DOI: 10.15468/6wyun5
title: Brevibacterium hankyongi sp. nov., isolated from compost

description: This dataset contains the digitized treatments in Plazi based on the original journal article Choi, Kang Duk, Siddiqi, Muhammad Zubair, Liu, Qingmei, Muhammad Shafi, Siddiqi, Durrani, Yasser, Lee, Soon Yeol, Kang, Myung-Suk, Im, Wan Taek (2018): Brevibacterium hankyongi sp. nov., isolated from compost. International Journal of Systematic and Evolutionary Microbiology 68 (9): 2783-2788, DOI: 10.1099/ijsem.0.002886, URL: http://dx.doi.org/10.1099/ijsem.0.002886AbstractA Gram-positive, strictly aerobic, non-motile, milky-white to creamy coloured and rod-shaped bacterium, designated BS05 T, was isolated from compost. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that the strain formed a distinct lineage within the genus Brevibacterium and was most closely related to Brevibacterium avium NCFB 3055 T (96.3 %), Brevibacterium oceani BBH 7 T (96.2 %) and Brevibacterium epidermidis NBRC 14811 T (96.1 %). The DNA G+C content was 62.3 mol%. The predominant quinone was MK-8(H 2). The major fatty acids were anteiso-C 15: 0, anteiso-C 17: 0, iso-C 16: 0 and iso-C. The cell-wall peptidoglycan of strain BS05 T contained meso -diaminopimelic acid. The major polar lipid was 15: 0 phosphatidylglycerol. Moreover, the low sequence similarity of the 16S rRNA gene sequencing, physiological, biochemical and chemotaxonomic analyses allowed the phenotypic and genotypic differentiation of strain BS05 T from the recognized species of the genus Brevibacterium. Therefore, strain BS05 T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium hankyongi sp. nov. is proposed, with the type strain BS05 T (= KACC 18875 T = LMG 29562 T).The genus Brevibacterium was first stated by Breed in 1953 [1], with the type species Brevibacterium linens, which was isolated from the surface of ripened cheese. At the time of description of B. linens, cells of the strain were Gram-positive, short rods, non-sporeforming and non-branching. In 2006 Collins [2] stated that the genus Brevibacterium has meso -diaminopimelic acid as major peptidoglycan and MK- 8(H 2) as major respiratory menaquinone. At the time of writing, the genus Brevibacterium comprises 49 species with validly published names (www.bacterio.net/brevibacterium. html). In addition to B. linens, recently the following species in the genus Brevibacterium have been reported: Brevibacterium ammoniilyticum [3] and Brevibacterium marinum [4]. In many cases, members of the genus Brevibacterium have been isolated from dairy products, including cheese and from human skin [5, 6]. In some cases, Brevibacterium strains have been recognized from places such as high salinity environments [4, 7], poultry [8] and a degraded thallus of a brown alga [9]. Members of the genus have been reported not only in relation to cheese production, but also in patents from biotechnological industry, including those for production of antibiotics and wastewater treatment [10, 11].During the course of a study to determine the types of aerobic bacteria in compost made from foodstuff waste in Jeju Island, Republic of Korea, the compost samples were collected from different locations of Jeju Island. For isolation of bacterial strains, 1/2 Reasoner’ s 2A (R 2A; Difco) agar was used. The serial dilution was made up to 10 ‒5 by dissolving 1 g sample in 10 ml 0.85 % sterile saline and 100 µl of each dilution was spread on the above medium and the plates were incubated aerobically at 30 Ǫ C for 5 days. Numerous types of colonies were observed and one, designated BS05 T, was chosen for taxonomic study. Strain BS05 T was routinely cultured on R 2A agar at 30 Ǫ C and was preserved at ‒80 Ǫ C in R 2A broth supplemented with 20 % (v/v) glycerol.Genomic DNA of strain BS05 T was extracted with a commercial genomic DNA extraction kit (Solgent). The