Ornithine L-Serine α-Ketoglutarate L-Malate

+

+

–

+

+

–

+ –

+ –

– +

Discussion

+ + + – – – – + + – – –

+ + + – – – – + – + – –

– – – + + + + – – + + +

Some microorganisms are initially isolated from artificial environments and only later isolated from sites that constitute their natural environments. Perhaps, the most famous example is the isolation of Legionella species from man-made environments such as hot water pipes and air-conditioning water towers and only later from natural environments such as water systems, geothermal water and soil [4, 13, 18]. The isolation of the São Gemil organisms followed this pattern, since Thermomonas haemolytica was isolated from an artificial environment, namely kaolin slurry at a paper pulp mill and a little later, a new species of the genus Thermomonas, represented by strains SGM-6T and SGM-7, was isolated from a natural thermal environment. Strains SGM-6T and SGM-7 represent a new species for several reasons; these organisms have a higher growth temperature range than Tm. haemolytica and there are large differences in the phenotypic characteristics, namely in single carbon source assimilation results. It noteworthy that Tm. haemolytica does not assimilate carbohydrates while strain SGM-6T and SGM-7 assimilates a few mono- and disaccharides, but we have no explanation for this phenomenon. The phylogenetic analysis based on 16S rRNA gene sequence shows that strain SGM-6T is closely related to the type strain of Tm. haemolytica but distinct in terms of sequence similarity (97.4%). Other isolates of this phylogenetic group, such as strains TJ7, S6 and BrG3 (Fig. 2) for which little information, other than a sequence deposited in the public databases, is available, could be considered to represent additional species of the genus Thermomonas since these organisms form a coherent clade.

+, positive result or growth; –, negative result or no growth; ND, not determined. All strains possessed catalase, cytocrome oxidase and α-glucosidase, alkaline and acid phosphatase, esterase, lipase, leucine arylamidase, valine arylamidase, chymotrypsin, naphtol-AS-BI-phosphohydrolase activities but did not have cysteine arylamidase, trypsin, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase or α-fucosidas activities. All strains degraded gelatin. Acetate and pyruvate were utilized by all strains. Strains SGM-6T and SGM-7 also assimilated D-furanose, L-lysine and glutamic acid. None of strains assimilated N-acetyl-D-glucosamine, Larabinose, D-fructose, D-galactose, D-mannose, D-melibiose, Lrhamnose, D-ribose, sucrose, D-xylose, adonitol, myo-inositol, mannitol, D-sorbitol, L-histidine, L-tryptophan, citrate, fumarate or lactate. Strains SGM-6T and SGM-7 did not assimilate D-arabinose, L-fucose, lactose, D-raffinose, L-sorbose, D-trehalose, D-arabitol, i-erythritol, ethanol, glycerol, methanol, ribitol, xylitol, L-alanine, L-arginine, L-asparagine, L-aspartate, L-cysteine, L-phenylalanine, glycine, L-isoleucine, L-leucine, L-methionine, benzoate, α-ketoglutarate, formate, L-malate, oxaloacetate, succinate, L-tyrosine, L-threonine or L-valine.

Fig. 2. Phylogenetic dendrogram based on 16S rRNA gene sequence comparisons. The dendrogram was reconstructed from evolutionary distances by using the neighbour-joining method.

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Table 2. Fatty acid composition of strains SGM-6T, SGM-7 and the type strain of Thermomonas haemolytica1. Fatty acids

% in –––––––––––––––––––––––––––––––––––––––––––––– SGM-7 Tm. SGM-6T haemolytica –––––––––––––– –––––––––––––– ––––––––––––– 37 °C 50 °C 37 °C 50 °C 37 °C 50 °C

10:0 iso 0.5 11:0 iso 5.0 11:0 anteiso 0.2 12:0 iso 0.4 11:0 iso 3OH 6.5 13:0 iso 1.0 12:0 iso 3OH 0.4 14:0 iso 5.3 14:1 ω5c 0.8 14:0 1.7 15:1 iso F 3.3 15:1 iso G – Summed feature 22 0.7 15:0 iso 41.9 15:0 anteiso 3.7 15:1 ω6c 0.9 15:0 0.8 16:1 iso H 0.5 16:0 iso 11.4 16:1 ω9c 0.6 Summed feature 43 4.6 16:0 2.3 iso 17:1 ω9c – Summed feature 54 0.2 17:0 iso 4.1 17:0 anteiso 0.2 16:0 3OH –

