Albidovulum Xiamenense Sp. Nov., a Moderately Thermophilic Bacterium from a Terrestrial Hot Spring

International Journal of Systematic and Evolutionary Microbiology (2012), 62, 1609–1612 DOI 10.1099/ijs.0.034454-0

Albidovulum xiamenense sp. nov., a moderately thermophilic bacterium from a terrestrial hot spring

Decui Yin,1,2 Jing Xiao,2 Jingqun Ao,2 Chunxiang Ai1 and Xinhua Chen2

Correspondence 1School of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, Fujian, Xinhua Chen PR China [email protected] 2Laboratory of Marine Biogenetic Resources of State Oceanic Administration, Chunxiang Ai Third Institute of Oceanography, Xiamen 361005, Fujian, PR China [email protected]

An aerobic, motile, moderately thermophilic, rod-shaped bacterium, strain YBY-7T, was isolated from a terrestrial hot spring of a garden exhibition located in Xiamen City, Fujian Province, People’s Republic of China. Cells of strain YBY-7T were Gram-negative, irregular rods, 2–6 mm long and 0.4–0.6 mm wide, with polar flagella, and the organism formed beige colonies. The temperature and pH ranges for growth of strain YBY-7T were 28–65 6C (optimum 50–58 6C) and pH 6.5–9.5 (optimum pH 7.5–8.5). Growth occurred in the presence of 5.5 % NaCl (optimum 3.0 %). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that Albidovulum inexpectatum FRR-10T was its closest neighbour (95.9 % similarity). Ubiquinone (Q-10) was the sole respiratory quinone and the DNA G+C content of strain YBY-7T was 70.6 mol%. The predominant polar lipids were phosphatidylcholine, phosphatidylglycerol and

phosphatidylethanolamine. The major fatty acids were C18 : 1v7c (29.3 %), C19 : 0 cyclo v8c

(25.6 %), C18 : 0 (23.6 %) and C16 : 0 (9.6 %). Based on its physiological characteristics and our phylogenetic analysis, we propose that strain YBY-7T represents a novel species of the genus Albidovulum, for which the name Albidovulum xiamenense sp. nov. is proposed, with the type strain YBY-7T (5MCCC 1A06317T 5CGMCC 1.10789T 5LMG 26247T).

The genus Albidovulum was proposed by Albuquerque 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 .6H2O, 5.94 g et al. (2002) for a non-pigmented, Gram-negative, short MgSO4 .7H2O and 1.3 g CaCl2 .2H2O (Bruns et al., rod-shaped proteobacterium. At the time of writing, it 2001). The strain was maintained routinely in the same comprised only one species, Albidovulum inexpectatum. medium and as a glycerol suspension (20 %, v/v) at Membersofthisgenusareslightlythermophilicand 270 uC. Culture characteristics were determined after halophilic and oxidase- and catalase-positive. The major 2 days at 55 uC. Morphological properties, spores and phospholipids are phosphatidylethanolamine (PE), phos- mycelium were examined by light microscopy (Nikon; phatidylcholine (PC) and phosphatidylglycerol (PG). HFX-DX) and transmission electron microscopy (JEOL Ubiquinone 10 (Q-10) is the sole respiratory quinone model apparatus JEM-1230; Fig. 1 and Fig. S1, available in (Albuquerque et al., 2002). IJSEM Online). Morphological observation of the strain T was done after 12–24 h of culture. After growth of strain Strain YBY-7 was isolated from water samples collected YBY-7T in 2216E medium, the cells were motile, irregular from a terrestrial hot spring (water temperature 87 uC; rods (2–6 mm long and 0.4–0.6 mm wide). Biomass for pH 7.6; salinity 17 %) of a garden exhibition located in chemical and molecular systematic studies was obtained by Xiamen City, Fujian Province, People’s Republic of China T cultivation at 55 uC for 24 h in flasks containing 2216E (24u 349 N 118u 049 E). Strain YBY-7 was isolated at 55 uC medium shaken at 160 r.p.m. Other biochemical tests from cultures grown on 2216E medium (5.0 g tryptone were performed using API 20E and API ZYM strips and 1.0 g yeast extract in 1 l synthetic seawater). The (bioMe´rieux) and Biolog GN2 MicroPlates according to synthetic seawater contained (per litre distilled water) the manufacturers’ instructions, except for adjusting the NaCl concentration to 3.0 % in all tests using distilled Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; seawater instead of distilled water. The type strain of A. PG, phosphatidylglycerol. inexpectatum, DSM 12048T, was tested at the same time for The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene comparison. These results are provided in the species sequence of strain YBY-7T is HQ709061. description and Table 1. Data for catalase and oxidase Two supplementary figures and a supplementary table are available with activities and API 20E, API ZYM and Biolog tests for strain T the online version of this paper. YBY-7 were obtained at the same time under the same

