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BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
Correspondence
I. Cleenwerck
Ilse.Cleenwerck{at}ugent.be
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The EMBL/GenBank/DDBJ accession number for the 16S rDNA sequence of strain LMG 4329T is AJ487013.
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for two aerobic freshwater spirilla with a G+C content of 63–65 mol% and the ability to reduce nitrate beyond the level of nitrite. Later studies on intra- and intergeneric relationships and chemotaxonomic characteristics of various species of Aquaspirillum showed that A. dispar should be removed from the genus Aquaspirillum and that a new genus should be created for organisms belonging to this species (Pot et al., 1992; Hamana et al., 1994; Sakane & Yokota, 1994). In this study, we determined the 16S rDNA sequence of [A.] dispar LMG 4329T, compared it with all accessible 16S rDNA sequences in the EMBL database and found a high level of similarity (99·4 %) to the sequence of Microvirgula aerodenitrificans SGLY2T (=LMG 18919T). M. aerodenitrificans was created by Patureau et al. (1998) for an aerobic, denitrifying Gram-negative bacterium that could not be assigned at that time to any recognized genus on the basis of its 16S rDNA sequence. In the present note, we report on a polyphasic taxonomic study that demonstrates that [A.] dispar and M. aerodenitrificans belong in the same species.
[A.] dispar strains LMG 4329T and LMG 4330 and M. aerodenitrificans LMG 18919T were grown on medium M8 (Janssens et al., 1998) or trypticase soy agar (TSA; BBL) and incubated at 28 °C. DNA for 16S rDNA sequence analysis was prepared by alkali extraction as described by Niemann et al. (1997). DNA for determination of the DNA base composition and hybridization analysis was prepared by the method of Pitcher et al. (1989). To obtain DNA solutions free of RNA, an RNase treatment was performed, followed by a chloroform extraction. The 16S rDNA sequence of [A.] dispar LMG 4329T was determined as described by Cleenwerck et al. (2002). The sequencing primers used were Gamma, *Gamma, PD, *PD, 3, *3 and *O (Coenye et al., 1999). The 16S rDNA sequence of LMG 4329T was compared with all accessible 16S rDNA sequences in the EMBL database and aligned with the 16S rDNA sequences of the phylogenetically closest species using the GeneCompar 2.1 software package (Applied Maths). Unknown bases were discarded from the calculations. The G+C content of the DNA was determined by HPLC according to the method of Mesbah et al. (1989) using a Waters Symmetry Shield RP8 column thermostatted at 37 °C. DNA–DNA hybridizations were performed using a modification of the microplate method described by Ezaki et al. (1989) (Goris et al., 1998; Cleenwerck et al., 2002). The hybridization temperature was 45 °C. Gram staining and oxidase and catalase activity were determined as described by Cleenwerck et al. (2002). API 20 NE (bioMérieux) and Biolog GN2 analyses were performed according to the manufacturers' instructions. Cellular fatty acid methyl ester extraction and analyses from cells grown on TSA (BBL) for 24 h at 28 °C were performed according to the recommendations of the MIDI system (Microbial Identification System).
The 16S rDNA sequence of [A.] dispar LMG 4329T (1490 bases) was 99·4 % similar to the 16S rDNA sequence of M. aerodenitrificans SGLY2T (accession no. U89333). Sequence similarities to other published taxa belonging to the 
-Proteobacteria were below 95 %. The closest taxa were Laribacter hongkongensis (accession no. AF389085; 94·3 %), Vogesella indigofera (accession no. AB021385; 91·8 %), Zoogloea ramigera (accession no. X74913; 90·4 %) and Chromobacterium violaceum (accession no. AJ247211; 86·3 %). The DNA G+C contents of [A.] dispar LMG 4329T and M. aerodenitrificans LMG 18919T were respectively 64·3 and 64·5 mol%. The DNA–DNA binding value between LMG 4329T and LMG 18919T was 84 %, showing that the two strains belong to the same species. In addition, the target site of a specific probe developed by Patureau et al. (1998) for M. aerodenitrificans LMG 18919T, within the V4 variable region of the 16S rRNA (positions 636–653; Escherichia coli numbering), was also present in the 16S rDNA sequence of [A.] dispar LMG 4329T.
Comparison of the morphological, physiological and biochemical characteristics of M. aerodenitrificans LMG 18919T and [A.] dispar strains LMG 4329T and LMG 4330 (the latter two strains showing 100 % DNA–DNA relatedness; Aragno et al., 1978) showed that these strains were phenotypically highly similar. Cells were Gram-negative, motile, curved to rod-shaped, occurring singly or in pairs. All three strains were oxidase- and catalase-positive. In API 20 NE tests, the three strains gave identical reactions. They were able to denitrify, assimilate caprate and malate and hydrolyse arginine. According to the API 20 NE identification system, the strains are phenotypically highly similar to Comamonas testosteroni and Pseudomonas alcaligenes, with only arginine dehydrolase as a differentiating feature. The three strains shared nearly identical carbon source oxidation patterns, as determined by the Biolog identification system. All strains were able to oxidize a large spectrum of carbon sources, except sugars. Some minor quantitative differences were found in the oxidation of glycogen, alaninamide, the organic acids cis-aconitic acid and 
-ketovaleric acid and the amino acids hydroxy-L-proline, D-serine and L-threonine. For M. aerodenitrificans LMG 18919T, the Biolog results were similar to those obtained by Patureau et al. (1998) except that -ketoglutaric acid, malonic acid, D-alanine and L-threonine were oxidized, while sebacic acid, L-leucine and glycerol were not. The cellular fatty acid profiles of LMG 4329T and LMG 18919T were very similar, with summed feature 3, 16 : 0 and 18 : 1 as the major non-polar fatty acid compounds and 12 : 0 3-OH as the only 3-hydroxy fatty acid (Table 1). Analogous results were obtained by Sakane & Yokota (1994) for [A.] dispar and, in their study, it was shown that this profile was not found in other Aquaspirillum species. Phenotypic characteristics that enable the differentiation of LMG 4329T, LMG 4330 and LMG 18919T from the phylogenetically and phenotypically most related species are given in Table 2.
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), the name Microvirgula aerodenitrificans must be retained for the unified taxon, since it is the type of the genus Microvirgula.
Emended description of Microvirgula aerodenitrificans Patureau et al. 1998
The description of the species is that given by Patureau et al. (1998), emended by the additional features described in the current paper. The species includes strains previously classified as Aquaspirillum dispar. The type strain is strain SGLY2T (=DSM 15089T=LMG 18919T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Cleenwerck, I., Vandemeulebroecke, K., Janssens, D. & Swings, J. (2002). Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int J Syst Evol Microbiol 52, 1551–1558.[Abstract]
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Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
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