Meiothermus rufus sp. nov., a new slightly thermophilic red-pigmented species and emended description of the genus Meiothermus

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Systematic and Applied Microbiology 32 (2009) 306–313 www.elsevier.de/syapm

Meiothermus rufus sp. nov., a new slightly thermophilic red-pigmented species and emended description of the genus Meiothermus$ Luciana Albuquerquea, Catarina Ferreiraa, David Tomaza, Igor Tiagoa, Anto´nio Verı´ssimoa, Milton S. da Costab, M. Fernanda Nobrea, a

Department of Zoology and Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal Department of Biochemistry, University of Coimbra, 3001-401 Coimbra, Portugal

b

Received 20 April 2009

Abstract Four red-pigmented isolates, with optimum growth temperatures of approximately 55–60 1C and an optimum pH for growth between 7.5 and 8.5, were recovered from hot springs in Central France. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented a new species of the genus Meiothermus. The new isolates could be distinguished from other strains of the species of the genus Meiothermus primarily by the glycolipid profile and fatty acid composition because these organisms lacked the hydroxy fatty acids and the glycolipid variant GL-1a found in all other isolates of the species of Meiothermus examined. On the basis of the results presented here we propose the name Meiothermus rufus for the new species, which is represented by strains CAL-4T ( ¼ DSM 22234T ¼ LMG 24878T) and CAL-12 ( ¼ DSM 22235 ¼ LMG 24879). We also propose emending the genus Meiothermus to include strains that have only one glycolipid instead of two glycolipid variants. r 2009 Elsevier GmbH. All rights reserved. Keywords: Meiothermus; M. rufus sp. nov.; Thermaceae; Taxonomy; Lipids

Introduction The species of the genera Thermus and Meiothermus, along with the recently described species of Marinithermus [25], Vulcanithermus [15] and Oceanithermus [14,16] isolated from abyssal hyperthermal vents, currently form the family Thermaceae [6]. The species Meiothermus ruber was initially included in the genus Thermus, $ Note: Nucleotide sequence data for the 16S rRNA gene are available in the DDBL/EMBL/GenBank databases under the accession numbers: CAL-4T (FN178496) and CAL-12 (FN178497). Corresponding author. Tel.: +351 239824024; fax: +351 239855789. E-mail address: [email protected] (M.F. Nobre).

0723-2020/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2009.05.002

but the description of other ‘‘low-temperature’’ species, clearly showed that these organisms belonged to a distinct genus, which was named Meiothermus [20]. The species of the genus Meiothermus, namely M. ruber [11], M. silvanus [26], M. chliarophilus [26], M. cerbereus [5], M. taiwanensis [4] and M. timidus [22] form, based on 16S rRNA gene sequence analysis, a separate line of descent within the genera of the family Thermaceae with which they share 85.2–86.6% sequence similarity [6,19]. The species of the genus Meiothermus have growth temperature ranges between approximately 35 and 68 1C, while the species of the other genera of the family Thermaceae grow between about 40 and 83 1C. The predominant aliphatic components of the cell membranes of the species of the family Thermaceae primarily

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possess iso- and anteiso-branched fatty acids. Only a few strains of the genus Thermus possess hydroxy fatty acids, namely 3-OH fatty acids, but 2-OH fatty acids have never been encountered in strains of this genus [3,18]. The species of the genera Marinithermus [25], Vulcanithermus [15] and Oceanithermus [14,16] do not appear to possess hydroxy fatty acids. In contrast to these organisms, branched chain 2-OH and 3-OH fatty acids have always been detected in Meiothermus strains [9,18]. The strains of the species of the genus Thermus possess one major phospholipid, designated PL-2, and a major glycolipid, designated GL-1 [3], while all strains of the species of the genus Meiothermus examined possess two major glycolipid variants on thin-layer chromatography (TLC), designated GL-1a and GL-1b, that have different mobilities on TLC due to the presence of amide-linked 2-OH fatty acids in GL-1a and amide-linked 3-OH iso-branched or an isobranched fatty acid in GL-1b [9]. The species of Meiothermus are generally red-pigmented, except for the yellow-pigmented strains of M. chliarophilus and M. timidus, while the other genera of the family Thermaceae consist of yellow- or non-pigmented strains. We recently recovered a large number of thermophilic and slightly thermophilic isolates from hot springs in the Chaude-Aigues hydrothermal area in Central France. Four red-pigmented isolates clearly belonged to a novel species of the genus Meiothermus. Phylogenetic analysis of the 16S rRNA gene sequence, physiological and biochemical characteristics clearly indicated that strains CAL-4T and CAL-12 belonged to a new species of the genus Meiothermus for which we propose the name Meiothermus rufus. Since these organisms possess only one glycolipid instead of two glycolipid variants we are of the opinion that the description of the genus Meiothermus must also be emended.

