DOI: 10.15468/ff3t7e
title: Lysobacter humi sp. nov., isolated from soil

description: This dataset contains the digitized treatments in Plazi based on the original journal article Lee, Dongwook, Jang, Jun Hyeong, Cha, Seho, Seo, Taegun (2017): Lysobacter humi sp. nov., isolated from soil. International Journal of Systematic and Evolutionary Microbiology 67 (4): 951-955, DOI: 10.1099/ijsem.0.001722, URL: http://dx.doi.org/10.1099/ijsem.0.001722AbstractA yellow-pigmented and strictly aerobic bacterial strain, designated FJY8 T, was isolated from the soil of Goyang, South Korea. The cells of FJY8 T were Gram-reaction-negative, non-motile rods. Colonies were circular, convex and transparent. Strain FJY8 T grew optimally at 30 Ǫ C, with 0 % (w/v) NaCl and at pH 8. Phylogenetic analysis of the 16S rRNA gene sequence of FJY8 T revealed a clear affiliation of this bacterium to the family Lysobacteraceae, and it was related to members of the genus Lysobacter, with Lysobacter xinjiangensis KCTC 22558 T being its closest relative (98.7 % sequence similarity). The DNA G+C content was 68.0±0.4 mol%. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol were identified as the major polar lipids, and an unidentified phospholipid and two unidentified aminophospholipids were also detected as the minor polar lipids. The major fatty acids were iso-C 16: 0, summed feature 9 (iso-C 17: 1 Ɯ 9 c and/or C 16: 0 10-methyl) and iso-C 15: 0. Only ubiquinone-8 (Q-8) was detected as the isoprenoid quinone. DNA – DNA hybridization values of strain FJY8 T with Lysobacter xinjiangensis RCML-52 T and Lysobacter mobilis 9NM-14 T were 55.8±2.0 and 45.2±4.8 %, respectively. On the basis of DNA – DNA hybridization, phylogenetic distinctiveness, and some physiological and biochemical tests, strain FJY8 T (= KCTC 42810 T = JCM 31019 T) represents a novel species of the genus Lysobacter, for which the name Lysobacter humi sp. nov. is proposed.Lysobacter is the type genus of the family Lysobacteraceae, members of which contain ubiquinone Q-8 as the predominant respiratory quinone. The genus Lysobacter was first proposed by Christensen and Cook [1] for non-fruiting bacteria with high guanine-plus-cytosine (G+C) contents in their deoxyribonucleic acids. According to LPSN (www.bacterio.net/lysobacter.html), at the time of writing the genus Lysobacter comprises 39 species with validly published names including the recently described species Lysobacter firmicutimachus [2], Lysobacter erysipheiresistens [3], Lysobacter hankyongensis and Lysobacter sediminicola [4]. Most of the species within the genus Lysobacter are frequently isolated from soil [5 – 9]. The present work is a continuation of our bacterial diversity and bioprospecting studies in different regions of South Korea. We report the characterization of a strain designated FJY8 T that represents a novel species within the family Lysobacteraceae.Strain FJY8 T was isolated from a soil sample collected in an arid area in Goyang, Gyeonggi Province, South Korea. For isolation, 5 g soil was suspended with 10 ml sterile NaCl (0.85 %, w/v) and serially diluted with the standard dilution technique. A 100-µl aliquot of each dilution was plated onto R 2A agar (MB cell) and then incubated at 30 Ǫ C for 3 days. Single colonies were then chosen for further purification. Purified colonies were stored at ‒70 Ǫ C in liquid R 2A broth (MB cell) supplemented with 25 % (v/v) glycerol. For morphological and biochemical characterization, strain FJY8 T was grown on R 2A agar. Lysobacter xinjiangensis RCML- 52 T and Lysobacter mobilis 9NM-14 T were respectively obtained from the KCTC (Daejeon, South Korea) and DSM (Braunschweig, Germany).The nearly complete 16S rRNA gene sequence of strain FJY8 T was amplified by colony PCR using the 27F, 785F, 800 R and 1492 R universal primers [10], and sequencing was performed by Solgent (Daejeon, South Korea). The 16S rRNA gene sequence of strain FJY8 T was assembled with SeqMan software (DNASTAR). The sequences obtained were compared with those of close relatives by BLAST searches [11] and by using the EzTaxon-e server (www. ezbiocloud.net/eztaxon/; [12]). Sequences were aligned by CLUSTAL W [13] within the BioEdit Sequence Alignment Editor, version 7.2.5 [14], and phylogenetic trees were reconstructed using MEGA version 6.06 [15, 16], by the neighbour-joining method