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1 HZI-Helmholtz Center for Infection Research, Department of Vaccinology, Inhoffenstrasse 7, D-38124 Braunschweig, Germany
2 CNRS & Université de Nice Sophia Antipolis, UMR 6543, Centre de Biochimie, Parc Valrose, F06108 Nice cedex 2, France
Correspondence
Ingrid Brettar
inb{at}gbf.de
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ABSTRACT |
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7c (73 %) as the predominant fatty acid; other major fatty acids were 19 : 0 cyclo 8c (10 %) and 16 : 0 (8 %). The pigment spectrum indicated the presence of carotenoids and unknown pigment(s) with an absorption maximum at 430 nm, but not bacteriochlorophyll a. According to phylogenetic analysis based on the 16S rRNA gene sequence and the phenotypic features, strain BA141T represents a new genus and species. The name Anderseniella baltica gen. nov., sp. nov. is proposed, with strain BA141T (=CIP 109499T=LMG 24028T) as the type strain.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences of strain BA141T are AM712634 and AM712635, respectively.
A table detailing results obtained using API and Biolog GN2 and tests for hydrolysis of and growth on various substrates for strain BA141T is available with the online version of this paper.
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). Members of the Alphaproteobacteria are considered to represent a major fraction of marine bacteria (Giovannoni & Rappé, 2000). Most of this knowledge is based on environmental sequence data rather than on cultured bacteria, because members of the Alphaproteobacteria tend to be more difficult to culture than members of the Gammaproteobacteria. Giovannoni & Rappé (2000) concluded that this was due to their high degree of adaptation to specific environmental conditions, which renders them less amenable to cultivation on the one hand, but very successful in marine ecosystems on the other.
The novel isolate studied here was most closely related to Rhodobium orientis. Garrity et al. (2004) proposed the family ‘Rhodobiaceae’ comprising the genera Rhodobium and Roseospirillum. Phylogenetic studies by Lee et al. (2005) showed that there were major inconsistencies in the formation of this family and suggested that additional taxonomic studies were needed before the classification of the genus Rhodobium could be confirmed.
Strain BA141T was isolated during a cruise onboard RV Aranda in September 1998 from the black anoxic surface of sediment (sediment top layer 0–1 cm depth, 5 °C, 11 
salinity) collected at a sampling site in the central Baltic Sea, station TEILI1 (5 ° 26' 07'' N 2 ° 30' 02'' E; at a depth of 165 m). The sediment sample was subsampled under sterile conditions from a large sample volume. Details of the environmental conditions, sampling and isolation procedures are given elsewhere (Brettar et al., 2002; Brettar & Rheinheimer, 1992; Höfle & Brettar, 1995). The sediment sample was serially diluted with sterile seawater, spread on agar plates and incubated in the dark for 3 weeks. The medium used for isolation was half-strength ZoBell agar (Oppenheimer & ZoBell, 1952). The strain grew well on half-strength ZoBell agar, in half-strength marine 2216 broth (Difco) and on marine agar (Difco).
The isolate was tested for a number of key characteristics using standard procedures (Gerhardt et al., 1994), such as Gram behaviour (KOH string test), cell size, motility and morphology (via phase-contrast microscopy, and electron microscopy after Pt/C shadow casting or thin sectioning) and cytochrome oxidase and catalase (3 % H2O2). Growth on 1 % yeast extract, 1 % peptone, 0.2 % 
-hydroxybutyrate and MacConkey agar was tested. In addition, the production of indole, nitrate reduction and hydrolysis of aesculin, casein, tyrosine, starch, gelatin and DNA were tested. Chitinase, cellulase and pectinase activities were tested as described by Atlas (1993).
For electron microscopy, samples from a liquid culture of strain BA141T were adsorbed onto carbon–Formvar foil, washed with water, blotted and air-dried. They were shadow-casted at 15 deg elevation with platinum–carbon and analysed using an energy-filter transmission electron microscope (CEM902; Zeiss), as described by Golyshina et al. (2000). Embedding and thin-sectioning were performed as described by Yakimov et al. (1998).
In addition, strain BA141T was characterized by using the whole test spectra of the identification systems API 50 CH, API 20NE, API ZYM (bioMérieux) and Biolog GN2 at 28 °C. Growth at different temperatures was assessed at 4, 10, 20, 25, 30, 37 and 42 °C. Growth at different salinities was tested at 0, 0.8, 1.0, 1.5, 3, 6 and 10 %, with salt being added as NaCl or Biomaris sea salt. Growth at different pH was tested at pH 5.7, 7 and 9.7 (pH adjusted using bicarbonate buffer or HCl, with growth assessed based on the occurrence of visible colonies on agar). For these tests we used half-strength marine broth or agar (Difco 2216), except for the salinity test for which half-strength salt-free ZoBell medium was supplemented with the respective amount of NaCl or Biomaris sea salt.
