Aeromicrobium halocynthiae sp. nov., a taurocholic acid-producing bacterium isolated from the marine ascidian Halocynthia roretzi

In an effort to investigate the chemical potential of microbes growing in marine environments, the microbial diversity associated with marine invertebrates and the production of secondary metabolites was investigated. During this survey, a new bile acid-producing, Gram-positive bacterium was isolated from the ascidian Halocynthia roretzi. NCBI nucleotide blast searches using the 16S rRNA gene sequence of the cultured bacterium revealed that the new isolate shared a close phylogenetic affiliation with members of the genus Aeromicrobium. It was most closely related to Aeromicrobium ginsengisoli with 16S rRNA gene sequence similarities of 97.7 %.

The genus Aeromicrobium, first proposed by Miller et al. (1991), belongs to the family Nocardioidaceae within the order Actinomycetales. Currently, nine recognized species of the genus Aeromicrobium have been reported including Aeromicrobium erythreum (Miller et al., 1991), A. fastidiosum (Tamura & Yokota, 1994), A. alkaliterrae (Yoon et al., 2005), A. panaciterrae (Cui et al., 2007) and A. ginsengisoli (Kim et al., 2008) from soil, A. marinum (Bruns et al., 2003), A. tamlense (Lee & Kim, 2007) and A. ponti (Lee & Lee, 2008) from the marine environment, and A. flavum (Tang et al., 2008) from air. Erythromycin is a representative secondary metabolite produced by A. erythreum (Miller et al., 1991). However, apart from erythromycin, no biologically valuable organic compounds have been reported from members of the genus Aeromicrobium.

The search for bioactive secondary metabolites in the culture broth of strain KME 001^T led to the isolation of taurocholic acid, a major secondary metabolite of this bacterium. Cholic acid is a bile acid produced by mammalian liver cells. The acid is synthesized in the liver from cholesterol and is generally found in a conjugated form with taurine and glycine. Taurocholic acid, taurine-conjugated cholic acid and its sodium salt have been used as cholagogues (agents which promote the flow of bile) and these compounds are currently manufactured commercially by purification from animal bile (Dewick, 2001). Currently, only 10 bacterial strains have been reported as bile acid-producing prokaryotes. Bile acids produced by these strains include cholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholic acid and cholic acid methyl ester (Park et al., 1995; Maneerat et al., 2005; Kim et al., 2007). To date, a taurocholic acid-producing bacterium has not been reported.

This paper describes the characterization of strain KME 001^T by means of a polyphasic taxonomic approach.

Strain KME 001^T was isolated from a marine ascidian, Halocynthia roretzi, collected at a depth of 15 m (water temperature: 17 °C) near Kyung-Po beach, Korea (3 ° 48′ 36.33″N, 12 ° 54′ 46.19″E), in June 2007. As soon as the ascidian was collected, it was washed with autoclaved seawater. The incurrent and excurrent siphon tissues were ground and diluted with autoclaved seawater (ratio of ground tissue to seawater 1 : 10). The diluted suspension (100 μl) was spread on an A1+C agar plate. The A1+C medium contained 10 g starch (Difco), 4 g peptone (Difco), 2 g yeast extract (Difco), 1 g calcium carbonate (Aldrich) and 18 g agar (Difco) in 1 l filtered seawater (pH 7.0). The plate was incubated for 3 weeks at 25 °C under aerobic conditions. Individual colonies were subcultured on A1+C agar medium before each pure bacterial strain was cultured in A1+C liquid medium (25 ml) while shaking at 200 r.p.m for 7 days at 25 °C. Stocks of each isolated bacterial strain were generated and stored at −80 °C in liquid culture medium containing 20 % (v/v) glycerol.

