DOI: 10.15468/sejq5f
title: Oceanisphaera avium sp. nov., isolated from the gut of the cinereous vulture, Aegypius monachus

description: This dataset contains the digitized treatments in Plazi based on the original journal article Sung, Hojun, Kim, Hyun Sik, Lee, June-Young, Kang, Woorim, Kim, Pil Soo, Hyun, Dong-Wook, Tak, Euon Jung, Jung, Mi-Ja, Yun, Ji-Hyun, Kim, Min-Soo, Shin, Na-Ri, Whon, Tae Woong, Rho, Jeong Rae, Park, Sun Duk, Shim, Hyung Eun, Bae, Jin-Woo (2018): Oceanisphaera avium sp. nov., isolated from the gut of the cinereous vulture, Aegypius monachus. International Journal of Systematic and Evolutionary Microbiology 68 (6): 2068-2073, DOI: 10.1099/ijsem.0.002797, URL: http://dx.doi.org/10.1099/ijsem.0.002797AbstractA Gram-stain-negative, aerobic, catalase- and oxidase-positive, rod-shaped, flagellated bacterial strain, designated AMac2203 T, was isolated from the gut of the cinereous vulture, Aegypiusmonachus, collected from the Seoul Grand Park Zoo, Republic of Korea. Strain AMac2203 T grew optimally at 15–25 Ǫ C, pH 7–8 and in the presence of 3–5 % (w/v) NaCl. Phylogenetic analysis revealed 97.4–97.9 % and 96.9–97.3 % sequence similarities of the 16S rRNA genes to its counterparts in Oceanisphaera profunda SM1222 T and Oceanisphaera ostreae T-w 6 T, respectively. The predominant fatty acids (&gt;10 %) of strain AMac2203 T were summed feature 3 (C 16: 0 Ɯ 7 c and/or C 16: 1 Ɯ 6 c, 33.6 %), summed feature 8 (C 18: 1 Ɯ 7 c, 24.5 %) and C 16: 0 (19.9 %). The primary isoprenoid quinone was ubiquinone-8. Polar lipids included phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified amino lipid and an unidentified lipid. Based on complete genome sequencing of strain AMac2203 T and the closest related type strain, O. profunda, the OrthoANI value is 77.5 %, which is below the 95 % cut-off for species demarcation. The genomic DNA G+C content of strain AMac2203 T is 47.1 mol%. Thus, strain AMac2203 T represents a novel species candidate of the genus Oceanisphaera. We propose the name Oceanisphaera avium sp. nov., with strain AMac2203 T (= KCTC 62118 T = JCM 32207 T) as the type strain.Scavengers perform pivotal roles in ecosystems by maintaining energy flow throughout the entire food web, and removing mammalian disease vectors by eating carcasses [1]. Vultures are scavengers that rarely attack living animals, preferring carcasses of dead animals. Even though vultures consume dead bodies infected with aggressive pathogens such as Clostridium botulinum and Bacillus anthracis, they digest carcasses with ease due to their extremely acidic stomachs and specialised immune systems [2]. To elucidate the vulture microbiome, various studies have investigated the pathogens among their intestinal microflora [3], and the hind gut microbiome of New World vulture species has received special attention [4]. Nevertheless, it is still unclear whether the gut microbiota of vultures affects the digestion of carrion or host physiology. To assess bacteria in the gut of the cinereous vulture, Aegypius monachus, faecal samples were collected from the Seoul Grand Park zoo (37 Ǫ 25′ 22″ N, 127 Ǫ 01′ 17.8″ E), Republic of Korea, from which bacterial isolation was performed by using a culture-dependent method.Faecal samples were diluted with filtered PBS and inoculated on Lactobacilli MRS agar (Difco), marine agar (Difco), MacConkey agar (BBL), Reasoner’ s 2A agar (Difco) and tryptic soy agar (Bacto) plates using the standard serial dilution plating method. Then, inoculated agar plates were incubated at 20 and 37 Ǫ C. Finally, 126 isolates were picked and spread on fresh medium at least three times to obtain pure cultures, and preserved at ‒80 Ǫ C in appropriate broth containing 40 % (v/v) glycerol. Among them, strain AMac2203 T, identified at 20 Ǫ C on MAC, was closely related to Oceanisphaera profunda SM1222 T and Oceanisphaera ostreae T-w6 T, based on phylogenetic analysis of 16S rRNA gene sequences. The genus Oceanisphaera in the class Gammaproteobacteria was first proposed by Romanenko [5] with the description of the type species Oceanisphaera litoralis. Subsequently, seven species belonging to the genus Oceanisphaera have been reported: Oceanisphaera donghaensis [6], Oceanisphaera ostreae [7], Oceanisphaera sediminis [8], Oceanisphaera arctica [9], Oceanisphaera profunda [10], Oceanisphaera marina [11] and Oceanisphaera psychrotolerans [12]. All previously reported species in the genus Oceanisphaera were isolated from marine-related environments. However, strain AMac2203 T was isolated from the avian gut. To verify the taxonomic position of strain AMac2203 T, we performed comparative studies based on chemotaxonomic, biochemical, phenotypic, genotypic and phylogenetic characteristics.For phylogenetic analysis, the 16S rRNA gene of the isolate was amplified by colony PCR using PCR pre-mix (i NtRON Biotechnology) with universal bacteria-specific primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′) [13]. Reaction mixtures were analysed using an automated system (PRISM 3730 DNA analyser, Applied Biosystems). The 16S rRNA gene fragments were assembled using the Seq- Man program (DNASTAR) and the obtained 16S rRNA gene sequence was compared with equivalents from other strains in the EzTaxon-e server [14]. Strain AMac2203 T was found to be closely related to the type species of O. profunda and O. ostreae.MacConkey agar was inappropriate for routine cultivation because it is mainly used for isolating and differentiating enteric bacterial organisms. Therefore, strain AMac2203 T was routinely cultivated on modified TYS agar consisting of 0.5 % tryptone (Oxoid), 0.1 % yeast extract (Oxoid) and 3 % NaCl (Duksan) at 20 Ǫ C according to the method described previously [10]. For a polyphasic approach to investigate further characteristics of strain AMac2203 T, both O. ostreae KCTC 23422 T and O. profunda KCTC 32510 T were obtained from the Korean Collection for Type Cultures (KCTC) and cultivated on the same modified TYS agar at 20 Ǫ C. Gram staining was performed using a commercial kit (bioḾerieux). Cell morphology was examined using an Eclipse 50i light microscope (Nikon). Flagellation and detailed cellular morphology were determined using a LIBRA 120 transmission electron microscope (Zeiss). Cell motility was examined by inoculating strain AMac2203 T into semi-solid modified TYS medium containing 0.4 % agar [15]. Oxygen requirement tests were performed in an anaerobic chamber under a nitrogen atmosphere composed of N 2 (90 %), H 2 (5 %) and CO 2 (5 %) on modified TYS agar for 7 days. To confirm the optimal growth conditions for strain AMac2203 T, growth tests were performed under various conditions. Bacterial cell growth was observed at 4, 10, 15, 20, 25, 30, 37, 45, 55 and 65 Ǫ C in modified TYS broth. Salinity tests were performed in non-NaCl modified TYS broth supplemented with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 % (w/v) NaCl. Growth was tested at pH 4, 5 and 6 using MES buffer, at pH 7, 8 and 9 using TAPS buffer and at pH 10 and 11 using Na 2 HPO 4. To monitor growth in all conditions, the culture turbidity was measured at 600 nm using a Synergy Mx spectrophotometer (BioTek) at 24 h, 48 h and 7 days after incubation. The results suggested that strain AMac2203 T was a Gram-stain-negative, aerobic, catalase- and oxidase-positive, DNase-negative, rod-shaped, motile bacterial species supported by a flagellum (Fig. S1, available in the online version