Abstract Three new spirilloid phototrophic purple non-sulfur bacteria were isolated in pure culture from threedifferent environments: strain CE2105 from a brackish la-goon in the Arcachon Bay (Atlantic coast, France), strainSE3104 from a saline sulfur spring in the Pyrenees(Navarra, Spain), and strain AT2115 a microbial mat(Tetiaroa Atoll, Society Islands). Single cells of the threestrains were spiral-shaped and highly motile. Their intra-cellular photosynthetic membranes were of the vesiculartype. Bacteriochlorophyll a and carotenoids of the normalspirilloxanthin series were present as photosynthetic pig-ments. Optimal growth occurred under photoheterotrophicconditions and in the presence of 0.5–4% w/v NaCl.These features are similar to those described for Roseospiramediosalina. Comparative sequence analysis of their 16SrRNA genes placed these strains within the α-subclass ofProteobacteria, in a cluster together with Roseospiramediosalina and Rhodospira trueperi. They form a closelyrelated group of slightly to moderately halophilic spiral-

shaped purple nonsulfur bacteria. However, the three newisolates exhibited some differences in their physiologyand genetic characteristics. Consequently, we propose thatthey are members of three new species within the genusRoseospira, Roseospira marina sp. nov., Roseospira navar-rensis sp. nov., and Roseospira thiosulfatophila sp. nov.,with strains CE2105, SE3104, and AT2115 as the typestrains, respectively. As a consequence, an emended de-scription of the genus Roseospira is also given.

Keywords Phototrophic purple nonsulfur bacteria ·Roseospira · Coastal lagoon · Saline sulfur spring ·Microbial mat · Moderate halophilic bacteria

Introduction

Purple nonsulfur bacteria are commonly encountered inanoxic waters and sediments exposed to light. Many pur-ple nonsulfur bacterial species are found in freshwaterhabitats rich in organic matter such as lake and river sedi-ments (Kaiser 1966), waste water ponds (Siefert et al.1978), wetlands (Burke et al. 1974) or rice fields (Okudaet al. 1957). Other species of the purple nonsulfur bacteriaare typical halophilic bacteria (Imhoff and Trüper 1992).

Among the spiral-shaped purple nonsulfur bacteria someare considered as halophilic bacteria (Caumette et al. 1999;Imhoff 2001). They have been isolated from hypersalinehabitats such as evaporitic seawater ponds (Drews 1981),salterns (Nissen and Dundas 1984; Pfennig et al. 1997;Glaeser and Overmann 1999), the Dead Sea (Mack et al.1993) or hot saline springs (Kompantseva and Gorlenko1985). These bacteria were described as new species andnamed Rhodothalassium salexigens, Rhodovibrio sali-narum, Rhodovibrio sodomense, Roseospira mediosalina(Imhoff et al. 1998), Rhodospira trueperi (Pfennig et al.1997), and Roseospirillum parvum (Glaeser and Over-mann 1999). The freshwater species are now grouped intothe genera Rhodospirillum, Phaeospirillum, and Rhodocista(Imhoff et al. 1998).

Rémy Guyoneaud · Sophie Mouné · Claire Eatock · Virginie Bothorel · Agnès Hirschler-Réa · John Willison ·Robert Duran · Werner Liesack · Rodney Herbert · Robert Matheron · Pierre Caumette

Characterization of three spiral-shaped purple nonsulfur bacteria isolated from coastal lagoon sediments, saline sulfur springs, and microbial mats: emended description of the genus Roseospiraand description of Roseospira marina sp. nov., Roseospira navarrensis sp. nov.,and Roseospira thiosulfatophila sp. nov.Received: 14 November 2001 / Revised: 17 May 2002 / Accepted: 21 May 2002 / Published online: 26 July 2002

ORIGINAL PAPER

R. Guyoneaud · S. Mouné · V. Bothorel · R. Duran ·P. Caumette (✉)Laboratoire d’Ecologie Moléculaire – Microbiologie, IBEAS,Université de Pau, BP 1155, 64013, Pau Cedex 13, Francee-mail: pierre.caumette@univ-pau.fr, Tel.: +33-5-59923146, Fax: +33-5-59808311

S. Mouné · J. WillisonLaboratoire de Biochimie et Biophysique des Systèmes Intégrés,DBMS, CEA Grenoble, 38054 Grenoble Cedex 9, France

C. Eatock · R. HerbertDepartment of Biological Sciences, University of Dundee,Dundee DD1 4HN, UK

V. Bothorel · A. Hirschler-Réa · R. MatheronMicrobiologie IMEP, Faculté des Sciences et Techniques St-Jérome, 13397 Marseille Cedex 20, France

W. LiesackMax-Planck-Institut für Terrestrische Mikrobiologie, Abteilung Biogeochemie, Karl-von-Frisch-Strasse, 35043 Marburg,Germany

Arch Microbiol (2002) 178 :315–324DOI 10.1007/s00203-002-0454-y

© Springer-Verlag 2002

Spiral-shaped purple nonsulfur bacteria were isolatedduring investigations on eutrophic coastal lagoons (Guy-oneaud et al. 1996), saline springs, and microbial mats(unpublished data). Strain CE2105 was isolated from theCertes Fishponds (Arcachon Bay, France), strain SE3104from the Salinas de Oro (Navarra Pyrénées, Spain), andstrain AT2115 from the Tetiaroa Atoll (French Polynesia);these three strains have been studied in detail. The presentpaper describes their phenotypic properties and their ge-netic relationships with halophilic species of the spiral-shaped purple nonsulfur bacteria. Based on comparativeanalyses of their 16S rRNA gene sequences, these threestrains cluster together with the species Roseospiramediosalina and Rhodospira trueperi. However, due todifferences in phenotypic characteristics and genetic data,the three strains can be readily separated from the existingspecies and from each other. Therefore, they are describedas new representatives of the genus Roseospira, with thenames Roseospira marina sp. nov. for strain CE2105,Roseospira navarrensis sp. nov. for strain SE3104, andRoseospira thiosulfatophila sp. nov. for strain AT2115. Asa result of their characteristics, an emended description ofthe genus Roseospira was necessary.

