Phenylobacterium falsum sp. nov., an Alphaproteobacterium isolated from a nonsaline alkaline groundwater, and emended description of the genus Phenylobacterium

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Systematic and Applied Microbiology 28 (2005) 295–302 www.elsevier.de/syapm

Phenylobacterium falsum sp. nov., an Alphaproteobacterium isolated from a nonsaline alkaline groundwater, and emended description of the genus Phenylobacterium$ Igor Tiagoa, Vı´ tor Mendesa, Carlos Piresa, Paula V. Moraisb, Anto´nio Verı´ ssimoa, a

Departamento de Zoologia and Centro de Neurocieˆncias de Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal b Departamento de Bioquı´mica and Instituto do Ambiente e Vida Universidade de Coimbra, 3004-517 Coimbra, Portugal Received 26 January 2005

Abstract A Gram-negative bacterium designated AC-49T was isolated from an alkaline groundwater with a pH 11.4. This organism formed rod-shaped cells, was strictly aerobic, catalase and oxidase positive, with an optimum growth temperature of 35 1C and an optimum pH value of 8.0. Strain AC-49T assimilated primarily amino acids and some Krebs cycle metabolites, did not use sugars for growth. The organism did not grow on L-phenylalanine or antipyrin. The G+C content of DNA was 66.9 mol%. The phylogenetic analyses based on the 16S rRNA sequencing showed that the closest relatives of strain AC-49T were Phenylobacterium lituiforme and Phenylobacterium immobile, indicating that the organism is a member of the order Caulobacterales of the Alphaproteobacteria. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strain AC-49T, represents a novel species of the genus Phenylobacterium for which we propose the name Phenylobacterium falsum sp. nov. r 2005 Elsevier GmbH. All rights reserved. Keywords: Alphaproteobacteria; Phenylobacterium falsum; Alkaline groundwater

Introduction Natural alkaline environments are uncommon geological features. Soda lakes and soda deserts represent the most stable naturally occurring alkaline environments on earth, with pH values generally higher than 10 and occasionally reaching pH 12 [12]. The alkalinity of these $

Nucleotide sequence data reported are available in the DDBJ/ EMBL/GenBank databases under the accession number(s): AJ717391 for strain AC-49T : Corresponding author. Tel.: +351 239824024; fax: +351 239826798. E-mail address: [email protected] (A. Verı´ ssimo). 0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2005.02.005

environments is generated by the accumulation of sodium carbonate. However, most of these sites contain high concentrations of NaCl and other salts giving rise to saline, and sometimes, hypersaline environments [12]. Nonsaline alkaline environments are much rarer. The genesis of these peculiar environments depends on complex geological formations that probably lead, in all cases to a single geochemical process known as serpentinization [27]. Basically this process can be depicted as the weathering, by CO2-charged waters, of silicate minerals present in mafic or ultramafic rocks. This rare process can be found in some of environments such as: groundwater associated to kimberlite formation as in Lake Timiskaming and Kirkland Lake kimberlite

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fields in Canada [22]; ground and surface water associated to some ophiolites in northern California [24], the Semail in Oman [4], Troodos ophiolictic complex in Cyprus [5], and the Maqarin site in Jordan [3]. Recently, serpentinization has been implicated in the formation of a new class of sea-floor hydrothermal system know as the Lost City Field, and considered as a potential environment for the emergence of life on the Earth’s ocean floor [16]. Remarkably, water associated with ophiolites and serpentinization activity has also been considered as habitat analogs on Mars [24]. The alkalinity of these water environments is generated by high levels of Ca(OH)2 and maintained, around pH 11, due to an equilibrium between the solid phase Ca(OH)2 and the soluble Ca2+ and OH
. Recently, we investigated the bacterial diversity of a groundwater at Cabec¸o de Vide, in Southern Portugal. The ophiolite-like geological background of this aquifer and its chemical characteristics, strongly suggests serpentinization activity. This groundwater has a high alkalinity (pH 11.4) associated with an extremely low ionic concentration where Ca2+ and OH
are major chemical constituents [27]. We isolated a large number of bacterial strains during a survey of the bacterial diversity present in the water samples collected from the Cabec¸o de Vide borehole which were mainly Gram-positive. Nevertheless, we also recovered a small number of Gram-negative isolates phylogenetically related to the caulobacters and particularly one organism closely related to the lineage containing the species Phenylobacterium immobile [18] and Phenylobacterium lituiforme [15]. In this study, we describe the morphological, physiological, chemotaxonomic and phylogenetic characteristics of this organism. On the basis of the data presented, strain AC-49T should be placed in the genus Phenylobacterium as a new species for which the name Phenylobacterium falsum sp. nov. (AC-49T ¼ LMG 22693T ¼ CIP 108469T) is proposed.

