Brevundimonas halotolerans sp. nov., Brevundimonas poindexterae sp. nov. and Brevundimonas staleyi sp. nov., prosthecate bacteria from aquatic habitats

Wolf-Rainer Abraham,1 Andre´ia B. Estrela,1 Dennis I. Nikitin,2 John Smit3 and Marc Vancanneyt4

Correspondence Wolf-Rainer Abraham [email protected]
1Helmholtz Center for Infection Research, Chemical Microbiology, Inhoffenstrasse 7, 38124 Braunschweig, Germany
2Institute of Microbiology, Russian Academy of Sciences, Prospect 60-Letiya Octyabrya 7, korp. 2, Moscow 117811, Russia
3University of British Columbia, Dept of Microbiology and Immunology, Vancouver, BC, Canada 4BCCM/LMG Bacteria Collection and Laboratory of Microbiology, Ghent University, Ghent, Belgium

Eight strains of Gram-negative, bacteroid-shaped, prosthecate bacteria, isolated from brackish water (MCS24T, MCS17 and MCS35), the marine environment (CM260, CM272 and CM282) and activated sludge (FWC40T and FWC43T), were characterized using a polyphasic approach. Analysis of 16S rRNA gene sequences determined that all strains were affiliated to the alphaproteobacterial genus Brevundimonas, forming three distinct phyletic lineages within the genus. The strains grew best with 5–30 g NaCl l”1 at 20–30 6C. DNA G+C contents for strains MCS24T, FWC40T and FWC43T were between 65 and 67 mol%, in accordance with values reported previously for other species of the genus. Moreover, chemotaxonomic data and physiological and biochemical tests allowed the phenotypic differentiation of three novel species within the genus Brevundimonas, for which the names Brevundimonas halotolerans sp. nov. (type strain MCS24T 5LMG 25346T 5CCUG 58273T), Brevundimonas poindexterae sp. nov. (type strain FWC40T 5LMG 25261T 5CCUG 57883T) and Brevundimonas staleyi sp. nov. (type strain FWC43T 5LMG 25262T 5CCUG 57884T) are proposed.

In a previous study, caulobacteria from a broad range of freshwater, brackish water, marine and soil habitats (Anast
& Smit, 1988; MacRae & Smit, 1991; Segers et al., 1994) were studied using a polyphasic approach. As a result, the descriptions of the genera Caulobacter and Brevundimonas

Belgium (LMG), and from one of the authors (J. S.) (CM, FWC and MCS strains) (Table 1). The strains were grown in freshwater Caulobacter medium PYEM (2 g peptone, 2 g yeast extract and 0.5 g NH4Cl per litre MQ water). After autoclaving and cooling, 5 ml sterile-filtered

were emended and a number of Caulobacter species were
riboflavin (0.2 mg ml
21
), 2 ml 50 % glucose (sterile), 1 ml

transferred to the genus Brevundimonas (Abraham et al., 1999). We report here on three novel species within the genus Brevundimonas which emerged from this study.
Strains used in this study were obtained from the American Type Culture Collection (ATCC), the Deutsche Sammlung fu¨r Mikroorganismen und Zellkulturen (DSMZ) and the Laboratorium voor Microbiologie, Universiteit Gent,
20 % MgSO4 (sterile) and 1 ml 10 % CaCl2 (sterile) were added. The strains were grown in 2 l flasks at 30 uC with shaking at 100 r.p.m. and biomass was harvested in the late exponential phase, after 72 h.
For the determination of DNA base compositions, genomic DNA was isolated from 2 ml culture using the DNeasy kit (Qiagen). DNA was digested enzymically and mean G+C

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains MCS24T, FWC40T, FWC43T, MCS17, CM260, CM272, CM282 and MCS35 are M83810, AJ227797–AJ227799 and FN397630–FN39763, respectively.
A 16S rRNA gene sequence-based UPGMA tree and details of phospho- and sulfolipids are available as supplementary material with the online version of this paper.
contents were determined by HPLC (Tamaoka &
Komagata, 1984). Calculations were carried out according to Mesbah et al. (1989), with non-methylated lambda phage DNA (Sigma) as a standard. For all strains, G+C contents between 64.6 and 67.0 mol% were found, within the range of those already reported for species of the genus Brevundimonas (Vancanneyt et al., 2005).