Gram-staining was performed according to the method described by Süßmuth et al. (1987). Cultures of strain KME 001^T grown at 25 °C for 5 days in A1+C medium were used to determine phenotypic characteristics. The ability of strain KME 001^T to grow on various agar media was evaluated using marine agar (MA) (Difco), A1+C agar, A1 agar (A1+C agar medium without calcium carbonate), R2A agar (Difco) and trypticase soy agar (TSA; Difco) for 5 days at 25 °C. Colony morphology and cell motility were investigated using a light microscope (Eclipse Te2000-U; Nikon). Cell morphology and the presence of flagella were observed using a scanning electron microscope (SEM; S-3500N, Hitachi). The requirement for and tolerance of NaCl was examined on A1+C agar plates that were prepared by using artificial seawater containing different concentrations of NaCl (0–10 %; w/v with intervals of 1 %). The ability of strain KME 001^T to grow at various temperatures was examined at 4, 7, 10, 15, 20, 25, 30, 37, 42 and 45 °C on A1+C agar plates. Growth at different pH values was evaluated using A1+C liquid medium for which the pH values were adjusted from pH 3.0 to pH 12.0 (at intervals of 1.0 pH unit). Oxidase activity was examined using oxidation of 1 % N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (Sigma-Aldrich). Catalase activity was determined using a 3 % (v/v) hydrogen peroxide solution.

The utilization of carbon sources was investigated using the API 50 CHE system (bioMérieux) according to the manufacturer's recommendations. API 20E and API ZYM tests (bioMérieux) were used to determine additional biochemical properties.

The diaminopimelic acid stereoisomers in the cell-wall peptidoglycan were determined by using the TLC method as described by Staneck & Roberts (1974). The bacterial menaquinones were extracted from freeze-dried biomass by using chloroform–methanol (2 : 1, v/v), and analysed by reverse-phase HPLC with a Spherisorb 5 μm ODS2 (250×4.6 mm) column as described previously (Tamaoka, 1986). The analysis of fatty acid methyl esters was carried out by GC (6890; Hewlett Packard) with an HP-1 (cross-linked methyl siloxane, 30 m×0.32 mm×0.25 μm) column using the MIDI Microbial Identification System (MIS; Microbial ID) (Miller, 1982).

Chromosomal DNA of strain KME 001^T was extracted using a G-spin Genomic DNA Extraction kit (iNtRON Biotechnology, Inc.). PCR amplification of the 16S rRNA gene was performed with the universal primers 27F and 1492R (Lane, 1991). The DNA sequencing reaction was carried out with an ABI Prism BigDye terminator cycle sequencing ready reaction kit v3.1 (Applied Biosystems). The sequencing product was purified using a Montage dye removal kit (Millipore) according to the manufacturer's protocol. Gene sequences were determined on a Perkin-Elmer capillary DNA Sequencer (model ABI 3730XL; Applied Biosystems). The 16S rRNA gene sequence of strain KME 001^T was edited using the BioEdit program (Hall, 1999) and compared with primary sequence information within the GenBank/DDBJ/EMBL nucleotide sequence database using blast (Altschul et al., 1997). Pairwise nucleotide sequence similarity values were determined by using the EzTaxon server 2.1 (; Chun et al., 2007). Multiple alignments were performed with the clustal w program 1.83 (Thompson et al., 1994) and evolutionary distances calculated by using the Kimura two-parameter model (Kimura, 1983). The phylogenetic tree was constructed by means of the neighbour-joining method (Saitou & Nei, 1987) with bootstrap values based on 1000 resamplings (Felsenstein, 1985) with the mega software package 4.0 (Kumar et al., 2008).

DNA–DNA hybridization was determined by the membrane filter method using the DIG High Prime DNA labelling and detection starter kit II (Roche Applied Science) according to the manufacturer's instructions. The DNA G+C content of strain KME 001^T was determined by HPLC of the P1 nuclease hydrolysate (Katayama-Fujimura et al., 1984).

