DOI: 10.15468/56nrrj
title: Melaminivora jejuensis sp. nov., isolated from Swinery waste

description: This dataset contains the digitized treatments in Plazi based on the original journal article Kim, Ji-Young, Park, So-Hyun, Lee, Dong-Heon, Song, Gwanpil, Kim, Young-Ju (2018): Melaminivora jejuensis sp. nov., isolated from Swinery waste. International Journal of Systematic and Evolutionary Microbiology 68 (1): 9-13, DOI: 10.1099/ijsem.0.002294, URL: http://dx.doi.org/10.1099/ijsem.0.002294AbstractA sulfur-oxidizing bacterium, designated strain KBB12 T, was isolated from swinery waste collected in Jeju, Republic of Korea. The cells were Gram-stain-negative, flagellated and rod-shaped. Growth occurred at 15 – 45 Ǫ C (optimum, 30 – 37 Ǫ C), at pH 6 – 9 (optimum, pH 7.0) and in the presence of 0 – 1 % (w/v) NaCl. The major cellular fatty acids were summed feature 3 (iso-C 15: 0 2-OH and/or C 16: 1 Ɯ 7 c, C 16: 0 and C 18: 1 Ɯ 7 c. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospholipid and an unidentified lipid. The major isoprenoid quinone was ubiquinone-8 (Q-8) and the DNA G+C content of the genomic DNA was 69.6 mol%. Phylogenetic analyses, based on 16S rRNA gene sequences, showed that the novel isolate belongs to the genus Melaminivora and was most closely related to Melaminivora alkalimesophila CY 1 T (97.2 % similarity). The DNA – DNA relatedness values between strain KBB12 T and M. alkalimesophila DSM 26005 T was 43.4+2.7 %. On the basis of phylogenetic and phenotypic evidence, it is proposed that strain KBB12 T represents a novel species of the genus Melaminivora, for which the name Melaminivora jejuensis sp. nov. is proposed. The type strain is KBB12 T (=KCTC 32230 T =JCM 18740 T).The genus Melaminivora belongs to the family Comamonadaceae within the class Betaproteobacteria, was established by Wang et al. [1]. At the time of writing, the genus comprises a single species with a validly pulished name, Melaminivora alkalimesophila, which was isolated from wastewater [1]. Cells of species of the genus Melaminivora are Gramnegative, rod-shaped, motile by means of a polar flagellum and catalase- and oxidase-positive. Ubiquinone-8 is the major quinone, with C 16: 1 Ɯ 7 c and/or C 16: 1 Ɯ 6 c, C 16: 0, C 10: 0 3-OH and C 18: 1 Ɯ 7 c and/or C 18: 1 Ɯ 6 c as the predominant fatty acids.An investigation of sulfur-oxidizing bacteria in microaerobic sulfur-rich waste water was performed with a survey combining molecular characterization of 16S rRNA and soxB genes [2 – 5]. The presence of a soxB gene has been used as an indicator for the presence of the Sox pathway and then for the ability of the organism to oxidize sulfur [4, 6, 7]. During the study on cultivable aerobic bacterial communities in the waste site of a swinery field in Jeju, Republic of Korea, a novel sulfur-oxidizing bacterium, designated KBB12 T, was recovered. The aim of the present study was to describe the isolation and determination of the taxonomic position of the novel isolate, on the basis of a polyphasic approach, which included phylogenetic and physiological analyses.Strain KBB12 T was isolated from swinery waste collected in Jeju, Republic of Korea. Enrichment and isolation were performed using a slightly modified version of the medium used for sulfur-oxidizing bacteria [8], designated BH medium (l ‒ 1 in distilled water: MgSO 4. 7H 2 O, 0.409 g; CaCl 2 .2H 2 O, 0.0265 g; KH 2 PO 4, 1 g; NH 4 NO 3, 1 g; Na 2 HPO 4 .12H 2 O, 6 g; FeCl 3. 6H 2 O, 0.0833 g; NaHCO 3, 0.2 g, and Na 2 S 2 O 3, 4.0 g, plus 1 ml trace element solution). The strain was subcultivated on tryptic soy agar (TSA; Difco) at 30 Ǫ C and preserved in a glycerol solution (20 %, v/v) at ‒80 Ǫ C.The 16S rRNA gene [9] and soxB gene [4] were amplified and cloned by using a TOPO vector kit (Invitrogen), according to the manufacturer’ s instructions. The cloned DNA was sequenced with ABIPRISM Dye Terminator Cycle sequencing kits (Applied Biosystems) at Genotech (Seoul, Korea). The full sequence of the 16S rRNA gene was compiled using SeqMan software (DNASTAR). Sequences of related taxa were obtained from the GenBank database and EzTaxon server [10]. The soxB nucleotide sequence was translated into a deduced amino