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

Arcicella aquatica NO-502^T, 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-502^T is 34·5 mol%; its genome size is 2·9x10⁹ Da. The genus Arcicella and its type species Arcicella aquatica (type strain NO-502^T=LMG 21963^T=CIP 107990^T) are proposed, and descriptions of this genus and species are given.

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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-502^T is AJ535729.

From a freshwater neuston film a slow-growing bacterial isolate, NO-502^T, 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., 1994); 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-502^T.

Strain NO-502^T was isolated on a medium containing 0·1 % peptone, yeast extract and glucose each at pH 7·07·2 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-502^T was determined as described previously (Abraham et al., 1999). It was found that an identical sequence was reported for isolate AH57 (accession no. AJ289964) from Lake Fuchskuhle, Germany (Glöckner et al., 2000). 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-502^T, from freshwater ecosystems. Next relatives of this genus are species of the genus Flectobacillus, as shown in Fig. 1. The 16S rDNA sequence similarity between strain NO-502^T 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. 1) was discussed by Nakagawa et al. (2002). Nikitin et al. (1994), however, listed further genomic data which, together with our data, require the separation of strain NO-502^T from the genus Flectobacillus. The G+C content of strain NO-502^T was found to be 34·5 %, while a G+C content of 39·540·3 mol% has been reported for Flectobacillus major (Gosink et al., 1998).

(25K): 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., 2003) and/or RDP (Cole et al., 2003) databases were obtained through the ARB database release June 2000 (). Sequence data were aligned with the FASTALIGNER of the ARB program and adjusted manually. JukesCantor 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, 1989). Bar, 10 nucleotide substitutions per 100 bases.

Using tandem mass spectrometry (MS) the structures of many polar lipids of strain NO-502^T could be elucidated using collision-induced decay MS by electrospray ionization (ESI) recorded in the negative mode [()-ESI] (Abraham et al., 1997). The phospholipids contained 2-N-(2'-D-hydroxyisopentadecanoyl)amino-3-hydroxyisoheptadeca-4(E)-ene-1-phosphate. The phosphate was detected as [M1] 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-502^T. 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 1). The latter compound has already been described from this strain and from Flectobacillus major (Batrakov et al., 1999). Furthermore, three ions at m/z 977, 1181 and 1310 that belonged to unknown lipids were found in the lipid extract of strain NO-502^T.

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-502^T 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-502^T, 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-502^T but present in Flectobacillus major (Batrakov et al., 2000). 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 C_{16 : 1}ω7/t, C_{18 : 1}ω6 and C_{18 : 1}ω7 were the main unsaturated fatty acids (Table 2). Strain NO-502^T has higher proportions of C_{14 : 0} and C_{16 : 0} than Flectobacillus major, while C_{15 : 1} and C_{17 : 1}, present in Flectobacillus major, are missing from strain NO-502^T (Nikitin et al., 1994). The cellular fatty acid composition of strain NO-502^T is distinct from those of many type strains of the family Spirosomaceae, as NO-502^T has low amounts of branched fatty acids (Urakami & Komagata, 1986). However, these branched fatty acids are part of the sphingolipids of this strain, making it a clearly distinct member of this family.

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 3. High activities of amino-acid-hydrolysing enzymes were found, even though strain NO-502^T could not grow on amino acids; no lipase activity was observed. In the API ZYM test, catalase, β-galactosidase and β-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 3). The nutritional requirements of strain NO-502^T and Flectobacillus major were determined on a mineral medium [0·01 g KH₂PO₄ l¹, 0·1 g K₂HPO₄ l¹, 0·2 g (NH₄)₂HPO₄ l¹, 0·03 g MgSO₄ l¹, 0·01 g NaCl l¹, 0·01 g CaCl₂ l¹, 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., 1994). The substrates used differently by these two strains have been listed in Table 3 and compared with other type species of the family Spirosomaceae.

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., 1998); 3, Flexibacter flexilis; 4, Runella slithyformis; 5, Spirosoma linguale (Raj & Maloy, 1990). 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., 1994); no data are available for these substrates for taxa 35.

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-502^T 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-502^T 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). Cells are vibrioid, measure 2·53·0 by 0·50·75 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 2830 °C; optimal pH is around 7. Grows with NaCl concentrations between 5 and 60 g l¹; 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 C_{16 : 0}, C_{14 : 0}, C_{18 : 1}ω6 and C_{18 : 0}. G+C content is 34 mol%. Lives in freshwater habitats.

Type species is Arcicella aquatica.