Table 1. Strains used in this study

Accession numbers of sequences determined in this study are in bold.

Strain Origin 16S rRNA gene sequence
accession no.

Brevundimonas halotolerans sp. nov.
CM260

J. Poindexter, Woods Hole, MA, USA

FN397630

CM272 J. Poindexter, Woods Hole, MA, USA FN397631
CM282 J. Poindexter, Woods Hole, MA, USA FN397632

MCS17
Brackish water slough adjacent to Arness Park,
Kingston, WA, USA
AJ227799

MCS24T
Brackish water creek flowing into salt water at
Carkeek Park, Seattle, WA, USA
M83810

MCS35 Water of the Baltic Sea north of Rostock, Germany FN397633

Brevundimonas poindexterae sp. nov.
FWC40T

Secondary treatment facility, activated sludge system,
Kelowna, Canada

AJ227797

Brevundimonas staleyi sp. nov.

FWC43T Secondary treatment facility, activated sludge system,
Calgary, Canada
AJ227798

The isolates were identified phylogenetically by sequencing of the 16S rRNA genes and by comparison of the sequences with those of type strains. For the PCR, DNA from single colonies was used which had been lysed by exposure to 100 ml TE buffer for about 10 min at 95 uC. Nearly complete 16S rRNA gene sequences were obtained as described previously (Yakimov et al., 2003). The reactions were evaluated on an Applied Biosystems 377 Genetic Analyzer. The program SEQUENCHER version 4.0.5 (Gene Codes Corporation) was used to analyse the sequences. Phylogenetic analysis was made using the CLUSTAL W software (Thompson et al., 1997) for the alignments and the neighbour-joining algorithm and bootstrap percentages based on 1000 replications (Fig. 1) and the UPMGA algorithm with Kimura’s two-parameter model was calculated in the software MEGA 3.1. (Kumar et al., 2004) (Supplementary Fig. S1, available in IJSEM Online) using sequences contained in the EMBL database (Kanz et al., 2005). The 16S rRNA gene sequences determined have been deposited in the EMBL nucleotide database under the accession numbers listed in Table 1. The similarity of 16S rRNA gene sequences was 97.8, 97.5 and 97.3 % between FWC40T and Brevundimonas lenta DS-18T, Brevundimonas subvibrioides CB81T and Brevundimonas bullata DSM 7126T, respectively, and 96.8 and 96.5 % between MCS24T and Brevundimonas variabilis ATCC 15255T and Brevundimonas bacteroides CB7T, respectively. The identity between the 16S rRNA gene sequences of FWC43T and B. bullata DSM 7126T (Kang et al., 2009) was 98.6 %, but the two strains differ sharply in their cell morphology.
For whole-cell fatty acid analysis, cells were saponified [15 % (w/v) NaOH, 30 min, 100 uC], methylated to fatty

extracted (hexane/methyl-tert-butyl ether; 1 : 1, v/v) as described in detail by Osterhout et al. (1991). Fatty acid methyl esters were analysed on an HP 5890A gas chromatograph. Separation of fatty acid methyl esters was achieved with a fused-silica capillary column (25 m by 0.2 mm) with cross-linked 5 % phenyl methyl silicone (film thickness 0.33 mm; HP Ultra 2). The computer- controlled parameters were the same as those described by Osterhout et al. (1991). The instrument was equipped with a flame-ionization detector and an autosampler (HP 7673). The main fatty acids were summed feature 7 (one or more of C18 : 1v7c, C18 : 1v9t and C18 : 1v12t) and C16 : 0 for all strains; the main hydroxy fatty acid was always C12 : 0 3- OH. Strain FWC40T had a rather large amount of 11- methyl-12-trans-octadecanoic acid (11-methyl C18 : 1v5t; ECL 18.080) (Abraham et al., 2008), the highest yet reported for a Brevundimonas strain (Table 2).
Polar lipid fatty acid analysis with fast-atom-bombardment mass spectrometry (FAB-MS) was performed in the negative mode on the first of two mass spectrometers of a tandem high-resolution instrument in an E1B1E2B2 configuration (JMS-HX/HX110A; JEOL) using the condi- tions reported by Abraham et al. (1997). In all strains, phosphatidylglycerol, 1,2-di-O-acyl-3-O-[b-D-glucopyra- nosyl-(1A4)-a-D-glucopyranuronosyl]glycerol, lyso-phos- phoglucolipid and 1,2-di-O-acyl-3-O-[69-(sn-10,20-di-O- acyl-glycero-30-phosphoryl)-a-D-glucopyranosyl]-sn-gly- cerol were present. Furthermore, sulfoquinovosyl dia- cylglycerols could be detected in strains CM260, CM272, CM280, MCS17 and MCS24T (Supplementary Table S1).
For phenotypic characterization, strains were grown in 20 ml PYEM medium amended with 0, 5, 10, 20, 30, 40, 60,

