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1 Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, Gent, Belgium
2 BCCM/LMG Bacteria Collection, Universiteit Gent, Gent, Belgium
3 Alfred-Wegener-Institut für Polar- und Meeresforschung, Bremerhaven, Germany
Correspondence
Stefanie Van Trappen
stefanie.vantrappen{at}ugent.be
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ABSTRACT |
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7c, summed feature 2 (comprising any combination of C12 : 0 aldehyde, an unknown fatty acid of equivalent chain length 10·928, C16 : 1 iso I and C14 : 0 3-OH) and summed feature 3 (C15 : 0 iso 2-OH and/or C16 : 17c) as the major fatty acid components. On the basis of their phylogenetic, genomic and phenotypic properties, the five novel strains can be assigned to the genus Alcaligenes as a novel species, for which the name Alcaligenes aquatilis sp. nov. is proposed. The type strain is LMG 22996T (=CCUG 50924T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LMG 22996T is AJ937889.
A dendrogram derived from the linkage of correlation coefficients between the protein patterns of the strains studied and a digitized representation of normalized rep-PCR profiles are available as supplementary figures in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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), Alcaligenes latus (Palleroni & Palleroni, 1978) and Alcaligenes defragrans (Foss et al., 1998). A. faecalis strains have been isolated from a wide variety of different habitats, e.g. soil, water and several clinical samples (Kersters & De Ley, 1984; Schroll et al., 2001). A. defragrans strains have been isolated from soil and are capable of using alkenoic monoterpenes as sole carbon sources (Foss et al., 1998). A. latus is considered a species incertae sedis and recent data have shown that this organism belongs to the family Comamonadaceae (Coenye et al., 2003). Recently, another novel subspecies, ‘A. faecalis subsp. phenolicus’, isolated from a greywater bioprocessor, has been described (Rehfuss & Urban, 2005). A polyphasic taxonomic study was performed on five isolates from different aquatic habitats to elucidate their taxonomic position. The results of the genotypic and phenotypic analyses showed that they belong to a novel Alcaligenes species.
Four nitrite-dissimilating strains (LMG 22996T, LMG 22997, LMG 22998 and LMG 22999) were isolated from Weser Estuary sediments in Germany (Rüger et al., 1983). Strain M3A (=ATCC 700596) was isolated from salt-marsh sediment from the bank of Shem Creek in Charleston Harbor (South Carolina, USA) (de Souza & Yoch, 1995). The reference strains A. faecalis subsp. faecalis LMG 1229T, A. faecalis subsp. parafaecalis LMG 22680T and A. defragrans LMG 18538T were included in some experiments. All strains were routinely cultivated on trypticase soy agar at 28 °C for 48 h, except when mentioned otherwise.
SDS-PAGE of whole-cell proteins of the five strains was performed and strains were grown on buffered nutrient agar for 48 h at 28 °C. Preparation of whole-cell proteins and SDS-PAGE were performed as described previously (Pot et al., 1994). Densitometric analysis, normalization and interpolation of the protein profiles, as well as numerical analysis using Pearson's product-moment correlation coefficient, were performed using the GELCOMPAR 4.2 software package (Applied Maths).
After numerical analysis and visual comparison of the profiles, one cluster could be delineated: strains LMG 22996T, LMG 22997, LMG 22998 and LMG 22999 showed almost identical profiles, whereas strains ATCC 700596, LMG 1229T (A. faecalis subsp. faecalis), LMG 18538T (A. defragrans) and LMG 22680T (A. faecalis subsp. parafaecalis) formed separate branches in the dendrogram (see Supplementary Fig. S1 available in IJSEM Online).
The five strains, together with the type strains of A. faecalis subsp. faecalis and A. faecalis subsp. parafaecalis, were arranged in similarity groups based upon the results of rep-PCR fingerprinting using the GTG5 and BOX primers (Versalovic et al., 1991; Rademaker & de Bruijn, 1997; Rademaker et al., 2000). Numerical analysis was carried out using the BioNumerics 4.0 software package, as described by the same authors. Strains LMG 22996T, LMG 22997, LMG 22998 and LMG 22999 showed virtually identical profiles, whereas strain ATCC 700596 and the type strains of the two A. faecalis subspecies formed separate branches (see Supplementary Fig. S2 available in IJSEM Online).
Small-scale DNA extracts were prepared using the method of Pitcher et al. (1989) and a 1082 nt fragment of the 16S rRNA gene of strain LMG 22996T was amplified by a PCR using conserved primers (Coenye et al., 1999). PCR products were purified using a QIAquick PCR purification kit (Qiagen) according to the instructions of the manufacturer. Sequence analysis was performed as described previously (Van Trappen et al., 2004). Evolutionary distances were calculated using the algorithm of Jukes & Cantor (1969) and a phylogenetic tree (Fig. 1) was constructed using the neighbour-joining method with the TREECON program (Van de Peer & De Wachter, 1994).
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illustrates the phylogenetic relationships within the genus Alcaligenes. Bootstrap analysis indicated that strains LMG 22996T and ATCC 700596 form a stable phylogenetic group (with a bootstrap value of 93 %).
The genomic relatedness between strains LMG 22996T and ATCC 700596 and the most closely related strains, A. faecalis subsp. faecalis LMG 1229T, A. faecalis subsp. parafaecalis LMG 22680T and A. defragrans LMG 18538T, was determined by DNA–DNA hybridization. DNA was prepared according to the method of Pitcher et al. (1989) and DNA–DNA hybridizations were carried out with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989), using a HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements. The hybridization temperature was 42 °C and reciprocal experiments were performed for every pair of strains. The level of hybridization between strains LMG 22996T and ATCC 700596 was 84 %, indicating that they belong to a single species (Wayne et al., 1987), whereas the DNA–DNA binding values with the other Alcaligenes strains were low (60 % with LMG 1229T, 59 % with LMG 22680T and 10 % with LMG 18538T). Differences between reciprocal experiments were less than 10 %. These results demonstrate that the five isolates are genotypically distinct from A. faecalis subsp. faecalis and A. faecalis subsp. parafaecalis, their nearest neighbours in phylogenetic terms, and constitute a novel species within the genus Alcaligenes.