Materials and methods Bacterial strains and culture conditions Strain AC-49T was isolated, as described previously, in 10  diluted Alkaline-Buffered Medium 2 (ABM2), adjusted to pH 7.0, at 37 1C [27]. After isolation the strain was routinely cultured in an altered version of R3A medium (designated R3A-V) adjusted to pH 8.0, at 35 1C, and maintained at
70 1C in the same medium supplemented with 15% glycerol. The R3A-V contained the following components per liter of media: yeast extract (Difco), 1.0 g; proteose peptone (Difco no. 3), 1.0 g; casamino acids, 1.0 g; glucose, 1.0 g; K2HPO4,

0.6 g; MgSO4  7H2O, 0.1 g; Na pyruvate, 0.05 g; agar (Difco), 15.0 g; 50 ml of a macronutrients solution 10  concentrated; 5 ml of a micronutrients solution 100  concentrated; and 100 ml of a specific buffer solution (listed below) at a concentration of 1 M, autoclaved separately and mixed after cooling. The 10  concentrated macronutrients solution contained per liter: nitrilotriacetic acid, 1.0 g; CaSO4  2H2O, 0.6 g; MgSO4  7H2O, 1.0 g; NaCl, 0.8 g; KNO3, 1.03 g; NaNO3, 6.89 g; NaHPO4, 1.11 g. The 100  concentrated micronutrients solution contained per liter: MnSO4  H2O, 0.22 g; ZnSO4  7H2O, 0.05 g; H3BO3, 0.05 g; CuSO4  5H2O, 0.0025 g; Na2MoO4  2H2O, 0.0025 g; CoCl2  6H2O, 0.0046 g. The following buffer solutions, prepared according to Gomory [9], were used to adjust the medium to different pH values: citrate buffer was used to adjust the pH to 6.5, phosphate buffer was used to adjust the pH to 7.0 and 7.5, tris (hydroxymethyl) aminomethane–Tris buffer was used for pHs between 8.0 and 8.5, carbonatebicarbonate buffer was used for pHs between 9.0 and 10.0 and carbonate-KOH was used to for pH values higher than 10.0. The pH value of each lot of solid medium was verified prior to using with a surface-testing Sentixs Sur pH electrode (WTW, Hoskin Scientific).

Morphological, physiological and biochemical tests Cell morphology and motility were examined by phase-contrast microscopy. Gram reaction, the presence of cytochrome oxidase and catalase were determined as described by Smibert and Krieg [25]. All tests were performed after 24 h of incubation at 35 1C on R3A-V, pH 8.0. The pH range for growth of strain AC-49T was examined by determining the turbidity (610 nm) of cultures incubated in 300 ml Erlenmeyer flasks containing 100 ml of R3A-V, between pH 6.0 and 10.0, in a reciprocal water bath (170 rpm) at 35 1C. The growth temperature range of strain AC-49T was examined in liquid medium at pH 7.0, 8.0 and 9.0 between 20 and 45 1C. Growth of AC-49T, in the presence NaCl (w/v) ranging up to 9.0%, was examined in liquid medium at pH value 8.0 and 35 1C. Single-carbon source assimilation was determined using API 50 CH test strips (Analytab Products Inc., Biomerieux, France), using 0.1 M tris (hydroxymethyl) aminomethane–Tris buffer pH 8.0 supplemented with 0.03 g agar (Difco); K2HPO4, 600 mg; MgSO4  7H2O, 100 mg; NH4Cl2, 500 mg; vitamin B12, 0.03 mg; 50 ml of macronutrients solution and 5 ml of micronutrients solution (described above) per liter. Microorganisms were resuspended in sterilized water with a turbidity corresponding to the McFarland No. 5 and 6 standard [25]. The cell suspensions (3 ml) were added to 60 ml of