Materials and methods

307

M. taiwanensis ( ¼ DSM 14542T), M. cerbereus GY-1 ( ¼ DSM 11376T), M. silvanus VI-R2 ( ¼ DSM 9946T), M. chliarophilus ALT-8 ( ¼ DSM 9957T), M. timidus SPS-243 ( ¼ LMG 22897T) and Thermus oshimai SPS-17 ( ¼ DSM 12092T) were used for comparative purposes.

Growth, morphology, physiological and biochemical characteristics Cell morphology and motility were examined by phase contrast microscopy during the exponential growth phase. Unless otherwise stated, all biochemical and tolerance tests were performed in Thermus liquid medium or on Thermus agar at 55 1C for up to 6 days, as described previously [22]. The growth temperature range of the strains was examined at 30, 35, 40, 45, 50, 55, 60, 65 and 70 1C by measuring the turbidity (610 nm) of cultures incubated in 300 ml metal-capped Erlenmeyer flasks, containing 100 ml of medium in a rotary water bath shaker at 150 rpm [1]. The pH range for growth was examined at 55 1C in the same medium by using 50 mM MES, HEPES, TAPS and CAPSO over a pH range from 5.5 to 10.5 in a rotary water bath shaker [1]. The salt tolerance of the organisms was determined in Thermus liquid medium, with NaCl ranging between 0.0% and 2.0% [1]. Catalase, oxidase and DNAse activities were examined as described previously [2]. Additional enzymatic activities were obtained using the API ZYM system (bioMe´rieux) at 55 1C. Anaerobic growth was assessed in cultures in Thermus medium, containing KNO3 (1.0 g l1) incubated in anaerobic chambers (GENbox anaer, bioMe´rieux). Single carbon source assimilation tests were performed in a medium composed of Thermus basal salts to which filtersterilized ammonium sulphate (0.5 g l1), yeast extract (0.1 g l1) and the carbon source (2.0 g l1) were added. Growth of the strains was examined by measuring the turbidity of cultures incubated at 55 1C in 20 ml screw capped tubes containing 10 ml medium for up to 6 days.

Isolation and bacterial strains Polar lipid, lipoquinone and fatty acid composition Strains CAL-4T, CAL-7, CAL-11 and CAL-12 were isolated from water samples at the Lavoire in the Chaudes-Aigues hydrothermal area in the Auvergne region of France. Water samples were transported and maintained without temperature control for 4 days, and then filtered through membrane filters (Gelman type GN-6; pore size 0.45 mm; diameter 47 mm). The filters were placed on the surface of agar-solidified Thermus medium [6,29], wrapped in plastic bags and incubated at 50 1C for up to 4 days. Cultures were purified by subculturing and the isolates were stored at 70 1C in Thermus medium with 15% (v/v) glycerol. The type strains of M. ruber Loginova 21 ( ¼ DSM 1279T),

The cultures for polar lipid and for fatty acid analysis were grown in 1 l Erlenmeyer flasks containing 250 ml Thermus medium at 45, 50, 55 and 60 1C in a rotary water bath shaker until the late exponential growth phase. Harvesting of the cultures, extraction of the lipids and single-dimensional thin-layer chromatography were performed as described previously [1]. Lipoquinones were extracted, purified by thin-layer chromatography and separated by high-performance liquid chromatography [28]. Fatty acid methyl esters (FAMEs) were obtained from fresh wet biomass and separated, identified and quantified with the standard MIS Library

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Generation Software (Microbial ID Inc.), as described previously [2].

