Curvibacter Fontana Sp. Nov., a Microaerobic Bacteria Isolated from Well Water

J. Gen. Appl. Microbiol., 56, 267‒271 (2010)

Short Communication

Curvibacter fontana sp. nov., a microaerobic bacteria isolated from well water

Linxian Ding1,2,* and Akira Yokota2

1 College of Chemistry and Life Sciences, Zhejiang Normal University, Yingbin Road 688, Jinhua 321004, China 2 Laboratory of Bioresources, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113‒0032, Japan

(Received November 16, 2009; Accepted February 5, 2010)

Key Words—bacterial isolates from well water; Curvibacter fontana sp. nov.

The genus Curvibacter was created by Ding and (Polypepton 1 g, yeast extract 0.2 g, MgSO4･7H2O Yokota (2004), within the family Comamonadaceae 1 g, brain heart infusion 2 g, agar 15 g, distilled water (Willems et al., 1991), to accommodate Gram-nega- 1,000 ml, pH 7.0). Because of the poor growth of these tive, heterotrophic, aerobic, curved rod-formed bacte- strains, we added 10% sterile supernatant of late loga- ria. At present, genus Curvibacter comprises three rec- rithmic growth phase culture broth of Micrococcus lu- ognized species, the type species Curvibacter gracilis, teus IAM 14879T, which was obtained by incubating at Curvibacter delicatus (formerly [Aquaspirillum] delica- 25‒30°C for 7‒10 days followed by centrifugation. The tum), and Curvibacter lanceolatus (formerly [Pseudomo- composition of the medium used for cultivating Micro- T nas] lanceolata) (Ding and Yokota, 2004). The major coccus luteus IAM 14879 was NH4Cl 4 g, KH2PO4 characteristics of the genus Curvibacter are the cell 1.4 g, biotin 5 mg, L-methionine 20 mg, thiamine 40 mg, shape is slightly curved rods, the fl agella arrangement inosine 1 g, MgSO4 70 mg, CuSO4 24 μg, MnCl2 0.5 mg, is polar or none, they are aerobic or microaerobic, the FeSO4 1 mg, Na2MoO4 25 μg, ZnSO4 50 μg, lithium L- colony pigmentation is yellow-brown, and the ubiqui- lactate 10 g, pH 7.5, autoclaving 121°C 25 min (Kapre- none is Q-8. During the survey of microbial diversity of lyants and Kell, 1992). These strains all slowly grew on aqueous environments, three novel bacterial strains, the agar plate under aerobic condition, but were better designated AQ9T, AQ10 and AQ12, were isolated from on the slant under microaerobic conditions. The poly- well water in Osaka, Japan, and studied using a poly- merase chain reaction (PCR) of 16S rRNA gene se- phasic taxonomic approach. On the basis of the re- quences amplifi cation was performed, and sequenc- sults of this study, a novel species Curvibacter fontana ing of the PCR products was carried out as described sp. nov. is proposed. previously (Ding and Yokota, 2004). The 16S rRNA The three strains were grown in PYMB medium gene sequences obtained from the DNA database were aligned using CLUSTAL_X, version 1.83 (Thomp- * Address reprint requests to: Dr. Linxian Ding, College of son et al., 1994). Alignment gaps and ambiguous bas- Chemistry and Life Sciences, Zhejiang Normal University, Ying- es were not taken into consideration. The evolutionary bin Road 688, Jinhua 321004, China. distances [distance options according to the Kimura’s Tel and Fax: +86‒579‒82282269 two-parameter model (Kimura, 1980)] and clustering E-mail: [email protected] The GenBank/EMBL/DDBJ accession numbers for the 16S with the neighbor-joining algorithm (Saitou and Nei, rRNA gene sequences of strains AQ9T, AQ10 and AQ12 are 1987) were determined by using bootstrap values for AB120963, AB120964 and AB120966, respectively. 1,000 replications (Felsenstein, 1985). The similarity 268 DING and YOKOTA Vol. 56 values were calculated using the same software. DNA sured by HPLC according to the method described by was prepared according to the method of Meyer and Mesbah et al. (1989). Biochemical tests were per- Schleifer (1978), and DNA‒DNA hybridization analysis formed with API 20NE test strips (bioMérieux). was performed in microplate wells (Black Maxisorp; The 16S rRNA gene sequence similarities among Nunc) using a fl uorometric method (Ezaki et al., 1989). the isolates, Curvibacter gracilis, C. lanceolatus and C. The fl uorescence intensity was detected by a fl uores- delicatus were 96.4‒97.8%; however, among these cence multi-well Plate Reader (Cytofl uor Series 4000; isolates they were 98.3‒99.9%. The 16S rRNA gene Per Septive Biosystems). The hybridization tempera- phylogenetic tree showed that these strains formed a ture was 53°C with photobiotin-labeled DNA. Cellular cluster with C. gracilis IAM 15033T, C. lanceolatus IAM fatty acids were extracted according to the protocol of 14947T and C. delicatus IAM 14955T with a higher the MIDI system, analysis by gas chromatography was bootstrap value (821), and they were separated from controled by MIS software (Microbial ID, Inc.) and the those of related and recently reported genera Pseu- peaks were automatically integrated and identifi ed by dorhodoferax (Bruland et al., 2009), Caenimonas (Ryu the Microbial Identifi cation software package (Sasser, et al., 2008), and Ramlibacter (Heulin et al., 2003) 1990). Isoprenoid quinones were extracted from (Fig. 1). Chromosomal DNA‒DNA hybridization stud- freeze-dried cells with chloroform/methanol (2：1, v/v) ies were performed to establish whether the isolates and were purifi ed by TLC by using n-hexane/diethyl AQ9T and other related species represent a distinct ether (85：15, v/v) as the solvent. The ubiquinone frac- species. Strain AQ9T displayed low levels of DNA‒DNA tion was extracted with acetone, dried under a nitro- reassociation (17%) with the type species C. gracilis gen gas stream and then analyzed by HPLC (model IAM 15033T of the genus Curvibacter; those with C. LC-10A apparatus; Shimadzu) with a Nacalai ODS lanceolatus IAM 14947T and C. delicatus IAM 14955T 5C18 column (4.6 × 150 mm). Genomic DNA was were 11% and 23%, respectively. These results are be- prepared according to the method of Sambrook et al. low the cut-off point recommended for the circum- (1989). The G+C content of the total DNA was mea- scription of bacterial genomic species by Wayne et al.

