Archives of Microbiology (2018) 200:439–444 https://doi.org/10.1007/s00203-017-1457-z ORIGINAL PAPER Lysobacter tongrenensis sp. nov., isolated from soil of a manganese factory Jingxin Li1 · Yushan Han1 · Wei Guo1 · Qian Wang1,3 · Shuijiao Liao1,2 · Gejiao Wang1 Received: 16 August 2017 / Accepted: 15 November 2017 / Published online: 29 November 2017 © Springer-Verlag GmbH Germany, part of Springer Nature 2017 Abstract A Gram-staining negative, aerobic, non-motile, rod-shaped bacterial strain, designated YS-37T, was isolated from soil in a manganese factory, People’s Republic of China. Based on16S rRNA gene sequence analysis, strain YS-37T was most closely related to Lysobacter pocheonensis Gsoil 193 T (97.0%), Lysobacter dokdonensis DS-58T (96.0%) and Lysobacter daecheongensis Dae08T (95.8%) and grouped together with L. pocheonensis Gsoil 193 T and Lysobacter dokdonensis DS- 58T. The DNA–DNA hybridization value between strain YS-37T and L. pocheonensis KCTC 12624T was 43.3% (± 1). The major respiratory quinone of strain YS-37T was ubiquinone-8, and the polar lipids were diphosphatidylglycerol, phosphati- dylethanolamine, phosphatidylglycerol, phospholipid, phosphatidylmethylethaolamine and two unknown lipids. Its major cellular fatty acids (> 5%) were iso-C15:0, iso-C17:1ω9c, iso-C16:0, iso-C11:0 3-OH and iso-C11:0 and the G + C content of the genomic DNA was 67.1 mol%. Strain YS-37T also showed some biophysical and biochemical differences with the related strains, especially in hydrolysis of casein. The results demonstrated that strain YS-37T belongs to genus Lysobacter and represents a novel Lysobacter species for which the name Lysobacter tongrenensis sp. nov. is proposed. The type strain is YS-37T (= CCTCC AB 2016052T = KCTC 52206T). Keywords Lysobacter tongrenensis · Soil · Polyphasic taxonomy · 16S rRNA gene Introduction Genus Lysobacter was first described by Christensen and Cook in 1978 with Lysobacter enzymogenes as the type spe- cies (Christensen and Cook 1978). This genus was classi- fied within family Xanthomonadaceae of Gamma-Proteo- Communicated by Erko Stackebrandt. bacteria (Saddler and Bradbury 2005) and the description was later emended by Park et al. (2008). To data, the genus Jingxin Li and Yushan Han contributed equally to this work. Lysobacter consists of 43 species (http://www.bacterio.net/ lysobacter.html). Lysobacter members are generally Gram- Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00203-017-1457-z) contains negative, aerobic, gliding and have a high DNA G + C con- supplementary material, which is available to authorized users. tents (61.7–70.7%), and the major quinones, fatty acids and polar lipids are of ubiquinone-8 (Q-8), iso-branched fatty * Gejiao Wang acids, and diphosphatidylglycerol (DPG), phosphatidyletha- [email protected] nolamine (PE) and phosphatidylglycerol (PG), respectively 1 State Key Laboratory of Agricultural Microbiology, (Christensen and Cook 1978; Christensen 2005; Weon et al. College of Life Science and Technology, Huazhong 2006; Ten et al. 2008, 2009; Srinivasan et al. 2010; Liu et al. Agricultural University, Wuhan, Hubei 430070, 2011; Wang et al. 2011; Oh et al. 2011; Luo et al. 2012; Ye People’s Republic of China et al. 2015; Ngo et al. 2015). Here we describe the polypha- 2 College of Basis Science, Huazhong Agricultural University, sic characteristics of strain YS-37T that related to Lysobacter Wuhan, Hubei 430070, People’s Republic of China members. 3 Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, MT 59717, USA Vol.:(0123456789)1 3 440 Archives of Microbiology (2018) 200:439–444 Materials and methods (Jiancheng Biotech, China) in combination with the 3% KOH method (Smibert and Krieg 1994). Gliding motil- Bacterial strain and culture conditions ity was tested on a fresh R2A broth culture using the hanging-drop method (Bernardet et al. 2002). Growth Strain YS-37T was isolated from soil of a manganese fac- was assessed at various temperatures (0, 4, 16, 20, 25, tory named Guizhou Dalong Manganese Industry (109°04ʹE, 28, 32, 37, 42 and 45 °C) on R2A agar, and in various 27°28ʹN) in Tongren city, Guizhou Province, People’s NaCl concentrations (0, 0.5, 1, 2, 3, 5, 6, and 7%, w/v) Republic of China. The soil sample was suspended in 0.85% in R2A liquid medium at 28 °C. The pH range (pH 4–10) NaCl (w/v) and the diluted solutions were spread on R2A was tested in R2A liquid medium with different buffer agar plates and incubated at 28 °C for 1 week. A total of 32 systems (0.1 M citric acid/0.1 M sodium citrate, for pH strains were isolated (data not shown) and a strain named 4.0–5.0; 0.1 M KH 2PO4/0.1 M NaOH, for pH 6.0–8.0; T YS-37 was selected for this study due to the preliminary 0.1 M NaHCO3/0.1 M Na2CO3, for pH 9–10). Growth low 16S rRNA gene sequence similarity. The resistance lev- under