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the medium and added to the API 50 CH test strip wells, as recommended by the manufacturer. Results were recorded after 24, 48 h and 5 days incubation at 35 1C. Additional single-carbon source assimilation tests were performed, in 20 ml screw capped tubes containing 10 ml of the same minimal medium. Growth was examined daily by measuring the turbidity of cultures incubated for a total of 5 days at 35 1C. Positive and negative control cultures were grown in R3A-V (pH 8.0) and in the minimal medium without carbon source. For comparison purposes, we evaluated the ability of strain AC-49T and type strain of Phenylobacterium immobile ET ( ¼ DSMZ 1986T, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany) to grow in L-phenylalanine, succinate, citrate and in the herbicide antipyrin (Sigma) as single carbon sources. These determinations were performed on the minimal medium describe above, and also in Phenylobacterium medium as described by Lingens et al. [18]. Strain DSM 1986T was cultured at pH 7.0 and incubated at 30 1C up to 15 days. Anaerobic growth was assessed in cultures grown in R3A-V (pH 8.0) medium incubated in anaerobic chambers with an H2/CO2 atmosphere (BioMerieux, Marcy L’Etoile, France). Nitrate reduction, indole production and the presence of b-galactosidase were determined using the API 20NE system with the minimal medium described above. Results were recorded after 24, 48 and 72 h of incubation at 35 1C. The hydrolysis of elastin, arbutin, gelatin, casein, esculin, starch and hippurate, the presence of urease, xylanase and DNAse, and the reduction of nitrate was assessed on R3A-V (pH 8.0) as described by Hudson et al. [11] and Smibert and Krieg [25] for up to 6 days.

Lipoquinones and fatty acid composition Lipoquinones were extracted from freeze dried cells, purified by thin layer chromatography and separated with a Gilson high-performance liquid chromatography apparatus, 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 [28]. Cultures for fatty acid analysis, of strain AC-49T, were grown on R3A-V plates adjusted at pH 8.0, in sealed plastic bags submerged in a water bath at 35 1C for 24 h. The fatty acid profile of type strain of Phenylobacterium immobile (DSM 1986T) was cultured on Plate Count Agar medium, at 30 1C for 48 h. Fatty acid methyl esters (FAMEs) were obtained from fresh wet biomass by saponification, methylation and extraction as described previously, and separated, identified and quantified with the standard MIS Library Generation Software (Microbial ID Inc., Newark, Delaware, USA) as described by the manufacturer.

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Antibiotic sensitivity Antibiotic susceptibility of strain AC-49T was examined with disks (BioMe´rieux) containing amoxicillin (25 mg), ceftriaxon (30 mg), cephalothin (30 mg), ceftazidin (30 mg), chloramphenicol (30 mg), colistin (50 mg), doxycyclin (30 mg), gentamicin (10 mg), kanamycin (30 mg), nalidixic acid (30 mg), ofloxacin (5 mg), penicillin G (10 U/IE), polymyxin B (300 U/IE), streptomycin (10 mg), tetracycline (30 mg) on R3A-V (pH 8.0) 35 1C for 72 h.

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 was isolated as described by Nielsen et al. [20]. The G+C content of DNA was determined by highperformance 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 and sequencing of the purified PCR products were carried out as described by Rainey et al. [21]. Purified reactions mixtures were electrophoresed using a model 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). The quality of 16S rRNA gene sequences was checked manually using Bioedit editor [10] and aligned against representative reference sequences of the most closely related members, obtained from the Ribossomal Database Project [6] and EMBL, using the multiple-alignment CLUSTAL X software package [26]. The method of Jukes and Cantor [14] was used to calculate evolutionary distances; phylogenetic dendrograms were constructed using the neighbor-joining method [23], and trees topologies were evaluated by performing bootstrap analysis [8] of 1000 data sets by using the MEGA2 package [17].