The optical rotation of the taurocholic acid was measured on a Perkin-Elmer polarimeter (model 343; Perkin-Elmer). NMR spectra were obtained in CD₃OD on a Varian UNITY Plus 500 MHz spectrometer. NMR chemical shifts were referenced to the residual solvent peaks (δ_H 3.30 and δ_C 45.3 for CD₃OD). Low-resolution electrospray ionisation mass spectroscopy (ESI-MS) was measured on an Agilent Technologies VS/Agilent 1100 system and TLC was performed by using Merck silica gel 60 F254. Silica gel 60 (Merck, 0.063–0.200 mm) was also used for flash column chromatography. Preparative HPLC separations were performed using a Gilson 321 HPLC system with a Phenomenex Luna C18(2) 10 μm column (10×250 mm) at a flow rate of 4 ml min⁻¹. HPLC-MS was performed with an Agilent 1100 LC-MS system using a Phenomenex Luna C18(2) 5 μm column (4.6×150 mm) at a flow rate of 0.7 ml min⁻¹. A Waters 1525 HPLC-PDA system with a Phenomenex Luna C18(2) 5 μm column (4.6×150 mm) at a flow rate of 1.0 ml min⁻¹ was used for the analysis of extracts and fractions.

Strain KME 001^T was a Gram-positive, aerobic, non-spore-forming and non-motile bacterium on A1+C medium at 25 °C. SEM observations of strain KME 001^T revealed rod-shaped bacterial cells. Cells were 4.1–6.0 μm in length and 0.4–0.5 μm in diameter (Fig. 1⇓). Colonies were circular, convex, smooth, light yellowish and 0.6–1 mm in diameter on A1+C agar plates after 5 days at 25 °C. Strain KME 001^T grew between 10 and 42 °C, with an optimal temperature for growth of 25 °C on A1+C agar plates. Strain KME 001^T also grew on marine agar, R2A agar and A1 agar plates at 25 °C. However, no growth was observed on TSA medium. Previous studies have shown that the type strains of all recognized species of the genus Aeromicrobium are capable of growth on TSA, apart from A. panaciterrae. The phenotypic characteristics of strain KME 001^T were compared with those of related species of the genus Aeromicrobium (Table 1⇓). Interestingly, only strain KME 001^T and the type strain of A. marinum were capable of using mannitol as a sole carbon source.

ll-Diaminopimelic acid was found to be the diagnostic diamino acid of the cell-wall peptidoglycan of strain KME 001^T. The predominant menaquinone was MK-9(H₄). These characteristics were similar to those of other type strains of species of the genus Aeromicrobium. The predominant fatty acids of KME 001^T (≥1.00 %) were C_18 : 1ω9c (35.24 %), C_16 : 0 (31.50 %), 10-methyl C_18 : 0 (21.05 %), C_18 : 0 (5.47 %), C_17 : 0 (1.47 %) and C_16 : 1ω9c (1.71 %) (see Supplementary Table S1 in IJSEM Online). The fatty acid composition of strain KME 001^T was similar to those of other species of the genus Aeromicrobium. However, strain KME 001^T was unique in that it lacked C_16 : 0 2-OH, a fatty acid detected in all other recognized species of the genus Aeromicrobium.

Phylogenetic analyses using the 16S rRNA gene sequence (1358 nt) of strain KME 001^T revealed that it was associated with the family Nocardioidaceae and was most closely related to members of the genus Aeromicrobium (Fig. 2⇓). Comparisons of the 16S rRNA gene sequence of strain KME 001^T with sequences from related genera, including Aeromicrobium, Marmoricola, Pimelobacter, Nocardioides and Lechevalieria, showed that strain KME 001^T was most closely related to members of the genus Aeromicrobium. Strain KME 001^T shared relatively high sequence similarity (96.5–97.7 %) with all other species of the genus Aeromicrobium species depicted on the phylogenetic tree (Fig. 2⇓). The 16S rRNA gene sequence similarity values between strain KME 001^T and the type strains of related Aeromicrobium species were: A. ginsengisoli (97.7 %), A. erythreum (97.6 %), A. ponti (97.5 %), A. alkaliterrae (97.4 %), A. marinum (97.4 %), A. panaciterrae (97.1 %), A. tamlense (96.5 %), A. fastidiosum (96.5 %) and A. flavum (96.2 %). Although the type strain of A. ginsengisoli appeared to have the highest gene sequence similarity with strain KME 001^T, it was located on another branch belonging to A. marinum and A. panaciterrae in the phylogenetic tree due to the higher levels of 16S rRNA sequence similarity with these species, 99.3 % and 99.0 %, respectively.

