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Int J Syst Evol Microbiol 54 (2004), 681-684; DOI  10.1099/ijs.0.02896-0
© 2004 International Union of Microbiological Societies

Phylogeny of the ring-forming bacterium Arcicella aquatica gen. nov., sp. nov. (ex Nikitin et al. 1994), from a freshwater neuston biofilm

Denis I. Nikitin1, Carsten Strömpl2, M. S. Oranskaya1 and Wolf-Rainer Abraham2

1 Institute of Microbiology, Russian Academy of Sciences, Prospect 60-Letiya Octyabrya 7, korp. 2, Moscow 117811, Russia
2 GBF – National Research Center for Biotechnology, Dept Environmental Microbiology, Mascheroder Weg 1, 38124 Braunschweig, Germany

Correspondence
Wolf-Rainer Abraham
wab{at}gbf.de


   ABSTRACT
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Arcicella aquatica NO-502T, obtained from a neuston film on a freshwater lake and belonging to the phylum Bacteroidetes, is characterized by ring-forming cells. The bacterium is a strict aerobe, with optimal growth between 28 and 30 °C. Carbohydrates, but no organic acids or amino acids, are used as substrates. The G+C content of strain NO-502T is 34·5 mol%; its genome size is 2·9x109 Da. The genus Arcicella and its type species Arcicella aquatica (type strain NO-502T=LMG 21963T=CIP 107990T) are proposed, and descriptions of this genus and species are given.

Abbreviations: ESI, electrospray ionization; MS, mass spectrometry

Published online ahead of print on 9 January 2004 as DOI 10.1099/ijs.0.02896-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Arcicella aquatica NO-502T is AJ535729.


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From a freshwater neuston film a slow-growing bacterial isolate, NO-502T, was obtained which formed rings consisting of several bacterial cells. Such a morphology is also known from some other bacterial genera, including Flectobacillus. In 1994, Nikitin and colleagues described this isolate as ‘Arcocella aquatica’ (Nikitin et al., 1994Go); however, no type species was deposited in a public culture collection and the description remained invalid. The aim of this note is to describe the genus and species validly and to give further information on the phylogenetic position of the type strain, NO-502T.

Strain NO-502T was isolated on a medium containing 0·1 % peptone, yeast extract and glucose each at pH 7·0–7·2 from a neuston film of a freshwater lake near Moscow, Russia. Cells are polymorphic, showing vibrioid, curved and spiral-shaped cells.

The sequence of the 16S rRNA gene of strain NO-502T was determined as described previously (Abraham et al., 1999Go). It was found that an identical sequence was reported for isolate AH57 (accession no. AJ289964) from Lake Fuchskuhle, Germany (Glöckner et al., 2000Go). The latter isolate together with the sequence of uncultured bacterium GKS2-216 (accession no. AJ290033) from the same study and the clone sequence GWF23A (accession no. AJ011696) from a cave water ecosystem could be considered to fall within the range of a common genus and to represent one or more additional species. These three undescribed organisms came, like strain NO-502T, from freshwater ecosystems. Next relatives of this genus are species of the genus Flectobacillus, as shown in Fig. 1Go. The 16S rDNA sequence similarity between strain NO-502T and the most similar published sequence of an organism with a validly published name, Flectobacillus major, is 93·52 %. Bootstrap analyses of neighbour-joining trees supported the phylogenetic association of Arcicella aquatica with Flectobacillus major (data not shown). The wide distribution of Flexibacter species throughout the phylum Bacteroidetes (Fig. 1Go) was discussed by Nakagawa et al. (2002)Go. Nikitin et al. (1994)Go, however, listed further genomic data which, together with our data, require the separation of strain NO-502T from the genus Flectobacillus. The G+C content of strain NO-502T was found to be 34·5 %, while a G+C content of 39·5–40·3 mol% has been reported for Flectobacillus major (Gosink et al., 1998Go).



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  		Fig. 1. Unrooted dendrogram based on the 16S rDNA sequence of Arcicella aquatica NO-502T within the context of the phylum Bacteroidetes. Sequence data of reference strains of other genera published in the GenBank/EMBL (Stoesser et al., 2003Go) and/or RDP (Cole et al., 2003Go) databases were obtained through the ARB database release June 2000 (http://www.arb-home.de). Sequence data were aligned with the FASTALIGNER of the ARB program and adjusted manually. Jukes–Cantor evolutionary distances were estimated using pair-wise sequence dissimilarities calculated with DNADIST, and the dendrogram was created with the FITCH program of the implemented PHYLIP package (Felsenstein, 1989Go). Bar, 10 nucleotide substitutions per 100 bases.