Results Isolation, morphological and biochemical characteristics of strains AC-49T Several bacteria were isolated by Tiago et al. [27] from the artesian borehole feeding the spa at Cabec¸o de Vide in Southern Portugal during a prokaryotic diversity study. The borehole water had a temperature of 20.5 1C and a pH of 11.4. Strain AC-49T, was one of the rare Gram-negative isolates recovered from the water samples analyzed. This organism formed rod-shaped (0.6 mm in width and 1.2–3.3 mm in length) cells, glistening white colonies, was nonmotile and aerobic. Strain AC-49T had

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an optimum growth temperature of about 35 1C, and did not grow at 20 or 45 1C (Fig. 1a). The optimum pH for growth of strain AC-49T was around 8.0, no growth was observed at pH 6.0 or at pH 10.0 (Fig. 1b). Curiously, the pH influences the specific growth rate but also the range of temperature for growth. Moreover, when the strain AC-49T was cultured at high pH values (9.0), the temperature range observed was narrower, varying between 20 and 40 1C (Fig. 1a). The specific growth rate of the strain was higher in R3A-V without NaCl, but the organism grew in medium containing up to 7.0% NaCl (results not shown). The strain was catalase and cytochrome oxidase positive. The organism was strictly aerobic and heterotrophic. Nitrate was not reduced to nitrite and the organism did not utilize sugars. Only some Krebs cycle metabolites and several amino acids were used as substrates (Table 1). However, strain AC-49T, did not use L-phenylalanine or the herbicide antipyrin as single carbon sources (Table 1). The type strain of P. immobile, however, used both substrates on minimal R3A-V and Phenylobacterium medium. Isolate AC-49T was resistant to the antibiotics tested, except for chloramphenicol, colistin, doxycyclin, gentamicin, kanamycin, ofloxacin, polymyxin B, streptomycin and tetracycline.

(approximately 50% of the total), 17:0 and 16:0. The same fatty acids were also predominant in the type strain (ET ¼ DSM 1986T) of P. immobile (Table 2).

16S rRNA gene sequence comparison and G+C content of DNA Partial 16S rRNA gene sequence comprising 1465 nucleotides was determined for strain AC-49T. Comparison of this sequence with representatives of the main lines of descent within the domain Bacteria indicated that this strain was a member of the order Caulobacterales, of the Alphaproteobacteria (Fig. 2). The most closely related organisms were the type strains of the species P. lituiforme (similarity value of 96.3%) and P. immobile (similarity value of 95.7%). The mean pairwise similarity of the 16S rRNA gene sequences determined between strain AC-49T and members of the genera Caulobacter, Brevundimonas and Asticcacaulis were much lower (94.9%, 93.1% and 92.1%, respectively). The G+C content of the DNA determined for strain AC-49T was 66.9 mol%.

Discussion Chemotaxonomic characteristics The major respiratory quinone found of strain AC49T was ubiquinone 10 (U10). The predominant cellular fatty acids detected in strain AC-49T were 18:1o7c

Specific growth rate (h−1)

(a)

0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

pH7 pH8 pH9

15

20

25

30 35 Temperature °C

40

45

50

Specific growth rate (h−1)

(b) 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 5

6

7

8 pH

9

10

11

Fig. 1. Effect of temperature (a) and pH determined at 35 1C (b) on the growth rate of strain AC-49T.

Phylogenetic analysis, based on 16S rRNA gene sequence of strain AC-49T, clearly shows that the organism is related to the type strains of the two validly described species P. lituiforme and P. immobile. Moreover, the values of 16S rRNA gene sequence similarities between our isolate and the other strains of the genus Phenylobacterium (around 96%), are, by itself, a justifiable reason to the inclusion of strain AC-49T, as a new species of the genus Phenylobacterium. The fatty acid profiles of P. immobile type strain and AC-49T clearly agree with the inclusion of these strains in the Caulobacter–Brevundimonas–Asticcacaulis branch. Indeed, all members of this phylogenetic group are characterized by predominant 16:0 and 18:1o7c [1,2], despite differences in the amounts of these, as well as, other fatty acids which may be important to distinguish strains within each species. The high level of 17:0, together with the low quantity of 15:0, and trace levels of 16:1o7c, (referred to as summed feature 4 by Abraham et al. [1,2]) constitute characteristic features that concur with our view that strain AC-49T represents a new species within the genus Phenylobacterium. Despite the close phylogenetic relationship with the type strains of P. lituiforme and P. immobile, strain AC49T can be clearly distinguished from these species by the cardinal temperatures for growth, by the optimum pH for growth and by the halotolerance (Table 1). Strain AC-49T was isolated from an environment with pH 11.4, but the optimum pH for growth under