with the Kimura two-parameter model [17, 18]. Bootstrap values were calculated based on 1000 replicates [19]. The min-mini heuristic algorithm was applied in maximum-parsimony analysis to compare with the neighbour-joining phylogenetic tree [20]. The sequence of Bacillus subtilis DSM 10 T was used as an outgroup. In the neighbour-joining phylogenetic tree, strain FJY8 T clearly belonged to the lineage defined by the genus Lysobacter of the family Lysobacteraceae (Fig. 1). Also, strain FJY8 T was in the same cluster as Lysobacter xinjiangensis RCML-52 T. Sequence comparisons based on the 16S rRNA gene revealed that the strain FJY8 T was most closely related to Lysobacter xinjiangensis RCML-52 T (98.7 % sequence similarity), Lysobacter mobilis 9NM-14 T (97.4 %), Lysobacter bugurensis ZLD-29 T (95.8 %) and Lysobacter korlensis ZLD-17 T (95.4 %). The 16S rRNA gene sequence comparisons by means of NCBI BLAST searches showed that strain FJY8 T had 93.9~98.7 % sequence similarity with type strains of validly named species of the genus Lysobacter.The Gram-staining reaction was determined following the non-staining method of Buck [21]. Morphological characteristics of strain FJY8 T were investigated by transmission electron microscopy (LIBRA 120, Carl Zeiss), and the 0.4 % agar method was used to determine cell motility. The NaCl tolerance of the isolate was assessed by growing the strain in R 2A medium supplemented with 0–10 % (w/v) NaCl, in increments of 0.5 %, for 10 days at 30 Ǫ C. The ability to grow in acid and alkaline media was assessed by adjusting media to pH 4–11 with acetic acid/sodium acetate (pH 4.0), 10 mM MES (pH 5.0–6.0), 10 mM Tris (pH 7.0–9.0) and sodium carbonate/sodium bicarbonate (pH 10.0–11.0). Growth at 30 Ǫ C was observed for 10 days on Luria–Bertani (Difco) agar, MacConkey agar (Difco), marine agar (Difco), nutrient agar (Difco) and tryptic soy agar (Difco). Growth at different temperatures (4, 10, 15, 20, 25, 30, 37, 42 and 45 Ǫ C) was assessed using R 2A agar (MB cell) after a 10-day incubation period. Tetramethyl- p -phenylenediamine and H 2 O 2 (3 %, w/v) were used to test for oxidase and catalase activity, respectively. A GasPak jar was used for anaerobic incubation (BBL). To identify additional biochemical characteristics, the API 20NE, API 32GN and API ZYM systems (bioMerieux) were used according to the manufacturer’ s instructions. After 2 days of incubation at 30 Ǫ C, strain FJY8 T cultured on R 2A agar yielded colonies that were circular, convex, transparent and yellow-coloured. The cells of strain FJY8 T were Gram-reaction-negative, non-motile, strictly aerobic and rod-shaped, with dimensions in the range 0.7–1.0×1.3–1.5 µm (see Fig. S1 available in the online Supplementary Material). The phenotypic characteristics of strain FJY8 T are given in the species description, and a comparison with the characteristics of other species of the genus Lysobacter is presented in Table 1.Fig. 1. Neighbour-joining phylogenetic tree reconstructed using the 16S rRNA gene sequences of strains FJY8 T and related taxa. Numbers at branch nodes present bootstrap values (&gt;50 %) obtained as percentages of 1000 replicates. Filled circles indicate the corresponding nodes recovered by using the maximum-parsimony algorithm. Bar, 0.02 substitutions per nucleotide position.Table 1. Characteristics that differentiate strain FJY8 T from phylogenetically related species of the genus LysobacterStrains: 1, FJY8 T (data from this study); 2, Lysobacter xinjiangensis RCML-52 T (data from this study); 3, Lysobacter mobilis 9NM-14 T (data from this study); 4, Lysobacter xinjiangensis ZLD-29 T (data from [27]); 5, Lysobacter xinjiangensis ZLD-17 T (data from [27]). +, Positive;, ‒ negative.Characteristic 1 2 3 4 5 Ranges for growth Temperature (Ǫ C) 20–42 20–42 15–37 10–37 10–37 NaCl (%, w/v) 0–0.5 0–2 0–1 0–3 0.5–4 pH 7–9 6–10 6–8 6–11 6–11 Oxidase + + ‒ + + Nitrate reduction ‒ ‒ ‒ + + Hydrolysis of aesculin + + ‒ + + Enzyme activities Cystine arylamidase ‒ + + ‒ ‒ Lipase (C14) + + ‒ ‒ ‒ Trypsin + + + ‒ + Valine arylamidase + + + ‒ ‒ a- Chymotrypsin + + ‒ + + Production of acid from glucose ‒ ‒ + + + DNA G+C content (mol%) 68.0 67.9 68.0 68.2 67.9Total lipids [22] extracted from strain FJY8 T and Lysobacter xinjiangensis RCML-52 T were examined by two-dimensional TLC with two developing solvents, chloroform/methanol/water (65: 25: 4, by vol.) and chloroform/methanol/ acetic acid/water (80: 12: 15: 4, by vol.). TLC plates were visualized with appropriate detection reagents [23]. Cells for fatty acid analysis were harvested from the third quadrant on R 2A agar (MB cell) after incubation at 30 Ǫ C for 3 days. Cellular fatty acids were extracted, saponified and methylated as reported by Kuykendall et al. [24]. The extract was analysed using the Sherlock Microbial Identification System v6.01 (MIS, database TSBA6, MIDI) and subsequently compared with those of other type species. To purify isoprenoid quinones, Sep-Pak Vac cartridges (Waters) were used, and the extract was analysed by HPLC as previously reported by Hiraishi et al., and Collins and Jones [25, 26]. TLC of polar lipids showed that strain FJY8 T contained phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified phospholipid and two unidentified aminophospholipids. Strain FJY8 T and Lysobacter xinjiangensis RCML-52 T had phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and two unidentified aminophospholipids in common. However, strain FJY8 T differed from the reference strain by the absence of the unidentified lipid (see Fig. S2). This result supported affiliation of strain FJY8 T to the genus Lysobacter; in particular, all members of this genus produce phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as major polar lipids [5, 7, 27]. The major fatty acids of strain FJY8 T were identified as iso-C 16: 0, summed feature 9 (iso-C 17: 1 Ɯ 9 c and/or C 16: 0 10-methyl) and iso- C 15: 0. Minor amount of fatty acids with less than 0.5 % are C 14: 0, C 16: 0 N-alcohol, iso-C 17: 0 3-OH, Summed feature 1 (iso-C 15: 1 H and/or iso-C 15: 1 I). iso-C 16: 0 was the most abundant fatty acid in all strains, indicating that the fatty acid profile of strain FJY8 T was in agreement with those of related type strains; detailed fatty acid compositions are given in Table S1. The only isoprenoid quinone detected in strain FJY8 T was ubiquinone 8 (Q-8), which corresponds to the description of the genus Lysobacter [9, 27, 28].Total genomic DNA was extracted according to the method of Ausubel et al. [29]. DNA–DNA hybridization was performed to assess the relatedness of the new isolate and related taxa on the basis of the thermal denaturation principles and equations of De Ley et al. [30] and Gillis et al. [31] and an optimized procedure evaluated by Loveland-Curtze et al. [32]. These experiments were performed three times independently. Hybridization values are expressed as the mean±SD of the three values. The DNA G+C content of strain FJY8 T was determined in triplicate using a simple fluorimetric method [33] and SYBR Green 1 (Life Technologies) and a real-time PCR thermocycler (Rotor-Gene Q, Qiagen). The genomic DNAs of Lactococcus lactis subsp. lactis KACC 13877 T, Bacillus subtilis subsp. subtilis KACC 17796, Bacillus licheniformis KACC 10476 T, Escherichia coli KACC 14818, Pseudomonas aeruginosa ATCC 15442 and Micrococcus luteus KACC 13377 were used for calibration. For genomic characterization, we performed DNA–DNA hybridization using the fluorimetric method. Strain FJY8 T was found to have 55.8±2.0 and 45.2±4.8 % DNA–DNA relatedness with Lysobacter xinjiangensis RCML-52 T and Lysobacter mobilis 9NM-14 T. These values support the conclusion that strain FJY8 T represents a novel species distinct from closely related species of the genus Lysobacter [34, 35]. Further, the DNA G+C content of strain FJY8 T was 68.0 ±0.4 mol%, a value that is similar to those of other members of the genus Lysobacter.Based on 16S rRNA gene sequence analysis, strain FJY8 T was most closely related to members of the genus Lysobacter in the family Lysobacteraceae. Common characteristics of members of the genus Lysobacter identified in this study were the presence of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as major polar lipids, Q-8 as a respiratory quinone and iso-C 16: 0 and summed feature 9 (iso-C 17: 1 Ɯ 9 c and/or C 16: 0 10-methyl) as abundant fatty acids. However, the results of DNA–DNA relatedness analysis and the polyphasic study clearly showed that strain FJY8 T can be distinguished from other species in the genus Lysobacter. Thus, strain FJY8 T represents a novel species within the genus Lysobacter, for which the name Lysobacter humi sp. nov. is proposed.