Marimicrobium Arenosum Gen. Nov., Sp. Nov., a Moderately Halophilic Bacterium Isolated from Sea Sand Maytiya Konkit, Jong-Hwa Kim and Wonyong Kim

International Journal of Systematic and Evolutionary Microbiology (2016), 66, 856–861 DOI 10.1099/ijsem.0.000803

Marimicrobium arenosum gen. nov., sp. nov., a moderately halophilic bacterium isolated from sea sand Maytiya Konkit, Jong-Hwa Kim and Wonyong Kim

Correspondence Department of Microbiology, Chung-Ang University College of Medicine, Seoul 06974, Wonyong Kim Republic of Korea [email protected] A Gram-stain-negative, non-pigmented, non-spore-forming, non-motile, strictly aerobic bacterial strain, designated CAU 1038T, was isolated from a sea sand sample in Modo, Republic of Korea, and its taxonomic position was examined using a polyphasic approach. Cells of strain CAU 1038T grew optimally at 30 8C, pH 7.5 in 2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CAU 1038T formed a distinct lineage within the class Gammaproteobacteria as a separate deep branch, with 95.2 % or lower sequence similarity to representatives of the genera Haliea, Halioglobus and Chromatocurvus, and 92.3 % or lower with Luminiphilus, Pseudohaliea and Congregibacter. The major cellular fatty T acids of strain CAU 1038 were C16 : 0,C16 : 1v7c and C18 : 1v7c. The polar lipid pattern of the isolate consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unidentiﬁed aminolipid and two unidentiﬁed lipids. The strain contained lipoquinone (Q-8) as the sole respiratory quinone. The G+C content of the genomic DNA was 65 mol%. On the basis of phenotypic and chemotaxonomic data, and phylogenetic inference, strain CAU 1038T represents a novel species of a new genus in the family Halieaceae, for which the name Marimicrobium arenosum gen. nov., sp. nov. is proposed. The type strain of the type species is CAU 1038T (5KCTC 42300T5NBRC 110727T).

Gammaproteobacteria are a class of numerous medically Modo is a small island located in Ongjingun, Incheon, in and ecologically essential groups of bacteria, such as Enter- the west sea of the Republic of Korea (378 329 050 N obacteriaceae, Pseudomonadaceae, Vibrionaceae, Alteromo- 1268 249 330 E). This area has an abundance of sediment nadaceae and Oceanospirillaceae. The family Halieaceae that is interesting for the isolation of a wide variety of (Spring et al., 2015) belongs to the order Cellvibrionales micro-organisms. Strain CAU 1038T was isolated from in the class Gammaproteobacteria. This family was found Modo Island and belongs to the class Gammaproteobac- in various habitats, such as tidal flat sediment, seawater, teria, order Cellvibrionales and family Halieaceae. The marine and marine coastal sediments. Members of the objective of this study was to evaluate the taxonomic pos- family Halieaceae are Gram-negative, rod-shaped and ition of this novel bacterial strain using a polyphasic non-spore-forming bacteria. Q-8 is the predominant iso- approach involving the determination of phenotypic, gen- prenoid quinone and the major fatty acid is unsaturated otypic and chemotaxonomic properties, and 16S rRNA fatty acid (C16 : 1 or C18 : 1). The G+C content of the geno- gene sequence analysis. mic DNA ranges from 52 to 66 mol% (Spring et al., 2015). A sea sand sample was serially diluted, plated on marine At the time of writing, the family Halieaceae comprises six agar 2216 (MA; Difco), supplemented with cycloheximide genera with validly published names: Chromatocurvus 2 2 (50 mg l 1) and nalidixic acid (20 mg l 1) to inhibit the (Csotonyi et al., 2011), Congregibacter (Spring et al., growth of fungi, and incubated at 30 8C for 7 days under 2009), Haliea (Urios et al., 2008), Halioglobus (Park aerobic conditions as described by Gordon & Mihm et al., 2012), Luminiphilus (Spring et al., 2013) and Pseudo- (1962). Colonies were randomly selected and a single haliea (Spring et al., 2013). colony was purified by subculturing on MA at 30 8C for 3 days. Strain CAU 1038T was preserved at 280 8Cin The GenBank/EMBL/DDBJ accession number for the 16S rRNA marine broth (MB; Difco) supplemented with 25 % (v/v) gene sequence of strain CAU 1038T is KP064188. glycerol. The type strains of species of the closely related T Three supplementary figures are available with the online Supplemen- genus Halioglobus (Halioglobus japonicus KCTC 23429 T tary Material. and Halioglobus pacificus KCTC 23430 ), were obtained