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conditions with A. inexpectatum DSM 12048T as a comparison. Antibiotic susceptibility tests were performed by the disc diffusion method according to Shieh et al. (2003). Lipoquinones were extracted from freeze-dried cells, separated and purified by TLC and identified as described by Collins et al. (1979) and analysed by HPLC as described by Groth et al. (1996). Q-10 was the sole respiratory quinone in strain YBY-7T. Polar lipids extracted by the method of Minnikin et al. (1979) were identified by two-dimensional TLC as described by Collins & Jones (1980). The results are shown in Fig. 2 and Fig. S2. Strain YBY-7T possessed PC, PG and PE as its predominant polar lipids and diphosphatidylglycerol as a minor polar lipid. These predominant polar lipids were the same as those of A. inexpectatum DSM 12048T. For fatty acid analysis, the strain was grown on marine agar 2216 (BD) medium at 55 uC for 24 h until the early Fig. 1. Transmission electron micrograph of a negatively stained exponential growth phase. Fatty acids were then extracted, cell of strain YBY-7T showing a polar flagellum. Bar, 1 mm. A saponified and esterified, followed by GC analysis of the second micrograph is available as Fig. S1. fatty acid methyl esters according to the instructions of the

Table 1. Characteristics that differentiate strain YBY-7T from A. inexpectatum DSM 12048T

Data were obtained in this study unless indicated. Cells of both strains were rods. Both strains were positive for nitrate reduction, arginine dihydrolase, urease, aesculin hydrolysis (b-glucosidase), b-galactosidase, lysine decarboxylase and ornithine decarboxylase. Both strains were negative for indole production from tryptophan, glucose fermentation, gelatin hydrolysis (protease), citrate utilization, H2S production, gelatinase and fermentation/oxidation of amygdalin. In API ZYM tests, both strains were positive for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, a-galactosidase, b-galactosidase, a-glucosidase and b-glucosidase, negative for a-fucosidase, a- mannosidase and N-acetyl-b-glucosaminidase and weakly positive for esterase (C4), lipase C14, trypsin, chymotrypsin and b-glucuronidase. The following carbon sources were utilized by both strains in the Biolog GN-II system: D-fructose, monomethyl succinate, acetic acid, b- and c- hydroxybutyric acids, succinamic acid, glycerol and thymidine. Both strains were sensitive to carbenicillin, vancomycin, tetracycline, streptomycin, rifampicin, piperacillin, polymyxin B, penicillin G, oxacillin, ofloxacin, norfloxacin, neomycin, metronidazole, minomycin, cefazolin, kanamycin, furazolidone, vibramycin, clindamycin, rocephin, gentamicin, cefalexin, ciprofloxacin, cefobid and cephradin and resistant to lincomycin and co- trimoxazole (see Table S1 for amounts tested). +, Positive; W, weakly positive; 2, negative.

Characteristic YBY-7T A. inexpectatum DSM 12048T

Cell length (mm) 2–6 1.4–2.2* DNA G+C content (mol%) 70.6 63.6* Tween 80 hydrolysis + 2 Gelatin hydrolysis (protease) + 2 Cystine arylamidase W + b-Galactosidase (ONPG) + 2 Utilization of: a-Cyclodextrin + 2 Glycogen W 2 L-Proline W + DL-Lactic acid + W Succinic acid 2 + Cellobiose + 2 Xylitol + 2 p-Nitrophenyl b-D-galactopyranoside 2 + Trisodium citrate 2 + Sucrose W +

*Data from Albuquerque et al. (2002).

