DOI: 10.15468/mev6ub
title: Arenibacillus arenosus gen. nov., sp. nov., a member of the family Rhodobacteraceae isolated from sea sand

description: This dataset contains the digitized treatments in Plazi based on the original journal article Kim, Jong-Hwa, Kanjanasuntree, Rungravee, Kim, Dae-Hoon, Lee, Jung-Sook, Sukhoom, Ampaitip, Kantachote, Duangporn, Kim, Wonyong (2019): Arenibacillus arenosus gen. nov., sp. nov., a member of the family Rhodobacteraceae isolated from sea sand. International Journal of Systematic and Evolutionary Microbiology 69 (1): 153-158, DOI: 10.1099/ijsem.0.003121, URL: http://dx.doi.org/10.1099/ijsem.0.003121AbstractA Gram-stain-negative, aerobic, non-motile, non-spore-forming, rod-shaped bacterial strain, designated CAU 1304 T, was isolated from sea sand. Strain CAU 1304 T grew optimally at pH 8.5 and 30 Ǫ C and in the presence of 1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CAU 1304 T belonged to the family Rhodobacteraceae, and was most closely related to Roseicitreum antarcticum ZS 2-28 T (96.54 % similarity) and Rhodobacter veldkampii ATCC 35703 T (96.46 %). The major fatty acid was C 7 c and the respiratory quinone was Q-10. The polar lipids 18: 1 Ɯ were composed of phosphatidylglycerol, phosphatidylcholine and an unidentified aminolipid. The DNA G+C content was 55.9 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analysis, it is proposed that strain CAU 1304 T represents a novel species of a new genus of the family Rhodobacteraceae, for which the name Arenibacillus arenosus gen. nov., sp. nov. is proposed. The type strain of Arenibacillus arenosus is CAU 1304 T (= KCTC 42827 T = NBRC 113022 T).The family Rhodobacteraceae, first described by Garrity et al. [1], belongs to the order Rhodobacterales in the class Alphaproteobacteria and includes more than 100 genera and 300 species [2]. The family Rhodobacteraceae comprises 128 validly described genera at the time of writing (http://www. bacterio.net/). The predominant respiratory quinone is Q-10, and the major fatty acid is C 18: 1 Ɯ 7 c (&gt;50 % of the total). The G+C content of the genomic DNA is in the range 47– 76 mol% [3]. During the course of the screening of bacteria from marine environments, strain CAU 1304 T was isolated from the sand of Eurwangni beach (37 Ǫ 26′ 55.6″ N 126 Ǫ 22′ 13.1″ E) in the Republic of Korea. Strain CAU 1304 T formed a monophyletic lineage within the family Rhodobacteraceae with 96.54 % or lower 16S rRNA gene sequence similarity to representatives of the genera Roseicitreum and Rhodobacter [4, 5] based on phylogenetic analysis. The purpose of this study was to establish the taxonomic position of this bacterial strain by using a polyphasic taxonomic investigation that included the determination of phenotypic and chemotaxonomic properties, and detailed phylogenetic investigation based on 16S rRNA gene sequence analysis.Strain CAU 1304 T was isolated from sea sand by a standard dilution-plating method on marine agar 2216 (MA; Difco) [6]. The sea sand sample was serially diluted with sterile distilled water and spread on MA. After incubation under aerobic conditions at 30 Ǫ C for 7 days, the strain was isolated from a single colony and deposited in the Korean Collection for Type Cultures (KCTC) and the Biological Resource Centre, National Institute of Technology and Evaluation (NITE) (NBRC), as KCTC 42827 T and NBRC 113022 T, respectively. The type strains of the most closely related species, Roseicitreum antarcticum LMG 24863 T and Rhodobacter veldkampii NBRC 16458 T, were obtained from the Belgian Coordinated Collections of Micro-organisms (BCCM, Gent, Belgium) and the Biological Resource Centre, NITE (NBRC; Chiba, Japan), and used as reference strains for biochemical characterization and fatty acid analysis.The 16S rRNA gene sequence of strain CAU 1304 T was amplified by PCR with universal bacteria-specific primers: 27F and 1525R [7]. Multiple alignments and calculation of levels of sequence similarity with a broad selection of type strains were carried by using the EzBioCloud (http://www. ezbiocloud.net/) [8] and CLUSTAL X 2.1 software [9]. Evolutionary distance matrices were generated by using the maximum-likelihood algorithm in the PHYLIP package [10]. Phylogenetic trees were reconstructed by using the neighbour-joining, maximum-likelihood and maximum-parsimony methods with Kimura’ s two-state parameter model in MEGA version 7 [11]. The tree topology was evaluated by the bootstrap resampling method (1000 replicates) and calculated using the CONSENSE and SEQBOOT programs from the PHYLIP package [12]. The G+C content of the genomic DNA was evaluated using the HPLC method [13].Author affiliations: 1 Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Republic of Korea; 2 Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Republic of Korea; 3 Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla, Thailand.*Correspondence: Wonyong Kim, kimwy@cau.ac.krKeywords: Arenibacillus arenosus; Rhodobacteraceae; Alphaproteobacteria.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CAU 1304 T is KT369807.Three supplementary figures are available with the online version of this article.The nearly complete 16S rRNA gene sequence of strain CAU 1304 T (1447 bp) was obtained and compared with the corresponding sequence of reference strains in the GenBank database. The maximum-likelihood tree indicated that strain CAU 1304 T formed a distinct lineage within the family Rhodobacteraceae, and clustered with members of the genera Roseicitreum and Rhodobacter as its closest relatives (Fig. 1). The trees obtained with the neighbour-joining and maximum-parsimony algorithms showed basically the same topology (data not shown). 16S rRNA gene sequence similarity calculations showed that strain CAU 1304 T was most closely related to Roseicitreum antarcticum ZS 2-28 T (96.54 % similarity), followed by Rhodobacter veldkampii ATCC 35703 T (96.46 %). The DNA G+C content of strain CAU 1304 T was 55.9 mol%, a value slightly lower than that of Roseicitreum antarcticum LMG 24863 T (63.3 mol%) [4] and Rhodobacter veldkampii NBRC 16458 T (64.4–67.5 mol%) [14] but in the range reported for members of the family Rhodobacteraceae [3].For the investigation of morphological, physiological and biochemical characteristics, strain CAU 1304 T was cultivated on MA at 30 Ǫ C. Cell morphology was examined by light microscopy (DM 1000; Leica) and transmission electron microscopy (JEM 1010; JEOL) using cells from an exponentially growing culture. The intracytoplasmic membrane systems were visualized using transmission electron microscopy of ultrathin sections. The Gram reaction was performed using the bioḾerieux Gram staining kit. Motility was examined on MA soft agar (0.3 % agar) and gliding motility was evaluated with cells on the microscope slides coated with MA (0.7 % agar) using the hanging drop method [15]. Oxidase activity was evaluated based on the oxidation of 0.1 % (w/v) tetramethyl- p -phenylenediamine and catalase activity was determined by bubble production in 3 % (v/v) H 2 O 2 solution [16]. Growth at various temperatures (4–45 Ǫ C) was determined in an aerobic incubator (MIR-253; Sanyo) and an anaerobic chamber (Bactron; Sheldon) after 3 days of incubation on MA. The pH value for growth was investigated at 30 Ǫ C in marine broth (MB) adjusted to pH 4.5–11.5 (at increments of 0.5 pH units) using three different buffers (final concentration, 50 mM): sodium acetate/acetic acid, Tris/HCl and Na 2 CO 3 buffers and values were confirmed again after autoclaving. Growth with NaCl was examined at 30 Ǫ C using a range of 0–15 % (w/v) (at increments of 1 %) by supplementing MB with the appropriate concentration of NaCl, prepared in accordance with the formula of the BD medium except that NaCl was excluded. Photolithoautotrophic or photo-organotrophic tests were performed under anaerobic conditions as previously described by Labrenz [14]. Bacteriochlorophyll a (Bchl a) detection was examined according to the methods of Wang [17]. Hydrolysis of casein, starch, urea and gelatin was determined according to the methods of Ĺanyí [18] and Smibert and Krieg [19]. Acid production from carbohydrates and other biochemical properties were evaluated using the API 20E, API 20NE and API ZYM systems (bio-Merieux ́) supplemented with 0.85 % (w/v) NaCl solution, according to the manufacturer’ s instructions. API 20E and API 20NE strips were read at 30 Ǫ C after 48 h and API ZYM strips at 30 Ǫ C after 24 h.Fig. 1. Maximum-likelihood phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationships of strain CAU 1304 T to members of related genera of the family Rhodobacteraceae. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the neighbour-joining and maximum-parsimony algorithms. The numbers at nodes indicate levels of bootstrap support based on a maximum-likelihood analysis of 1000 resampled datasets; only values&gt;70 % are given. Bar, 0.01 substitutions per nucleotide position. Pelagicola litoralis CL-ES 2 T (EF192392) was used as an outgroup organism.Table 1. Differential properties between strain CAU 1304T and the type strains of the most closely related members of the genera Roseicitreum and Rhodobacter Strains: 1, CAU 1304T; 2, Roseicitreum antarcticum LMG 24863T; 3, Rhodobacter veldkampii NBRC 16458T. Data were obtained in this study unless indicated otherwise. All strains were non-motile and non-spore-forming. All strains were positive for catalase and oxidase; enzyme activity of alkaline phosphatase, esterase (C4), leucine arylamidase and naphthol-AS-BI-phosphatase; and utilization of malic acid. All strains were negative for indole production; hydrolysis of casein; enzyme activity of lipase (C14), trypsin, a- chymotrypsin, a- galactosidase, b- glucoronidase, N -acetyl-b- glucosaminidase, a- mannosidase and a- fucosidase; and utilization of D- fucose, potassium gluconate, capric acid, adipic acid, trisodium citrate and phenylacetic acid. +, Positive; ‒, negative; ND, not determined.Characteristic 1 2 * 3 * Isolation source Marine sand Sandy intertidal sediment a Freshwater b Colour of colony Cream Pink a Yellowish brown b Cell morphology Temperature range (Ǫ C) Rod-shaped 20–37 Lemon-shaped a 25–27 a Ovoids, rod-shaped b 30–35 b pH range (optimum) 5.0–9.5 (8.5) 5.0–9.5 (7.0) a (7.5)c ND NaCl tolerance (%, w/v) (optimum) 0–4 (1) 0–15 (7–8) a Not required c Hydrolysis of: Gelatin ‒ ‒ + Urease ‒ + ‒ Enzyme activities: Arginine dihydrolase ‒ + ‒ Lysine decarboxylase ‒ + ‒ Ornithine decarboxylase ‒ + + Esterase lipase (C8) + + ‒ Valine arylamidase + + ‒ Crystine arylamidase ‒ ‒ + Acid phosphatase ‒ + ‒ b- Galactosidase ‒ + + a- Glucosidase + ‒ ‒ b- Glucosidase + + ‒ Utilization of: D- Glucose ‒ + + L- Arabinose + + ‒ D- Mannitol + + ‒ Maltose ‒ + ‒ Inositol ‒ ‒ + L- Rhamnose ‒ + ‒ Sucrose ‒ + ‒ Polar lipids PG, PC, 1AL PG, PE, PC, 1AL a DPG, PG, PE, PC, GL, 2L a DNA G+C content (mol%) 55.9 63.3 a 64.4–67.5 b*Data taken from: a, Yu et al. [4]; b, Hansen et al. [14]; c, Ramana et al. [23]. DPG, Diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; AL, aminolipid; GL, glycolipid, L; lipid.Cells of strain CAU 1304 T were Gram-stain-negative, nonspore-forming, non-motile and rod-shaped (Fig. S1, available in the online version of this article). Colonies were cream-coloured, smooth and circular with diameters of 0.1– 0.5 mm after 3 days of cultivation on MA at 30 Ǫ C. Cells of strain CAU 1304 T grew at 20–37 Ǫ C (optimum 30 Ǫ C), at pH 5.0–9.5 (optimum pH 8.5) and with 0–4 % (w/v) NaCl (optimum 1 %). Bchl a and anaerobic growth under photoautotrophic and photoheterotrophic conditions showed negative results. An intracytoplasmic membrane system was absent (Fig. S2). Strain CAU 1304 T differed from its closest relative, Roseicitreum antarcticum ZS 2-28 T, based on no production of bchl a, observation of anaerobic growth both photoautotrophically and photoheterotrophically, and absence of an intracytoplasmic membrane system. The phenotypic properties of strain CAU 1304 T are listed in Table 1. Strain CAU 1304 T hydrolysed aesculin and had enzyme activities for alkaline phosphatase, arginine dihydrolase, esterase lipase, leucine arylamidase, valine arylamidase, naphthol-AS-BIphosphohydrolse, a- glucosidase and b- glucosidase. Citrate was utilized. D- Mannitol, L- arabinose, D- ribose and D- xylose were utilized as a sole