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1 GBF – German Research Centre for Biotechnology, Department of Environmental Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany
2 UMR 6543 CNRS and Université de Nice Sophia Antipolis, Centre de Biochimie, Parc Valrose, F-06108 Nice cedex 2, France
Correspondence
Ingrid Brettar
inb{at}gbf.de
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ABSTRACT |
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7c (24–33 %), 17 : 18c (14–18 %) and 18 : 17c (9–12 %). Based on the data presented, BA131T is proposed as the type strain of a novel species of the genus Rheinheimera, Rheinheimera perlucida sp. nov. The type strain is BA131T (=LMG 23581T=CIP 109200T).
Tables detailing positive results using the API and Biolog test systems and for substrate hydrolysis of strain BA131T and micrographs of cells of strain BA131T are available as supplementary material in IJSEM Online.
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). Members of the Gammaproteobacteria are considered to represent a large fraction of the marine surface water bacteria that are able to grow and to degrade rapidly the more easily degradable organic fraction of marine organic matter (Bianchi & Bianchi, 1995; Pinhassi & Berman, 2003; Poretsky et al., 2005). Recent studies have shown such a role for the genus Rheinheimera, members of which are found abundantly in marine and estuarine environments (Alavi et al., 2001; Giovannoni & Stingl, 2005). The new isolate can be included within a recently described branch of the Gammaproteobacteria, which so far comprises the genera Alishewanella (Fonnesbech Vogel et al., 2000), Rheinheimera (Brettar et al., 2002; Romanenko et al., 2003) and ‘Alkalimonas’ (Ma et al., 2004). Most of the isolates and environmental 16S rRNA gene sequences of members of this branch are of marine or aquatic origin, with the exception of Alishewanella fetalis and a few environmental sequences in the close phylogenetic neighbourhood of this species.
Strain BA131T was isolated during a cruise onboard RV Aranda in September 1998 from surface water (5 m depth, 15 °C, 7 
S, pH 8.4) from a site in the central Baltic Sea, station TEILI1 (59° 26' 07'' N 21° 30' 02'' E). All details on environmental conditions, sampling and isolation procedures have been given previously (Brettar et al., 2002; Brettar & Rheinheimer, 1992; Brettar & Höfle, 1993; Höfle & Brettar, 1995). The medium for isolation was ZoBell agar (Oppenheimer & ZoBell, 1952). The strain grew well on half-strength ZoBell agar and marine broth or agar (Difco 2216).
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 and morphology (via phase-contrast microscopy, and electron microscopy after Pt/C shadow casting) and cytochrome oxidase and catalase (3 % H2O2). Furthermore, production of hydrogen sulfide (Dye, 1968), aminopeptidase (Merck Bactident test), haemolysis and hydrolysis of starch, gelatin, Tween 80 and lecithin were tested. The strain was additionally characterized by the whole test spectrum of the API 50CH, API 20NE, API ZYM (bioMérieux) and Biolog GN2 identification systems at 28 °C. Growth at different temperatures was assessed at 4, 10, 20, 25, 30, 33, 37 and 40 °C. Growth at different salinities was tested at 0, 0.8, 1.0, 1.5, 3, 6, 8 and 10 % NaCl. Growth at different pH was tested at pH 5.7, 7, 9 and 10 (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.
Genomic DNA was prepared from individual colonies as described by Moore et al. (1996). 16S rRNA genes were amplified by PCR (Mullis & Faloona, 1987) and 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 on servers of the NCBI (nr database), EBI (EMBL database) and Infobiogen (NucAll database) with filter option set to false; these queries identified 98 most similar sequences having a 16S rRNA gene sequence that was clearly described in the Feature lines. These sequences were included and aligned within a local database of 122 000 previously aligned and analysed bacterial 16S rRNA gene sequences. In a first analysis, these 98 sequences were carefully aligned (manual adjustments) and analysed by phylogeny (bioNJ, as detailed below). This allowed us to select a set of 28 sequences belonging to a clade including that of strain BA131T. These 28 sequences were further analysed 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 were from PHYLIP (Felsenstein, 1993). The phylogenetic trees were drawn using NJPLOT (Perrière & Gouy, 1996). Bootstrap analyses were performed using bioNJ based on 1000 replications. From the 28 sequences, eight obtained from cultured strains (including the six sequences of the type strains of this branch of the Gammaproteobacteria) plus the environmental clone most closely related to the sequence of BA131T were chosen for a final analysis; all three methods and a bootstrap analysis were performed as described above, using nucleotide positions 159–1445 of the BA131T sequence (no problems of alignment, and no missing parts in any sequence). The result of this final phylogenetic analysis is shown in Fig. 1.