acid methyl esters (methanolic HCl, 10 min, 80 uC) and 80 or 100 g NaCl l21 at 30 uC. The OD600 of the cell

Fig. 1. Unrooted neighbour-joining dendro- gram of the phylogenetic relationships
between Brevundimonas poindexterae sp. nov. FWC40T, Brevundimonas staleyi sp. nov. FWC43T, Brevundimonas halotoler- ans sp. nov. MCS24T (and five other strains) and all recognized type strains of the genus Brevundimonas based on a distance-matrix analysis of 16S rRNA gene sequences. GenBank accession numbers are given in parentheses. The sequence of Hirschia baltica ATCC 49814T was used as an outgroup (not shown). Bootstrap percentages .50 % are indicated at tree branching points. Bar, 0.005 substitutions per nucleotide position.

suspension was determined at the beginning of the experiment and after 2 days. The difference between these two measurements was used to determine salt tolerance. All
growth, albeit slow, with salt concentrations up to 80 g l Strain FWC40T showed reduced growth without NaCl.
21
.

strains could grow with salt concentrations of 5–30 g l21 but not with 100 g l21. Strain MCS24T also showed
Substrate specificity tests were conducted by the use of API Biotype 100 and API 20 NE test strips (bioMe´rieux) using

Table 2. Fatty acid contents of whole-cell hydrolysates of Brevundimonas strains

Strains: 1, B. alba ATCC 15265T; 2, B. aurantiaca ATCC 15266T; 3, B. bacteroides LMG 15096T; 4, B. diminuta LMG 2089T; 5, B. intermedia ATCC 15262T; 6–10, B. halotolerans sp. nov. strains CM260 (6), CM272 (7), CM282 (8), MCS17 (9) and MCS24T (10); 11, B. poindexterae sp. nov. FWC40T; 10, B. staleyi sp. nov. FWC43T; 13, B. subvibrioides LMG 14903T; 14, B. variabilis ATCC 15255T; 15, B. vesicularis LMG 2350T; 16, B. bullata DSM 7126T. Data were obtained in this study. Values are percentages of total fatty acids; only fatty acids accounting for more than 1.0 % (mean amount) are indicated. The following strains contained significant amounts (.1.0 %) of additional fatty acids: B. subvibrioides LMG 14903T also contained 20 : 2v6,9c (1.6 %); B. alba also contained 16 : 1v9c (2.7 %). tr, Trace amount (,1.0 %); 2, not detected; ECL, unknown fatty acid identified by equivalent chain length.