 
Using tandem mass spectrometry (MS) the structures of many polar lipids of strain NO-502T could be elucidated using collision-induced decay MS by electrospray ionization (ESI) recorded in the negative mode [(–)-ESI] (Abraham et al., 1997Go). The phospholipids contained 2-N-(2'-D-hydroxyisopentadecanoyl)amino-3-hydroxyisoheptadeca-4(E)-ene-1-phosphate. The phosphate was detected as [M–1]– ion at m/z 604 in (–)-ESI MS. The corresponding aminoethyl derivative was detected at m/z 647 and the choline compound at m/z 689. In addition, homologues to these phospholipids were detected. 2-D-(2'-D-Hydroxyisopentadecanoyl)amino-3-D-hydroxyisoheptadecane-1-sulfonic acid was the only sulfolipid found in strain NO-502T. The ion of a glycolipid was seen at m/z 729 and identified as 2-N-(2''-hydroxyisopentadecanoyl)amino-3-hydroxyisoheptadeca-4(E)-ene-1-hydroxycarbonyl-6-deoxy-6-amino-mannopyranoside (Table 1Go). The latter compound has already been described from this strain and from Flectobacillus major (Batrakov et al., 1999Go). Furthermore, three ions at m/z 977, 1181 and 1310 that belonged to unknown lipids were found in the lipid extract of strain NO-502T.


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  		Table 1. Polar lipids of Arcicella aquatica NO-502T as determined by negative ESI quadrupole time-of-flight (ESI-QTOF) MS

 
In the lipid extract of Flectobacillus major, phosphatidylethylamine was found but not fully characterized; hence, a comparison with the three different compounds of this phospholipid from strain NO-502T was not possible. While 2-D-(2'-D-hydroxyisopentadecanoyl)amino-3-D-hydroxyisoheptadecane-1-sulfonic acid and 2-N-(2'-hydroxyisopentadecanoyl)amino-3-hydroxyisoheptadeca-4(E)-ene-1-(2''-aminoethyl)-phosphate were detected both in Flectobacillus major and in strain NO-502T, their corresponding 2'-deoxy derivatives, the lipoamino acid N-[3-D-(isoheptadecanoyloxy)isoheptadecanoyl]-glycine and the lipopeptide N-{N'-[3''-D-(isoheptadecanoyloxy)isoheptadecanoyl]glycyl}-L-serine were absent from strain NO-502T but present in Flectobacillus major (Batrakov et al., 2000Go). The homologue 2-N-(2'-hydroxyisopentadecanoyl)amino-3-hydroxyoctadeca-4(E)-ene-1-(2''-aminoethyl)-phosphate not known from Flectobacillus major is reported here for the first time.

The cellular fatty acids consisted mainly of even-numbered saturated fatty acids, while C16 : 1{omega}7/t, C18 : 1{omega}6 and C18 : 1{omega}7 were the main unsaturated fatty acids (Table 2Go). Strain NO-502T has higher proportions of C14 : 0 and C16 : 0 than Flectobacillus major, while C15 : 1 and C17 : 1, present in Flectobacillus major, are missing from strain NO-502T (Nikitin et al., 1994Go). The cellular fatty acid composition of strain NO-502T is distinct from those of many type strains of the family Spirosomaceae, as NO-502T has low amounts of branched fatty acids (Urakami & Komagata, 1986Go). However, these branched fatty acids are part of the sphingolipids of this strain, making it a clearly distinct member of this family.


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  		Table 2. Polar lipid fatty acids (PL) and cellular fatty acids (TL) of strain NO-502T

Values are given as a percentage of total fatty acids.

 
Enzyme activities were determined using the API ZYM test and the results are given in Table 3Go. High activities of amino-acid-hydrolysing enzymes were found, even though strain NO-502T could not grow on amino acids; no lipase activity was observed. In the API ZYM test, catalase, {beta}-galactosidase and {beta}-glucosidase activities were observed, but no nitrate was reduced, no acids were produced by fermentation on glucose and no protease nor urease activity was found (Table 3Go). The nutritional requirements of strain NO-502T and Flectobacillus major were determined on a mineral medium [0·01 g KH2PO4 l–1, 0·1 g K2HPO4 l–1, 0·2 g (NH4)2HPO4 l–1, 0·03 g MgSO4 l–1, 0·01 g NaCl l–1, 0·01 g CaCl2 l–1, trace amounts of MnSO4 and FeSO4, 0·005 % yeast extract; pH 7·2] with concentrations of 0·1 % of the carbon sources (Nikitin et al., 1994Go). The substrates used differently by these two strains have been listed in Table 3Go and compared with other type species of the family Spirosomaceae.