– 6.1 – – 7.7 – – 1.1 – 1.1 0.6 – – 56.2 1.6 – 0.9 – 5.1 – 0.8 3.8 6.4 – 8.6 0.3 –

0.5 4.8 0.3 0.4 6.3 0.6 0.4 4.0 0.6 1.4 2.5 – 0.6 37.7 4.0 0.8 0.7 0.5 15.1 0.5 4.8 3.3 5.3 0.1 4.7 0.6 0.3

– 5.6 – – 6.0 – – 0.9 – 1.0 0.5 – – 55.8 1.8 – 0.9 – 5.4 – 0.9 4.8 7.0 – 9.5 0.3 –

0.2 7.0 – 0.1 9.2 0.6 0.1 5.0 – 0.3 2.6 – 0.5 62.9 0.4 – 0.1 – 5.3 – 0.2 0.3 3.9 0.1 1.5 – –

– 6.1 0.2 – 9.0 0.3 – 6.5 – 1.6 _ 1.6 – 50.5 4.7 – 5.0 – 7.1 – 0.2 3.4 1.1 0.9 2.0 – –

from a hot spring, although not a very hot one). Thermomonas hydrothermalis forms Gram-negative rodshaped cells 0.6 to 0.9 µm in width by 2.0 to 4.0 µm in length. Cells are non-motile. This species forms lightbrown colored colonies 0.5 to 2.0 mm in diameter and a diffusible brown pigment in older cultures. The optimum temperature for growth is about 50 °C; growth at 30 and at 60 °C is very poor. The optimum pH for growth ranges from about 6.5 to 8.5; growth does not takes place at pH 4.5 or 9.5. Catalase and cytochrome oxidase positive. Strictly aerobic and organotrophic. Strains SGM-6T and SGM-7 degrade proteins and lipids. The major polar lipids are phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The major respiratory quinone is ubiquinone 8 and the major fatty acids are 15:0 iso, 16:0 iso, 11:0 iso and 11:0 3OH iso. Yeast extract is necessary for growth. The type strain assimilates D-glucose, D-maltose, D-cellobiose, D-furanose, L-glutamate, L-glutamine, L-lysine, L-proline, L-ornithine, acetate, L-glutamic acid and pyruvate. Strain SGM-7 also assimilates L-serine but not L-ornithine. The G + C ratio of the DNA is 64.7 mol%. The organism was isolated from the hot spring at São Gemil in Central Portugal. Strain SGM-6T has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany as DSM 14834 and in the American Type Culture Collection, Manassas, USA as ATCC BAA-470. Acknowledgements This work was supported, in part by the PRAXIS XXI Program (PRAXIS/PCNA/BIO/46/96) and POCTI 35029/99, Portugal. FAR was in part supported by NSF award DEB-971427.

1

All organisms were grown on half-strength TSA medium for 24 hours. 2 Group of fatty acids (15:1 iso H and/or 13:0 3OH) that could not be separated by this method. 3 Group of fatty acids (16:1 ω7c and/or 15:0 iso 2OH) that could not be separated by this method. 4 Group of fatty acids (17:1 iso I and/or 17:1 anteiso B) that could not be separated by this method.

Within a short period of time two slightly thermophilic species, Thermomonas haemolytica and Pseudomonas thermotolerans, have been described within the γ-subclass of the Proteobacteria [3, 17]. Of these organisms, the species represented by strains SGM-6T and SGM-7 appears to have a growth temperature range only slightly higher than the other two organisms. The results presented here leads us to propose that strains SGM-6T and SGM-7 represent a new species of the γ-subclass of the Proteobacteria for which we propose the name Thermomonas hydrothermalis. Description of Thermomonas hydrothermalis sp. nov. Thermomonas hydrothermalis (hy. dro. ther. ma´ lis, Gr. n. hydros, water; Gr. adj. thermos, hot; N. L. adj. hydrothermalis, to indicate that the organism originated

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Corresponding author: Milton S. da Costa, Departamento de Bioquímica, Universidade de Coimbra, 3001-401 Coimbra, Portugal Tel.: +351-239824024; Fax: +351-239826798; e-mail: [email protected]