RAPD analysis of the isolates, DNA G+C content and 16S rRNA gene sequence determination, and phylogenetic analyses Random amplified polymorphic DNA (RAPD) analysis was performed as a primary method to group the isolates, as previously described by Tiago et al. [27]. The DNA for the determination of the G+C content of the DNA was isolated as described previously [17]. The G+C content of DNA was determined by highperformance liquid chromatography, as described by Mesbah et al. [13]. The 16S rRNA gene was amplified by PCR and sequenced as described by Rainey et al. [23]. Phylogenetic analyses were performed using the ARB software package [12]. Phylogenetic trees were constructed using the neighbor-joining [24], maximum parsimony [8] and maximum likelihood [21] algorithms included in the ARB software package. The topology of the tree generated from distance matrices calculated with Jukes– Cantor correction [10] using the neighbor-joining algorithm was evaluated by performing bootstrap analysis [7] of 1000 resamplings of the dataset.

Morphology, growth, biochemical and physiological characteristics Strains CAL-4T and CAL-12 formed Gram-negative rod-shaped cells of variable length and were 0.5–0.8 mm wide, long filaments were also present and they were not motile. Colonies were red-pigmented on Thermus medium. The organisms had an optimum growth temperature between 55 and 60 1C and did not grow at 30 or 70 1C. The optimum pH of strains was between 7.5 and 8.5 and no growth was detected at pH 6.0 or 10.0. No growth occurred in medium containing 1.0% NaCl (w/v). These organisms were oxidase, catalase and DNAse positive. Other enzyme activities are listed in the species description. Hippurate, aesculin, arbutin, elastin, starch, gelatine, casein and xylan were hydrolysed. A small amount of yeast extract (0.1 g l1) was necessary for growth of the organisms. CAL-4T and CAL-12 had practically identical single carbon source assimilation patterns but they were different from the six species of the genus Meiothermus. Several carbohydrates, polyols, organic acids and amino acids were used as single carbon and energy sources. Nitrate was reduced to nitrite and anaerobic growth with nitrate as an electron acceptor was not observed (Table 1).

Polar lipids, respiratory quinones and fatty acids

Results Isolation of strains Eighty-five isolates were recovered from several hot springs in the Chaudes-Aigues area and they were grouped by fatty acid analysis. A total of 490 bases were sequenced between positions 27 and 519 (E. coli sequence used as reference) of the 16S rRNA gene of two or three isolates from each fatty acid group. Four isolates of T. scotoductus and four isolates of T. brockianus had sequence similarities identical to the respective type strains. Four isolates had identical partial 16S rRNA gene sequences to M. cerbereus or M. silvanus. Ten red-pigmented isolates belonged to species of Porphyrobacter. Strains CAL-4T, CAL-7, CAL-11 and CAL-12 had partial 16S rRNA gene sequence similarities that were between 88.3% and 95.3% to the known species of the genus Meiothermus. These red-pigmented organisms were recovered from one sample of the Lavoire runoff at a temperature of 67 1C and pH 7.5–8.0. RAPD analysis indicated that all four strains had a clonal origin (data not shown), and strains CAL-4T and CAL-12 were selected for further characterization of this novel species.