acid sequence, which was compared to data available from the GenBank database by using the BLAST program (tblastn; http://www.ncbi.nlm.nih. gov/blast). Multiple alignments were performed using the CLUSTAL X program (version 1.83) [11] and gaps were edited by using the BioEdit program [12]. A phylogenetic tree was reconstructed by using the neighbour-joining [13], maximum-likelihood [14] and maximum parsimony [15] algorithms contained in MEGA version 5.0 [16]. Bootstrapping analysis, based on 1000 resamplings, was used to evaluate tree topology [17]. DNA–DNA hybridization values were determined fluorometrically by the method of Ezaki et al. [18] using photobiotin-labelled probes and microplate wells. Hybridizations were replicated five times for each sample.Author affiliations: 1 Research institute for basic science, Jeju National University, Jeju 63241, Republic of Korea; 2 Department of Aquatic Life Medicine, College of Ocean Science, Jeju 63241, Republic of Korea; 3 Jeju Biological Resource Co., Ltd, CTC Business Incubator, Jeju 63242, Republic of Korea; 4 Marine and Environmental Research Institute of Jeju National University, Jeju, 63241, Republic of Korea.*Correspondence: Ji-Young Kim, jym1006@jejunu.ac.kr; Young-Ju Kim, youngju40@naver.comKeywords: sulfur oxidizing bacterium; Melaminivora; family Comamonadaceae.Abbreviations: DPG, Diphosphatidylglycerol; L, Lipid; PE, Phosphatidylethanolamine; PG, Phosphatidylglycerol; PL, Phospholipid.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KBB12 T is JX997988. The GenBank accession number for the soxB gene amino acid sequence of strain KBB12 T is KC295221.Three supplementary figures are available with the online version of this article.The morphology of bacterial cells was observed by light microscopy (Nikon) and scanning electron microscopy (Mini SEM; SEC). The presence of flagella was studied by transmission electron microscopy (JEM-161 101; Jeol), with cells grown on TSA for 2 days at 30 Ǫ C. Gram-staining was performed using a Gram-stain kit (BD Bioscience) according to the manufacturer’ s instructions. The temperature and pH for the growth of strain BB12 T were examined on TSA at 5–50 Ǫ C (4, 10, 13, 15, 20, 25, 30, 35, 37, 40, 45, 47 and 50 Ǫ C) and in TSB at pH 4.0–11.0 (at intervals of 0.5 pH unit), respectively. The pH was adjusted prior to sterilization by the addition of 1M HCl or 1M NaOH. Salt tolerance was examined after incubation for 7 days on TSA supplemented with 1–10 % (w/v) NaCl (at increments of 1 %). Catalase activity was observed from bubble production in a 3 % solution (v/v) of hydrogen peroxide and oxidase activity was determined from the oxidation of 1 % (w/v) tetramethyl p -phenylenediamine (Merck). Hydrolysis of casein, starch and Tween 20, 40, 60, and 80 was performed on TSA using the substrate concentrations described previously [19]. DNase activity was examined using DNase test agar (Difco) with methyl green. Growth under anaerobic conditions was determined after incubation for 4 weeks in an AnaeroPack (Oxoid) on TSA and on TSA supplemented with potassium nitrate (0.1 %, w/v). Other physiological and biochemical properties were tested using the API20E, API20NE, and APIZYM kits (BioḾerieux), according to the manufacturer’ s instructions. Utilization of different carbon sources was assessed using GN2 microplates (Biolog), according to the manufacturer’ s instructions. Susceptibility to antibiotics was tested on TSA plates using antibiotic discs (BBL) containing the following: ampicillin (10 µg), cephalothin (30 µg), chloramphenicol (30 µg), gentamicin (10 µg), erythromycin (15 µg), kanamycin (30 µg), lincomycin (2 µg), nalidixic acid (30 µg), neomycin (30 µg), novobiocin (30 µg), oleandomycin (15 µg), penicillinG (10 IU), polymyxinB (300 IU), rifampicin (5 µg), streptomycin (10 µg) and tetracycline (30 µg).Cell masses of strains KBB12 T and M. alkalimesophila DSM 26006 T for cellular fatty acid and polar lipid analysis were obtained after growth on TSA at 30 Ǫ C for 2 days. Fatty acid methyl esters were prepared according to the standard protocol of the Sherlock Microbial Identification System (MIDI; version 4.5) [20] and analyzed by gas chromatography (GC 6850, Agilent) using