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1 Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
2 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 26–32 (IFZ), D-35392 Giessen, Germany
3 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität Wien, Veterinärplatz 1, A-1210 Wien, Austria
Correspondence
Andreas Stolz
Andreas.Stolz{at}imb.Uni-Stuttgart.de
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8c. On the basis of low 16S rRNA gene sequence similarity to other members of the Alcaligenaceae, fatty acid and polar lipid profiles, and other unique phenotypic properties of strain BN9T, the creation of a new genus and species with the name Pusillimonas noertemannii gen. nov., sp. nov. is proposed. The type strain is BN9T (=DSM 10065T=NCIMB 14020T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Pusillimonas noertemannii BN9T is AY695828.
Two-dimensional chromatograph of polar lipids of different members of the Alcaligenaceae is available as supplementary material in IJSEM Online.
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). The metabolism of 5AS by strain BN9T was analysed in detail and it was shown that the strain converted 5AS by an extraordinary ring-fission dioxygenase to a non-aromatic aminated ring-fission product (Stolz et al., 1992; Stolz & Knackmuss, 1993a). The strain also degraded 3-aminobenzoate via 5AS and 2,3-, 2,4- and 2,5-dihydroxybenzoate via different degradative pathways (Russ et al., 1994; Stolz, 1989; Stolz & Knackmuss, 1993b).
The organism was originally tentatively identified as a pseudomonad, but an analysis of its quinone system (Q-8), polyamine pattern (mainly putrescine, spermidine and 2-hydroxyputrescine) and a 158 bp sequence of its 16S rRNA suggested that the strain was closely related to the genus Alcaligenes (Busse et al., 1992). Here we report a detailed characterization of strain BN9T.
Strain BN9T showed only weak growth on all complex [Luria–Bertani (LB) and nutrient broth (NB)] or mineral media tested. Generally, the best growth was observed on chocolate blood agar (Columbia agar base; Oxoid CM331, prepared as recommended by the manufacturer, after cooling to 45 °C, supplemented with 10 % defibrinated sheep blood and boiled) after 3–4 days incubation at 37 °C, which resulted in a colony diameter of approximately 2–3 mm. Growth was slightly slower on blood agar (Oxoid 331, prepared as described above but supplemented with 5 % defibrinated sheep blood and without boiling) under the same conditions. On complex media such as NB or peptone yeast extract (PYE; 0·3 % yeast extract, 0·3 % peptone, 1·5 % agar, pH 7·2) the colonies reached a diameter of about 1–2 mm within 5–6 days at 30 °C. The colonies were brownish, circular with an entire margin. Growth at 37 °C was slightly faster than at 30 °C. In the presence of increased CO2 concentrations (5 %) no visible differences compared with growth at normal O2 and CO2 pressures were observed (at 37 °C). Other growth characteristics are given in the species description below.
The 16S rRNA gene sequence of strain BN9T, containing a continuous sequence of 1524 bp (positions 46–1502 according to the Escherichia coli numbering scheme; Brosius et al., 1978), was used to search the GenBank library. The results of the sequence comparisons demonstrated that strain BN9T was indeed most closely related to different members within the Alcaligenaceae. Phylogenetic trees were generated by using the maximum-parsimony, maximum-likelihood and neighbour-joining methods from the PHYLIP program package (Felsenstein, 1993). In these trees, strain BN9T grouped separately from the clades formed by the genera Achromobacter, Alcaligenes, Bordetella and Kerstersia. A closer relationship was suggested by the sequence comparison programs between strain BN9T and members of the genera Taylorella, Pelistega and Advenella (Fig. 1). Furthermore, all phylogenetic trees suggested that Alcaligenes faecalis DSM 30030T and Alcaligenes defragrans DSM 12141T (Foss et al., 1998) do not form a coherent group within the clade and therefore presumably do not belong to the same genus.
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; Vandamme et al., 1996; Busse & Stolz, 2004). However, the presence of the major compound C19 : 0 cyclo 8c was specific for strain BN9T (Table 1; Busse & Stolz, 2004).