856 000803 G 2015 IUMS Printed in Great Britain Marimicrobium arenosum gen. nov., sp. nov. from the Korean Collection for Type Cultures (KCTC) and tetramethyl-p-phenylenediamine (Cappuccino & Sherman, used as reference strains in most analyses. 2002). Catalase activity was determined by observing bubble production in 3 % (v/v) H O solution. Hydrolysis Genomic DNA of strain CAU 1038T was extracted by the 2 2 of casein, starch and urea were determined using SYPHC method of Marmur (1961). PCR amplification of the 16S medium with 2 % NaCl. Acid production and assimilation rRNA gene of strain CAU 1038T was examined according from carbohydrates, enzyme activity, and other physiologi- to Lane (1991) and the conditions described by Nam cal and biochemical features were tested as described by et al. (2004). The amplified 16S rRNA gene was directly Leifson (1963) using API 20NE, API 20E, API 50CHE, sequenced using a BigDye Terminator Cycle Sequencing and API ZYM strips (bioMe´rieux). kit and an automated 3730 DNA sequencer (Applied Bio- systems). Multiple sequence alignments of the 16S rRNA For the determination of fatty acid composition, the cell T gene sequence of strain CAU 1038 with the corresponding biomass of strain CAU 1038T, Halioglobus japonicus sequences from a broad selection of closely related strains, KCTC 23429T and Halioglobus pacificus KCTC 23430T and calculations of the levels of sequence similarity, were were harvested from MA after cultivation for 3 days at made using the EzTaxon-e server (Kim et al., 2012; 30 8C. The physiological age of the biomass used for fatty http://www.ezbiocloud.net/eztaxon) and CLUSTAL X 2.1 soft- acid analysis was chosen according to the standard MIDI ware (Larkin et al. 2007). Evolutionary distance matrices Sherlock Microbial Identification System (MIDI) method- were generated by the neighbour-joining method described ology. Cellular fatty acid methyl esters were obtained and by Jukes and Cantor (1969). Phylogenetic trees were reon- separated on a 6890N gas chromatograph fitted with a structed using the neighbour-joining (Saitou & Nei, 1987), 7683 autosampler (Agilent Technologies). Peaks were least-squares (Fitch & Margoliash, 1967), maximum-likeli- identified using the Microbial Identification software pack- hood (Felsenstein, 1981) and maximum-parsimony (Fitch, age [MOORE version 5.0 (Agilent Technologies); MIDI 1971) algorithms in the PHYLIP package (Felsenstein, 1989). database TSBA6]. The polar lipids of strain CAU 1038T Branch support in the neighbour-joining tree was esti- were identified using two-dimensional thin-layer chroma- mated by the bootstrap resampling method (1000 replica- tography by the method of Minnikin et al. (1984). The + tions) (Felsenstein, 1985). The mol% G C content of plate was sprayed with 10 % ethanolic molybdatophospho- the DNA was performed using reversed-phase HPLC fol- ric acid (for total lipids), molybdenum blue (for phospho- lowing the method of Tamaoka & Komagata (1984). For lipids), ninhydrin (for aminolipids) and a-naphthol/ detection of photosynthesis genes pufL and pufM sulphuric acid reagent (for glycolipids). Ubiquinones (encoding subunits of the photosynthetic reaction were separated by HPLC using an isocratic solvent centre), PCR amplification was performed using primer system [methanol/isopropyl ether (3 : 1, v/v)] and flow 2 set pufL2/pufMR2 and the following cycle conditions: rate of (1 ml min 1) (Komagata & Suzuki, 1987). initial denaturation at 98 8C for 3 min, 35 cycles of denaturation at 98 8C for 15 s, annealing at 56 8C for 25 s The nearly complete 16S rRNA gene sequence of strain and extension at 72 8C for 1.5 min, followed by final exten- CAU 1038T (1515 bp) was determined and compared sion at 72 8C for 10 min (Spring et al., 2013). with corresponding sequences of related bacterial strains in the GenBank database. Phylogenetic analysis showed For the investigation of morphological, physiological and that strain CAU 1038T was distinct from a clade rep- biochemical characteristics, strain CAU 1038T was culti- resented by the members of the genera Microbulbifer, Sac- vated on MA at 30 8C for 3 days. Cell morphology was charophagus, Spongiibacter, Dasania, Marinimicrobium, investigated under a DM 1000 light microscope (Leica Teredinibacter, Haliea, Luminiphilus, Chromatocurvus, Microsystems) and a JEM-1200EX transmission electron Pseudohaliea, Congregibacter and Halioglobus. In pairwise microscope (JEOL) using cells from an exponentially grow- analyses, Haliea salexigens DSM 19537T (95.2 % 16S ing culture. Gram staining was carried out using the bio- rRNA gene sequence similarity), Halioglobus japonicus S1- Me´rieux Gram staining kit. Motility was assessed in MB 36T (94.6 %), Halioglobus pacificus S1-72T (93.7 %), Chro- culture using the hanging-drop method (Bowman, 2000). matocurvus halotolerans EG19T (93.6 %), Haliea mediterra- Growth between 4 8C and 45 8C was examined by growing nea 7SM29T (93.2 %), Luminiphilus syltensis NOR5-1BT the isolate on MA for 3 days in a MIR-253 aerobic incuba- (92.3 %), Pseudohaliea rubra CM41_15aT (92.2 %) and tor (Sanyo Electric Biomedical). The pH range for growth Congregibacter litoralis KT71T (90.5 %) were the recognised was investigated in MB that had been adjusted to pH 4.5– species that appeared most closely related to strain CAU T 12.0 (at 0.5 pH unit intervals) by using CH3COONa/ 1038 . The trees generated using the least-squares, maxi- CH3COOH and Na2CO3 buffers. The NaCl concentration mum-likelihood, and maximum-parsimony algorithms for growth was determined using modified SYPHC (Fig. S1, available in the online Supplementary Material) medium as used by Spring et al. (2013), adjusting the con- show similar topologies to neighbour-joining tree centration of NaCl from 0 to 15 % (w/v). Anaerobic (Fig. 1). The G+C content of the DNA of strain CAU growth was checked in a Bactron anaerobic chamber 1038T was 65 mol%. The pufL and pufM genes were not (Sheldon Manufacturing) under the optimal conditions. detected in strains CAU1038T based on PCR amplification Oxidase activity was evaluated with 0.1 % (w/v) of these photosynthesis genes. http://ijs.microbiologyresearch.org 857 M. Konkit, J.-H. Kim and W. Kim