The polar lipid pattern of strains CAL-4Tand CAL-12 was composed of one major phospholipid (PL-2) and only one major glycolipid corresponding to GL-1b of other Meiothermus species (Fig. 1). This glycolipid also corresponded to glycolipid 1 of Thermus species. Three temperatures were used to examine the polar lipid and fatty acid composition, but glycolipid variant GL-1a was unable to be detected at any of the temperatures. However, all the type strains of the other species of the genus Meiothermus had the two glycolipid variants, although GL-1a decreased as the growth temperature was raised (Fig. S1). The two glycolipids visible on TLC between PL-2 and GL-1a and GL-1b in M. chliarophilus corresponded to precursors of glycolipid variants GL-1a and GL-1b lacking the terminal hexose of the polar head group. The fatty acid composition of these new organisms was similar to that of the other strains of the species of the genus Meiothermus being predominantly composed of iso- and anteiso-branched fatty acids (Table 2). However, unlike the fatty acid composition of the other strains of the genus Meiothermus, 2-OH and 3-OH fatty acids were not detected during growth at any of the temperatures examined (Table S1). A decrease in the levels of 2-OH fatty acids of M. cerbereus and M. chliarophilus (Table S1) was reflected in the amount of variant GL-1a detected by TLC (Fig. S1). The major

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Table 1.

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Physiological and biochemical characteristics that distinguish strains of the genus Meiothermus. Strains

Pigmentation Growth with 1% NaCl  Reduction of NO 3 to NO2 Presence of Catalase Hydrolysis of Starch Xylan Utilization of L-rhamnose D-ribose D-xylose L-Arabinose Sucrose D-trehalose D-cellobiose D-raffinose D-melizitose D-sorbitol D-mannitol Ribitol Xylitol Myo-inositol Glycerol a-Ketoglutarate Pyruvate Succinate Aspartate Malate Acetate L-proline L-serine L-asparagine L-arginine L-glutamine L-lisine L-leucine L-ornithine

CAL-4T

CAL-12

M. silvanus VI-R2T

M. rubera,b Loginova 21T

M. taiwanensisc WR-30T

M. cerbereusb GY-1T

M. chliarophilusd ALT-8T

M. timidusd SPS-243T

Red – +

Red – +

Red – +

Red + –

Red (+) ()

Red (+) –

Yellow + +

Yellow (+) +

+

+

–

+

+

–

–

+

+ +

+ +

+ –

– –

– –

– –

+ –

+ –

– – + + + + + – – + + – – – – – + + + + – – – + + + – – +

– – + + + + + – – + + – – – + – + + – + – – – + + + + – –

– + + – – – – – + + + + + – + + + – – + – + + + + + + + +

– () + – + + + + (+) + + – () + + (+) – + (+) + (+) + + + + + (+) (+) (+)

+ () + + + – + + (+) + + + () + – (+) + + () – (+) (+) – + + + () (+) (+)

– () – – + + + – () – – – () – – (+) + – (+) () () + – – – – () () ()

– () + – + + + + () + + – () () + () + – () () – + + + + + () () ()

– () + + + + + + () + + – () – – (+) + + (+) (+) () + + + + + (+) (+) (+)

+, Positive result; , negative result; (+) or (), our result. All of the organisms hydrolyzed DNA, casein, gelatine, arbutin and aesculin. D-glucose, D-fructose, D-galactose, D-mannose, maltose and lactose were utilized by all of the strains. None of the strains utilized L-fucose, erythritol and citrate. Strains CAL-4T and CAL-12 utilized D-arabitol, glutamate, lactate and fumarate but did not utilize L-sorbose, D-arabinose, L-arabitol, benzoate, formate, D-gluconate, D-glucuronate, L-alanine, glycine, Lhistidine, valine, L-phenylalanine, L-isoleucine and methionine. a Data from [26]. b Data from [5]. c Data from [4]. d Data from [22].

respiratory quinone of all strains was menaquinone 8 (MK-8).

16S rRNA gene sequence comparison and G+C content of DNA Partial 16S rRNA gene sequences comprising 1487 nucleotides were determined for strains CAL-4T and

CAL-12 that shared a sequence similarity of 100%. Comparison of these sequences with representatives of the main lines of descent within the domain Bacteria indicated that these strains were members of the family Thermaceae (Fig. 2), and clearly members of the genus Meiothermus. Strains CAL-4T and CAL-12 represented an isolated branch within this genus, with higher phylogenetic relationships with the cluster comprising M. cerbereus, M. taiwanensis and M. ruber with which

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PL-2 GL-2b GL-2a PGL GL-1b GL-1a