Fatty acid 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
12 : 0 3-OH 1.1 2.3 2.3 1.5 1.7 1.3 1.2 1.2 1.2 1.0 3.1 2.2 2.8 2.3 1.9 1.3
12 : 1 3-OH tr tr 2 tr 2 2 2 2 2 2 2 2 2 2 2 tr
14: 0 1.0 3.4 3.0 tr 1.5 1.5 1.4 1.4 1.0 1.3 1.7 3.2 4.6 2.5 2.4 tr
15: 0 5.4 3.0 tr 7.6 2.8 6.5 6.7 7.7 6.7 1.2 2.8 6.4 1.5 5.4 4.0 5.1
ECL 15.275 2.0 2 2 2 2 tr tr tr 1.5 1.2 2 2 2 2 2 2
16: 0 16.8 21.3 12.8 10.1 24.3 16.7 16.3 16.4 12.3 13.8 19.2 14.8 15.9 13.5 20.7 18.9
Summed feature 4* 5.9 4.0 6.7 1.0 7.5 5.6 5.9 5.2 6.4 12.8 4.1 10.6 11.2 6.2 5.1 4.5
17: 1v6c 2.5 1.7 tr 8.5 1.6 2.8 2.6 3.6 3 tr tr 1.6 tr 2.2 2.4 1.6
17: 1v8c 1.5 1.0 tr 6.2 1.1 1.6 1.7 1.9 3 tr tr 2.2 tr 2.5 1.4 1.5
17: 1 7.7 1.6 1.4 10.8 2.0 4.1 4.2 4.6 7.5 1.6 2.6 5.6 1.8 5.7 2.6 3.3
ECL 17.897 1.4 1.3 tr 1.1 tr 2 2 2 1.5 tr 1.3 1.1 tr tr 1.1 1.1
18: 0 tr tr tr tr tr 2 tr tr 2 tr 2 2 1.4 tr tr tr
Summed feature 7* 43.2 56.5 69.4 38.7 49.4 51.5 50.0 48.4 51.7 60.4 52.8 50.2 56.7 55.8 53.7 55.5
18: 1v9c 1.0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
11-Methyl 18 : 1v5t 2.7 3.5 tr 2 6.3 3.3 4.0 3.6 1.1 1.9 8.2 tr tr 2 4.5 1.6
ECL 18.797 tr 2 2 3.1 2 tr tr tr 2 2 2 tr 2 1.0 2 tr
19: 0 cyclo v8c 2.4 2 2 6.2 2 2 2 2 2 2 2 2 2 2 2 1.1

*Summed features consist of one or more fatty acids that could not be separated by the Microbial Identification System. Summed feature 4, one or more of iso-15 : 0 2-OH, 16 : 1v7c and 16 : 1v7t; summed feature 7, one or more of 18 : 1v7c, 18 : 1v9t and 18 : 1v12t.

the protocols supplied by the manufacturer. The test strips were incubated at 30 uC for 14 days and monitored three times a week. A test was considered positive if the interface between sample well and air was visibly turbid due to bacterial growth after incubation for 14 days (Ru¨ger &
Krambeck, 1994). Only strain MCS24T could reduce nitrate to nitrite. The results for individual strains are given in the species descriptions.

For enzyme activity tests, API ZYM test strips (bioMe´rieux) were used according to the protocol supplied by the manufacturer. Strains MCS24T and FWC43T showed weak lipase (C14) activity, which was absent from FWC40T. Leucine arylamidase activity was strong in all strains tested. Cystine arylamidase activity was strong in MCS24T,
moderate in FWC43T but weak in FWC40T; acid phosphatase activity was weak in MCS24T but strong in all other tested strains. The strains also differed in a- glucosidase activity, which was strong in MCS24T and FWC43T but absent from FWC40T. b-Glucosidase activity

Due to the heterogeneity of the isolates, three different taxa can be discerned and proposals are made of three novel species of Brevundimonas. Discriminating characteristics of the newly proposed species and their nearest neighbours are summarized in Table 3.

Description of Brevundimonas halotolerans sp. nov.
Brevundimonas halotolerans (ha.lo.to9le.rans. Gr. n. hal, halos salt; L. part. adj. tolerans tolerating; N.L. part. adj. halotolerans salt tolerating).
The description is the same as that given for the genus (Segers et al., 1994; emended by Abraham et al., 1999), with the following additional characteristics. Gram-negative, aerobic, non-spore-forming, prosthecate cells; colonies white coloured, cells bacteroid. B. halotolerans has complex substrate requirements and shows optimal growth on peptone-yeast extract medium with 0–40 g NaCl l21. With 60–80 g NaCl l21, growth is observed, although reduced;

was found only in FWC43T.
no growth is found with 100 g NaCl l
21
. Grows optimally