Microbulbifer salipaludis SM-1T (AF479688) 0.1 •95 Microbulbifer hydrolyticus DSM 11525T (AJ608704) • 75 Microbulbifer elongatus DSM 6810T (AF500006) 92 • 74 • T Microbulbifer agarilyticus JAMB A3 (AB158515) ISL-39T (EF486352) • 100 Microbulbifer celer Microbulbifer halophilus YIM 91118T (EF674853) • 82 Microbulbifer thermotolerans JAMB A94T (AB124836) • 82 T • Microbulbifer maritimus TF-17 (AY377986) Saccharophagus degradans 2-40T (AF055269) • Teredinibacter turnerae T7902T (AY028398) • 79 Simiduia agarivorans SA1T (EF617350) • Marinimicrobium koreense M9T (AY839869) • 100 Marinimicrobium agarilyticum M18T (AY839870) Dasania marina DSM 21967T (AY771747)

Spongiibacter marinus HAL40bT (AM117932) • 100 Marimicrobium arenosum CAU 1038T (KP064188)

94 Halioglobus pacificus S1-72T (AB602430) • 74 Halioglobus japonicus S1-36T (AB602427) Luminiphilus syltensis NOR5-1BT (ACCY01000005) 73 Haliea mediterranea 7SM29T (FN398053) Haliea salexigens DSM 19537T (AY576769) Chromatocurvus halotolerans EG19T (AM691086) Pseudohaliea rubra CM41_15aT (EU161717) • 98 Congregibacter litoralis KT71T (AY007676) Escherichia coli ATCC 11775T (X80725)

Fig. 1. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the position of strain CAU 1038T. Filled circles indicate that the corresponding nodes were recovered in the trees generated with the maximum-likelihood and least-squares algorithms. Bootstrap values .70 %, based on a neighbour-joining analysis of 1000 resampled datasets, are shown at nodes. Escherichia coli ATCC 11775T (GenBank accession no. X80725) was used as an outgroup organism. Bar, 0.1 substitutions per nucleotide position.

The differential morphological, cultural, physiological and Chromatocurvus (7–40 8C), Congregibacter (9–33 8C), biochemical characteristics of strain CAU 1038T are pre- Luminiphilus (12–32 8C) and Pseudohaliea (15–44 8C). sented in Table 1. Strain CAU 1038T is strictly aerobic, In addition, the salt tolerance range distinguished strain non-pigmented and can grow at temperatures from 20– CAU 1038T (0–3.0 %) from the genera Haliea (0.7– 37 8C (optimum 30 8C) and at pH 6.5–8.5 (optimum, 7.0 %), Chromatocurvus (0–18.0 %) and Congregibacter pH 7.5). Cells were short, rod-shaped, approximately (1.0–7.0 %). Strain CAU 1038T differed from the close 0.3–0.5 mm in diameter and 1.0–1.1 mm in length, non- relative, Haliea salexigens DSM 19537T (Urios et al., motile and did not have flagella (Fig. S2). As strain CAU 2008) by the ability to utilize glucose, and from members 1038T was isolated from a saline environment, it required of the genus Halioglobus by the inability to utilize fructose NaCl for growth around 0–3 % (w/v) (optimum 2 %). and sucrose (Table 1). The oxidase and catalase tests were positive but urease was negative, and nitrate is reduced to nitrite but not to The pattern of polar lipids of strain CAU 1038T was phos- nitrogen. Esterase enzyme is produced. Strain CAU 1038T phatidylethanolamine, diphosphatidylglycerol, phosphati- could utilize glucose and ribose, but was unable to utilize dylglycerol, an unidentified aminolipid and two arabinose or maltose. The growth temperature range unidentified lipids (Fig. S3). The single isoprenoid quio- distinguished strain CAU 1038T (20–37 8C) from the none detected was Q-8. The fatty acid profile of strain genera Haliea (10–37 8C), Halioglobus (15–30 8C), CAU 1038T is shown in Table 2. The strain contained

858 International Journal of Systematic and Evolutionary Microbiology 66 http://ijs.microbiologyresearch.org

Table 1. Differential characteristics of strain CAU 1038T and type strains of closely related species of the family Halieaceae

Strain: 1, CAU 1038T (data from this study); 2, Haliea salexigens 3X/A02/235T (Urios et al. 2008); 3, Halioglobus japonicus KCTC 23429T (this study); 4, Halioglobus paciﬁcus KCTC 23430T (this study); 5, Chromatocurvus halotolerans EG19T (Spring et al., 2013); 6, Congregibacter litoralis KT71T (Spring et al., 2013); 7, Luminiphilus syltensis NOR5-1BT (Spring et al., 2013); 8, Pseudohaliea rubra CM41_15aT (Spring et al., 2013).+, Positive; 2, negative; DPG, Diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL, phospholipid.