DNA–DNA hybridization was performed to evaluate the genomic DNA relatedness between strain KME 001^T and its nearest neighbours, A. ginsengisoli KCTC 19207^T, A. erythreum KCCM 41104^T and A. ponti KACC 20565^T. Strains with more than 70 % DNA–DNA relatedness are generally assumed to be members of the same species (Wayne et al., 1987). The DNA–DNA relatedness values between strain KME 001^T and A. ginsengisoli KCTC 19207^T, A. erythreum KCCM 41104^T and A. ponti KACC 20565^T were 49.6 %, 57.1 % and 63.5 %, respectively. The DNA G+C content of strain KME 001^T was 75.9 mol%. This value appeared to be slightly higher than that found for other members of the genus Aeromicrobium, which range from 65.5 to 74.0 mol%.

On the basis of these phenotypic and phylogenetic data, it is proposed that strain KME 001^T represents a new member of the genus Aeromicrobium. The name Aeromicrobium halocynthiae sp. nov. is proposed.

In order to investigate secondary metabolite production by strain KME 001^T, the strain was cultured in ten 1 l Erlenmeyer flasks each containing 500 ml A1+C broth. The flasks were incubated at 25 °C for 7 days with shaking at 200 r.p.m. Secondary metabolites in the culture broth were analysed every day (from days 3–7) by using an Agilent 1100 LC-MS system using a Phenomenex Luna 5 μm C-18(2) analytical column (4.6×150 mm) (see Supplementary Fig. S1 in IJSEM Online). At the end of the culture period (day 7), 20 g l⁻¹ Amberlite XAD-7 adsorbent resin was added to each flask, followed by shaking for two additional hours. The resin was then collected by filtration through cheesecloth, washed with deionized water and eluted twice with acetone. The acetone solution was concentrated under reduced pressure to yield 1.2 g crude extract. The acetone extract was fractionated using silica gel flash column chromatography and a step gradient elution with methylene chloride and methanol (20 : 1, 10 : 1, 3 : 1 and methanol) to give four subfractions. The 100 % methanol fraction (374 mg) was dried in vacuo and fractionated by reverse-phase HPLC using an isocratic elution of 23 % acetonitrile in water [flow rate 4 ml min⁻¹, Phenomenex Luna C18(2) 10 μm 10×250 mm]. Taurocholic acid (3 mg) was purified by repeated HPLC in the same manner as the 100 % methanol fraction. Taurocholic acid was obtained as an optically active, pale yellowish powder. The structure of the taurocholic acid was characterized by comparing the physicochemical properties and spectroscopic data (see Supplementary Figs S2 and S3) with values previously reported in the literature (Gowda et al., 2006; Ijare et al., 2005). Cholic acid is synthesized from cholesterol in the mammalian liver. To determine whether cholesterol, the precursor of many cholic acids, was present in the A1+C liquid medium used to cultivate strain KME 001^T, TLC analysis of the A1+C liquid medium alone were performed and compared with authentic cholesterol (see Supplementary Figure S4). No cholesterol was detected in the medium, suggesting that strain KME 001^T was capable of producing taurocholic acid de novo. To the authors' knowledge, strain KME 001^T is the first example of a taurocholic acid-producing bacterium.