Material and methods

Source of strains

Strain CE2105 was isolated during ecological studies on the brack-ish Certes Fishponds (Arcachon Bay, French Atlantic coast). Theseman-made brackish shallow lagoons are periodically flooded withseawater from the Arcachon Bay (Guyoneaud et al. 1996). Liquidenrichment cultures were prepared from the upper layer of theanoxic sediments.

Strain SE3104 was isolated from the surface of a sulfide-richsediment in a small saline pond in the Spanish Pyrenees. This pondresults from the outflow of a saline spring (Salinas de Oro,Navarra, Spain) with salinity varying from 2 to 10% (total salinity).This spring water is rich in chloride (46% w/v), sodium (28% w/v),sulfate (15% w/v), calcium (5% w/v), and potassium (4% w/v).

Strain AT2115 was isolated from microbial mats in FrenchPolynesia (Tetiaroa Atoll, Society Islands). These mats consistedof stratified structures dominated by cyanobacteria at the surfaceand profuse anoxygenic phototrophs in the deeper layers (Mao Cheet al. 2001).

Media, isolation, and culture conditions

During enrichment and isolation procedures for strain CE2105,cultures were obtained by using a basal medium containing: fil-tered (0.2 µm pore size) seawater, 750 ml; distilled water, 250 ml;NH4Cl, 0.035% (w/v); yeast extract, 0.04% (w/v); and Fe-citrate,0.001% (w/v). The medium was autoclaved and cooled under a gasmixture of N2/CO2 (90/10, v/v). Vitamin V7 solution (Pfennig andTrüper 1992; 1 ml.l–1), phosphate buffer (0.1 M, pH 6.8, 36 ml.l–1),and Na-ascorbate/cysteine-HCl (0.25% (w/v)/0.5% (w/v) solutionat pH 7.0, 0.2 ml.l–1) were then aseptically added to the medium.For enrichment and isolation of strain SE3104, the culture mediumwas prepared according to the method of Pfennig and Trüper(1992) and supplemented with 5% (w/v) NaCl and 1% (w/v)MgCl2

.6H2O. The culture medium used for enrichment and isola-tion of strain AT2115 contained: filtered (0.2-µm pore size) sea-water, 1,000 ml; NH4Cl, 0.05% (w/v); KH2PO4, 0.02% (w/v); yeastextract 0.05% (w/v). The medium was autoclaved and cooled un-der N2/CO2 (90/10, v/v). Vitamin V7 solution (1 ml.l–1), NaHCO3

(0.15% w/v), and Na2S.9H2O (0.02% w/v) were then asepticallyadded to the medium.

The final pH for all media was adjusted to 6.8. The media weredispensed into sterile 50-ml screw-capped bottles. Organic sub-strates (5 mM sodium acetate and 5 mM di-sodium succinate) wereadded just before utilization.

Pure cultures were obtained by repeated application of the deep-agar dilution method (Pfennig 1978). Deep-agar tubes were incu-bated at 25°C under a light/dark cycle (16 h light/8 h dark) usingtungsten lamps. Purity of the strains was checked by both micro-scopic observations and growth tests in deep-agar AC medium(Difco) supplemented with NaCl (2% or 5% w/v), sodium thiosul-fate (0.05% w/v), and glucose (0.05% w/v) and incubated in thedark under air or a N2/CO2 (90/10, v/v) atmosphere.

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Table 1 Utilization of substrates as electron donors and/or carbonsources by strains CE2105, SE3401, and AT2115. Unless other-wise indicated, the concentration was 5 mM. Growth was checkedby optical density measurements at 650 nm against a control with-out substrate: – no utilization (similar or less than control), (+) poorutilization (1–1.5 times more than control), + good utilization (morethan 1.5 times more than control), nd not determined

Substrates CE2105 SE3401 AT2115

Sulfide (1 mM) – + +Sulfur (0.05%) – – –Thiosulfate – – +Sulfite – – –Formate (+) – –Acetate + + +Propionate + + –Butyrate + + +Valerate + + +Crotonate + + +Caprylate (2 mM) – – –Pelargonate (2 mM) – – –Lactate + + +Glycolate – – –Benzoate (2 mM) – + –Tartrate (2 mM) – – –Pyruvate + + +Malate + + –Fumarate + + –Succinate + + –Citrate – + –2-oxoglutarate + + +Glucose – – +Fructose + – –Gluconate + + +Glycerol + + +Mannitol + + –Cysteine (2 mM) (+) nd ndMethionine (2 mM) – – ndAspartate + + +Glutamate + + +Peptone (0.05%) + + +Casamino acids (0.05%) + + +Yeast extract (0.05%) + + +

The following substrates (concentration in mM) were tested butnot utilized by strains CE2105 and SE3401: tetrathionate (2), thio-glycolate (2), thioacetamide (2), glycine-betaine (2), palmitate (2),cyclohexane-carboxylate (2), nicotinate (2), ascorbate (2), gallate(2), trehalose (2), sucrose (2), methanol (5), ethanol (5), propanol(5), butanol (5), catechol (2)

The composition of final synthetic media for maintenance andcharacterization of the pure cultures contained (per liter of distilledwater): KH2PO4, 0.03% (w/v); NH4Cl, 0.05% (w/v); CaCl2

.2H2O,0.005% (w/v); MgCl2

.6H20, 0.1% (w/v) (0.3% w/v for strainSE3104); MgSO4

.7H2O, 0.05% (w/v) (0.2% w/v for strain SE3104);NaCl, 2% (w/v) (5% w/v for strain SE3104); trace element solu-tion SL12 (Overmann et al. 1992), 1 ml; yeast extract, 0.05% (w/v).Media were autoclaved and cooled under N2/CO2 (90/10, v/v).Vitamin V7 solution (1 ml.l–1), Na-ascorbate (0.05% w/v), andNaHCO3, (0.15% w/v) were then aseptically added to the medium.The final pH was adjusted to 6.8–7.0 and the medium was dis-pensed into sterile 50-ml screw-capped bottles. Organic substrates(5 mM Na-acetate and/or 5 mM di-Na-succinate) were added assubstrates before use. In addition, for strains SE3104 and AT2115,Na2S.9H2O (0.02% w/v) was also added to the medium prior to uti-lization. Pure cultures were grown in 50-ml screw-capped bottlesand stored at +4°C in the dark for preservation.