Pigment analysis was performed according to Hiraishi et al. (1995, 2000), i.e. the same procedure as used for analysis of the genus Rhodobium (Hiraishi et al., 1995). Briefly, after growth in half-strength marine broth, cells were washed three times in PBS and either (i) resuspended in 50 mM phosphate buffer (pH 7.0), sonicated for 3 min and centrifuged to remove cell debris or (ii) extracted in ice-cold acetone/methanol solution (7 : 2, v/v) in the dark for 12 h. Absorption spectra of the resultant cell extracts were recorded spectrophotometrically from 300–900 nm.
Genomic DNA was prepared from individual colonies as described by Moore et al. (1996). 16S rRNA genes were amplified by PCR according to Mullis & Faloona (1987). The gyrase B (gyrB) gene sequence was amplified by PCR as described by Yamamoto & Harayama (1995). The PCR-products were sequenced directly as described by Moore et al. (1999).
For phylogenetic analysis based on the 16S rRNA gene sequence, the most similar sequences were identified by running BLAST queries at NCBI (nr database), with the filter option set to false. These sequences were included and aligned within a local database of 289 000 previously aligned and analysed bacterial 16S rRNA gene sequences. In a first analysis, new sequences were carefully aligned (manual adjustments) and analysed by phylogeny (BIONJ, as detailed below). This was done to demonstrate that the novel strain was not related to any previously described genus. Among the 117 related sequences, 22 sequences of known type strains were selected and analysed further using three phylogenetic methods (BIONJ, maximum-likelihood and maximum-parsimony). For the neighbour-joining analysis, distance matrices were calculated using the Kimura two-parameter correction, BIONJ was used according to Gascuel (1997), and maximum-likelihood and maximum-parsimony (using the Global option) were from PHYLIP (Felsenstein, 1993). Bootstrap analyses were performed using BIONJ based on 1000 replications. The phylogenetic trees were drawn using TreeDyn (Chevenet et al., 2006). The results of the final phylogenetic analysis are shown in Fig. 1. Phylogenetic analysis based on gyrB gene sequences was not successful due to a lack of available gyrB gene sequences in the phylogenetic neighbourhood of strain BA141T. Therefore, only the GenBank/EMBL/DDBJ number for the gyrB gene sequence of strain BA141T is provided.
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and Mesbah et al. (1989).
For analysis of the cellular fatty acid profile, strain BA141T was grown on half-strength marine agar 2216 for 24 h at 28 °C. Fatty acid methyl esters were obtained from washed cells by saponification, methylation and extraction. Gas chromatography analysis was controlled by MIS software (Microbial ID) and the peaks were automatically integrated and identified using the microbial identification software package (Sasser, 1990).
Cells of strain BA141T were Gram-negative, non-motile, pleomorphic rods, 1–5 µm in length and 0.5–1 µm in width (Fig. 2a–c). On marine agar, strain BA141T formed small dark-red-pigmented opaque colonies. When grown in half-strength marine broth, the culture had a light to intense orange–red colour. Cells harvested from liquid culture were bright-red in colour. Strain BA141T was slow-growing, with irregular lag phases after inoculation. To obtain visible growth on agar or in liquid culture, an incubation time of about 1 week at room temperature was required, with 2–4 weeks incubation for a densely grown culture.
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and c–e). In liquid culture, cells formed star-like cell aggregates. Ultrastructural analysis using electron microscopy revealed the presence of polyhydroxyalkanoate (PHA) deposits. Enlargement of the cells was often observed in the vicinity of the PHA deposits. Neither budding cells, flagella nor intracytoplasmic membranes were observed, as was the case for Rhodobium orientis (Hiraishi et al., 1995). All details on the physiological and biochemical traits are summarized in the species description and in Supplementary Table S1 (available in IJSEM Online). In general, strain BA141T showed no substrate use in the API 50 CH and API 20NE test systems and a rather limited and mostly weak substrate use was recorded using the API ZYM test system. Substrates used in the Biolog GN2 test systems were mostly organic acids plus L-arabinose, alaninamide and glycerol. As a general rule, phenotypic features were rated as positive when a weak or more pronounced signal was obtained (for details see Supplementary Table S1).
The pigment spectrum of the sonicated cell extract showed a major peak at 430 nm, with shoulder peaks around 470, 500 and 530 nm (Fig. 3). In addition, a minor peak occurred at 365 nm. No peaks above 600 nm were observed, indicating that strain BA141T did not contain bacteriochlorophyll a. The large peak at 430 nm was not identified; the shoulder peaks on the large peak between 470 and 530 nm were considered to indicate the presence of carotenoids. The pigment spectrum was very different from that of Rhodobium orientis, which showed pronounced peaks for bacteriochlorophyll a at 800 and 870 nm, but no peak at 430 nm (Hiraishi et al., 1995). In the acetone/methanol extract of strain BA141T a broad peak from 450 to 550 nm was observed, supporting the presence of carotenoids.
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). Differences were observed in the presence of flagella, formation of budding cells, presence of bacteriochlorophyll a and intracytoplasmic membranes, and loss of pigmentation in oxic cultures. In terms of physiological features, they differed regarding denitrification, and the use of sugars and alcohols (Hiraishi et al., 1995). The two species were able to use a broad spectrum of organic acids.