of this article). The strain grew optimally at 15–25 Ǫ C, pH 7–8 and in the presence of 3–5 % (w/v) NaCl.To confirm the physiological and biochemical characteristics of strain AMac2203 T, enzyme activities were determined using API 20NE and API ZYM test strips (bioḾerieux). Acid production from carbohydrates was determined using API 50 CH test strips (bioḾerieux). Carbon source utilisation was determined using GN2 Micro- Plates (Biolog) with inoculating fluid supplemented with 5 % NaCl according to the manufacturer’ s instructions. Catalase activity was determined by monitoring bubble production in 3 % (v/v) hydrogen peroxide solution, and oxidase activity was confirmed using 1 % (w/v) tetramethyl- p -phenylenediamine reagent (bioḾerieux). Hydrolysis of DNA was tested on DNase test agar (BBL). Detailed biochemical characteristics of strain AMac2203 T and the two reference species are listed in Table 1.For analysis of cellular fatty acids, isoprenoid quinones and polar lipids, cellular biomass of strain AMac2203 T and the two reference species was harvested from modified TYS medium after 48 h under optimal growth conditions. Cellular fatty acids were determined as described in the Sherlock Microbial Identification System operating manual version 4.5 [16]. Extracted fatty acids were analysed by gas chromatography on an HP Agilent 6890 GC System (Hewlett Packard) and identified using the Sherlock version 6.2 Microbial Identification software package based on the TSBA6 library. The major (&gt;10 %) fatty acid components of strain AMac2203 T were summed feature 3 (C 16: 0 Ɯ 7 c and/or C Ɯ 6 c, 33.6 %), summed feature 8 (C 18: 1 Ɯ 7 c, 24.5 %) and C 16: 0 (19.9 %; Table 2). Isoprenoid quinones were extracted from freeze-dried cell biomass and determined as described by Collins and Jones [17] using a Finnigan Surveyor high-performance liquid chromatograph (Thermo Scientific) with a Thermo ODS Hypersil (250× 4.6 mm) column [18]. The major quinone of strain AMac2203 T was ubiquinone-8 (Q-8), which was in common with the two reference strains. Polar lipids were extracted as previously described [19], separated by twodimensional thin-layer chromatography (TLC) on a silica gel 60 F 254 plate (Merck) and detected by spraying with appropriate detection reagents [20] consisting of 5 % (v/v) ethanolic molybdophosphoric acid (Sigma-Aldrich) for total lipids, ninhydrin for aminolipids, Oi -naphthol for glycolipids and Zinzade reagent for phospholipids. Two distinct solvents were used for each dimensional development: chloroform–methanol–water mixture (65: 24: 4, by vol.) was used for the first dimension, followed by chloroform–acetic acid–methanol–water mixture (80: 15: 12: 4, by vol.). Phospholipid components were separated with reference to standard phospholipids phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine (Sigma-Aldrich) by one-dimensional TLC on a silica gel 60 F 254 plate using chloroform–acetic acid–ethanol–water mixture (50: 6: 6: 1, by vol.) as solvent. The polar lipid components of strain AMac2203 T were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified amino lipid and an unidentified lipid. Compared with the polar lipid profiles of the two reference strains, all of the type strains had mainly phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine, which were typical polar lipid components in the genus Oceanisphaera [5 – 12].16: 1Table 1. Phenotypic characteristics of strain AMac2203 T and the type strain of two closely related speciesStrain 1, strain AMac2203 T; 2, O. profunda KCTC 32510 T; 3, O. ostreae KCTC 23422 T. All data were from the current study unless indicated otherwise (* and †). All strains were motile and possess the ability to hydrolyse