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1 Institut für Angewandte Mikrobiologie, Justus Liebig Universität, IFZ – Heinrich-Buff-Ring 26–32, D-35392 Giessen, Germany
2 Department of Biology, The University of Konstanz, D-78457 Konstanz, Germany
3 Department of Biological Sciences, Korean Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejon, 305-701, Republic of Korea
4 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität Wien, Veterinärplatz 1, A-1210 Wien, Austria
Correspondence
P. Kämpfer
peter.kaempfer{at}agrar.uni-giessen.de
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains NKNTAUT and TJ4 are U82826 and AF508102, respectively.
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. After numerous reclassifications, the authentic genus now encompasses three species: the type species of the genus, Alcaligenes faecalis, with the three subspecies A. faecalis subsp. faecalis, A. faecalis subsp. parafaecalis (Schroll et al., 2001) and A. faecalis subsp. phenolicus (Rehfuss & Urban, 2005), Alcaligenes aquatilis (Van Trappen et al., 2005) and Alcaligenes defragrans (Foss et al., 1998). The species Alcaligenes latus has been shown to belong to the family Comamonadaceae (Coenye et al., 2003b), and its transfer to the novel genus Azohydromonas was proposed recently (Xie & Yokota, 2005). Strains of A. defragrans have been isolated from soil and are capable of using alkenoic monoterpenes as sole carbon sources (Foss et al., 1998). Additional isolates (strains TJ4 and NKNTAUT), presumptively identified as A. defragrans, have been shown to degrade phenol (Baek et al., 2003) and taurine (Denger et al., 1997; Ruff et al., 2003) under denitrifying conditions.
Members of the authentic genus Alcaligenes are Gram-negative, strictly aerobic rods or coccobacilli that are motile. They possess oxidase and catalase. They grow well on complex media such as nutrient agar. The predominant fatty acids are C16 : 0, C16 : 1
7c, C17 : 0 cyclo, C18 : 17c and/or C14 : 0 3-OH (Foss et al., 1998; Schroll et al., 2001; Coenye et al., 2003b). A. faecalis has been reported to possess, among its polar lipids, an ornithine lipid (Yabuuchi et al., 1995). It has been shown in a previous report (Stolz et al., 2005), however, that the fatty acid profiles and polar lipids of A. defragrans differ from those of the subspecies of A. faecalis. In order to establish the exact taxonomic position of A. defragrans, we included the type strain of A. defragrans in our polyphasic taxonomic study on strains NKNTAUT and TJ4.
A. defragrans DSM 12141T was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany); isolation of strains NKNTAUT and TJ4 was described previously (Denger et al., 1997; Baek et al., 2003). Subculturing was done on nutrient agar (Oxoid) at 30 °C for 24 h. Gram-staining was performed as described by Gerhardt et al. (1994). Cell morphology was examined with a Zeiss light microscope at 1000x magnification, using cells that had been grown for 24 h at 30 °C on nutrient agar. The results regarding cell morphology are given in the formal species descriptions. The 16S rRNA gene was analysed as described previously (Kämpfer et al., 2003). Analysis of sequence data was performed using the software package MEGA, version 2.1 (Kumar et al., 2001) after multiple alignment of the data using CLUSTAL X (Thompson et al., 1997). A distance-matrix method (with distance options according to the Kimura two-parameter model) was employed, using clustering obtained with the neighbour-joining method (Fig. 1) as well as a discrete character-based maximum-parsimony method (data not shown). For each method, bootstrap values were calculated on the basis of 1000 replications. Sequence-similarity calculations indicated that strains NKNTAUT and TJ4 are closely related (99·9 %). The highest level of 16S rRNA gene sequence similarity (98·2 %) to a species with a validly published name was found with A. defragrans (Foss et al., 1998). The next most similar species were Pusillimonas noertemannii (<96·5 %) and A. faecalis (all subspecies) (<94 %).