Table 3. Characteristics that are useful in discerning the novel species

Strains: 1, B. poindexterae sp. nov. FWC40T; 2, B. staleyi sp. nov. FWC43T; 3, B. halotolerans sp. nov. MCS24T; 4, B. terrae KSL-145T (data from Yoon et al., 2006); 5, B. diminuta LMG 2089T; 6, B. bullata DSM 7126T (data in columns 5 and 6 from Fritz, 2000); 7, B. variabilis ATCC 15255T; 8, B. bacteroides CB7T; 9, B. lenta DS-18T (data in columns 7–9 from Yoon et al., 2007). ++, Strongly positive; +, positive; W, weakly positive; 2, negative; ND, no data available.

Characteristic 1 2 3 4 5 6 7 8 9
Utilization of:
a-D-Glucose + + 2 2 2 + 2 2 2
D-Galactose 2 2 2 2 2 2 2 W 2
D-Mannose 2 + 2 2 2 2 2 2 2
Maltotriose + 2 2 ND 2 2 + 2 ND
Maltose + 2 2 2 2 2 2 2 2
Cellobiose + + 2 2 2 2 2 2 2
Aesculin + + + 2 2 ND + + +
DL-Lactate 2 + 2 2 2 2 + 2 2
Succinate + + 2 2 2 2 2 2 2
Fumarate + + 2 ND 2 2 2 + ND
L-Proline + + 2 + ++ + 2 2 +
D-Alanine 2 2 2 ND + + 2 2 2
L-Alanine 2 W 2 ND + + 2 2 2
Activity of:
Lipase (C14) 2 ++ W ND 2 2 W 2 2
Valine arylamidase + ++ ++ ND 2 ++ + + 2
Cystine arylamidase W + ++ ND 2 2 2 2 2
a-Chymotrypsin W + 2 ND W W W W 2
Acid phosphatase ++ ++ W ND ++ ++ W W +
Naphthol-AS-BI-phosphohydrolase ++ ++ ++ ND ++ ++ W W +
a-Glucosidase 2 + ++ ND 2 W 2 2 +
b-Glucosidase 2 + 2 ND 2 2 2 2 2
Protease 2 + + ND ND + ND ND +
b-Galactosidase 2 + 2 2 ND 2 ND ND 2
Reduction of nitrates to nitrites 2 2 + 2 ND 2 ND ND 2
DNA G+C content (mol%) 67.0 66.5 64.6 61.8 67 66.7 ND 66 68.7

at 20–40 uC; slow growth at 10 uC and no growth at 5 or 50 uC. Nitrate is reduced to nitrite but not to nitrogen. Shows strong activities of alkaline phosphatase, esterase (C4), esterase/lipase (C8), naphthol-AS-BI-phosphohydro- lase, leucine, valine and cystine arylamidases, trypsin, a- glucosidase and protease and weak activities of lipase (C14) and acid phosphatase. All strains are characterized by two major fatty acids, C18 : 1v7 and C16 : 0; minor fatty acids are C15 : 0, C16 : 1v7, iso-C17 : 1v8, C17 : 0 and 11-methyl C18 : 1v5c. Polar lipids are a-D-glucopyranosyl, a-D-glucur- onopyranosyl, D-glucopyranosyl-(1A4)-a-D-glucopyra-
nuronosyl, sulfoquinovosyl, phosphatidyl and 6-
phosphatidyl-a-D-glucopyranosyl diacylglycerols. The G+C content of the type strain is 64.6 mol%. Known isolates have been obtained from brackish water or seawater.
The type strain, MCS24T (5LMG 25346T 5CCUG 58273T), was isolated from brackish water of a creek flowing into salt water at Carkeek Park, Seattle, WA, USA. The species is widespread in the marine environment and is known from the Canadian Pacific coast (strains MCS17 and MCS24T), the North American Atlantic coast (strains CM260, CM272 and CM282), the Tasman Sea near New Zealand (strains CDF5, CDF18 and CDF35) (Fenton, 1994) and the Baltic Sea (strain MCS35). Its ability to grow both in fresh water and in ocean water may be one reason for this wide distribution.