GL-1

1

2

3

4

5

6

7

8

9

Fig. 1. Monodimensional thin-layer chromatography of polar lipids of M. ruber Loginova 21T (1), M. taiwanensis WR-30T (2), M. cerbereus GY-1T (3), M. silvanus VI-R2T (4), M. chliarophilus ALT-8T (5), M. timidus SPS-243T (6), CAL-4T (7), CAL-12 (8) and Thermus oshimai SPS-17T (9) at the optimum growth temperature. GL-1, glycolipid 1; GL-1a, glycolipid 1a; GL-1b, glycolipid 1b; GL-2a, glycolipid 2a; GL2b, glycolipid 2b; PGL, phosphoglycolipid; PL-2, phospholipid 2.

they shared a sequence similarity of 95.0%, 94.6% and 95.3%, respectively. Furthermore, strains CAL-4T and CAL-12 shared a sequence similarity between 88.5% and 90.9% with other members of genus Meiothermus. The G+C content of the DNA of strains CAL-4T and CAL-12 was 64.170.4 and 63.670.5 mol%, respectively.

Discussion Strains CAL-4Tand CAL-12 clearly belong to the genus Meiothermus based on the phylogenetic analysis of the 16S rRNA gene sequence and the low growth temperature range. The phylogenetic analysis shows that the new species, represented by strains CAL-4T and CAL-12, are most closely related to M. cerbereus, M. taiwanensis and M. ruber with which they share approximately 95.0% 16S rRNA gene sequence similarity. Several characteristics distinguish the new organism from the most closely related species, namely those of the M. ruber group. However, the most striking characteristic of the new organism is the lack of the glycolipid variant designated GL-1a. It was found that all strains of the Meiothermus species, unlike those of the genus Thermus, had two glycolipid variants migrating very close to each other [9]. It was also found that glycolipid variant Gl-1a always contained a 2-OH fatty acid amide linked to the hexosamine of the polar head group [9]. Glycolipid variant GL-1b, however, like the Thermus glycolipid GL-1, contained branched chain iso15 or iso-17 amide linked to the glucosamine of the

polar head group of GL-1 or a 3-OH fatty acid when galactosamine was present [3,9]. The examination of the polar lipids of six strains belonging to all the species of Meiothermus showed that there were no exceptions to the presence of the two glycolipid variant pattern using TLC. However, it was clear to us that other glycolipid patterns would eventually be encountered, since the different mobility of glycolipid variant GL-1a on TLC was exclusively due to the synthesis of a 2-OH fatty acid. It was, therefore, not surprising when we found that the absence of glycolipid variant GL-1a was linked to the lack of 2-OH fatty acids in the organisms of the new species represented by CAL-4Tand CAL-12. The fatty acid composition of all the type strains of the genus Meiothermus were examined in order to investigate if the absence of 2-OH fatty acids was, perhaps, related to the growth temperature and it was found that the growth temperature did indeed lead to different levels of 2-OH fatty acids. Moreover, these changes in 2-OH fatty acids were related to the amount of variant GL-1a visible with TLC. These results show that the inability to synthesize one fatty acid can reflect the overall lipid composition of Meiothermus strains. It also shows that the absence of one characteristic can affect another characteristic and that this also has taxonomic significance. Most species of the genus Meiothermus are redpigmented, although M. timidus and M. chliarophilus are yellow-pigmented. This characteristic, allied to the deep phylogenetic branch that separates the yellow-pigmented species from the red-pigmented species, could be misconstrued as a valid reason to propose a new genus. However, there is no conceivable reason to propose a new genus for M. timidus and M. chliarophilus without additional results of taxonomic significance. Differences in carbon source assimilations, the lack of hydroxy fatty acids and the 16S rRNA gene sequence analysis lead us to propose a new species, named M. rufus for strains CAL-4T ( ¼ DSM 22234T ¼ LMG 24878T) and CAL-12 ( ¼ DSM 22235 ¼ LMG 24879).