Microscopy

Microscopy observations and photomicrographs were made withan Olympus OM 2 photomicroscope according to the method ofPfennig and Wagener (1986). Flagella were observed by transmis-sion electron microscopy after negative staining with 1% phospho-tungstic acid neutralised to pH 7.2. The fine structure of the cellswas studied by electron microscopy after fixation of a cell pellet bythe method of Ryter and Kellenberger (1958) and ultrathin section-ing of the cells according to Glazer et al. (1971). The observationswere made with a JEOL 1200 ES electron microscope.

Pigments

In vivo absorption spectra were analyzed with a Perkin ElmerLambda 12 spectrophotometer after suspension of a cell pellet in asucrose solution (Pfennig and Trüper 1992). Absorption spectra af-ter ethanol or acetone extraction were recorded with the samespectrophotometer. The precise carotenoid composition was deter-mined by HPLC analysis according to the method of Buffan-Dubauet al. (1996). For identification of carotenoids, strains with knowncarotenoid composition were used as reference.

Physiological tests

Optimal NaCl concentrations, pH, temperature, light intensity, andsulfide tolerance were determined with the final synthetic media.

Utilization of carbon sources and electron donors was tested withthe final synthetic media without substrates and supplemented withonly 0.01% yeast extract. Substrates were added aseptically fromstock solutions to the final concentrations indicated in Table 1. Theexperiments were carried out in triplicate in completely filled 25-ml screw-capped tubes. Growth was measured by optical den-sity at 650 nm over a period of 10–15 days.

Capacity for aerobic or micro-aerobic growth and anaerobicgrowth in the dark was tested according to the method of Kämpfand Pfennig (1980). Vitamin requirements were tested after fourconsecutive transfers, in the final synthetic media without yeastextract, according to an experimental protocol of Goupy (1988).Assimilatory sulfate-reduction was tested through five consecutivetransfers in 60-ml screw-capped bottles filled with final syntheticmedium with sulfate as the only sulfur source. Hydrogen utiliza-tion was tested in aluminum-cap-sealed bottles with gas-imperme-able butyl stoppers. Bottles were filled one third with liquid me-dium without electron donor and two-thirds with H2/CO2 (80/20,v/v, 100 kPa). Capacity for dinitrogen fixation was checked in rub-ber-stopper bottles half-filled with synthetic media lacking fixednitrogen sources under a N2/CO2 (90/10, v/v) gas atmosphere; fourconsecutive transfers were tested. Catalase was checked by addinga few drops of 3% (v/v) H2O2 to a cell pellet obtained by centrifu-gation.

Determination of the genomic DNA G+C content, comparative 16S rDNA analysis, and DNA-DNA hybridization

The G+C content of the DNA of the three strains was determinedby HPLC according to the methods of Mesbah et al. (1989) andTamaoka and Komagata (1984).

Genomic DNA was isolated and the almost complete 16S rDNAwas amplified via PCR as described by Liesack and Finster (1994),Mouné et al. (2000), and Guyoneaud et al. (1998) for strainsCE2105, SE3104, and AT2115, respectively. The 16S rDNA/rRNAsequences of 13 related species, including representatives of thepurple nonsulfur bacteria and the non-photosynthetic species Aqua-spirillum itersonii, obtained from the EMBL database were used inthe phylogenetic analysis (Table 2). The 16S rDNA sequences werealigned with the CLUSTAL W program (Thompson et al. 1994)from position 71 to 1,372 according to the Escherichia coli num-bering scheme, including gaps. Distance matrices were calculatedusing the DNADIST program with the algorithm of Jukes andCantor (1969) within the PHYLIP package (Felsenstein 1993). Thephylogenetic tree was inferred from evolutionary distances withthe FITCH program of the PHYLIP package (Fitch and Margoliash1967). The Allochromatium vinosum 16S rDNA sequence was in-

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Table 2 Strain designationsand accession numbers of 16S rDNA sequences for allorganisms used in the phylo-genetic analysis

Organism Strain designation Accession number

Roseospira marina CE2105 ATCC BAA-447 AJ298879Roseospira navarrensis SE3104 ATCC BAA-448 AJ298880Roseospira thiosulphatophila AT2115 ATCC BAA-449 AJ401208Roseospira mediosalina BN280T AJ000989Rhodospira trueperi ATCC 700224T X99671Rhodospirillum photometricum E 11 D30777Rhodospirillum rubrum ATCC 11170T D30778Roseospirillum parvum DSM 12498T AJ011919Phaeospirillum fulvum DSM 113T D14433Phaeospirillum molischianum ATCC 14031T M59067Rhodocista centenaria ATCC 43720T D12701Rhodothalassium salexigens ATCC 35888T D14431Rhodovibrio salinarum ATCC 35394T M59069Aquaspirillum intersonii NCIMB 9070 Z29620Rhodovibrio sodomensis DS1 M59072Allochromatium vinosum DSM 180T M26629

cluded to root the tree. The confidence level of the phylogenetictree topology was evaluated by performing 100 bootstrap replica-tions with the programs SEQBOOK and CONSENSE of the samepackage. The three sequences obtained in the present work weredeposited with EMBL (Table 2).

For DNA-DNA hybridization studies, DNA was isolated as de-scribed by Cashion et al. (1977). DNA-DNA hybridization wascarried out as described by De Ley et al. (1970), with the modifi-cations of Huss et al. (1983) and Escara and Hutton (1980), usinga Gilford System model 2600 spectrometer equipped with a Gil-ford model 2527-R thermoprogrammer and plotter. Renaturationrates were computed with the TRANSFER.BAS program by Jahnke(1992).

