DOI: 10.15468/mhz95w
title: Hymenobacter aquatilis sp. nov., isolated from a mesotrophic artificial lake

description: This dataset contains the digitized treatments in Plazi based on the original journal article Kang, Heeyoung, Cha, Inseong, Kim, Haneul, Joh, Kiseong (2018): Hymenobacter aquatilis sp. nov., isolated from a mesotrophic artificial lake. International Journal of Systematic and Evolutionary Microbiology 68 (6): 2036-2041, DOI: 10.1099/ijsem.0.002792, URL: http://dx.doi.org/10.1099/ijsem.0.002792AbstractA novel strain, designated HMF3095 T, isolated from freshwater of a mesotrophic artificial lake in the Republic of Korea, was characterized by polyphasic taxonomy. The cells were Gram-stain-negative, aerobic, non-motile, straight rods and formed reddish colonies. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain HMF3095 T fell within the cluster of the genus Hymenobacter and was most closely related to Hymenobacter seoulensis 16F7G T and Hymenobacter tenuis POB 6 T (96.7 % sequence similarity). Sequence similarities to all other type strains were 96.3 % or less. The major fatty acids were iso-C 15: 0, C 16: 1 Ɯ 5 c, summed feature 4 (iso-C 17: 1 I and/or anteiso-C 17: 1 B), summed feature 3 (C 16: 1 Ɯ 7 c and/or C 16: 1 Ɯ 6 c) and anteiso-C 15: 0. The major isoprenoid quinone was menaquinone 7. The major polar lipids were phosphatidylethanolamine, three unidentified aminophospholipids and one unidentified phospholipid. The DNA G+C content was 58.9 mol%. On the basis of the evidence presented in this study, strain HMF3095 T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter aquatilis sp. nov. is proposed. The type strain is HMF3095 T (= KCTC 52398 T = NBRC 112669 T).The genus Hymenobacter, a member of the family Cytophagaceae [1], comprises 61 reported species, including the recently identified species Hymenobacter cavernae, Hymenobacter rutilus, Hymenobacter coccineus, Hymenobacter lapidarius, Hymenobacter glacialis, Hymenobacter frigidus, Hymenobacter seoulensis, Hymenobacter aquaticus, Hymenobacter gummosus, Hymenobacter pallidus, Hymenobacter tenuis and Hymenobacter defluvii [2 – 11]. Members of the genus Hymenobacter have been isolated from environments such as soil and glacial ice of a polar area, desert, cave, air, freshwater, seawater, and wastewater [2, 3, 11, 12]. Some species of the genus Hymenobacter can survive under extreme conditions such as desiccation and radiation, and tolerate high levels of oil and heavy metals [3, 13]. Members of the genus Hymenobacter are generally Gram-stain-negative, aerobic, non-motile, reddishpigmented and rod-shaped bacteria. These bacteria chemotaxonomically contain iso-C 15: 0, anteiso-C 15: 0, summed feature 3 (C 16: 1 Ɯ 7 c and/or C 16: 1 Ɯ 6 c) and summed feature 4 (iso- C 17: 1 I and/or anteiso-C 17: 1 B) as major fatty acids, menaquinone 7 as the predominant respiratory quinone, and polar lipid profile including phosphatidylethanolamine as the major component. The DNA G+C content ranges from 55 to 70 mol% [2, 3, 5, 7, 8, 12 – 14].To study bacterial diversity, freshwater was collected from a mesotrophic artificial lake within the campus of Hankuk University of Foreign Studies, Yong-in, Republic of Korea (37 Ǫ 20′ 18″ N 127 Ǫ 16′ 11″ E). The water sample was diluted using tenfold dilution series methods, spread on an R 2A agar (Becton Dickinson) plate and incubated at 30 Ǫ C for 7 days. Strain HMF3095 T was purified by streaking three times on R 2A agar. The isolate was routinely cultured on R 2A at 30 Ǫ C for 2 days. The bacterial culture was stored at ―80 Ǫ C in distilled water supplemented with 20 % (v/v) glycerol and also preserved using lyophilization.The 16S rRNA gene region of strain HMF3095 T was amplified by PCR using the universal primers 27F and 1492 R [15]. Sequencing was performed using an Automatic Sequencer 3730XL (Applied Biosystems) with universal primers (337F, 785F and 518 R). Identification of phylogenetic neighbours, similarity calculation and alignment of pairwise 16S rRNA gene sequences were performed by using the EzBioCloud server and the SILVA Incremental Aligner [16, 17]. Phylogenetic analysis was performed using the software package MEGA version 7.0 [18], and clustering with the neighbour-joining [19], maximum-parsimony [20] and maximum-likelihood [21] methods. The topology of trees were evaluated by using the bootstrap method with 1000 replicates. A neighbour-joining tree was reconstructed by using the Jukes–Cantor model [22]. A