Description of Brevundimonas poindexterae sp. nov.
Brevundimonas poindexterae (poin.dex9ter.ae. N.L. gen. n. poindexterae of Poindexter, named to honour Jeanne S. Poindexter, who contributed much to our current understanding of the Caulobacterales).
The description is the same as that given for the genus (Segers et al., 1994; emended by Abraham et al., 1999), with the following additional characteristics. Gram-negative, aerobic, non-spore-forming, prosthecate cells; colonies tan coloured, cells bacteroid. No S layer is detected and multiple bands of polysaccharides are observed (Walker et al., 1992). The species can grow on peptone-yeast extract

tagatose, glycerol, myo-inositol, D-mannitol, maltitol, (+)- turanose, D-sorbitol, adonitol, D-lyxose, i-erythritol, 1-O- methyl and 3-O-methyl a-D-glucopyranoside, saccharate, mucate, (+)-L-, (2)-D- and meso-tartrate, (+)-D-malate, cis- and trans-aconitate, tricarballylate, citrate, D- glucuronate, 2-keto-D-gluconate, N-acetyl-D-glucosamine, D-gluconate, phenylacetate, protocatechuate, 4-hydroxy-
benzoate, (2)-quinate, gentisate, 3-hydroxybenzoate, benzoate, m-coumarate, trigonelline, betaine, putrescine, 4-aminobutryate, histamine, DL-lactate, glutarate, DL-gly- cerate, 5-aminovalerate, ethanolamine, tryptamine, itaco- nate, L-aspartate, L-glutamate, D- and L-alanine, L-serine, malonate, L-tyrosine and 2-ketoglutarate are not used. Enzymic activity of alkaline and acid phosphatases, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase and b-glucosidase is present. Nitrate is not reduced to nitrite or to nitrogen. Polar lipids are a-D-glucopyranosyl,
a-D-glucuronopyranosyl, D-glucopyranosyl-(1A4)-a-D- glucopyranuronosyl, phosphatidyl and 6-phosphatidyl-a- D-glucopyranosyl diacylglycerols. Characterized by two major fatty acids, C18 : 1v7 and C16 : 0; minor fatty acids are C15 : 0, C16 : 1v7, C17 : 1v8 and 11-methyl C18 : 1v5. The main hydroxy-fatty acid is C12 : 0 3-OH. The G+C content of the type strain is 67.0 mol%.

The type strain is FWC40T (5LMG 25261T 5CCUG 57883T), isolated from activated sludge from a secondary treatment facility at Kelowna, British Columbia, Canada.

Description of Brevundimonas staleyi sp. nov.
Brevundimonas staleyi (sta9ley.i. N.L. gen. n. staleyi of Staley, named to honour the American microbiologist James T. Staley for his contribution to the knowledge of the caulobacteria).

The description is the same as that given for the genus (Segers et al., 1994; emended by Abraham et al., 1999), with the following additional characteristics. Gram-negative, aerobic, non-spore-forming, prosthecate cells; colonies bright yellow coloured, cells bacteroid. No S layer is detected and multiple bands of polysaccharides are observed (Walker et al., 1992). Can grow on peptone-

medium without NaCl, but optimal growth occurs with 5–
yeast extract medium with 0–40 g NaCl l
21
, with optimal

30 g NaCl l
21
. Can not tolerate salt concentrations above
growth at 5–30 g NaCl l
21
. Does not tolerate salt