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Table 1. Characteristics of strain AC-49T and comparison with the type strains of Phenylobacterium immobile (ET) and P. lituiforme (FaiI3T) Characteristic

AC49T

P. immobilea

P. lituiformeb

Source Motility O2 requirement Optimum growth temperature (1C) Optimum pH range Halotolerance

Alkaline groundwater Non-motile Obligate aerobe 30–35 7.5–8 Optimal at 0% No growth at 8% +

Soil Non-motile Obligate aerobe 28–30 6.8–7 Optimal at 0% No growth at 1% (weak) positive

Water from subsurface aquifer Motile Facultative aerobe 40–41 6–6.5 Optimal at 0% No growth at 1%



+
+

NDc
ND ND

ND ND ND




+ +


ND
ND ND

+ ND + ND ND







+





+ + + + + + + +
e +e
e
e 66.9







+ + + +

+ (Slow) ND ND ND ND ND ND
e + (Slow)e +e +e 65.5

+ ND ND ND ND ND ND + + + ND 66.5

Oxidase Presence of Xylanase Urease DNAse b-Galactosidase Hydrolyze Casein Elastin Gelatin Arbutin Hippurate Reduction of nitrate to Nitrite Utilization ofd Glucose Sucrose Xylose Glycerol a-ketoglutarate Pyruvate Glutamate Alanine Isoleucine Proline Glutamine Arginine Citrate Succinate Phenylalanine Antipyrin G+C (mol%)

All strains were catalase positive, esculin, starch and indol negative. a Data from Lingens et al [18]. b Data from Kanso and Patel [15]. c Not determined. d Strain AC-49T did not utilize any single carbon sources included in API 20NE and in API 50CH test strips. Aspartate, aspargine, cysteine, glycine, histidine, lysine, methionine, serine, threomine, valine and ornithine were not utilized. e Results obtained in R3A-V minimal medium and in Phenylobacterium medium.

laboratory conditions was only 8.0 and growth did not occur at 10.0. However, it is not uncommon that isolates from extreme alkaline environments, under laboratory conditions, only grow at moderate pH values [7,12,13,27]. Other noticeable differences that distinguish the species of this genus pertain to the ability to use single carbon sources for growth; P. lituiforme strain FaiI3T

uses a wide variety of organic substrates, including some sugars, while P. immobile strain ET only uses a very limited number of specialized substrates which include; succinate, L-phenylalanine, antipyrin and other chemically related herbicides [15,18]. On the other hand, strain AC-49T uses primarily amino acids and some Krebs cycle metabolites as organic substrates but, like P. immobile does not use sugars for growth. Moreover,

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the most significant characteristic of AC-49T is the inability to use L-phenylalanine, the rare distinctive characteristic of the other two species of genus Phenylobacterium [15,18]. On the basis of these findings, Table 2. Major fatty acid composition of strain AC-49T and type strain of Phenylobacterium immobile (ET) Total fatty acid (%)a AC49T 11:0 12:0 13:0 12:1 3OH 12:0 3OH 14:0 a 15:0 15:0 16:1 o11c 16:1 o7c 16:0 a 17:0 i 17:0 Unknown 16.762 17:1 o8c 17:1 o6c 17:0 Unknown 17.607 i 18:0 18:1 o7c 18:0 18:1 o7c 11 methyl Unknown 18.806 19:0 cyclo o8c

0.5 1.3 0.3 1.4 0.3 —b — 1.7 2.7 0.2 7.6 — 1.7 4.3 4.3 4.5 12.1 1.2 — 49.9 0.7 3.7 3.6 —

Phenylobacterium immobile 0.5 4.1 0.5 3.2 1.3 0.5 0.5 6.9 — 3.7 17.4 1.1 — — 1.9 2.3 7.0 — 0.9 41.1 0.6 0.7 0.3 4.4

a Values for fatty acids present at levels of less than 0.5% in both strains are not shown. b Not detected.

we propose that strain AC-49T represents a new species for which we recommend the name P. falsum sp. nov.