Characteristic 1 2 3 4 5 6 7 8

Shape Short rod Short rod Coccus Coccus Pleomorphic Pleomorphic Straight- Straight rods to-bent Motility 2 + 22 ++22 Growth temperature 20–37 10–37 15–30 15–30 7–40 9–33 12–32 15–44 (8C) range optimum 30 30 20–25 20–25 37 28 28 30 pH range optimum 6.5–8.5 5.0–9.0 6.0–9.0 6.0–10.0 7.0-12.0 6.5–9.0 7.0–9.0 5.0–9.0 7.5 8.0 7.0–8.0 7.0–8.0 7.0 7.5–8.0 8.0 8.0 NaCl tolerance (%) 0–3.0 0.7–7.0 1.0–4.0 1.0–5.0 0–18.0 1.0–7.0 1.0–9.0 0.7–4.2 range optimum 2.0 4.0 2.0 2.0 4.0 2.0 3.0 3.5 Catalase ++22 ++++ Oxidase ++++ ++++ Utilization of: Glucose + 2 ++ 222+ Fructose 22++ 2222 Sucrose 22++ 2 + 22 Photosynthetic pigment 2222 ++++

Polar lipids DPG, PE, PG DPG,PG DPG, PE, PG, PL DPG, PG, PL PE, PG PE, PG PE, PG PE, PG arenosum Marimicrobium Major cellular fatty acids C16 : 0 ,C16 : 1 v7c, C16 : 1 v7c,C17 : 1 v8c, C16 : 0 ,C16 : 1 v7c, C16 : 1 v7c, C17 : 1 v8c, C16 : 1 v7c,C17 : 1 v8c, C16 : 1 v7c, C16 : 0 , C16 : 0, C16 : 1 v7c, C18 : 1 v7c C18 : 1 v7c C18 : 1 v7c C18 : 1 v7c C18 : 1 v7c C18 : 1 v7c C16 : 1 v7c C18 : 1 v7c DNA G+C content 65 61 60 59 63 58 57 65 (mol%) e.nv,s.nov. sp. nov., gen. 859 M. Konkit, J.-H. Kim and W. Kim

Table 2. Cellular fatty acids (%) of strain CAU 1038T and chemotaxonomic and phenotypic characteristics with clo- type strains of closely related species of the genus sely related taxa were observed (Table 1). The novel isolate Halioglobus could be distinguished from members of the genera of the family Halieaceae by its phylogenetic relationship, and phe- Strains: 1, CAU 1038T;2,Halioglobus japonicus KCTC 23429T;3, T notypic and chemotaxonomic characteristics. Therefore, Halioglobus pacificus KCTC 23430 . All data are from this study we propose that strain CAU 1038T be classified as a repre- with all strains cultured under identical conditions (marine agar for sentative of a novel genus and species, Marimicrobium are- 8 3 days at 30 C) and analysed using the Microbial Identification nosum gen. nov., sp. nov. System. Fatty acids amounting to .0.5 % of the total fatty acids in all strains are shown. 2, Not detected; TR, trace (,0.5 %). Description of Marimicrobium gen. nov. Fatty acid 1 2 3 Marimicrobium (Ma.ri.mi.cro9bi.um. L. n. mare the sea; Hydroxy N.L. neut. n. microbium microbe; N.L. neut. n. Marimicro- 2 C10 : 0 3-OH 2.8 2.7 bium small, marine microbe). C11 : 0 3-OH 2 0.6 1.5 Cells are Gram-stain-negative, non-pigmented, strictly C12 : 0 3-OH 2.4 TR 1.5 aerobic, oxidase- and catalase-positive, rod-shaped, non- C12 : 0 2-OH 1.7 22 C 3-OH 222spore-forming and non-motile. The temperature range 12 : 1 u C13 : 0 3-OH 222for growth is 20–37 C. Growth occurs in the presence of Saturated 0–3 % (w/v) NaCl. The pH range for growth is 6.5–8.5.