Emended description of the genus Meiothermus Nobre et al. [20]; Nobre and da Costa [19] Characteristics are as given for the description of the genus by Nobre et al. [20] and Nobre and da Costa [19]. Some strains have only one major glycolipid corresponding to GL-1b, as other strains of Meiothermus, or glycolipid GL-1 of the species of the genus Thermus. 2-hydroxy fatty acids are not detected in some strains.

Description of Meiothermus rufus sp. nov. M. rufus (ru’fus. L. masc. adj. rufus, red, ruddy). M. rufus forms rod-shaped cells of variable length but with a minimum length of about 3.0 mm and 0.5–0.8 mm

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Table 2. Mean fatty acid composition of type strains of M. ruber, M. taiwanensis, M. silvanus, M. cerbereus, M. chliarophilus, M. timidus, CAL-4T and CAL-12 grown at 55 1C. Fatty acids

13:0 iso 14:0 iso 14:0 13:0 iso 3OH 15:1 iso Fa 15:0 iso 15:0 anteiso 15:1 o8c 15:0 16:1 o7t alcohol 16:0 iso Summed featureb 3 16:0 Unknown diol 15:0 iso 3OH 15:0 2OH Summed feature 9 Summed feature 4 17:1 anteiso o9c 17:0 iso 17:0 anteiso 17:1 o8c 17:1 o6c 17:0 16:0 iso 3OH 16:0 2OH 16:0 3OH 18:0 iso 17:0 iso 2OH 17:0 anteiso 2OH 17:0 iso 30H 17:0 2OH 17:0 3OH 19:0 iso 19:0 anteiso 18:0 iso diol 18:0 anteiso diol

Percentage of the total in CAL-4T

CAL-12

M. ruber Loginova 21T

M. taiwanensis WR-30T

M. cerbereus GY-1T

M. silvanus VI-R2T

M. chliarophilus ALT-8T

M. timidus SPS-243T

0.470.0 0.570.0 0.370.0 0.370.0 0.370.0 39.070.1 13.070.1 – 2.470.1 – 2.770.1 0.570.0 4.870.2 1.470.1 – – 0.770.1 tr tr 19.770.2 10.470.1 – – 1.070.0 – – – tr – – tr – – tr tr 0.970.1 tr

0.370.0 1.070.0 0.370.0 0.270.0 0.270.0 32.870.5 11.370.2 tr 3.570.1 tr 5.970.1 0.370.0 5.570.2 1.470.1 – – 0.770.0 – tr 20.270.4 10.470.1 tr – 1.870.1 – – – 0.370.0 – – tr – – tr tr 1.270.1 tr

0.670.0 0.870.1 0.370.0 0.270.0 1.870.0 41.770.3 9.970.3 0.270.0 1.570.1 0.470.0 2.970.1 0.370.0 3.570.1 0.770.0 0.370.0 0.470.1 3.270.0 0.370.0 0.470.1 13.970.2 4.470.2 0.270.0 0.670.0 0.670.1 tr 0.270.0 tr 0.370.0 5.670.2 0.470.1 2.070.1 0.670.0 – – – 0.670.1 –

0.670.1 1.070.3 tr 0.870.1 0.270.1 40.171.7 3.270.6 – 1.870.3 tr 5.270.1 – 5.270.2 0.570.1 – tr 0.870.1 – – 22.273.0 2.970.3 tr 0.570.1 1.470.3 – 0.270.1 – 0.370.1 8.571.3 0.270.8 0.870.3 0.470.1 – 0.370.1 – 1.370.1 –

1.470.1 3.570.7 0.470.1 – 3.070.5 36.071.3 11.070.5 0.570.1 2.670.4 1.870.1 5.371.0 0.570.0 3.470.2 0.970.0 0.870.0 0.370.0 3.170.9 0.270.1 0.670.2 4.570.2 2.270.3 0.470.1 0.670.1 0.270.0 1.170.2 tr 0.270.0 – 2.870.0 tr 7.570.5 1.770.3 0.470.0 – – tr –

0.470.0 0.670.0 tr 1.070.1 – 25.872.3 31.172.4 – 0.370.0 – 1.870.1 0.370.1 4.670.2 2.670.3 – tr – – – 7.470.0 7.170.9 – 0.870.0 0.270.0 – – – 0.270.0 8.371.4 2.770.1 tr – tr 0.770.0 0.770.1 2.570.1 0.570.1