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), polar lipids (Tindall, 1990) and fatty acids (Kämpfer & Kroppenstedt, 1996). Quinones, polar lipids and polyamines were extracted from biomass grown for 72 h on PYE medium (0·3 % yeast extract, 0·3 % peptone from casein, pH 7·2). The quinone system of A. defragrans DSM 12141T, strain NKNTAUT and strain TJ4 consisted predominantly of ubiquinone Q-8 (98 %), with small amounts of Q-7 and Q-9. The three strains exhibited a homogeneous polyamine pattern consisting predominantly of putrescine [48–70 µmol (g dry weight)–1], with moderate amounts of 2-hydroxyputrescine [5–9 µmol (g dry weight)–1] and minor amounts of 1,3-diaminopropane [<0·5 µmol (g dry weight)–1], cadaverine [<0·2 µmol (g dry weight)–1], spermidine [<2·5 µmol (g dry weight)–1], sym-homospermidine [<0·1 µmol (g dry weight)–1] and spermine [<0·5 µmol (g dry weight)–1]. Both the quinone systems and polyamine patterns are in excellent agreement with those of members of the Betaproteobacteria (Busse & Auling, 1988; Auling et al., 1991; Hamana & Takeuchi, 1998).
Strains TJ4 and NKNTAUT exhibited identical polar lipid profiles, containing a predominance of phosphatidylethanolamine, moderate amounts of diphosphatidylglycerol and phosphatidylglycerol and minor amounts of two unknown aminolipids (AL3 and AL1) and an unknown polar lipid (L7) (data not shown). These lipids were also detected in A. defragrans DSM 12141T (Stolz et al., 2005). However, the polar lipid compositions of the two strains were less complex than those of A. defragrans DSM 12141T. Hence, the polar lipid profiles appear to be useful for the differentiation of TJ4 and NKNTAUT from A. defragrans DSM 12141T.
The fatty acid profiles of strains NKNTAUT and TJ4 are given in Table 1. The predominant fatty acids are C16 : 0, C16 : 17c, C17 : 0 cyclo, C18 : 17c and/or C14 : 0 3-OH. The fatty acid C12 : 0 3-OH, present in all subspecies of A. faecalis, could not be detected in strain DSM 12141T or in strains NKNTAUT and TJ4.
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; Schroll et al., 2001) are given in the species description. All of the strains under study were able to utilize a number of organic acids and amino acids. Sugars and sugar alcohols (polyols) were not utilized. Table 2 shows the differentiating characteristics.
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, except that for nick translation, 2 µg DNA was labelled during a 3 h incubation at 15 °C. Strains NKNTAUT and TJ4 showed DNA–DNA relatedness values of 80 % (reciprocal analysis, 74 %), clearly indicating that the two strains belong to the same species. Strains NKNTAUT and TJ4 showed relatively low levels of DNA–DNA relatedness to A. defragrans DSM 12141T (30 %, reciprocal 43 %; 43 %, reciprocal 37 %, respectively). The standard deviation for all of the DNA–DNA hybridizations was 8·5 %. These results clearly showed that NKNTAUT and TJ4 are members of a single species that is separate from A. defragrans. On the basis of the phylogenetic and chemotaxonomic data, A. defragrans is clearly different from strains belonging to the genus Alcaligenes. For this reason, a novel genus is proposed for the A. defragrans lineage. Moreover, detailed investigations on strains NKNTAUT and TJ4 show that, on the basis of genotypic and phenotypic results, the two strains represent a novel species of the new genus, for which the name Castellaniella gen. nov. is proposed.
Description of Castellaniella gen. nov.
Castellaniella (Cas.tel.la'ni.el.la. N.L. fem. dim. n. Castellaniella named after Sir Aldo Castellani, a British–Italian bacteriologist, who first described the bacterial genus Alcaligenes in 1919).
Gram-negative, facultatively anaerobic and denitrifying. Cells are motile, short rods, 1·3–2·0 µm long and 0·2–0·8 µm wide. Good growth occurs after 24 h incubation on nutrient agar at 25–30 °C. The polar lipid profile contains the major compound phosphatidylethanolamine, moderate amounts of phosphatidylglycerol and diphosphatidylglycerol and minor amounts of two unknown aminolipids and an unknown polar lipid. The polyamines consist predominantly of putrescine with moderate amounts of 2-hydroxyputrescine; the main component of the quinone system is ubiquinone Q-8. The major fatty acids are C16 : 0, C16 : 17c, C17 : 0 cyclo, C18 : 17c and/or C14 : 0 3-OH. The DNA G+C content of the type species, Castellaniella defragrans, is 66·9 mol%. As currently circumscribed, the genus contains two species, Castellaniella defragrans and Castellaniella denitrificans.