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and Fig. A (supplementary material, IJSEM Online). Each of the five strains showed a distinct polar lipid profile, including the known polar lipids diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as well as several unknown aminophospholipids, aminolipids, phospholipids and further polar lipids. The most pronounced characteristics of strain BN9T were the presence of almost equal amounts of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and rather high amounts of the two characteristic aminolipids AL1 and AL2. In addition to the presence and absence of several minor lipids these characteristics clearly distinguished strain BN9T from the other four strains. In contrast to a previous report of Yabuuchi et al. (1995), who described for Alcaligenes faecalis EY 1056T only the presence of phosphatidylethanolamine (54 %), phosphatidylglycerol (26 %) and ornithine lipid OL-1 (20 %), in the present study diphosphatidylglycerol and several unknown aminophospholipids, phospholipids, aminolipids and polar lipids were also detected [see supplementary Fig. A(II) in IJSEM Online]. Achromobacter denitrificans DSM 30026T and Achromobacter xylosoxidans KM543T were more closely related in their polar lipid profile [see supplementary Fig. A(IV) and (V) in IJSEM Online] to each other than to any other strain studied here, although they differed with regard to the presence of phospholipid PL6 and aminophospholipid 3 and with regard to the relative proportion of phospholipid PL3. The differences observed between Achromobacter xylosoxidans KM543T and Achromobacter denitrificans DSM 30026T supported the view of Coenye et al. (2003a) that the two strains represent different species and not two subspecies of Achromobacter xylosoxidans as favoured by Yabuuchi et al. (1998). The polar lipid profile of Alcaligenes defragrans DSM 12141T showed several unique characteristics [Table 2 and see Supplementary Fig. A(III), IJSEM Online] that might be useful for a future reclassification of this species within a novel genus of the family Alcaligenaceae.
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). Only simple aliphatic and aromatic organic acids were utilized as sources of carbon and energy (for details, see species description below).
The family Alcaligenaceae currently consists of the recognized affiliated genera Alcaligenes, Achromobacter, Advenella, Bordetella and Pigmentiphaga. However, according to 16S rRNA gene sequence comparisons the genera Brackiella, Kerstersia, Oligella, Pelistega, Sutterella and Taylorella should also be included in this family (Busse & Stolz, 2004; Willems et al., 2002; Coenye et al., 2005). Phylogenetically, these genera form together with strain BN9T a homogeneous cluster separate from other taxa within the ‘Betaproteobacteria’. The affiliation of strain BN9T to this family was unambiguously demonstrated by 16S rRNA gene sequence comparisons and phylogenetic analyses. This affiliation was also confirmed based on the presence of ubiquinone Q-8, putrescine and 2-hydroxyputrescine in the polyamine pattern (Busse & Auling, 1988; Busse et al., 1992), and large amounts of 16 : 0 and 17 : 0 cyclo fatty acids present in strain BN9T.
Two subgroups can be defined within the Alcaligenaceae based on the G+C content of their genomic DNA. Members of the genera Alcaligenes, Achromobacter and Pigmentiphaga have G+C contents ranging from 54 to 69 mol%. In contrast, members of the genera Oligella, Taylorella and Pelistega have only relatively low G+C contents of 36·5–47·5 mol% (Busse & Stolz, 2004). The G+C content of strain BN9T was previously determined as 61·8±0·2 mol% (Busse et al., 1992) and thus would clearly suggest a closer relationship of strain BN9T with the free-living members of the genera Alcaligenes, Achromobacter and Pigmentiphaga than with the genera Oligella, Taylorella and Pelistega. This was in clear contrast to the results of the 16S rRNA gene sequence comparisons, which placed strain BN9T close to the ‘low-G+C members' of the Alcaligenaceae.
Thus, strain BN9T could not be affiliated to any of the recognized genera within the family Alcaligenaceae. Furthermore, 16S rRNA gene sequence similarity values between strain BN9T and all other described species within the family Alcaligenaceae were 
96 %. It was previously stated that 16S rRNA gene sequence similarity values among Achromobacter and Bordetella species are 97–98 % and that it could be assumed from these values that the threshold for the differentiation of genera within the family Alcaligenaceae is at <97 % similarity (Blümel et al., 2001). Strain BN9T was also clearly separated from all other members of the Alcaligenaceae based on the presence of trace amounts of 3-OH C16 : 0 and by the large amounts of the fatty acid C19 : 0 cyclo 8c. On the basis of these results, we consider that strain BN9T is representative of a new taxon, for which the name Pusillimonas noertemannii gen. nov., sp. nov. is proposed.