1.770.1 0.870.3 0.370.1 – – 48.770.5 10.371.2 – 1.670.1 – 1.570.9 – 7.971.2 0.670.1 1.370.1 – – – – 18.771.1 2.870.1 – – 1.0070.1 – – – 0.270.0 2.270.1 – 0.570.1 – – 0.370.1 – – –

1.070.2 0.570.1 0.370.1 – – 43.971.8 7.771.0 – 1.370.2 – 1.170.1 0.270.0 5.871.5 0.570.2 1.270.5 – – – – 24.675.7 5.270.7 – – 0.770.1 – – – tr 2.970.9 0.370.0 0.770.1 tr – 0.370.2 tr 1.070.1 –

7, SD, the results are the mean plus the standard deviation of two to four analyses; tr, trace (o0.2%); -, not detected. a The double-bond position of this fatty acid is not known. b A summed feature represents groups of two or three fatty acids that could not be separated by GLC with the MIDI System. Summed feature 3 comprises 16:1 o7c and/or 16:1 o6c; Summed feature 9 comprises 17:1 iso o9c and/or 16:0 10-methyl; Summed feature 4 comprises 17:1 anteiso B and/or iso I.

in width. Long filaments are also present. Gram stain is negative. The cells are non-motile and spores are not formed. Colonies on Thermus medium are red-pigmented and 0.5–1 mm in diameter after 72 h growth. The optimum growth temperature is between 55 and 60 1C; growth does not occur at 30 or 70 1C. The optimum pH is between 7.5 and 8.5; growth does not occur at pH 6.0 or 10.0. The major respiratory quinone is menaquinone 8. The major polar lipids are PL-2 and GL-1. The major fatty acids are 15:0 iso, 17:0 iso, 15:0 anteiso and 17:0 anteiso. Aerobic and heterotrophic. Oxidase, catalase and DNAse positive. Nitrate is reduced to nitrite.

Degradation of hippurate, aesculin, arbutin, elastin, starch, gelatine, casein and xylan is positive. Alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase, acid phosphatase, naphthol-AS-BIphosphohydrolase, b-galactosidase, a-glucosidase and b-glucosidase test positive in API ZYM; other activities test negative. Strains CAL-4T and CAL-12 assimilated D-glucose, D-fructose, D-galactose, D-mannose, D-xylose, D-maltose, L-arabinose, sucrose, maltose, lactose, Dcellobiose, D-trehalose, D-sorbitol, D-mannitol, D-arabitol, lactate, pyruvate, succinate, malate, fumarate, glutamate, L-asparagine, L-arginine and L-glutamine.

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Fig. 2. Phylogenetic dendrogram based on a comparison of the 16S rRNA gene sequences of strains CAL-4T and CAL-12, and the closest phylogenetic relatives. The trees were created using the neighbor-joining method. The numbers on the tree indicate the percentages of bootstrap sampling derived from 1000 replications. Symbol (K) indicates node branches conserved when the tree was reconstructed using maximum parsimony and maximum likelihood algorithms. The isolates characterized in this study are indicated in bold. Scale bar, 1 inferred nucleotide substitution per 100 nucleotides.

The DNA of strain CAL-4T has a G+C content of 64.170.4 mol% (HPLC method). The type strain CAL-4T ( ¼ DSM 22234T ¼ LMG 24878T) was isolated from the Lavoire fountain in Chaudes-Aigues, France. Strain CAL-12 ( ¼ DSM 22235 ¼ LMG 24879) is an additional strain of this species.

[4]

[5]

Acknowledgement We are indebted to Prof. J. P. Euze´by (E´cole National Ve´te´rinaire, Toulouse, France) for the etymology of the new organisms’ name.

[6]

[7]

Appendix A. Supporting Information Supplementary data associated with this article can be found in the online version at doi:10.1016/ j.syapm.2009.05.002.

[8] [9]

[10]

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