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). In addition, the bootstrap support of the neighbour-joining analysis was low (52 %; Fig. 1). This is a strong indication that strain BA141T represents a new genus. Rhodobium orientis (GenBank no. D30792) had a 16S rRNA gene sequence similarity of 92.5 % with strain BA141T. Also, with most species of the genus Mesorhizobium, a 16S rRNA gene sequence similarity of 92 % or below was observed. In comparison to 16S rRNA gene sequences derived from cultured bacteria, 16S rRNA gene sequence similarities of above 93 % were not observed.
The DNA G+C content of strain BA141T was 61.2 mol% (Table 1). Values for strains of the related species Rhodobium orientis ranged from 61.5 to 65.7 % and are therefore within the same range as that for strain BA141T.
The fatty acid profile was dominated by unsaturated fatty acids (>86 %) and showed a low number of detected fatty acids. The fatty acid profile was composed of 14 : 0 (0.5 %), 16 : 111c (1.0 %), 16 : 0 (8.2 %), 16 : 0 2-OH (1.0 %), 18 : 19c (1.8 %), 18 : 17c (73.3 %), 19 : 0 cyclo 8c (9.9 %) and an unknown component (ECL 11.799, 4.3 %). Thus the fatty acid spectrum was strongly dominated by 18 : 17c. The fatty acid profile of strain BA141T was similar to those of members of the genus Mesorhizobium, with the closest similarity with Mesorhizobium plurifarium (Tighe et al., 2000).
Based on 16S rRNA gene sequence analysis, strain BA141T belongs to the Alphaproteobacteria, but is clearly identified as representing a new genus. Also, from a phenotypic perspective, the most closely related species and the only described representative of the genus Rhodobium at the time of writing, Rhodobium orientis, showed many features that were different from those of strain BA141T, i.e. presence of flagella, formation of budding cells, presence of bacteriochlorophyll a and intracytoplasmic membranes, loss of pigmentation in oxic cultures, and many physiological features, such as denitrification, and broader use of sugars and alcohols (Hiraishi et al., 1995).
Based on the polyphasic approach used, we propose a new genus and species, Anderseniella baltica gen. nov., sp. nov., to accommodate the novel Baltic Sea isolate, with strain BA141T as the type strain.
Description of Anderseniella gen. nov.
Anderseniella (An.der.sen.i.el'la. N.L. dim. n. Anderseniella named in honour of the late marine scientist Valérie Andersen, Observatoire Océanologique de Villefranche sur Mer, France, in recognition of her valuable work on marine ecosytems).
Cells are Gram-negative, rod-shaped to pleomorphic, and oxidase- and catalase-positive. Non-flagellated and red-pigmented. Growth is heterotrophic and aerobic. Predominant fatty acid is 18 : 17c. Other major fatty acids are 16 : 0 and 19 : 0 cyclo 8c. Cells contain carotenoids but not bacteriochlorophyll a. Cells contain PHA. Member of the Alphaproteobacteria. The type species is Anderseniella baltica.
Description of Anderseniella baltica sp. nov.
Anderseniella baltica (bal'ti.ca. N.L. fem. adj. baltica from the Baltic Sea, referring to the source of the type strain).
Exhibits the following properties in addition to those given in the species description. Colonies are small, opaque, circular, smooth, convex and entire. Forms dark-red colonies and light to intense orange–red liquid cultures (>1 week, 25 °C, half-strength marine broth). Cells are partially branching (width 0.5–1.0 µm, length 1.0–5.0 µm). Cells form star-like aggregates in liquid culture. Grows at oxic to low oxic conditions. Does not reduce nitrate to nitrite. Temperature for growth ranges from 10 to 37 °C, with optimum growth at 25–30 °C. Salinity range for growth is from 0.8 to 6 % of final concentration of artificial sea salt; addition of NaCl alone is not sufficient for growth. Grows at pH 7–7.5; no growth occurs at pH 5.7 or 9.7. Grows on 1 % yeast extract and 1 % peptone, but not on aesculin, casein or -hydroxybutyrate. Does not hydrolyse starch, gelatin, tyrosine, DNA, cellulose, chitin or pectin. Does not produce indole. Does not grow on MacConkey agar. Acid production is not observed using the API 50 CH test system. All API 20NE tests are negative. In the API ZYM test system, alkaline phosphatase, esterases (C4 and C8) and leucine arylamidase are positive; all other tests are negative. In the Biolog GN2 system, positive results are obtained for L-arabinose, glycerol, alaninamide and the organic acids succinic acid monomethyl ester, acetic acid, formic acid, 
-hydroxybutyric acid, -hydroxybutyric acid, -ketobutyric acid, DL-lactic acid, succinamic acid and urocanic acid; all other tests are negative. In general, the use of organic acids is better than for other substrates. All unmentioned tests of the API and Biolog GN2 test systems are negative. A detailed overview of all positive and negative test results for the biochemical tests is given in Supplementary Table S1 (in IJSEM Online). The DNA G+C content of the type strain is 61.2 mol%.
The type strain, BA141T (=CIP 109499T=LMG 24028T), was isolated from the surface layer of anoxic sediment in the central Baltic Sea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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