aesculin. All strains were negative for acid production from glycerol, erythritol, D- arabinose, L- arabinose, D- ribose, D- xylose, L- xylose, D- adonitol, methyl β -D- xylopyranoside, D- galactose, D- glucose, D- fructose, D- mannose, L- sorbose, L- rhamnose, dulcitol, inositol, D- mannitol, D- sorbitol, methyl Oi -D- mannopyranoside, methyl Oi -D- glucopyranoside, N -acetyl-glucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D- lyxose, D- tagatose, D- fucose, L- fucose, D- arabitol, L- arabitol, gluconate, 2-keto-gluconate and 5-keto-gluconate. All strains assimilated pyruvic acid methyl ester, acetic acid, formic acid, succinic acid, bromosuccinic acid, L- alaninamide, D- alanine, L- alanine, L- asparagine, L- aspartic acid, L- glutamic acid, L- histidine, L- leucine, L- ornithine, L- phenylalanine, L- serine, L- threonine and malic acid. All strains possessed catalase, oxidase, esterase lipase (C8) and leucine arylamidase enzyme activities. Reactions for which all strains were negative are not presented. +, Positive; –, negative; W, weakly positive; ND, no data available.Characteristic 1 2 3 Isolation source Gut of a cinereous vulture Deep-sea sediment, South China Sea* Seawater from an oyster farm, South Sea, Republic of Korea† Temperature for growth (Ǫ C): Range 4–30 4–35* 4–37† Optimum 15–25 25–30* 20–25† pH for growth: Range 6.0–9.0 5.5–10.5* 5.0‒ND† Optimum 7.0–8.0 7.5–8.0* 7.0–8.0† NaCl concentration tolerance (%, w/v): Range 0–8 0.5–8* 0–10† Optimum 3–5 2–3* 2† Nitrate reduction – + + Urea hydrolysis – + + Assimilation of: Glycogen, Tween 40, Tween 80, Oi -hydroxybutyric acid, β -hydroxybutyric acid, Oi -ketobutyric acid, Oi -ketoglutaric acid, L- alanyl glycine, D- serine, Ƴ -amino butyric acid – – + Hydroxy-L- proline, trisodium citrate – + – Succinic acid monomethyl ester, succinamic acid, L- proline, – + + urocanic acid D,L- Lactic acid + – + Enzyme activity: Alkaline phosphatase – W + Esterase (C4) + + – Valine arylamidase – W – Acid phosphatase, naphthol-AS-BI-phosphohydrolase – + – DNA G+C content (mol%) 47.1 49.8 56.6† Data from: *Xu [10] and †Choi [7].For complete genome sequencing, genomic DNA from strain AMac2203 T and O. profunda KCTC 32510 T was extracted according to the instructions supplied with the MG Genomic DNA purification kit (Macrogen). The concentration of extracted genomic DNA was determined using a NanoDrop spectrophotometer (Thermo Scientific) and a Qubit fluorimeter (Life Technology). For PacBio RS sequencing, 8 µg of input genomic DNA was used for 20 kb library preparation. For genomic DNA where the size range was less than 17 kb, a Bioanalyzer 2100 (Agilent) was used to determine the actual size distribution. To ensure library insert sizes were in the optimal size range, genomic DNA was sheared with a g-TUBE device (Covaris) and purified using AMPure PB magnetic beads (Beckman Coulter) if the apparent size was greater than 40 kb. The 10 µl library was prepared using a PacBio DNA Template Prep Kit 1.0. Next, SMRTbell templates were annealed using a PacBio DNA/ Polymerase Binding Kit P6. The PacBio DNA Sequencing Kit 4.0 and eight SMRT cells were used for sequencing. SMRT cells (Pacific Biosciences) using C4 chemistry and 240 min movies were captured for each SMRT cell using the PacBio RS II (Pacific Biosciences) sequencing platform (Macrogen). As a result, the complete circular genomic DNA was obtained. From the complete genome of strain AMac2203 T, full lengths of 16S rRNA gene sequences were determined and there was heterogeneity between 16S rRNA gene sequences. Considering the sequence similarity between them, four types of 16S rRNA gene sequences were finally confirmed. Each 16S rRNA gene sequence consisted of 1559, 1547, 1546 and 1559 