Analysis of compatible solutes by 13C-NMR spectroscopy

The strains were grown in 8-l batch cultures in basal medium sup-plemented with 100 mM Na-acetate and 6% (w/v) NaCl. Cultureswere incubated at room temperature and continuously sparged withoxygen-free nitrogen to maintain anoxic conditions. Mid-exponen-tial phase (OD650=0.7) cultures were harvested, extracted and ana-lyzed by 13C-NMR spectroscopy according to the methods of Welshand Herbert (1994).

Results

Natural habitat and isolation

Strain CE2105

The black silty sediments in the man-made fishponds ofCertes are rich in organic matter and sulfide, mainly FeSand FeS2. During ecological investigations in 1992, en-richment cultures obtained from the top layers of the sed-iments and incubated in the light gave rise to the growthof spiral-shaped purple nonsulfur bacteria (Guyoneaud etal. 1996). These enrichments served as an inoculum foragar-dilution series. Strain CE2105 was isolated in pureculture and maintained for further characterization.

Strain SE3104

In 1994, a sample of black sediment was collected on theshore of a saline pond (Salinas de Oro) and enriched incompletely filled 120-ml screw-capped bottles containingsynthetic medium. After 1–2 weeks, a red bloom of pho-totrophic bacteria appeared in the bottles. Microscopy ob-servations revealed the presence of vibrioid and highlymotile cells often forming half circles. Several strains havebeen isolated from these enrichments. Strain SE3104,which grew the best, was further characterized.

Strain AT2115

Samples from a Polynesian microbial mat were collectedin 1997 and directly inoculated in agar-dilution series.Some of the red colonies obtained after 15 days of incu-bation consisted of spiral-shaped phototrophic bacteria.Several strains were isolated from different agar series

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Fig.1 Phase-contrast photomicrographs of Roseospira marinastrain CE2105 (A), Roseospira navarrensis strain SE3104 (B), andRoseospira thiosulfatophila strain AT2115 (C). Bar 4 µm

and isolated in pure culture. Strain AT2115 was furthercharacterized.

Morphology and fine structure

Individual cells of strain CE2105 were spiral-shaped,0.4–0.8 µm wide (Fig.1A). The length of the cells variedfrom 1.5–2.5 µm (curved cells) to 4.0–6.0 µm (one com-plete turn of a spiral). Longer cells (10–20 µm) rarely oc-curred. In liquid medium, the strain mostly developed assingle cells but occasionally occurred in aggregates depend-ing on the substrate. The cells of strain SE3104 were vib-rioid rods, 0.6–0.9 µm wide and 3.5–6.5 µm long, oftenby pair, forming half to complete circles (Fig.1B). Cellsrarely occurred in rosettes and inclusions were often visi-ble in the cells (Fig.1B). Individual cells of strain AT2115were spiral-shaped, 0.5–0.8 µm wide and 2.5–6.5 µm long(Fig.1C).

The three strains divided by binary fission. Cells werehighly motile by means of bipolar tufts of flagella in strainsCE2105 and SE3104 or polar tuft of flagella in strainAT2115. In thin sections, a vesicular intracellular mem-brane system was visible. All three strains stained Gram-negative and a cell wall typical of Gram-negative bacteriawas visible on ultrathin section.

Photosynthetic pigment composition

Cell suspensions of strains CE2105 and AT2115 were redwhile suspensions of strain SE3104 were brown-red. Theabsorption spectra of living cells (Fig.2) showed similarcharacteristic absorption peaks of bacteriochlorophyll a(Bchl a) with maxima at 370/376, 589/591, 799/805 and863/864 nm for strains CE2105 and SE3104, respectively.Strain AT2115, by contrast, exhibited a rather unusual invivo absorption spectrum (Fig. 2C), with, in addition to thecharacteristic Bchl a peaks at 370, 590, 800, and 883 nm,a large shoulder in the infra-red at 909/910 nm. The ace-tone extracts spectrum revealed that normal Bchl a withmaximum absorption at 770 nm was present (not shown).

Absorption peaks of carotenoids ranged from 470 to540 nm with a maximum at about 500 nm for all strains,indicating the presence of carotenoids of the normal spi-rilloxanthin series. HPLC analyses confirmed the biosyn-thetic pathway with rhodovibrin or rhodopine as majorcarotenoids depending on the strains (Table 3).

Physiological properties

Optimal growth for all strains occurred photoheterotroph-ically under anoxic conditions in the light with various or-ganic substrates (Table 1). Strain CE2105 was not capableof photolithotrophy. In contrast, sulfide could serve as elec-tron donor for strains SE3104 and AT2115, the latter be-ing also able to use thiosulfate. These two strains neededa reduced sulfur source whereas strain CE2105 was able

to use sulfate as sole sulfur source. The strains toleratedfree sulfide concentrations up to 3 mM for strain CE2105,2 mM for strain SE3104, and 3.5 mM for strain AT2115.

Chemoorganotrophic growth in the dark with acetatewas possible under oxic conditions in strain CE2105 andmicro-oxic conditions in strains SE3104 and AT2115.Catalase was present in the three strains and strongly pos-itive in strain CE2105 when grown under oxic conditions.No respiratory or fermentative metabolism was observedunder anoxic conditions in the dark.

Strain CE2105 required niacin, thiamine, and p-amino-benzoic acid as growth factors but addition of yeast extract(0.01% w/v) strongly increased growth. Strains SE3104and AT2115 required yeast extract as a growth factor. Al-though not confirmed by acetylene reduction, the strainsgrew phototrophically with N2 as sole nitrogen source af-ter four consecutive transfers, indicating their ability to fixdinitrogen.

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Fig. 2 In vivo absorption spectra of Roseospira marina strainCE2105 (A), Roseospira navarrensis strain SE3104 (B), andRoseospira thiosulfatophila strain AT2115 (C)

The three strains have similar ecophysiological require-ments. They grow, for at least three consecutive transfersat the same salinities, at a NaCl concentration between 0.5and 10% (optimum 2–4%) for strain CE2105, between 1and 10% (optimum 3–4%) for strain SE3104, and be-tween 0.2 and 5% (optimum 0.5%) for strain AT2115. Thethree strains exhibited the same NMR spectrum with re-spect to the compatible solutes accumulated, with ectoineand trehalose as the principal components. Their optimumpH was 6.7–6.8 (pH range, 5.3–8.4) for strain CE2105,6.8–7.0 (pH range, 6.0–8.5) for strain SE3104, and 6.8–7.0 (pH range, 5.6–8.6) for strain AT2115. The strainswere mesophilic with optimum temperature of about 30–35°C. When grown under optimal conditions, the growthrates of the three strains ranged from 0.06 h–1 to 0.08 h–1.