Emended description of Phenylobacterium (Lingens et al. [18]) Kanso and Patel [15]. Characteristics as in Lingens et al. [18], and Kanso and Patel [15] except that some members of the genus Phenylobacterium do not grow on L-phenylalanine.

Description of Phenylobacterium falsum sp. nov. Phenylobacterium falsum (fal’. sum; L. neut. adj. falsum, false, impostor, a false Phenylobacterium species because it does not degrade phenylalanine). Phenylobacterium falsum forms small rod cells, 0.6 mm in width by 1.2–3.3 mm in length, nonmotile, Gram stain is negative, strictly aerobic and heterotrophic, colonies are small and white, oxidase and catalase positive. The optimum temperature for growth is about 35 1C, no growth occurs at 20 1C or 45 1C; the optimum pH is 8.0, no growth occurs at pH 6.0 or 10.0; growth is faster without added NaCl, but grows up to 7.0% NaCl. The respiratory quinone is ubiquinone 10 (U10). The predominant fatty acids are 18:1o7c, 17:0 and 16:0. Does not reduce nitrate. Does not hydrolyze casein, elastin, gelatin, starch or esculin. Hydrolyzes arbutin and hippurate. Urease and b-galactosidase are detected. DNAse and xylanase are not detected. Is sensitive to chloramphenicol, colistin, doxycyclin, gentamicin, kanamycin, ofloxacin, polymyxin B, streptomycin and tetracycline. The organism assimilates succinate, aketoglutarate, pyruvate, glutamate, alanine, isoleucine, proline, glutamine and arginine. Sugars are not utilized. Growth does not occur on L-phenylalanine or on antipyrin. The mole G+C ratio of the DNA of the type strain is 66.9 mol%. This bacterium was isolated

Brevundimonas intermedia ACM 2608T (AJ007802) Brevundimonas vesicularis LMG 2350T (AJ227780) Brevundimonas aurantiaca ATCC 15254T (AJ227787) Brevundimonas alba ATCC 15265T (AJ227785) 95 Brevundimonas bacteroides ATCC 15254T (AJ227782) Brevundimonas diminuta LMG 2089T (AJ227778) Caulobacter fusiformis ATCC 15257T (AJ227759) Caulobacter henricii ATCC 15253T (AJ227758) Caulobacter vibrioides DSM 9893T (AJ009957) Asticcacaulis biprosthecium DSM 4723T (AJ247193) Asticcacaulis excentricus DSM 4724T (AJ247194) Phenylobacterium immobile ATCC 35973T (Y18216) AC-49T (AJ717391) Phenylobacterium lituiforme DSM 14363T (AY534887) Woodsholea maritima LMG 21817T (AJ578476)

87 87 100 99

88 89 99 99

0.01

Fig. 2. Phylogenetic dendrogram based on a comparison of the 16S rDNA sequences of strains AC-49T, and the closest phylogenetic relatives. Scale bar, 10 inferred nucleotide substitutions per 100 nucleotides.

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from groundwater taken from the borehole at Cabec¸o de Vide in Southern Portugal. The type strain, AC-49T, has been deposited in the Collection of the Institute Pasteur, Paris, France, as strain CIP 108469T and in the BCCM/LMG Bacteria Collection, Ghent, Belgium as strain LMG 22693T.

[8]

[9] [10]

Acknowledgements This research was funded in part by FCT/FEDER project POCTI/BSE/42732/2001. We are indebted to Prof. J. Euze´by (E´cole National Ve´te´rinaire, Toulouse, France) for the etymology of the new organisms’ names. We thank Dr. Fernanda Nobre and Prof. Milton da Costa (Universidade de Coimbra, Portugal) for helping in the FAME analysis and critical reading of the manuscript. We also thank to the Junta de Freguesia de Cabec¸o de Vide and Mr. Manuel Fontainhas for permission to collect the water samples.

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