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Extraction of genomic DNA and amplification of the 16S rRNA gene were performed as described by Rainey et al. (1996), followed by ligation to the vector pMD18-T and transformation into Escherichia coli DH5a.An almost-complete 16S rRNA gene sequence of strain YBY-7T, comprising 1376 bp, was obtained and compared with sequences of type strains retrieved from the EzTaxon server (http://www.eztaxon.org/; Chun et al., 2007) and the GenBank database. Strain YBY-7T showed the highest 16S rRNA gene sequence similarity (95.9 %) to A. inexpectatum T FRR-10 . Phylogenetic analysis was performed using MEGA version 4 (Tamura et al., 2007) after multiple alignment of data by using DNAMAN version 5.2 (Lynnon Biosoft). Distances (distance options according to Kimura’s two- parameter model) and clustering with the neighbour-joining method (Saitou & Nei, 1987) and minimum-evolution method (Rzhetsky & Nei, 1993) were determined. Boot- strapping analysis was used to evaluate the tree topology of Fig. 2. Two-dimensional TLC of a polar lipid extract from strain the data obtained from the two algorithms based on 1000 YBY-7T, stained with molybdatophosphoric acid. DPG, Diphos- resamplings (Fig. 3). phatidylglycerol; PC, phosphatidylcholine; PE, phosphatidyletha- + nolamine; PG, phosphatidylglycerol. See Fig. S2 for the results of The G C content of the chromosomal DNA was deter- one-dimensional TLC of polar lipids of strain YBY-7T and A. mined according to the methods described by Mesbah & inexpectatum DSM 12048T. Whitman (1989) using reversed-phase HPLC. The DNA G+C content of isolate YBY-7T was 70.6 mol%, higher than the value reported for the type strain of A. inexpectatum (63.6 mol%; Albuquerque et al., 2002). MIDI system (Sherlock 6.0) (Sasser, 2001). The GC analysis On the basis of its morphological, physiological and was performed with an Agilent 6850 and the database used chemotaxonomic characteristics, strain YBY-7T represents to identify the fatty acids was TSBA6 6.00. The fatty acid a novel species in the genus Albidovulum, for which the profile of A. inexpectatum DSM 12048T was determined in name Albidovulum xiamenense sp. nov. is proposed. parallel with strain YBY-7T in this study, and the results are shown in Table 2. The major fatty acid of strain YBY-7T Description of Albidovulum xiamenense sp. nov. was C18 : 1v7c, which accounted for 29.3 % of the total fatty acids. The major fatty acid of A. inexpectatum DSM 12048T Albidovulum xiamenense (xi.a.men.en9se. N.L. neut. adj. was also C18 : 1v7c in this study, but the proportion xiamenense pertaining to Xiamen, the city where the (65.8 %) was much larger than that in strain YBY-7T. organism was first isolated). The organism forms irregular rods, 2–6 mm long and 0.4– 0.6 mm wide, and is non-spore-forming. The organism stains Gram-negative and is motile. The optimum growth Table 2. Fatty acid compositions of strain YBY-7T and A. temperature is 50–58 uC, with no growth at 25 or 70 uC. inexpectatum DSM 12048T The optimum pH is 7.5–8.5. Growth occurs in the presence Data were obtained in this study. Values are percentages of total fatty of 0.25–5.50 % NaCl (optimum, 3.0 %); the organism acids. requires NaCl for growth. Tests for cytochrome oxidase and catalase are positive. Strictly aerobic: growth does not Peak name YBY-7T A. inexpectatum DSM occur under anaerobic conditions in the dark or light. The T 12048 predominant fatty acids are C18 : 1v7c and C19 : 0 cyclo v8c. Nitrate is reduced to nitrite, but not nitrogen. Positive for C 9.6 3.8 16 : 0 arginine dihydrolase, urease, aesculin hydrolysis (b-gluco- C17 : 0 1.5 0.7 sidase), b-galactosidase (p-nitrophenyl b-D-galactopyrano- C18 : 0 23.8 9.1 C 3-OH 0 4.0 sidase), lysine decarboxylase and ornithine decarboxylase. 10 : 0 Negative for indole production from tryptophan, glucose C16 : 0 2-OH 1.3 1.3 fermentation, gelatin hydrolysis (protease), b-galactosidase C18 : 0 3-OH 0.3 1.3 b D C18 : 1v7c 29.3 67.9 (o-nitrophenyl - -galactopyranosidase), utilization of C20 : 1v7c 1.5 0 citrate, H2S production, gelatinase and fermentation/ 11-Methyl C18 : 1v7c 0.3 7.4 oxidation of amygdalin. In the API ZYM test, positive for C19 : 0 cyclo v8c 25.6 0 alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, a-galactosidase,

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Fig. 3. Phylogenetic placement of strain YBY- 7T according to 16S rRNA gene sequence analysis. The tree was reconstructed using the neighbour-joining method, and genetic distances were computed from Kimura’s two- parameter model. Bootstrap values, expressed as percentages of 1000 replications, are given at branching points. Bar, 0.01 substitutions per nucleotide position.

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