G+C content of genomic DNA, phylogenetic analysisand DNA-DNA hybridization

The DNA base compositions were 69.1±0.3 mol% G+Cfor strain CE2105, 66.8 mol% G+C for strain SE3104,and 72.1±0.2 mol% G+C for strain AT2115.

Comparative 16S rDNA sequence analysis placed strainsCE2105, SE3104, and AT2115 within the α-subclass of theProteobacteria, which comprises most of the spiral-shaped

purple nonsulfur bacteria. Within this subgroup, the threestrains formed a separate line of descent together withRoseospira mediosalina and Rhodospira trueperi (Fig.3).The 16S rDNA sequences of strains CE2105, SE3104,and AT2115 differed by 4.4%, 4.6%, and 5.3%, respec-tively, from that of Roseospira mediosalina. The strainswere more distantly related to Rhodospira trueperi, withdifferences in the 16S rDNA sequences of 6.0%, 6.2%,and 6.4%, respectively. Between the three strains, the per-cent differences in the 16S rDNA ranged from 2.4 to3.5%, values around the limits for reliable differentiationbetween species (Stackebrandt and Goebel 1994).

DNA-DNA hybridization values were 28% betweenstrains CE2105 and SE3104, 40% between strains CE2105and AT2115, and 54% between strains AT2115 andSE3104.

Discussion

Strains CE2105, SE3104, and AT2115 are typical mem-bers of the purple nonsulfur bacteria (Imhoff and Trüper1989). According to phylogenetic relatedness, they belongto the α-subclass of the Proteobacteria within the spiril-loid purple nonsulfur bacteria. These bacteria, primarilygrouped in the genus Rhodospirillum, were reclassified

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Table 3 Main characteristics of Roseospira marina strain CE2105,Roseospira navarrensis strain SE3104, Roseospira thiosulfatophilastrain AT2115, and Roseospira mediosalina. Data for R. medios-

alina are from Kompantseva and Gorlenko (1985). (a) µ Growthunder micro-oxic conditions, + growth under oxic conditions

Strain CE2105R. marina

Strain AT2115R. thiosulfatophila

Strain SE3401R. navarrensis

Roseospiramediosalina

Cell size (mm) 0.4–0.8¥1.5–6.0 0.5–0.8¥2.5–6.5 0.6–0.9¥3.5–6.5 0.8–1.0¥2.2–6.0

Color of cell suspensions Red Red Brown-red Brown-red

Main carotenoids Rhodovibrine,rhodopine

Rhodovibrine,spirilloxanthin

Rhodopine,lycopene

Rhodopine,lycopene

Intracytoplasmic membrane system Vesicular Vesicular Vesicular Vesicular

DNA G+C content (mol%) 68.8–69.4 71.9–72.3 66.8 66.6

Growth factors Niacin, thiamine,p-aminobenzoate

Yeast extract Yeast extract Niacin, thiamine,p-aminobenzoate

Optimal NaCl concentration (range) 2–4% (0.5–10) 0.5% (0.2–5) 3–4% (1–10) 4–7% (0.5–15)

Assimilatory SO42– reduction + – – +

Dark aerobic growth(a) + m m m

Photolithoautotrophic growth(electron donor)

– + (H2S, S2O3) + (H2S) + (H2S)

Photoorganotrophic growth on: Fumarate, malate, succinate + – + + Citrate – – + – Propionate + – + + Benzoate – – + – Glucose – + – – Fructose + – – – Mannitol + – + –

into several genera on the basis of comparative 16S rDNAsequence analyses (Imhoff et al. 1998). The genera can bedifferentiated according to their salt requirements (Imhoff2001): Rhodospirillum, Phaeospirillum, and Rhodocistacomprise the freshwater to halotolerant bacteria. Roseo-spira, Rhodospira, and Roseospirillum contain the slightlyhalophilic bacteria. Rhodovibrio and Rhodothalassiumcomprise moderate halophiles isolated from hypersalineenvironments. Strains CE2105, SE3104, and AT2115 donot exhibit high NaCl requirements and must be consideredas slightly halophilic bacteria. The absence of close phy-logenetic relationships between these isolates and fresh-water or moderate halophilic species confirms that salt re-quirement is a strong taxonomic feature at the genus levelfor the spiral-shaped purple nonsulfur bacteria (Imhoff etal. 1998). The dendrogram constructed (Fig.3) from the16S rDNA comparisons revealed that the new isolates areclosely related to the slightly halophilic species Roseo-spira mediosalina, and to a lesser extent Rhodospiratrueperi. In contrast, they were more distantly related withRoseospirillum parvum.

Rhodospira trueperi is a spiral-shaped marine purplenonsulfur bacterium containing bacteriochlorophyll b andtetrahydrospirilloxanthin as main pigments (Pfennig et al.1997). This difference in pigment composition with ourthree isolates, which contain Bchl a and carotenoids of thenormal spirilloxanthin series, confirms, in addition to thephylogenetic relatedness, that the three new strains cannotbe included in the genus Rhodospira.