carbon and energy source. Some biochemical characteristics of strain CAU 1304 T were similar with previously published data for Roseicitreum antarcticum ZS 2-28 T [4] and Rhodobacter veldkampii NBRC 16458 T [5]. However, strain CAU 1304 T differed from Roseicitreum antarcticum ZS 2-28 T and Rhodobacter veldkampii NBRC 16458 T by its utilization of D- glucose and enzyme activities of ornithine decarboxylase, b- galactosidase and a-glucosidase.Table 2. Cellular fatty acid contents (%) of strain CAU 1304T and the type strains of the most closely related Roseicitreum and Rhodobacter species Strains: 1, CAU 1304T; 2, Roseicitreum antarcticum LMG 24863T; 3, Rhodobacter veldkampii NBRC 16458T. Fatty acids that represented &lt;0.5 % of the total in all strains are not shown. TR, Trace amount.Fatty acid 1 2 3 Saturated C14:0 – 0.5 – C16:0 0.6 8.8 8.7 C17:0 0.9 TR TR C18:0 1.4 TR 7.0 Unsaturated C17:1 Ɯ 6 c 0.6 – – C17:1 Ɯ 8 c 0.9 TR TR C17:0 cyclo – 1.0 – C18:1 Ɯ 7 c 11-methyl 6.0 8.4 4.7 C19:0 10-methyl 0.6 – – C19:0 cyclo Ɯ 8 c – 1.1 – C18:1 Ɯ 7 c 83.3 72.6 56.7 Hydroxy C10:0 3-OH 2.1 1.8 3.5 C11:0 3-OH TR – TR C18:0 3-OH 0.9 – 1.0 iso-C11: 0 3-OH TR – – Summed feature* 3 2.5 4.6 17.2For the analysis of fatty acid composition, the cell masses of strain CAU 1304 T, Roseicitreum antarcticum ZS 2-28 T and Rhodobacter veldkampii NBRC 16458 T were harvested from MA after cultivation for 3 days at 30 Ǫ C. The physiological age of the biomasses harvested for fatty acid analysis was standardized by observing growth development during incubation of the cultures and choosing the moment of harvesting according to the standard MIDI protocol (Sherlock Microbial Identification System version 6.1). Cellular fatty acid methyl esters were obtained by using the method of Minnikin [20] and separated in a 6890 N gas chromatograph (Agilent) fitted with a 7683 autosampler (Agilent). Peaks were identified by using the Microbial Identification software package (MOORE library ver. 5.0; MIDI database TSBA6). The polar lipids of strain CAU 1304 T were identified by using two-dimensional TLC [21]. The plate was sprayed with 10 % ethanolic molybdatophosphoric acid (for total lipids), molybdenum blue (for phospholipids), ninhydrin (for aminolipids), a- naphthol/sulphuric acid reagent (for glycolipids) and Dragendorff’ s reagent (for phosphatidylcholine; Sigma-Aldrich). Respiratory quinones were separated by HPLC using an isocratic solvent system [methanol/isopropyl ether (3: 1, v/v)] and a flow rate of 1 ml min ‒1 [22].The fatty acid profile of strain CAU 1304 T is shown in Table 2, together with Roseicitreum antarcticum ZS 2-28 T data obtained from this study. The strain contained saturated, unsaturated and hydroxy fatty acids. The major fatty acid (&gt;10 %) was C 18: 1 Ɯ 7 c (83.3 %). The fatty acids C 17: 0 Ɯ 6 c, C 19: 0 10-methyl, C 11: 0 3-OH and iso-C 11: 0 3-OH were also present in strain CAU 1304 T but were absent from the reference strains. This fatty acid profile was similar to those of the closely related Roseicitreum antarcticum ZS 2-28 T and Rhodobacter veldkampii NBRC 16458 T in that the major fatty acid C 18: 1 Ɯ 7 c was predominant, although there were differences in the proportionsof some fatty acids. However, strain CAU 1304 T could be distinguished from Roseicitreum antarcticum ZS 2-28 T and Rhodobacter veldkampii NBRC 16458 T based on the presence of C 17: 1 Ɯ 6 c, C 19: 0 10-methyl, C 11: 0 3-OH and iso-C 11: 0 3-OH. The polar lipid profile of strain CAU 1304 T comprised phosphatidylglycerol, phosphatidylcholine and an unidentified aminolipid (Fig. S3). The polar lipid pattern of strain CAU 1304 T was different from those of Roseicitreum antarcticum LMG 24863 T and Rhodobacter veldkampii NBRC 16458 T based on the absence of phosphatidylethanolamine, diphosphatidylglycerol, glycolipid and lipid. The only respiratory quinone of strain CAU 1304 T was Q-10, in agreement with data for the family Rhodobacteraceae. In conclusion, strain CAU 1304 T is classified as representing a novel species of a new genus, for which the name the name Arenibacillus arenosus gen. nov., sp. nov. is proposed.