the TSBA5 library for identification of the resultant peaks. Polar lipids were extracted according to the procedure described by Minnikin et al. [21] and analyzed by two-dimensional TLC. Individual polar lipids were identified by spraying with molybdophosphoric acid, ninhydrin, Zinzade and a- naphthol reagents and through co-migration with authentic standards [21, 22]. The DNA G+C content was determined at the Korean Culture Centre of Microorganisms (KCCM) by using HPLC analysis [23]. The respiratory quinones were analysed by reversed-phase high performance liquid chromatography [23].16S rRNA gene sequence analysis by Blast search with related species using the EzTaxon server [10] revealed that the closest relative of strain KBB12 T was M. alkalimesophila CY 1 T (97.2 %). Phylogenetic analysis using the neighbourjoining algorithm showed that strain KBB12 T formed a phylogenetic clade with members of the genus Melaminivora within the family Comamonadaceae (Fig. 1). The topologies of the phylogenetic trees reconstructed using the maximum-likelihood and maximum-parsimony algorithms also showed that strain KBB12 T has a clear phylogenetic relationship within the family Comamonadaceae. The novel isolate and reference strains formed a monophyletic cluster with bootstrap support of 100 % (Fig. 1).The DNA–DNA relatedness value between strain KBB12 T and M. alkalimesophila DSM 26006 T was 43.4+2.7 %, which is well below the 70 % cut-off value recommended for the assignment of a strain to the same species [24]. The genomic DNA G+C content of strain KBB12 T was 69.6 mol%, a value similar to those reported for members of the genus Melaminivora (Table 1).In the soxB gene based phylogenetic tree, strains KBB12 T formed a cluster with representatives of Betaproteobacteria having sulfur-oxidizing ability. The soxB gene of strain KBB12 T showed the greatest degrees of similarity to those of Comamonas thiooxydans S23 T (ABR67367; 76.4 %) and Thiobacillus denitrificans DSM 807 T (ABR67367; 76.4 %) (Fig. S1, available in the online version of this article).Strain KBB12 T was aerobic, Gram-stain-negative and rodshaped. Colonies were pale yellow, circular, rough and reached up to 1.5 mm in diameter after 3 days incubaction on TSA. Cells were regular rods with a length of 2.0–3.5 µm and a width of 0.5–0.7 µm and were motile by means of a polar flagellum (Fig. S2). It grew at temperatures of 15– 45 Ǫ C and pH 6–9. Growth did not occur below 10 Ǫ C or above 50 Ǫ C and below pH 5 or above pH 10. The other physiological and biochemical characteristics of strain KBB12 T are presented in Table 1 and the species description. The fatty acids of strain KBB12 T indicated high amounts of summed feature 3 (iso-C 15: 0 2-OH and/or C 16: 1 Ɯ 7 c; 42.9 %), C 16: 0 (25.2 %) and C 18: Ɯ 7 c (16.1 %). The total fatty acid profile was similar to those of the related genus Melaminivora, although there were differences in the proportions of some fatty acids (Table 2) [1]. The polar lipids comprised phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phospholipid(PL) and an unidentified lipid (L) (Fig. S3). Strain KBB12 T exhibited a very similar polar lipid profile to its closest relative, M. alkalimesophila CY 1 T, with all strains containing PE, PG, DPG and PL. However, it differed from those of other species of the genus Melaminivora due to the absence of aminophospholipid. The predominant respiratory quinone of strain KBB12 T was ubiquinone-8 (Q-8), a feature typical of the vast majority of the family Comamonadaceae.Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the position of strain KBB12 T within the family Comamonadaceae. Bootstrap values (&gt;70 %) based on 1000 replications are shown at the nodes. Burkholderia cepacia ATCC 25416 T was used as an out-group. Black circles indicate that the corresponding branches were also recovered in trees obtained by the maximum parsimony and the maximum likelihood methods. Bar, 0.01 substitutions per nucleotide position.Based on data from phylogenetic, phenotypic and genotypic analyses strain KBB12 T is considered to represent a novel species of the genus Melaminivora, for which the name Melaminivora jejuensis sp. nov. is proposed.