C10 : 0 5.9 1.7 1.8 The respiratory lipoquinone is Q-8. The polar lipids consist C11 : 0 2 0.8 0.6 of diphosphatidylglycerol, phosphatidylethanolamine, C12 : 0 2 0.8 0.8 phosphatidylglycerol, an unidentiﬁed aminolipid and two 22 C13 : 0 0.8 unidentiﬁed lipids. The major fatty acids are C16 : 0, C14 : 0 1.1 2.0 1.4 C16 : 1v7c and C18 : 1v7c. The type species is Marimicro- 222 C15 : 0 bium arenosum. iso-C15 : 0 2 TR 2 C16 : 0 27.0 9.4 3.4 Marimicrobium arenosum C17 : 0 2 2.4 2.2 Description of sp. nov. 2 C18 : 0 0.6 TR Marimicrobium arenosum (a.re.no9sum. L. neut. adj. areno- Unsaturated sum sandy, dwelling in marine sediment sand). C15 : 1v6c 2 0.5 1.2 v 2 C15 : 1 8c 0.9 1.6 Cells are approximately 0.3–0.5 mm in diameter and v 2 C16 : 1 6c TR TR 1.0–1.1 mm in length. Colonies on MA are cream and cir- v C16 : 1 7c 42.3 35.2 31.4 cular, and cells are small, straight, regular rods after incu- v 2 C17 : 1 6c TR TR bation for 3 days at 30 uC. Optimum growth occurs at v 2 C17 : 1 8c 4.2 13.1 30 uC, at pH 7.5 and with 2 % (w/v) NaCl. Gelatin, v C18 : 1 7c 14.7 34.7 28.0 casein, and starch are not hydrolysed. Nitrate is reduced C v6c 222 19 : 1 to nitrite but not to nitrogen. Assimilates D-mannitol and trisodium citrate, but not L-arabinose, D-mannose, maltose, N-acetylglucosamine, potassium gluconate, capric acid, adipic acid, malic acid or phenylacetic acid. Uses hydroxyl, saturated and unsaturated fatty acids. The major D-ribose, D-galactose and D-glucose as sole carbon sources, cellular fatty acids (.10 %) were C16 : 0 (27.0 %), but not erythritol, L-arabinose, L-xylose, L-sorbose, C16 : 1v7c (42.3 %), and C18 : 1v7c (14.7 %). These fatty L-rhamnose, L-fucose, L-arabitol, D-arabinose, D-xylose, acids were present in higher amounts than those recorded D-adonitol, D-fructose, D-mannose, dulcitol, inositol, for the type strains of the type species of the genera Haliea, D-mannitol, D-sorbitol, cellobiose, sucrose, melezitose, Halioglobus, Chromatocurvus, Luminiphilus, Pseudohaliea turanose, D-lyxose, D-tagarose, D-fucose, D-arabitol, and Congregibacter. The fatty acids C12 : 0 3-OH, C12 : 0 2- T methyl a-D-glucopyranoside, methyl b-D-xylopyranoside, OH, C and C were present in strain CAU 1038 10 : 0 14 : 0 N-acetylglucosamine, aesculin ferric citrate, starch, glyco- at .1%. gen, xylitol, potassium gluconate, potassium 2-ketogluco- In conclusion, it is evident from the phenotypic, chemo- nate or potassium 5-ketogluconate. In the API ZYM taxonomic and genotypic data that strain CAU 1038T posi- system, alkaline phosphatase, esterase (C4), esterase lipase tioned within the family Halieaceae, which encompasses (C8), a-chymotrypsin, acid phosphatase, and naphthol- the genera Haliea and Halioglobus (Fig. 1). Strain CAU AS-BI-phosphohydrolase activities are positive, but lipase 1038T showed very low 16s rRNA gene sequence similarity (C14), leucine arylamidase, valine arylamidase, crystine (j95.2 %) with respect to members of the family Haliea- arylamidase, trypsin, a-galactosidase, b-glucuronidase, ceae, which supports the placement of the novel isolate b-galactosidase, b-glucosidase, a-glucosidase, a-mannosi- within a separate genus. In addition, differences in dase, a-fucosidase and N-acetyl-b-glucosaminidase

860 International Journal of Systematic and Evolutionary Microbiology 66 Marimicrobium arenosum gen. nov., sp. nov.

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