Phylogenetic analysis (Fig.3) suggested a common an-cestor for strains CE2105, SE3104, and AT2115 togetherwith Roseospira mediosalina. Our isolates, which havesimilar salt requirements (Table 3), are phylogeneticallyclosely related, with dissimilarity values ranging from2.4% to 3.5% (Fig.3). In contrast, Roseospira medio-salina exhibits a higher optimal salinity (Table 3) and is4.4–5.3% distant from the new isolates. Because of strongsimilarities between our three isolates and Roseospiramediosalina, we must consider them as representatives ofthe genus Roseospira. However, on the basis of phyloge-netic properties, these three isolates are distant enoughfrom the type species Roseospira mediosalina to be con-

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Fig.3 Dendrogram showingthe relationships between 16SrDNA sequences of Roseospiramarina strain CE2105,Roseospira navarrensis strainSE3104, Roseospira thio-sulfatophila strain AT2115,and other phototrophic purplenon sulfur bacteria (strainnumbers and EMBL accessionnumbers are indicated). The16S rDNA sequence ofAllochromatium vinosum(DSM 180T) was included inthe sequence analysis to rootthe tree (not shown). Bar indi-cates 5% difference in nu-cleotide sequence. The num-bers in the dendrogram indi-cate the significance (percentof outcomes) of the branches(bootstrap analysis, seemethods)

sidered as different species. In addition, the results of theDNA-DNA hybridization studies between our three strains,showing very low percentages of homology, are consis-tent with the description of the three isolates as three newspecies.

The three isolates also showed differences in theirmorphology, pigment composition, G+C content of theDNA, and physiological capacities (Tables 1 and 3). StrainCE2105 is not capable of photolithotrophic growth butcan grow chemoorganotrophically in the dark under oxicconditions. In contrast, strain SE3104 uses sulfide as elec-tron donor for photolithotrophic growth but is unable togrow chemoorganotrophically under full oxic conditions.Strain AT2115 is able to use sulfide and thiosulfate aselectron donors for photolithotrophic growth. In addition,strain AT2115 exhibits unusual Bchl a absorption in theinfra-red with a large band from 883 nm to 909 nm. Suchabsorption in the infra-red was found in Roseospirillumparvum (Glaeser and Overmann 1999), which is distantlyrelated with our isolate. Finally, differences in the assimi-lation of some organic substrates were found between thethree strains and Roseospira mediosalina (Table 3). There-fore, according to these phenotypic and genetic differences,we propose to describe strain CE2105, strain SE3104, andstrain AT2115 as members of three new species of thegenus Roseospira, with the names Roseospira marina sp.nov., Roseospira navarrensis sp. nov., and Roseospirathiosulfatophila sp. nov., respectively. An emended de-scription of the genus Roseospira is given below.

Roseospira gen. emend.

Full description as in Imhoff et al. (1998), with additionalfeatures

Cells are vibrioid to spiral-shaped, 0.4–1.0 µm wide,motile by means of polar or bipolar flagella. DNA G+Ccontent between 65 and 72.3 mol%. Slightly halophilicbacteria that require NaCl or sea salt for growth. Salt con-centration optima for growth between 0.5 and 7% (w/v)NaCl. Growth is possible under micro-oxic to oxic condi-tions in the dark.

Description of Roseospira marina sp. nov.

ma.ri’na L. fem. adj. marina marineCells are spiral to vibrioid-shaped, 0.4–0.8 µm wide,

1.5–6.0 µm long. Multiplication by binary fission. Gram-negative. Motile by bipolar tufts of flagella (2–5 fibrils).Intracytoplasmic membrane system is of vesicular type.Color of anaerobically grown cultures is red. Containsbacteriochlorophyll a and carotenoids of the normal spi-rilloxanthin series with rhodovibrin as major carotenoid.

Photoorganotrophic under anoxic conditions; substratesused are pyruvate, malate, succinate, fumarate, 2-oxoglu-tarate, formate, acetate, propionate, butyrate, valerate, cro-tonate, lactate, fructose, gluconate, mannitol, glycerol, as-partate, glutamate, cysteine. Peptone, yeast extract, and

casamino acids also used as substrates. Not capable ofphotolithotrophic growth. Chemotrophic growth under oxicconditions. Not capable of anaerobic chemotrophic growth.Niacin, thiamine, and p-aminobenzoic acid required asgrowth factors. Optimal pH for growth 6.7–6.8, range 5.3–8.4. Optimal NaCl concentration for growth 2–4 %, range0.5–10%. Optimal temperature for growth 30–35°C.

DNA base composition of the type strain is 68.8–69.4 mol% G+C (HPLC).

Habitat: anoxic sediments and water from coastal andmarine environments.

Type strain: Strain CE2105T (ATCC number BAA-447)isolated from the anoxic sediments of the brackish lagoonsof Certes, Arcachon Bay, France. 16S rDNA sequence de-posited in EMBL under accession number AJ298879.

Description of Roseospira navarrensis sp. nov.

na.var’ren.sis M.L. fem. adj. navarrensis pertaining toNavarra, a Spanish region

Cells are vibrioid-rods, often in pairs, 0.6–0.9 µm wide,3.5–6.5 µm long. Multiplication by binary fission. Gram-negative. Motile by bipolar tufts of flagella. Intracytoplas-mic membrane system is of vesicular type. Color of anaer-obically grown cultures is brown-red. Contains bacterio-chlorophyll a and carotenoids of the normal spirilloxan-thin series with rhodopine as major carotenoid.

Photoorganotrophic growth under anoxic conditions;substrates used are pyruvate, malate, succinate, fumarate,citrate, 2-oxoglutarate, acetate, propionate, butyrate, valer-ate, crotonate, benzoate, lactate, gluconate, mannitol, glyc-erol, aspartate, glutamate. Peptone, yeast extract, andcasamino acids also used as substrates. Photoautotrophicgrowth with sulfide as electron donor. Chemoorgano-trophic growth under micro-oxic conditions. Not capableof anaerobic chemotrophic growth. Not capable of assim-ilatory sulfate reduction. Yeast extract required as growthfactor. Optimal pH for growth 6.8–7.0, range 6.0–8.5. Op-timal NaCl concentration for growth 3–4%, range 1–10%.Optimal temperature for growth 30–35°C.

DNA base composition of the type strain is 66.8 mol%G+C (HPLC).

Habitat: anoxic sediments exposed to light in inlandsaline springs.

Type strain: Strain SE3104T (ATCC number BAA-448)isolated from the spring Salinas de oro, Navarra, Spain.16S rDNA sequence deposited in EMBL under accessionnumber AJ298880.