maximum-likelihood tree was reconstructed with the K2+G+I model, after estimation of the best fit. The maximum-parsimony tree was inferred using subtree-pruning and regrafting with a default setting of 10 trees.A nearly complete 16S rRNA gene sequence (1448 nt) of strain HMF3095 T showed highest similarity to Hymenobacter seoulensis 16F7G T (96.7 %), Hymenobacter tenuis POB 6 T (96.7 %), Hymenobacter pallidus LYH-12 T (96.3 %), Hymenobacter luteus YIM 77921 T (96.3 %) and Hymenobacter latericoloratus YIM 77920 T (96.3 %). In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain HMF3095 T fell within the clade comprising species of the genus Hymenobacter. Strain HMF3095 T was clustered with H. pallidus LYH-12 T (Fig. 1). This relationship was also maintained in the tree topology reconstructed using maximum-likelihood and maximum-parsimony algorithms. This phylogenetic inference, coupled with 16S rRNA gene sequence similarities of &lt;97 % [23, 24] between strain HMF3095 T and other species of the genus Hymenobacter, indicated that the strain HMF3095 T should be assigned to the genus Hymenobacter as a representative of a novel species. According to phylogenetic analysis and sequence similarities of the 16S rRNA genes, H. seoulensis KCTC 52197 T, H. tenuis KCTC 52271 T, H. pallidus KCTC 42898 T, H. luteus DSM 26718 T, H. latericoloratus DSM 26701 T and H. xinjiangensis KACC 14201 T were obtained from the Korean Collection for Type Cultures (KCTC), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and the Korean Agricultural Culture Colletion (KACC), respectively, and used as reference strains in the following tests.The optimal growth temperature was tested at different temperatures (4, 10, 15, 20, 25, 30, 37 and 42 Ǫ C) on R 2A agar (Becton Dickinson). Growth at various pH values (pH 4.0– 10.0 at intervals of 1.0 pH unit) and NaCl concentrations (0–5.0 % at increments of 0.5 % intervals, w/v) was determined at 30 Ǫ C in R 2A broth. The pH values were obtained by using 0.1 M sodium acetate/acetic acid (pH 4.0–6.0), 0.1M phosphate (pH 6.0–8.0), 0.1 M Tris/HCl (pH 7.0–9.0) and 0.1 M Na 2 CO 3 (pH 9.0 and 10.0) buffers, respectively. Growth under anaerobic conditions was determined on R 2A agar at 30 Ǫ C for 5 weeks by using the GasPak EZ Anaerobic container System (Becton Dickinson). Growth was tested on nutrient agar, marine agar 2216, tryptic soy agar (TSA), blood and MacConkey agars (all Becton Dickinson). DNase activity was assessed on DNase test agar (Becton Dickinson). Hydrolysis of casein (3 % skimmed milk w/v; Becton Dickinson), carboxymethylcellulose (1 % CMcellulose w/v; Sigma), cellulose (1 % filter paper w/v; Whatman no. 1), dextrin (1 % dextrin w/v; Sigma) and starch (1 % soluble starch w/v; Sigma) was tested using 1/15 strength R 2A agar as the basal medium and evaluated after 3 days of incubation at 30 Ǫ C.Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of strain HMF3095 T in the genus Hymenobacter. Bootstrap percentages (&gt;70 %) from neighbour-joining (below nodes) analyses are shown. Filled and open circles indicate nodes recovered by all three treeing methods or with two treeing methods, respectively. Adhaeribacter aerophilus 6424 S-25 T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.Cell morphology and size of HMF3095 T were investigated using a transmission electron microscope (LIBRA120; Carl Zeiss). Gliding motility was tested using the hanging-drop method [25]. Gram-staining performed according to the method of Hucker [26]. Oxidase and catalase activity evaluated by using 1 % N, N, N’, N -tetramethyl- p -phenylenediamine dihydrochloride (Sigma) and adding 3 % (v/v) H 2 O 2 to freshly grown colonies, respectively. Flexirubin-type pigments were investigated by using the bathochromatic shift test with a 20 % (w/v) KOH solution [27]. API 20NE and API ZYM kits (bioMerieux) were used to determine the assimilation of carbon sources and to reveal the presence of important enzymes. Additionally, oxidation of carbon compounds was evaluated with GN2 MicroPlates (Biolog) according to the manufacturer’ s instructions. Antibiotic susceptibilities were tested by the paper disc method, after spreading the cell suspension of HMF3095 T onto R 2A agar plates. The antibiotic discs contained the following antibiotics (µg per disc, unless stated otherwise; Liofilchem): ampicillin (10), chloramphenicol (30), erythromycin (15), gentamycin (10), kanamycin (30), penicillin G (10 IU), rifampicin (30), streptomycin (10), tetracycline (30) and vancomycin (30). Susceptibility to antibiotics was determined according to the criteria for family Enterobacteriaceae of the Clinical and Laboratory Standards Institute [28]. Cells of strain HMF3095 T were strictly aerobic, Gram-stainnegative, non-motile, straight rod shaped (Fig. S1, available in the online version of this article), and oxidase- and catalase-positive. Strain HMF3095 T grew well on R 2A and nutrient agars. Casein (skimmed milk) and starch were hydrolysed. The characteristics that differentiate strain HMF3095 T and the closely related species of Hymenobacter are summarized in Tables 1 and S1.For genomic DNA G+C content calculations, DNA of strain HMF3095 T was prepared using a Bacteria Genomic DNA prep kit (Biofact). The G+C content of the DNA was determined using the thermal denaturation fluorometric method [29]. The DNA G+C content of strain HMF3095 T was 58.9 mol%, a value within the range reported for Hymenobacter strains [30].Characteristic 1 2 3 4 5 6 7 Temperature range for growth (Ǫ C) (optimum) 10–37 (25–30) 15–30 (30) 10–30 (30) 10–37 (25–30) 10–37 (25-30) 10–37 (25–30) 10–37 (30) NaCl range for growth (%, w/v) 0–0.5 0–1.0 0–1.0 0–0.5 0–0.5 0–0.5 0–0.5 Growth on TSA ― ― ― + + ― + Hydrolysis of: Dextrin ― ― ― + ― ― + Soluble starch + ― ― + + + + CM-cellulose ― ― ― + ― ― + Aesculin (API 20NE) + + + + + ― + Gelatin + + ― + + + + Enzyme activities: β -Galactosidase (PNPG) + ― + ― + + + Esterase (C4) + + ― + + + + Cystine arylamidase + + + + + + ― Trypsin + + + + + + ― Oi -Glucosidase + ― + + + + ― β -Glucosidase ― ― ― ― ― ― + DNA G+C content (mol%) 58.9 56.6 a 61.0 b 64.3 c 59.6 d 57.5 d 54.0eCellular fatty acid analysis was performed with cells grown to late-exponential phase on R 2A agar (Becton Dickinson) at 30 Ǫ C. Fatty acid methyl esters of strain HMF3095 T and six reference strains were extracted by using standard procedures [31]. The fatty acid methyl esters mixtures were separated by using the Microbial Identification System (MIDI; Sherlock version 6.0), which consists of a gas chromatograph (Agilent 7890A system). Peaks were automatically integrated and fatty acid names and percentages were determined using the RTSBA6 database. The major fatty acids (&gt;5 %) of strain HMF3095 T were iso-C 15: 0 (41.4 %), C 16: 1 Ɯ 5 c (11.0 %), summed feature 4 (iso-C I and/or anteiso- C 17: 1 B; 8.4 %), summed feature 3 (C 16: 1 Ɯ 7 c and/or C 16: 1 Ɯ 6 c; 7.4 %) and anteiso-C 15: 0 (6.4 %). This result was similar to that of the related reference strains and the characteristic fatty acid composition of previously reported species of the genus Hymenobacter [2, 6]. Differences in the fatty acid profiles among strain HMF3095 T and related reference strains are listed in Table 2.17: 1For analysis of respiratory quinones and polar lipids, freezedried cells of strain HMF3095 T were prepared from cultures grown in R 2A agar at 30 Ǫ C for 2 days. The extraction of respiratory quinones was performed according to the method Minnikin et al. [32]. The extracted quinones were purified by thin-layer chromatography (TLC) and analysed by high- performance liquid chromatography (HPLC) with an eclipse XDB-C18 column (Agilent) [33]. The major respiratory quinone of HMF3095 T was menaquinone 7, in line with all other members of the genus Hymenobacter [3, 6].The polar lipids were extracted and separated by twodimensional TLC. Chromatography was carried out according to Minnikin et al. [32]. Lipid spots were detected by spraying the following reagents: 10 % ethanolic molybdophosphoric acid (for total lipid), ninhydrin reagent (for aminolipids), molybdenum blue (for phospholipids) and Oi - naphthol sulphuric acid (for glycolipids). The polar lipid profile of strain HMF3095 T is shown Fig. S2, and included phosphatidylethanolamine, three unidentified aminophospholipids, one unidentified phospholipid and four unidentified polar lipids. The polar lipid profile of strain HMF3095 T was similar to those of the other closely related species of the genus Hymenobacter with respect to the phosphatidylethanolamine, unidentified aminophospholipids and unidentified phospholipid. Strain HMF3095 T mainly differed from the related reference strains by the absence of an unidentified aminolipid [6, 9, 10, 13, 34].On the basis of the phylogenetic relationship and the phenotypic and chemotaxonomic traits, strain HMF3095 T represents the novel species of the genus Hymenobacter for which the name Hymenobacter aquatilis sp. nov., is proposed.