nt, respectively. The sequence similarities between the four 16S rRNA gene sequences were found to be 99.6–99.8 % through pair-wise comparison analysis. The 16S rRNA genes of strain AMac2203 T shares 97.4–97.9 and 96.9–97.3 % sequence similarities to the 16S rRNA genes of O. profunda KCTC 32510 T and O. ostreae KCTC 23422 T, respectively. To indicate phylogenetic relationships among species closely related to strain AMac2203 T, a phylogenetic consensus tree was reconstructed. Multiple sequence alignment was conducted using CLUSTAL_W [21], and the 16S rRNA gene sequences of the isolate and related species were aligned based on the EzTaxon-e server. Including four different 16S rRNA gene sequences, phylogenetic distances were then calculated using MEGA version 7 [22] using neighbour-joining [23], maximum-parsimony [24] and maximum-likelihood [25] algorithms based on 1000 bootstrap replicates. The phylogenetic consensus tree indicated that strain AMac2203 T is closely related to the type species of O. profunda and O. ostreae, within the phylogenetic radiation comprising the genus Oceanisphaera (Fig. 1).Table 2. Cellular fatty acid profiles (%) of strain AMac2203T and the type strains of two closely related species Strains: 1, strain AMac2203T; 2, O. profunda KCTC 32510T; 3, O. ostreae KCTC 23422T. All data were performed from the current study. Values were expressed as a percentage of total fatty acids, and excluded when values of all fatty acid profiles were less than 0.5 %. Trace val- ues below 0.5 % are indicated as TR. The values of fatty acid profiles above 10 % are indicated as bold text. ‒, Not detected.Fatty acid (%) 1 2 3 Saturated: C12: 0 8.0 6.6 6.0 C14: 0 0.7 TR TR C16: 0 19.9 19.3 10.5 C17: 0 1.2 0.9 1.7 C18: 0 2.0 0.8 0.5 Unsaturated: C17: 1 Ɯ 8 c 0.7 0.6 2.3 C17: 1 Ɯ 6 c TR – 0.6 Summed features:* 1 TR – 0.7 2 6.1 6.2 4.9 3 33.6 40.6 45.5 8 24.5 21.9 25.1 9 – 0.6 –Considering the features of the complete genome, neither strain AMac2203 T nor O. profunda KCTC 32510 T harboured any additional plasmid chromosomes, which had only 1 contig, and the lengths of the circular genomes were 2 902 896 and 3 508 860 bp, respectively. The N50 of assembled genomes was the same as their total genome length. The genomic DNA G+C contents of strain AMac2203 T and O. profunda KCTC 32510 T were 47.1 and 49.8 mol%, respectively. The sequencing depth of coverage of their genomes was 372× and 263×, respectively. According to Chun et al. [26], the overall genome related index (OGRI) was checked to determine the genomic relatedness through comparison of genomic DNA sequence similarity. The average nucleotide identity (ANI) value, belonging to the OGRI, was calculated to estimate relatedness using the OrthoANI algorithm [27] provided by the EzTaxon-e server. The calculated OrthoANI value was 77.5 %, which is lower than the 95 % cut-off value for species demarcation. Because the ANI value is considered to provide reliable evidence for species demarcation, this confirmed that strain AMac2203 T is a novel species that differs from the closely related O. profunda KCTC 32510 T.*Summed features represent two or three fatty acids that cannot be separated by the MIDI system. Summed feature 1 contains C13: 0 3-OH and/or C15: 1 i H; summed feature 2 contains C14: 0 3-OH and/or iso- C16: 1 I; summed feature 3 contains C16: 1 Ɯ 7 c and/or C16: 1 Ɯ 6 c; summed feature 8 contains C18: 1 Ɯ 7 c; summed feature 9 contains iso- C17: 1 Ɯ 9 c.Phylogenetic, biochemical, chemotaxonomic, phenotypic and genotypic characteristics determined in the present work suggest that strain AMac2203 T represents a novel species affiliated to the genus Oceanisphaera, for which the name Oceanisphaera avium sp. nov. is proposed.