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and Mesbah et al. (1989).
For analysis of the cellular fatty acid profile, strain BA131T was grown on half-strength marine agar (Difco 2216) for 24 h at 28 °C. For comparison with Alishewanella fetalis, strain BA131T and Rheinheimera baltica OSBAC1T were grown additionally at 30 °C for 2 days on blood agar. Fatty acid methyl esters were obtained from washed cells by saponification, methylation and extraction. Analysis by gas chromatography was controlled by MIS software (Microbial ID) and peaks were automatically integrated and identified by the Microbial Identification software package (Sasser, 1990).
Cells of strain BA131T were Gram-negative rods (see Supplementary Fig. S1 available in IJSEM Online) and formed non-pigmented transparent colonies. All details on morphological, physiological and biochemical traits are summarized in the species description below and in Supplementary Table S1. In general, strain BA131T showed rather limited substrate use in the API 50CH, API 20NE and Biolog GN2 test systems. By contrast, the APIZYM test revealed a broader set of nine positive enzyme activities. 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).
In terms of phenotypic features, strain BA131T could be differentiated from R. baltica based on colony pigmentation, reduction of nitrate, NaCl tolerance, acid production from various carbohydrates (D-glucose, cellobiose, maltose and gentiobiose), assimilation of glucose and maltose, four positive enzyme activities and utilization of seven substrates (Table 1). It could be differentiated from Rheinheimera pacifica by the number and insertion site of the flagella, reduction of nitrate, activity of acid phosphatase, assimilation of arabinose, citrate and maltose and utilization of three amino acids, plus acetic acid and glycerol. Strain BA131T could be differentiated from all members of the genus Rheinheimera by the reduction of nitrate, acid phosphatase and the assimilation of maltose. It could be differentiated from Alishewanella fetalis by the presence of a flagellum, the requirement for NaCl for growth, the optimum temperature and temperature range for growth, reduction of nitrite, thiosulfate and trimethylamine N-oxide (TMAO), hydrolysis of starch and assimilation of N-acetylglucosamine. Compared with members of the genus ‘Alkalimonas’, strain BA131T had a different temperature range and optimum for growth, a different pH range and optimum for growth and differed in the use of 12 substrates (Table 1). In general, strain BA131T had a rather restricted spectrum of organic substrate use.
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). However, within this clade, the position of strain BA131T was unstable, being clustered with Rheinheimera or Alishewanella, depending upon the outgroups chosen and the method used to construct the phylogenetic tree. This might indicate that BA131T represents a new genus. With Serratia entomophila DSM 12358T as the outgroup, strain BA131T clustered more closely with the genus Rheinheimera (Fig. 1). Strain BA131T showed highest 16S rRNA gene sequence similarity (98 %) to a bacterium recovered from methane-hydrate-bearing deep marine sediments (uncultured bacterial clone MB-A2-102; Reed et al., 2002). Strain BA131T showed sequence similarities ranging between 96.6 and 95.5 % with strains of R. baltica and of 96.4 % with R. pacifica KMM 1406T.
The DNA G+C content of strain BA131T was 48.9 mol% (Table 1). Values for the related species R. baltica, R. pacifica and Alishewanella fetalis ranged from 48 to 51 mol%.