Description of Castellaniella defragrans (Foss et al. 1998) comb. nov.
Basonym: Alcaligenes defragrans Foss et al. 1998.
The description is essentially that of Foss et al. (1998), with the addition of the information obtained in this study. In addition to the characteristics listed in the genus description, the polar lipid profile contains minor amounts of three unknown aminolipids, two unknown phospholipids, two unknown aminophospholipids and four unknown polar lipids (Stolz et al., 2005). The polyamine pattern consists of major amounts of putrescine [69·9 µmol (g dry weight)–1], moderate amounts of 2-hydroxyputrescine [9·2 µmol (g dry weight)–1], minor amounts of spermidine [2·4 µmol (g dry weight)–1] and traces [<0·5 µmol (g dry weight)–1] of 1,3-diaminopropane, cadaverine, sym-homospermidine and spermine. The quinone system comprises ubiquinone Q-8. The type strain is strain 54PinT (=CCUG 39790T=CIP 105602T=DSM 12141T=LMG 18538T).
Description of Castellaniella denitrificans sp. nov.
Castellaniella denitrificans [de.ni.tri.fi'cans. N.L. part. adj. denitrificans (from N.L. v. denitrifico) denitrifying].
General characteristics are as described for the genus. On nutrient agar, colonies are beige and circular with an entire margin. Grows at 30, 37 and 42 °C; no growth at 4 °C. On the basis of the method described by Kämpfer et al. (1991), L-alanine p-nitroanilide (pNA) is hydrolysed. The following compounds are not hydrolysed: p-nitrophenyl (pNP) 
-D-galactopyranoside, pNP -D-glucuronide, pNP 
-D-glucopyranoside, pNP -D-glucopyranoside, pNP -D-xylopyranoside, bis-pNP phosphate, bis-pNP phenylphosphonate, bis-pNP phosphorylcholine, L-aniline pNA, 
-L-glutamate pna and L-proline pNA. On the basis of the method described by Kämpfer et al. (1991), the following compounds are used as sole sources of carbon: D-gluconate (weak), acetate, propionate, cis-aconitate, trans-aconitate, 4-aminobutyrate, citrate, fumarate, glutarate, DL-3-hydroxybutyrate, itaconate, DL-lactate, L-malate, mesaconate, 2-oxoglutarate, pyruvate, L-alanine, -alanine, L-aspartate, L-leucine, L-ornithine, L-proline and L-serine. The following compounds are not assimilated: N-acetylgalactosamine, N-acetylglucosamine, L-arabinose, L-arbutin, D-cellobiose, D-fructose, D-galactose, D-glucose, D-maltose, D-mannose, -D-melibiose, L-rhamnose, D-ribose, D-sucrose, salicin, D-trehalose, D-xylose, adonitol, i-inositol, maltitol, D-mannitol, D-sorbitol, putrescine, adipate, azelate, suberate, L-histidine, L-phenylalanine, L-serine, L-tryptophan, 3-hydroxybenzoate and phenylacetate. No acids are produced from glucose, lactose, sucrose, D-mannitol, dulcitol, salicin, adonitol, inositol, sorbitol, L-arabinose, raffinose, rhamnose, maltose, D-xylose, trehalose, cellobiose, methyl D-glucoside, erythritol, melibiose, D-arabitol or D-mannose. The quinone system, the polyamine pattern and the polar lipid profile are identical to those listed in the genus description. The polyamine pattern contains putrescine [48·1–53·1 µmol (g dry weight)–1], moderate amounts of 2-hydroxyputrescine [5·3–5·7 µmol (g dry weight)–1], spermidine [0·7–1·3 µmol (g dry weight)–1] and traces [<0·5 µmol (g dry weight)–1] of 1,3-diaminopropane, cadaverine, sym-homospermidine and spermine.
The type strain, NKNTAUT (=DSM 11046T=CCUG 39541T), was isolated from an anaerobic enrichment culture with taurine as sole source of carbon and an inoculum from an anaerobic sludge digestor (Denger et al., 1997). Isolate TJ4, which represents a second strain of this species, was isolated from an environmental sample in Korea, enriched under denitrifying conditions with phenol as the sole source of carbon (Baek et al., 2003).
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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-cis-9,10-methylenehexadecanoic acid; C16 : 0 2-OH, 2-hydroxyhexadecanoic acid; C18 : 1