The DNA G+C contents of the isolates were determined using an HPLC method as described by Van Trappen et al. (2003): the values for strains LMG 22996T, LMG 22997, LMG 22998, LMG 22999 and ATCC 700596 are respectively 56, 57, 56, 56 and 55 mol%. These values are consistent with the G+C contents of the genera Alcaligenes, Achromobacter and Pigmentiphaga, which range from 54 to 69 mol% (Stolz et al., 2005).
The cellular fatty acid patterns of the strains were determined as described by Mergaert et al. (2001). The five strains showed very similar fatty acid profiles and the mean composition was 2·3 % C10 : 0, 2·6 % C12 : 0 2-OH, 32·7 % C16 : 0, 13·3 % C17 : 0 cyclo, 1·0 % C18 : 0, 9·1 % C18 : 17c, 11·3 % summed feature 2 (which comprises any combination of C12 : 0 aldehyde, an unknown fatty acid of equivalent chain length 10·928, C16 : 1 iso I and C14 : 0 3-OH) and 24·6 % summed feature 3 (C15 : 0 iso 2-OH and/or C16 : 17c). In Table 1, the fatty acid compositions of several Alcaligenes species are compared: the profiles of the novel strains resemble those determined for the other members of the genus Alcaligenes. The dominant fatty acids are C16 : 0, C17 : 0 cyclo, C18 : 17c, summed feature 2 and summed feature 3; the novel strains differ from the other Alcaligenes species in their relative amounts of C14 : 0 3-OH, C17 : 0 cyclo and summed feature 3.
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; de Souza & Yoch, 1995) and are listed below. Tests in the commercial API 20NE and API ID 32 GN systems (bioMérieux) were performed according to the instructions of the manufacturer. Anaerobic growth of the strains in the presence of nitrate and nitrite has been investigated and the reduction of nitrate and nitrite under anaerobic conditions has been tested according to the protocol of Smibert & Krieg (1994). Alkali production from some organic salts and amides has been tested using the medium of Hugh and Leifson (see species description).
On the basis of this polyphasic taxonomic analysis, the five strains can be clearly differentiated from the other species within the genus Alcaligenes (Table 2) and should be assigned to a novel species, for which the name Alcaligenes aquatilis sp. nov. is proposed.
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) indicate that it would be more appropriate to allocate this taxon to a distinct genus in the family Alcaligenaceae.
Description of Alcaligenes aquatilis sp. nov.
Alcaligenes aquatilis (a.qua'ti.lis. L. masc. adj. aquatilis living, growing or found in, or near, water, aquatic).
Cells are Gram-negative short rods (0·7–1·1 µmx1·0–2·5 µm) and are motile via peritrichous flagella. They form non-pigmented or yellow-pigmented (strains LMG 22999 and ATCC 700596), circular, low-convex and smooth colonies with spreading, irregular edges and a diameter of 2·0–5·0 mm on trypticase soy agar plates after 2 days incubation at 28 °C. The growth temperature is in the range 4–35 °C, while the optimal growth temperature ranges from 18 to 24 °C. Tests for catalase and cytochrome oxidase are positive (except for strain ATCC 700596, which is catalase-negative). Nitrate and nitrite are not reduced under anaerobic conditions, but slight growth is observed under these conditions after 5 days incubation at 28 °C. Positive for the alkalinization of asparagine, citrate and glutamine. Positive for the utilization of sodium acetate, sodium citrate and malonate for most of the strains (with the exception of strain ATCC 700596, which is negative for utilization of sodium citrate and malonate). 
-Galactosidase activity is not detected. Indole and hydrogen sulfide are not produced, and starch, gelatin, aesculin and urea are not degraded. Strains LMG 22996T, LMG 22997, LMG 22998 and LMG 22999 are able to reduce nitrite but not nitrate. No acids are produced from carbohydrates (except for strain ATCC 700596, which produces acid from maltose, mannitol, xylose and L-arabinose). No activity detected for arginine dihydrolase (variable for strain ATCC 700596), lysine decarboxylase or ornithine decarboxylase. Growth is not observed on glucose, L-arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, adipate, salicin, D-melibiose, L-fucose, D-sorbitol, propionate, 2-ketogluconate, 3-hydroxybutyrate, 4-hydroxybenzoate, L-proline, rhamnose, D-ribose, inositol, D-sucrose, itaconate, malonate, L-alanine or 5-ketogluconate. Growth is observed on caprate (except for strain ATCC 700596), malate, phenylacetate, valerate, citrate, histidine, suberate, acetate, DL-lactate, glycogen, 3-hydroxybenzoate and L-serine. Cells contain the fatty acids C16 : 0, C17 : 0 cyclo, C18 : 17c, summed feature 2 (comprising any combination of C12 : 0 aldehyde, an unknown fatty acid of equivalent chain length 10·928, C16 : 1 iso I and C14 : 0 3-OH) and summed feature 3 (C15 : 0 iso 2-OH and/or C16 : 17c) as the main constituents. The DNA G+C content of the strains is 56 mol%.
The type strain is LMG 22996T (=CCUG 50924T). Reference strains are LMG 22997, LMG 22998 and LMG 22999, isolated from sediments of the Weser Estuary, Germany, as was the type strain, and M3A (=ATCC 700596), isolated from sediments of Shem Creek, Charleston Harbor, USA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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