60 g NaCl l21. Grows optimally at 20–30 uC; slow growth
concentrations above 60 g NaCl l
21
. Grows best at 20–

at 40 uC, no growth at 10 or 50 uC. Can use pyruvate, maltotriose, maltose, 1-O-methyl a-galactopyranoside, cellobiose, aesculin, xylose, glucose, rhamnose, malate, galacturonate, succinate, fumarate, 3-hydroxybutyrate, asparagine, L-glutamate and L-proline. In contrast, b- (+)-D-fructose, (+)-D-galactose, trehalose, (+)-D-man- nose, (+)-L-sorbose, (+)-melibiose, sucrose, (+)-raffi- nose, lactose, lactulose, 1-O-methyl b-galactopyranoside, (+)-gentiobiose, 1-O-methyl b-glucopyranoside, (2)-D- ribose, (+)-L-arabinose, palatinose, a-L-fucose, (+)-mele- zitose, (+)-D- and (2)-L-arabitol, xylitol, dulcitol, D-
40 uC; slow growth at 10 uC and no growth at 5 or 50 uC. D-Glucose, D-mannose, cellobiose, gentiobiose, 1-O-methyl b-glucopyranoside, aesculin, a-L-rhamnose, gentisate, DL- lactate, succinate, fumarate, 3-hydroxybutyrate, L-aspar- tate, L-glutamate, L-proline, L-alanine, malonate and L- tyrosine are used as substrates. b-(+)-D-Fructose, (+)-D- galactose, (+)-trehalose, (+)-L-sorbose, (+)-melibiose, sucrose, (+)-raffinose, maltotriose, maltose, lactose, lac- tulose, 1-O-methyl a- and b-galactopyranoside, (+)- gentiobiose, (2)-D-ribose, (+)-L-arabinose, (+)-D-xylose, palatinose, a-L-fucose, (+)-melezitose, (+)-D- and (2)-L-

arabitol, xylitol, dulcitol, D-tagatose, glycerol, myo-inositol, D-mannitol, maltitol, (+)-turanose, D-sorbitol, adonitol, D-lyxose, i-erythritol, 1-O-methyl and 3-O-methyl a-D- glucopyranoside, saccharate, mucate, (+)-L-, (2)-D- and meso-tartrate, (+)-D- and (2)-L-malate, cis- and trans- aconitate, tricarballylate, citrate, D-glucuronate, D-galac- turonate, 2-keto-D-gluconate, N-acetyl-D-glucosamine, D- gluconate, phenylacetate, protocatechuate, 4-hydroxy- benzoate, (2)-quinate, gentisate, 3-hydroxybenzoate, benzoate, m-coumarate, trigonelline, betaine, putrescine, 4-aminobutyrate, histamine, glutarate, DL-glycerate, 5- aminovalerate, ethanolamine, tryptamine, itaconate, L- glutamate, D-alanine, L-serine, malonate, L-tyrosine and 2-ketoglutarate are not oxidized. Activities of alkaline and acid phosphatases, esterase (C4), esterase lipase (C8), leucine and valine arylamidases, trypsin, phosphatase, naphthol-AS-BI-phosphohydrolase, a-glucosidase, b-glu- cosidase, b-galactosidase and oxidase and weak activities of lipase (C14), cystine arylamidase and a-chymotrypsin are present. Nitrate is not reduced to nitrite or to nitrogen. Polar lipids are a-D-glucopyranosyl, a-D-glucuronopyrano- syl, D-glucopyranosyl-(1A4)-a-D-glucopyranuronosyl, phosphatidyl and 6-phosphatidyl-a-D-glucopyranosyl dia- cylglycerols. Characterized by two major fatty acids, C18 : 1v7 and C16 : 0; minor fatty acids are C15 : 0, C16 : 1v7, C17 : 1v8, C14 : 0 and C12 : 0 3-OH. The G+C content of the type strain is 66.5 mol%; genome size of the type strain is 2.26109 Da. Closely related to B. bullata by 16S rRNA gene sequence similarity, but clearly distinct by morpho- logy and cell cycle.

The type strain is FWC43T (5LMG 25262T 5CCUG 57884T), isolated from activated sludge of a secondary treatment facility at Calgary, Alberta, Canada.

Acknowledgements
We are indebted to Dagmar Wenderoth, Annette Kru¨ger and Peter Wolff for their excellent technical assistance. This work was supported by grants of the German Federal Ministry for Science, Education and Research (projects no. 0319433C and 01 KI 07 96) and the European Union within the T-project ‘High Resolution Automated Microbial Identification and Application to Biotechnologically Relevant Ecosystems’.

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