Description of Pusillimonas gen. nov.
Pusillimonas (Pu.sil.li.mo'nas. L. adj. pusillus very small/minute; Gr. fem n. monas unit/monad; N.L. fem. n. Pusillimonas very small monad/unicell, referring to the small size of cells and colonies of the type species).
Cells are Gram-negative, oxidase-positive and have an oxidative metabolism. G+C content of the DNA is 61·8 mol%. Pusillimonas belongs to the family Alcaligenaceae. The major isoprenoid quinone is ubiquinone Q-8. Putrescine, spermidine and 2-hydroxyputrescine are the major polyamines. Major polar lipids are two unknown aminolipids, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. Major non-polar fatty acids are C17 : 0 cyclo, C19 : cyclo 8c, C16 : 0 and summed feature 2 (C16 : 1 iso I/3-OH C14 : 0).
The type species is Pusillimonas noertemannii.
Description of Pusillimonas noertemannii sp. nov.
Pusillimonas noertemannii (noer.te.mann'i.i. N.L. gen. n. noertemannii of Nörtemann, in honour of Bernd Nörtemann, who isolated this and various other bacterial strains that had extraordinary degradative abilities).
The description of the species is as given for the genus above. Cells are rod-shaped (1–1·5 µm in length and 0·5–0·8 µm in diameter). They are motile by means of polarly inserted flagella. On nutrient agar, colonies are brownish, circular and with an entire margin. Grows at 30, 37 and 42 °C, but not at 4 °C. L-Alanine–p-nitroanilide is hydrolysed on the basis of the method described by Kämpfer et al. (1991). The following compounds are not hydrolysed: p-nitrophenyl 
-D-galactopyranoside, p-nitrophenyl -D-glucuronide, p-nitrophenyl 
-D-glucopyranoside, p-nitrophenyl -D-glucopyranoside, p-nitrophenyl -D-xylopyranoside, bis-p-nitrophenyl phosphate, bis-p-nitrophenyl-phenyl phosphonate, bis-p-nitrophenyl-phosphoryl choline, L-aniline–p-nitroanilide, 
-L-glutamate–p-nitroanilide and L-proline–p-nitroanilide. The following compounds are used as sole source of carbon: acetate, propionate, DL-3-hydroxybutyrate, DL-lactate, 2-oxoglutarate, pyruvate and 4-hydroxybenzoate, on the basis of the method described by Kämpfer et al. (1991). The following compounds are not assimilated: N-acetylgalactosamine, N-acetylglucosamine, L-arabinose, L-arbutin, D-cellobiose, D-fructose, D-galactose, gluconate, 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, cis-aconitate, trans-aconitate, adipate, 4-aminobutyrate, azelate, citrate, fumarate, glutarate, itaconate, L-malate, mesaconate, suberate, L-alanine, L-aspartate, L-histidine, L-leucine, L-ornithine, 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. Quinone system, polyamine pattern and polar lipid profile are identical to those listed under the genus description. The fatty acid profile consists of C10 : 0 (trace), C12 : 0 (4·4 %), 2-OH C12 : 0 (3·3 %), C13 : 1 AT 12–13 (trace), summed feature 1 (C12 : 0 alde?) (1·2 %), summed feature 4 (C16 : 1 iso I and/or 3-OH C14 : 0) (8·0 %), C16 : 17c (trace), C16 : 0 (18·5 %), C17 : 0 cyclo (30·9 %), 2-OH C16 : 0 (trace), 3-OH C16 : 0 (trace), C18 : 17c (trace), C18 : 17c 11 methyl (trace), C18 : 0 (2·5 %), C19 : 0 iso (0·9 %), C19 : 0 cyclo 8c (26·8 %) and C20 : 26,9c (trace).
The type strain (BN9T=DSM 10065T=NCIMB 14020T) was isolated from the River Elbe in Germany after enrichment of a mixed bacterial culture with 6-aminonaphthalene-2-sulphonate. The G+C content of the DNA of the type strain is 61·8 mol% (Tm).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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-cis-9,10-methylenehexadecanoic acid; 2-OH C16 : 0, 2-hydroxyhexadecanoic acid; C18 : 1