Description of Roseospira thiosulfatophila sp. nov.

thi.o.sul.fa.to’phi.la. M.L. n. thiosulfatum thiosulfate; Gr. adj. philos loving; M. L. fem. adj. thiosulfatophilathiosulfate-loving

Cells are spiral to vibrioid-shaped, 0.5–0.8 µm wide,2.5–6.5 µm long. Multiplication by binary fission. Gram-negative. Highly motile by polar tufts of flagella. Intracy-

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toplasmic membrane system is of vesicular type. Color ofanaerobically grown cultures is red. Contains bacterio-chlorophyll a with unusual absorption shoulder at 909 nm.Carotenoids are of the normal spirilloxanthin series.

Photoorganotrophic under anoxic conditions; substratesused are pyruvate, 2-oxoglutarate, acetate, butyrate, lac-tate, glucose, glycerol, glutamate. Peptone, yeast extract,and casamino acids also used as substrates. Photoau-totrophic growth with sulfide and thiosulfate as electrondonors. Chemotrophic growth under micro-oxic condi-tions. Not capable of anaerobic chemotrophic growth.Yeast extract required as growth factor. Optimal pH forgrowth 6.8–7.0, range 5.6–8.6. Optimal NaCl concentra-tion for growth 0.5%, range 0.2–5%. Optimal temperaturefor growth 30–35°C.

DNA base composition of the type strain is 71.9–72.3 mol% G+C (HPLC).

Habitat: Microbial mats in coastal and marine environ-ments.

Type strain: Strain AT2115T (ATCC number BAA-449)isolated from a microbial mat located in a large pond inTetiaroa Atoll, French Polynesia. 16S rDNA sequence de-posited in EMBL under accession number AJ401208.

Acknowledgements The authors are indebted to Dr. R. Le Menn(Electron Microscopy Laboratory, Bordeaux I University, France)for the electron microscopic studies and Prof. M. Urdaci, Prof. C.Payri, and Dr. L. Mao Che for providing samples from the Spanishsprings and the Polynesian Microbial mats. We also thank LineBourassou and Sonja Fleissner for their technical assistance. Thiswork was financed by a joint EU project on Coastal Lagoon Eu-trophication and Anaerobic processes (CLEAN), contract numberEV5V-CT92–0080, by an environmental project (KOPARA) ofthe French Polynesia Ministry for Environment Protection, and bythe Regional Council of Aquitaine.

References

Buffan-Dubau E, De Witt R, Castel J (1996) Feeding selectivity ofthe Harpacticoid copepod Canuella perplexa in benthic muddyenvironments demonstrated by HPLC analyses of chlorin andcarotenoid pigments. Mar Ecol Prog Ser 137:71–82

Burke M E, Gorham E, Pratt D C (1974) Distribution of purplephotosynthetic bacteria in wetland and woodland habitat ofcentral and northern Minnesota. J Bacteriol 117:826–833

Cashion P, Holder-Franklin MA, Mc Cully J, Franklin M (1977) Arapid method for base ratio determination of bacterial DNA.Anal Biochem 81:461–466

Caumette P, Matheron R, Welsh DT, Herbert REM, de Wit R(1999) Ecology and osmoadaptation of halophilic Chromati-aceae in hypersaline environments. In: Peschek GA, Löffel-hardt W, Schmetterer G (eds) The phototrophic prokaryotes.Kluwer Academic/Plenum, New-York, pp 707–713

De Ley J, Cattoir H, Reynaerts A (1970) The quantitative mea-surements of DNA hybridization from renaturation rates. Eur JBiochem 12:133–142

Drews (1981) Rhodospirillum salexigens, spec. nov., an obligatoryhalophilic phototrophic bacterium. Arch Microbiol 130:325–337

Escara JF, Hutton JR (1980) Thermal stability and renaturation ofDNA in dimethylsulfoxide solutions: acceleration of renatura-tion rate. Biopolymers 19:1315–1327

Felsenstein J (1993) PHYLIP (Phylogeny Inference Package), ver-sion 3.5c. University of Washington, Seattle USA

Fitch WM, Margoliash E (1967) Construction of phylogenetictrees: a method based on mutation distances as estimated fromcytochrome c sequences is of general applicability. Science155:279–284

Glaeser J, Overmann J (1999) Selective enrichment and character-ization of Roseospirillum parvum, gen. nov. and sp. nov., anew purple nonsulfur bacterium with unusual light absorptionproperties. Arch Microbiol 171:405–416

Glazer AN, Cohen-Bazire G, Stanier RY (1971) Characterizationof phycoerythrin from a Cryptomonas species. Arch Microbiol80:1–18

Goupy J (1988) La méthode des plans d’expérience. Dunod, ParisGuyoneaud R, Matheron R, Baulaigue R, Podeur K, Hirschler A,

Caumette P (1996) Anoxygenic Phototrophic Bacteria in Eu-trophic Coastal Lagoons of the French Mediterranean andAtlantic Coasts (Prévost Lagoon, Arcachon Bay, Certes Fish-ponds). Hydrobiologia 329:33–43

Guyoneaud R, Süling J, Petri R, Matheron R, Caumette P, ImhoffJ (1998) Taxonomic rearrangements of the genera Thiocapsaand Amoebobacter on the basis of 16S rDNA sequence analy-ses, and description of Thiolamprovum gen. nov. Int J SystBacteriol 48:957–964

Huss VAR, Festl H, Schleifer KH (1983) Studies on the spectro-metric determination of DNA hybridization from renaturationrates. J Syst Appl Microbiol 4:184–192

Imhoff JF (2001) True marine and halophilic anoxygenic photo-trophic bacteria. Arch Microbiol 176:243–254

Imhoff JF, Trüper HG (1989) Genus Rhodospirillum. In: StaleyJT, Bryant MP, Pfennig N, Holt JG (eds). Bergey’s manual of bacteriology, vol 3. Williams and Wilkins, Baltimore, pp 1658–1682

Imhoff JF, Trüper HG (1992) The genus Rhodospirillum and re-lated genera. In: Balows A, Trüper HG, Dworkin M, Harder W,Schleifer KH (eds) The prokaryotes. Springer, Berlin Heidle-berg New York, pp 2141–2155