bacterial universal primer sets 800 R, 1492 R, 27F and 518F were used to amplify the 16S rRNA gene sequence. The purified PCR product was sequenced by Genotech according to Kim et al. [12]. The nearly full-length sequence of the 16S rRNA gene was compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database and EzBioCloud [13]. Multiple sequence alignments were performed via the CLUSTAL_X program [14]. Gaps were edited in the BioEdit program [15] and 1370 nucleotides were used for phylogenetic tree reconstruction. For maximum-likelihood tree analysis, evolutionary distances were calculated using Kimura’ s twoparameter model (KM2) [16] and the gaps were treated by complete deletion. The neighbour-joining tree was reconstructed by using the same model (KM2) with complete deletion of gaps [17]. Similarly, the maximum-parsimony tree was made with the subtree-pruning-regrafting heuristic method and the gaps were edited with complete deletion [18] using the MEGA6 program [19] with bootstrap values based on 1000 replications [20].The almost-complete 16S rRNA gene sequence of strain BS05 T (1499 nt) was determined and subjected to comparative analysis. Phylogenetic analysis using the maximumlikelihood method based on 16S rRNA gene sequences indicated that strain BS05 T are clustered within the genus Brevibacterium (Fig. 1) and formed a monophyletic clade with Brevibacterium avium JCM 11680 T.The sequence similarity calculation showed that the closest relative strain of BS05 T was B. avium JCM 11680 T (96.3 %). Lower sequence similarities (&lt;96.3 %) were found to Brevibacterium oceani JCM 21798 T (96.2 %) and Brevibacterium epidermidis DSM 20660 T (96.1 %). The phylogenetic tree (Fig. 1) based on the maximum-likelihood algorithm showed that strain BS05 T clustered within the genus Brevibacterium and first joined B. avium JCM 11680 T. The tree topologies obtained using the other two (neighbour-joining and maximum-parsimony) algorithms were similar to that obtained when the maximum-likelihood algorithm was used (Fig. 1). Based on the EzBioCloud results of 16S rRNA gene sequence similarity and phylogenetic tree results, the strain BS05 T did not show a very close similarity to the type species B. linens DSM 20425 T (95.8 %) or the other members of genus Brevibacterium as compared to the above said species (B. avium JCM 11680 T, B. oceani JCM 21798 T and B. epidermidis DSM 20660 T). However, phylogenetic tree reconstruction (using 30 sequences of the type strains of the genus Brevibacterium and ten sequences of different genera: Kocuria, Arthrobacter, Nesterenkonia, Xylanimicrobium and Spelaeicoccus) indicated that strain BS05 T held a distinct position in the tree with B. avium JCM 11680 T, which was used as a reference for comparison with the isolated strain (Fig. S1, available in the online version of this article). Similarly, based on 16S rRNA gene sequence similarity next to B. avium JCM 11680 T, B. oceani JCM 21798 T and B. epidermidis DSM 20660 T were selected as the the closest recognized neighbour of strain BS05 T and used as reference strains in most of the subsequent phenotypic analysis.Fig. 1. Phylogenetic relationship between strain BS05 T and other related species of the genus Brevibacterium. The tree was reconstructed using the maximum-likelihood method based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branch points. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with maximum-parsimony and neighbour-joining algorithms. Spelaeicoccus albus D3-40 T (HF570029) was used as an outgroup. Bar, 0.005 substitutions per nucleotide position.The Gram-reaction was determined using the non-staining method, as described previously [21]. Cell motility was determined using hanging-drop method, while cell morphology was examined with the transmission electron microscope (SU-3500, Hitachi), using cells grown for 2 days at 30 Ǫ C on marine agar (MA) medium. Oxidase activity was determined using 1 % (w/v) N,N,N,N -tetramethyl-1,4-phenylenediamine reagent (bioḾerieux). Catalase activity was determined by the production of bubbles from 3 % (v/v) H 2 O 2 solution. Hydrolysis activity was tested using the following substrates: starch, casein, DNase (DNase agar medium, Sharlau), Tween 80 and CMC (carboxy methyl cellulose) [22]. All tests were performed and evaluated after 2 days of incubation at 30 Ǫ C. Biochemical tests using the commercial API kits (API ZYM, API 20NE and API 32GN; bioMerieux) were generally performed according to the manufacturer’ s instructions. The API ZYM test strips were read after 4 h of incubation at 37 Ǫ C, and the other API strips were examined after 2 days of incubation at 30 Ǫ C. Growth at different temperatures (4, 10, 15, 20, 25, 30, 37 and 42 Ǫ C) and various pH values (pH 4–10 at intervals of 0.5 and 1 pH units) was assessed after 3 days of incubation at 30 Ǫ C. The following buffers (final concentration, 50 mM) were used to adjust the pH of nutrient broth. Acetate buffer was used for pH 4.0–5.5, phosphate buffer was used for pH 6.0–8.0 and Tris buffer was used for pH 8.5–10.0. Salt tolerance test was evaluated on R 2A agar medium supplemented with 0–8 % (w/v at intervals of 0.5 and 1 % unit) NaCl and growth was assessed after 7 days of incubation at 30 Ǫ C. Growth on different media was also tested by using nutrient agar (NA, Difco), trypticase soy agar (TSA, Difco), lysogeny broth (LB) agar (Difco) and MacConkey agar (Difco) at 30 Ǫ C for 1 week. Anaerobic growth was observed in serum bottles by adding thioglycolate (1 g l –1) to R 2A broth and substituting the upper air layer with nitrogen gas.Cells of strain BS05 T were Gram-positive, strictly aerobic, non-motile and rod-shaped (Fig. S2). Colonies of strain BS05 T grown on R 2A agar were circular, convex, opaque and milky-white to creamy colour after 48 h of incubation at 30 Ǫ C. The isolate did not grow on LB or MacConkey agar, but grew weakly on NA or TSA at 30 Ǫ C. Strain BS05 T was negative for the hydrolysis of starch, casein, cellulose and DNase. Furthermore, physiological characteristics of strain BS05 T aresummarized in the species description and a comparison of selective characteristics of the isolated strain and related type strains is given in Table 1.To analyse the DNA G+C content of strain BS05 T, the genomic DNA was extracted and purified as previously described [23], and was degraded enzymatically into nucleosides, then the DNA G+C content was determined as described by Mesbah et al. [24] using reverse-phase high-performance liquid chromatography (HPLC). Strain BS05 T was examined for their polar lipid contents as described by Minnikin et al. [25]. One hundred milligrams of freeze-dried cells were suspend in methanol–water (10: 1, v/v) and stirred well. Then, 2 ml petroleum ether was added and mixed well by vortexing. The sample was centrifuged. After centrifugation, the upper layer was discarded, and the lipid residues were taken twice with the first extraction as chloroform–methanol–water (90: 100: 30, by volume) and the second extraction as (50: 100: 40, by volume). Chloroform and 0.3 % NaCl (1.3 ml each) solution were added to the combined supernatant, mixed thoroughly and centrifuged. Then the upper layer was discarded with a Pasteur pipette and only the bottom layer was used. Polar lipids were dissolved in chloroform–methanol (2: 1, by volume), and then the samples were spotted on the corner of two-dimensional thin layer chromatography (2D- TLC) apparatus using TLC Kiesel gel 60F 254 (Merck) plates (10× 10 cm). The polar lipids were developed in the first direction by using chloroform–methanol–water (65: 25: 4, v/v); while in the second direction, it was developed by chloroform–acetic acid–methanol–water (80: 15: 12: 4, by volume).Isoprenoid quinone of the isolate was extracted with chloroform–methanol (2: 1, by volume), evaporated under vacuum condition, and re-extracted in n-hexane/water (1: 1, by volume). The crude n-hexane–quinone solution was purified using Sep-Pak Vac cartridges silica (Waters) and subsequently analysed by HPLC as previously described [26]. Cellular fatty acid profiles were determined after 48 h of growth at 30 Ǫ C on R 2A agar medium. The cellular fatty acids were saponified, methylated and extracted according to the method described by the Sherlock Microbial Identification System (MIDI). The fatty acids analysed by a gas chromatograph (Hewlett Packard 6890) and identified by the Microbial Identification software package based on the Sherlock Aerobic Bacterial Database (TSBA60) [27]. Cell-wall peptidoglycan analysis of strain BS05 T was performed by using TLC after hydrolysis with 6 M HCl at 100 Ǫ C for 18 h, as described previously [28].DNA G+C contents of the strain BS05 T was 62.3 mol%, which was within the range of the genus Brevibacterium (Table 1). The main detected polar lipid of strain BS05 T was phosphatidylglycerol; the minor polar lipids were diphosphatidylglycerol and phosphatidylinositol mannosides (Fig. S3). Based on the polar lipid analysis, strain BS05 T shared a similar polar lipid profile with the described species of the genus Brevibacterium [7, 29]. The detected quinones were MK-8 (H 2) as a major isoprenoid quinine and MK-7(H 2) as a minor isoprenoid quinone. The fatty acid profile of strain BS05 T was compared with closely related species of the genus Brevibacterium. The major fatty acids of strain BS05 T were anteiso-C 17: 0 (32.4 %), anteiso-C 15: 0 (27.7 %), iso-C 16: 0 (22.6 %) and iso-C 15: 0 (13.0 %), which is a typical profile of members of the genus Brevibacterium. However, some differences in the values of fatty acids distinguished strain BS05 T from the closely related strains genus Brevibacterium (Table S1). Strain BS05 T contained meso -diaminopimelic acid as the cell-wall diamino acid.Characteristic 1 2 3 4 Isolation source Compost Poultry a Sea sediment b Skin c Colony colour Milky-white to creamy Creamy a Pale orange b Pale yellow c Temperature range for growth (Ǫ C) 18–42 22–37 a 10–35 b ND NaCl tolerance (%, w/v) 6 ND 12 b 15 c Oxidase activity + + a b ‒ c ‒ Enzyme activity: Arginine dihydrolase ‒ ‒ ‒ + Cystine arylamidase + ‒ + ‒ Aesculin hydrolysis ‒ + ‒ ‒ Esterase (C4) + + ‒ ‒ Leucine arylamidase + ‒ + ‒ Naphthol-AS-BI-phosphohydrolase ‒ + + + Nitrate reduction ‒ + ‒ + Assimilation of: Adipate ‒ + + + L- Arabinose ‒ + ‒ + Caprate + ‒ + + Citrate + ‒ + + D- Glucose ‒ + + + Gluconate ‒ ‒ + + 3-Hydroxy-butyrate + + ‒ ‒ 4-Hydroxy-benzoate + + ‒ ‒ Itaconate ‒ + + ‒ D- Mannose ‒ ‒ + + Malate + ‒ + + D- Mannitol ‒ ‒ ‒ + Melibiose ‒ + ‒ ‒ Phenyl-acetate + ‒ + + Propionate + ‒ + + L- Proline ‒ ‒ + + G+C content (mol%) 62.3 ND 62.5 b 66.2–67.2 Data from a, Brevibacterium avium JCM 11680T [8]; b, Brevibacterium oceani JCM 21798T [7] and c, Brevibacterium epidermidis DSM 20660T [5].On the basis of phylogenetic position, and phenotypic characteristics including chemotaxonomy characteristics, a new taxon in the genus Brevibacterium is created for strain BS05 T with the name, Brevibacterium hankyongi sp. nov. Phylogenetically the novel isolate is related to the genus Brevibacterium and shares several common features with members of this genus, e.g. MK-8(H 2) as the major respiratory quinone, phosphatidylglycerol as the major polar lipid and branched fatty acids with odd numbers of carbons (anteiso- C 17: 0 and anteiso-C 15: 0). However, not only a significant distance of 16S rRNA gene sequence (&lt;96.5 %) but also the fatty acid (iso-C) and some phenotypic characteristics distinguish the strain BS05 T from the closest species of the genus Brevibacterium.