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  		Table 3. Biochemical characteristics of Arcicella aquatica NO-502T compared with related taxa

Taxa: 1, Arcicella aquatica NO-502T; 2, Flectobacillus major (Gosink et al., 1998Go); 3, Flexibacter flexilis; 4, Runella slithyformis; 5, Spirosoma linguale (Raj & Maloy, 1990Go). Scale is from 0 (no reaction) to 5 (strong reaction); W, weak reaction; –, no reaction; +, growth; ++, abundant growth. NA, Not available. Strain NO-502T was unable to grow on monomethylamine, ethanol, alanine, glycine, valine, leucine, arginine, lasine, proline, cystine or tryptophan, whereas Flectobacillus major could (Nikitin et al., 1994Go); no data are available for these substrates for taxa 3–5.

 
The 16S rRNA gene sequence similarity (93·5 % and below) to related genera, and the differences in the polar lipids, the cellular fatty acids and biochemical and physiological characteristics between Flectobacillus major and strain NO-502T together with the differences in their G+C contents do not allow the placement of these species in the same genus. Therefore, for strain NO-502T Arcicella gen. nov. is proposed, with this strain as the type strain of Arcicella aquatica sp. nov.

Description of the genus Arcicella gen. nov.
Arcicella (Ar.ci.cel'la. L. masc. n. arcus the arc; L. fem. cella cell; N.L. fem. n. Arcicella arc-shaped cell).

The description is based on the one published by Nikitin et al. (1994)Go. Cells are vibrioid, measure 2·5–3·0 by 0·5–0·75 µm, and are stretched spirals or S-shaped. Colonies on solid media are mucous and light-orange pigmented. Growth occurs between 4 and 40 °C; the optimal growth temperature is 28–30 °C; optimal pH is around 7. Grows with NaCl concentrations between 5 and 60 g l–1; above these concentrations and without NaCl no growth is observed. Aerobic, do not grow on C1 compounds, amino acids or organic acids, but do grow on a wide range of carbohydrates. Biopolymers are not hydrolysed. Nitrate is not reduced. Main polar lipids are 2-N-(2'-hydroxyisopentadecanoyl)amino-3-hydroxyisoheptadeca-4(E)-ene-1-(2''-aminoethyl)-phosphate and its 3-hydroxyoctadeca-4(E)-ene homologues, phosphatidyl ethylamine, 2-D-(2'-D-hydroxyisopentadecanoyl)amino-3-D-hydroxyisoheptadecane-1-sulfonic acid and 2-N-(2''-hydroxyisopentadecanoyl)amino-3-hydroxyisoheptadeca-4(E)-ene-1-hydroxycarbonyl-6-deoxy-6-amino-mannopyranoside. Main fatty acids are C16 : 0, C14 : 0, C18 : 1{omega}6 and C18 : 0. G+C content is 34 mol%. Lives in freshwater habitats.

Type species is Arcicella aquatica.


   Description of Arcicella aquatica sp. nov.
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 MAIN TEXT
 Description of Arcicella...
REFERENCES
 
Arcicella aquatica (a.qua.ti'ca. L. adj. aquatica aquatic; Arcicella species from water).

The description is the same as given for the genus with the following additions. Main phospholipids are 2-N-(2'-hydroxyisopentadecanoyl)amino-3-hydroxyisoheptadeca-4(E)-ene-1-(2''-aminoethyl)-phosphate, 2-N-(2'-hydroxyisopentadecanoyl)amino-3-hydroxyoctadeca-4(E)-ene-1-(2''-aminoethyl)-phosphate, 1-pentadecanoyl-2-hexadecenoylphosphatidyl-2'-ethylamine, 1,2-bis-hexadecenoylphosphatidyl-2'-ethylamine and 1-hexadecanoyl-2-hexadecenoylphosphatidyl-2'-ethylamine. Cells display high activities of alkaline phosphatase, leucine and valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, {beta}-galactosidase, {alpha}-glucosidase, catalase and N-acetyl-{beta}-glucosaminidase, weak activities of esterase lipase (C8), {alpha}-chymotrypsin, {alpha}-galactosidase, {beta}-glucosidase and {alpha}-fucosidase, and no activities of lipase (C14), {alpha}-glucuronidase or {alpha}-mannosidase. Nitrate is not reduced to nitrite or nitrogen. Grows well on glucose, fructose, lactose, maltose, rhamnose, galactose, arabinose, ribose, sucrose, cellobiose and inulin; shows some growth on mannitol, sorbitol, acetate and aspartate, but no growth on sorbose, dulcitol, ethanol, methanol, formate, propionate, pyruvate, monomethylamine, citrate, oxalate, succinate, malate or amino acids, with the exception of aspartate.