Imhoff JF, Petri R, Süling J (1998) Reclassification of the spiral-shaped phototrophic purple non-sulfur bacteria of the α-Pro-teobacteria: description of the new genera Phaeospirillum gen.nov., Rhodovibrio gen. nov., Rhodothalassium gen. nov. andRoseospira gen. nov. as well as transfer of Rhodospirillum ful-vum to Phaeospirillum fulvum comb. nov., of Rhodospirillummolischianum to Phaeospirillum molischianum comb. nov., ofRhodospirillum salinarum to Rhodovibrio salinarum comb.nov., of Rhodospirillum sodomense to Rhodovibrio sodomensiscomb. nov., of Rhodospirillum salexigens to Rhodothalassiumsalexigens comb. nov. and of Rhodospirillum mediosalinum toRoseospira mediosalina comb. nov. Arch Microbiol 48:793–798

Jahnke KD (1992) Basic computer program for evaluation of spec-troscopic DNA renaturation data from GILFORD System 2600spectrometer on PC/XT/AT type personal computer. J Micro-biol Methods 15:61–73

Jukes TH, Cantor CR (1969) Evolution of protein molecules. In:Munro HN (ed) Mammalian protein metabolism, vol 3. Acade-mic, New York, pp 21–132

Kaiser P (1966) Ecologie des bactéries photosynthétiques. RevEcol Biol Sol 3:409–472

Kämpf C, Pfennig N (1980) Capacity of Chromatiaceae forchemotrophic growth. Specific respiration rates of Thiocystisviolacea and Chromatium vinosum. Arch Microbiol 127:125–135

Kompantseva EI, Gorlenko VM (1985) A new species of moder-ately halophilic purple bacterium Rhodospirillum mediosal-inum sp. nov. Microbiology (Mikrobiologiya) 53:775–781

Liesack W, Finster K (1994) Phylogenetic analysis of five strainsof gram-negative, obligately anaerobic, sulfur-reducing bacte-ria and description of Desulfuromusa gen. nov., includingDesulfuromusa kysingii sp. nov., Desulfuromusa bakii sp. nov.,and Desulfuromusa succinoxidans sp. nov. Int J Syst Bacteriol44:753–758

323

Mack EE, Mandelco L, Woese CR, Madigan MT (1993) Rho-dospirillum sodomense, sp. nov. a dead sea Rhodospirillumspecies. Arch Microbiol 160:363–371

Mao Che L, Andrefouet S, Bothorel V, Guezennec M, RougeauxH, Guezennec J, Deslandes E, Trichet J, Matheron R, Le Cam-pion T, Payri C, Caumette P (2001) Physical, chemical and mi-crobiological characteristics of microbial mats (KOPARA) inthe south pacific atolls of French Polynesia. Can J Microbiol47:994–1012

Mesbah M, Premachandran U, Whitman W (1989) Precise mea-surement of the G+C content of desoxyribonucleic acid by highperfermance liquid chromatography. Int J Syst Bacteriol 39:159–167

Mouné S, Eatock C, Matheron R, Willison JC, Hirschler A, Her-bert R, Caumette P (2000) Orenia salinaria sp. nov., a fermen-tative bacterium isolated from anaerobic sediments of Mediter-ranean salterns. Int J Syst Evol Microbiol 50:721–729

Nissen H, Dundas ID (1984). Rhodospirillum salinarum sp. nov., ahalophilic photosynthetic bacterium isolated from a Portuguesesaltern. Arch Microbiol 138:251–256

Okuda A, Yamaguchi M, Kamata S (1957) Nitrogen-fixing mi-croorganisms in paddy soils. Part 3: distribution of non-sulfurpurple bacteria in paddy soils. Soil Plant Food 2:131–133

Overmann J, Fischer U, Pfennig N (1992) A new purple sulfurbacterium from saline littoral sediments, Thiorhodovibriowinogradskyi gen. nov. and sp. nov. Arch Microbiol 157:329–335

Pfennig N (1978) Rhodocyclus purpureus gen. nov. and sp. nov., a ring-shaped, vitamin B12-requiring ember of the family Rho-dospirillaceae. Int J Syst Bacteriol 28:283–288

Pfennig N, Wagener S (1986) An improved method of preparingwet mounts for the photomicrography of microorganisms. J Microbiol Methods 4:303–306

Pfennig N, Trüper HG (1992) The family Chromatiaceae. In:Balows A, Trüper HG, Dworkin M, Harder W, Schleifer KH(eds) The prokaryotes. Springer, Berlin Heidelberg New-York,pp 3200–3221

Pfennig N, Lünsdorf H, Süling J, Imhoff JF (1997) Rhodospiratrueperi gen. nov., spec.nov., a new phototrophic proteobac-terium of the alpha group. Arch Microbiol 168:39–45

Ryter A, Kellenberger E (1958) Etude au microscope électroniquede plasmas contenant de l’acide désoxyribonucléique. I. Lesnucléotides des bactéries en croissance active. Z Naturforsch13b:597–605

Siefert E, Irgens R L, Pfennig N (1978) Phototrophic purple andgreen bacteria in a sewage treatment plant. Appl Environ Mi-crobiol 35:38–44

Stackebrandt E, Goebel BM (1994) Taxonomic note: a place forDNA-DNA reassociation and 16S rRNA sequence analysis inthe present species definition in bacteriology. Int J Syst Bacte-riol 44:846–849

Tamaoka J, Komagata K (1984) Determination of DNA base com-position by reversed-phase high performance liquid chromatog-raphy. FEMS Microbiol Lett 25:125–128

Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: im-proving the sensitivity of progressive multiple sequence align-ment though sequence weighting, position-specific gap penal-ties and weight matrix choice. Nucleic Acids Res 22:4673–4680

Welsh DT, Herbert RA (1994) Identification of organic solutes ac-cumulated in purple and green sulfur bacteria during osmoticstress using natural abundance 13C-nuclear magnetic spectros-copy. FEMS Microbiol Ecol 13:745–750

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