Fig. 1. Phylogenetic tree displaying the relationships among the strains AQ9T, AQ10 and AQ12, and the members of the families Comamonadaceae. Bootstrap values of 1,000 resamplings are shown at the branch points, and only bootstrap values above 500 are shown. 2010 Curvibacter fontana a microaerobic bacteria 269

(1987), and conﬁ rm the separation of the isolates AQ9T Q-8, and the G + C content of the DNA of strains AQ9T, from C. gracilis, C. lanceolatus and C. delicatus. At the AQ10 and AQ12 were 66.6, 66.0 and 66.7 mol%, re- same time, the results of DNA‒DNA hybridization stud- spectively. The classiﬁ cation of isolates AQ9T, AQ10 ies showed that the binding values of DNA‒DNA of the and AQ12 within the genus Curvibacter is supported strain AQ9T and AQ10 was 95%, while strain AQ9T and by phenotypic, morphological and biochemical char- AQ12 was 98%. Thus, the high level of DNA‒DNA re- acteristics. Table 1 summarizes morphological and latedness strongly suggests that the three strains were physiological characteristics of the isolates and the members of a single species. Furthermore, the major members of the genus Curvibacter. Table 2 shows ma- quinone type of these isolates were all the ubiquinone jor fatty acids of these strains were C15:0, C16:0, C16:1

Table 1. Morphological and physiological characteristics of the members of the genus Curvibacter.

C. fontana C. fontana C. fontana C. gracilis C. delicatus C. lanceolatus Characteristic AQ9T AQ10 AQ12 IAM 15033T IAM 14955T IAM 14947T Cell size (μm) 0.4 × 1.8 0.4 × 1.8 0.4 × 1.8 0.5 × 1.4 0.3 × 0.7a 0.6 × 1.8b Optimal temperature for growth (°C) 25‒30 25‒30 25‒30 25‒30 30‒32a 20‒30b Optimal pH for growth 7 7 7 5.0‒8.0 5.5‒8.5a neutrophilicb API 20 NE test: L-Arginine - + - + -- Urea + (+) - + - + Esculin ----+ - Gelatin ----+ - Glucose + + - (+) (+) - D-Mannose - + ---(+) Mannitol + ---(+) - Maltose + - + - (+) - Gluconate + - ++-- DNA G+C content (mol%) 66.6 66.0 66.7 66.2 62.2 66.0

Symbols used: +, positive; -, negative; (+), weak positive. Data from: a, Krieg (1984); b, Leifson (1962), others were obtained in this study.

Table 2. The fatty acid compositions of isolates and related species of the genus Curvibacter.

C. fontana C. fontana C. fontana C. gracilisa C. delicatusb C. lanceolatusa Strains AQ9T AQ10 AQ12 IAM 15033T IAM 14955T IAM 14947T

C12:0 ND ND ND 3.0 1.0 3.6 C14:0 4.1 3.2 3.7 0.9 ND 1.2 C15:0 11.4 12.6 5.3 0.7 ND ND C16:0 21.7 22.4 43.1 19.7 32.0 16.9 C17:0 3.4 4.4 1.5 ND 2.0 ND C19:0 ND ND ND ND 8.0 ND C16:1 ω7c and/or C15:0 iso 2-OH 29.4 24.8 28.9 44.6 35 (C16:1) 41.9 C15:1 ω6c 3.1 3.3 1.1 ND ND ND C17:1 ω6c 3.3 ND ND 1.5 ND ND C18:1 ω6c ND ND ND 3.4 ND ND C18:1 ND ND ND ND 23.0 ND C18:1 ω7c 9.2 8.6 14.5 25.0 ND 35.5 C8:0 3-OH ND ND ND 0.7 present 0.9 C10:0 3-OH 5.3 4.9 9.1 ND ND ND C17:0 CYCLO 5.7 11.3 17.3 ND ND ND

Data from: a, Ding and Yokota (2004); b, Sakane and Yokota (1994), others were obtained in this study. ND (not detected). 270 DING and YOKOTA Vol. 56

Table 3. Differential characteristics of the members of the genus Curvibacter.

Characteristic C. fontana C. gracilis C. delicatus C. lanceolatus Cell size (μm) 0.4 × 1.8 0.5 × 1.4 0.3 × 0.7a 0.6 × 1.8b Optimal temperature for growth (°C) 25‒30 25‒30 30‒32a 20‒30b Optimal pH for growth 7 5.0‒8.0 5.5‒8.5a neutrophilicb API 20 NE test: Esculin --+ - Gelatin --+ - Fatty acid: C12:0 - +++ C15:1 ω6c + --- C17:0 CYCLO + --- C8:0 3-OH - +++ C10:0 3-OH + --- DNA G+C (mol %) 66.0‒66.7 66.2 62.2 66.0

Symbols used: +, positive; -, negative; (+), weak positive. Data from: a, Krieg (1984); b, Leifson (1962), others were obtained in this study.

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