Type strain is NO-502T (=LMG 21963T=CIP 107990T). G+C content is 34·5 mol%. Genome size is 2·9x109 Da. Isolated from a neuston film in the central part of Lake Trostenskoe near Zvenigorod in the Moscow region.


   ACKNOWLEDGEMENTS
 
We thank Ina Buchholz and Jennifer Skerra for their skilful technical assistance. This work was supported by the grant ‘Soil functions' of the HGF strategic funds.


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Abraham, W.-R., Meyer, H., Lindholst, S., Vancanneyt, M. & Smit, J. (1997). Phospho- and sulfolipids as biomarkers of Caulobacter sensu lato, Brevundimonas, and Hyphomonas. Syst Appl Microbiol 20, 522–539.

Abraham, W.-R., Strömpl, C., Meyer, H. & 8 other authors (1999). Phylogeny and polyphasic taxonomy of Caulobacter species. Proposal of Maricaulis gen. nov. with Maricaulis maris (Poindexter) comb. nov. as the type species, and emended description of the genera Brevundimonas and Caulobacter. Int J Syst Bacteriol 49, 1053–1073.[Abstract/Free Full Text]

Batrakov, S. G., Sheichenko, V. I. & Nikitin, D. I. (1999). A novel glycosphingolipid from Gram-negative aquatic bacteria. Biochim Biophys Acta 1440, 163–175.[Medline]

Batrakov, S. G., Mosezhnyi, A. E., Ruzhitsky, A. O., Sheichenko, V. I. & Nikitin, D. I. (2000). The polar-lipid composition of the sphingolipid-producing bacterium Flectobacillus major. Biochim Biophys Acta 1484, 225–240.[Medline]

Cole, J. R., Chai, B., Marsh, T. L. & 8 other authors (2003). The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res 31, 442–443.[Abstract/Free Full Text]

Felsenstein, J. (1989). PHYLIP – Phylogeny inference package (version 3.2). Cladistics 5, 164–166.

Glöckner, F. O., Zaichikov, E., Belkova, N., Denissova, L., Pernthaler, J., Pernthaler, A. & Amann, R. (2000). Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an abundant group of actinobacteria. Appl Environ Microbiol 66, 5053–5065.[Abstract/Free Full Text]

Gosink, J. J., Woese, C. R. & Staley, J. T. (1998). Polaribacter gen. nov., with three new species, P. irgensii sp. nov., P. franzmannii sp. nov. and P. filamentus sp. nov., gas vacuolate polar marine bacteria of the Cytophaga–Flavobacterium–Bacteroides group and reclassification of ‘Flectobacillus glomeratus’ as Polaribacter glomeratus comb. nov. Int J Syst Bacteriol 48, 223–235.[Abstract/Free Full Text]

Larkin, J. M. & Borrall, R. (1984). Genus III. Flectobacillus Larkin, Williams and Taylor 1977, 152AL. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 129–132. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.

Nakagawa, Y., Sakane, T., Suzuki, M. & Hatano, K. (2002). Phylogenetic structure of the genera Flexibacter, Flexithrix, and Microscilla deduced from 16S rRNA sequence analysis. J Gen Appl Microbiol 48, 155–165.

Nikitin, D. I., Oranskaya, M. S., Pitryuk, I. A., Chernykh, N. A. & Lysenko, A. M. (1994). A new ring-forming bacterium Arcocella aquatica gen. et sp. nov. Mikrobiologiya 63, 152–159 (in Russian).

Raj, H. D. & Maloy, S. R. (1990). Proposal of Cyclobacterium marinus gen. nov., comb. nov., for a marine bacterium previously assigned to the genus Flectobacillus. Int J Syst Bacteriol 40, 337–347.[Abstract/Free Full Text]

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