Table 2 details the fatty acid composition of strain BA131T together with the phylogenetically related species R. baltica, R. pacifica, Alishewanella fetalis, ‘Alkalimonas amylolytica’ and ‘Alkalimonas delamerensis’. To enable a comparison with Alishewanella fetalis, fatty acids are given for R. baltica OSBAC1T and strain BA131T after growth on blood agar (30 °C, 2 days) in addition to cultivation on marine agar (half-strength, 28 °C). Major fatty acid components (>10 %) of strain BA131T were 16 : 0, 16 : 17c, 17 : 18c and 18 : 17c. Unsaturated fatty acids formed the major fraction of the total, representing 63.7 % when grown on marine agar and 61.4 % on blood agar. Compared with phylogenetically related species, the fatty acid composition of BA131T was most similar to that of R. baltica (e.g. for the major components 16 : 0 and 16 : 17c). A major distinction was the lower abundance of 17 : 18c for R. baltica. The overall fatty acid profile of strain BA131T for the two growth conditions (marine agar, blood agar) was similar; the major change was the disappearance of 15 : 0 when grown on blood agar, which was also the case for R. baltica OSBAC1T. Based on the fatty acid profile following growth on blood agar, Alishewanella fetalis could be differentiated from BA131T and R. baltica by the presence of 15 : 0 and lower proportions of 16 : 0.
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) but with some instability associated with its attribution to either of these genera. The fatty acid profiles for strain BA131T and the genera Rheinheimera and Alishewanella are similar. However, distinction of BA131T and R. baltica from Alishewanella fetalis is possible based on the two fatty acids 15 : 0 and 16 : 0. Features such as the occurrence of a flagellum, the range and optimum temperature and salinity for growth, the spectrum of utilizable electron acceptors and organic substrates differentiate strain BA131T from Alishewanella fetalis, and these features are not consistent with the description of the genus Alishewanella as given by Fonnesbech Vogel et al. (2000). Strain BA131T could be differentiated phenotypically from members of the genus Rheinheimera based on only three traits, reduction of nitrate and acid phosphatase and the assimilation of maltose, and fits well with the description of the genus. By inclusion of strain BA131T in the genus Rheinheimera, the genus Alishewanella can be distinguished by the presence of flagella, the salinity range for growth, the requirement for NaCl, the temperature range for growth (growth at 4, 10 and 41 °C), the use of thiosulfate and nitrite as electron acceptors, the assimilation of N-acetylglucosamine and the hydrolysis of starch. Based on the polyphasic data presented here, we propose to assign BA131T to the genus Rheinheimera as the type strain of a novel species, Rheinheimera perlucida sp. nov.
Description of Rheinheimera perlucida sp. nov.
Rheinheimera perlucida (per.lu'ci.da. L. fem. adj. perlucida transparent, referring to the transparent and colourless colonies, to distinguish R. perlucida from the type species R. baltica, which forms blue-coloured colonies).
Colonies are circular, smooth, convex, non-pigmented and entire. They are transparent, becoming slightly opaque with ongoing incubation (>2 weeks, 25 °C, on half-strength marine agar). Cells are Gram-negative rods (width 0.6–1.2 µm, length 0.9–2.4 µm) and oxidase- and catalase-positive. Able to reduce nitrate to nitrite. Temperature for growth ranges from 4 to 37 °C, with an optimum around 25 °C. NaCl is not required for growth; growth occurs from 0 to 8 % NaCl, with an optimum at 1–3 %. The strain grows from pH 5.7 to 10, with an optimum around pH 7. Hydrolyses gelatin, lecithin, starch and Tween 80. Does not produce sulfide and does not haemolyse bovine blood. In the API 50CH test system, acid is produced from N-acetylglucosamine, starch and glycogen; all other API 50CH test results are negative. In the API 20NE test system, it shows assimilation of N-acetylglucosamine and positive enzymic activities for 
-glucosidase and protease. Positive for alkaline and acid phosphatase, esterase (C4, C8), leucine arylamidase, trypsin, chymotrypsin, naphthol phosphohydrolase and N-acetyl--glucosaminase in the API ZYM test system. All other tests with the API ZYM are negative (for details see Supplementary Table S1). In the Biolog GN2 test system, it utilizes L-alanine, -hydroxybutyric acid, 
-cyclodextrin, L-alanyl glycine and L-threonine as substrates (all other Biolog GN2 substrates are negative; for details see Supplementary Table S1). In general, it shows a very limited spectrum of organic substrate use. The DNA G+C content of the type strain is 48.9 mol%.
The type strain, BA131T (=LMG 23581T=CIP 109200T), was isolated from surface water of the central Baltic Sea.
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ACKNOWLEDGEMENTS |
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