anaerobic condition was determined by incubation els of strain YS-37T for multi-metal(loids) were tested with in an anaerobic chamber at 28 °C for 2 weeks on R2A agar. the minimal inhibition concentration (MIC) on R2A agar Oxidase and catalase activities were determined using oxi- plates using MnCl 2, Na3AsO3, K2Sb2(C4H2O6)2, K2CrO4, dase reagent and 3% (v/v) H2O2, respectively (Smibert T CdCl2 and PbCl2. Strains L. pocheonensis KCTC 12624 , L. and Krieg 1994). Nitrate and nitrite reduction tests were dokdonensis KCTC 12822 T and L. daecheongensis KCTC performed as described by Lányí (1987). Hydrogen sulfide 12600T were purchased from the Korean Centre of Type production, indole test and hydrolyses of casein (5%, Cultures and used as reference strains. w/v), gelatin (15%, w/v), Tween 20, 40, 60, 80 and starch (0.2%, w/v) were determined according to Smibert and Krieg (1994). Acid production from carbohydrates were also tested (Dong and Cai 2001). The API 20 NE, API ID 32GN and API ZYM systems (bioMérieux) were used to Phylogenetic analysis determine biochemical properties, utilization of carbohy- drates and enzyme activities according to the manufac- Genomic DNA of strain YS-37T was extracted accord- turer’s protocols, and if necessary, in combination with ing to standard procedures (Sambrook and Russell 2001). traditional methods. The primers 27F and 1492R were used for amplification of the 16S rRNA gene fragment as described by Brosius et al. (1978). The PCR product was purified and cloned into pGEM-T vector (Promega), subsequently PCR amplified Chemotaxonomic characterization using primers T7 and SP6 and sequenced in Tsingke Com- pany (Beijing, China). The nearly completed 16S rRNA gene For whole-cell fatty acid analysis, cells of strain YS-37T sequence (1506 bp) of strain YS-37T was aligned with those and the three reference strains were inoculated on R2A from EzTaxon database (http://eztaxon-e.ezbiocloud.net) agar and harvested when the cells growth reached the (Chun et al. 2007) using the CLUSTAL X program (Thomp- exponential phase determined by the quadrant streak pat- son et al. 1997). Phylogenetic analyses were performed with tern method according to the protocol of MIDI (Sherlock MEGA 6 (Tamura et al. 2013) using neighbor-joining (Sai- Microbial Identification System, MIDI) and analyzed by tou and Nei 1987), maximum-parsimony (Fitch 1971) and gas chromatography (MIDI Sherlock version 4.5; MIDI maximum-likelihood (Felsenstein 1981) algorithms. For database TSBA40 4.10) (Sasser 1990). For the extraction each method, bootstrap values were calculated based on of respiratory quinones and polar lipids, the strains were 1000 replications (Felsenstein 1985). cultured in R2A medium and freeze-dried after harvest- ing. Respiratory quinone analysis was performed by HPLC as described by Minnikin et al. (1984). Polar lipid analy- ses of strain YS-37T and L. dokdonensis KCTC 12822T were determined by two-dimensional TLC method as Morphological, physiological, described by Tindall (1990). The DNA G + C content was and biochemical characterization determined by HPLC according to the method of Mesbah et al. (1989). Genomic DNA was extracted as described by Cellular morphology was observed by scanning elec- Pitcher and Saunders (1989) for DNA–DNA hybridization tron microscopy (SEM, JSM-6390; JEOL) (Fig. S1). analysis, which was performed using the thermal denatura- Gram reaction was determined using a Gram-staining kit tion and renaturation method of De Ley et al. (1970). 1 3 Archives of Microbiology (2018) 200:439–444 441 Results and discussion for urease activity, indole production and hydrogen sulfide production. These characteristics are consistent with the The 16S rRNA gene sequence of strain YS-37T was closely strains of the genus Lysobacter (Weon et al. 2006; Luo related to Lysobacter pocheonensis Gsoil 193T (97.0%), et al. 2012; Ye et al. 2015). The flexirubin-type pigment T Lysobacter dokdonensis DS-58T (96.0%) and Lysobacter was absent. Strain YS-37 showed some biophysical and daecheongensis Dae08T (95.8%), and showed similari- biochemical differences with the related strains, especially ties less than 95.4% with the other members of the genus in hydrolysis of casein (Table 1, supplementary material Lysobacter including the type species strain Lysobacter Table S1). enzymogenes (93.9%). In the phylogenetic tree based on The DNA–DNA hybridization value between strain YS- T neighbor-joining algorithm, strain YS-37 T was closely 37 and the closely related strain L. pocheonensis KCTC T related to the Lysobacter members and formed a branch 12624 was 43.3% (± 1, n = 2), which is below the thresh- with L. pocheonensis Gsoil 193T and L. dokdonensis DS- old value of 70% recommended for species delineation 58T (Fig. 1). The phylogenetic trees based on the max- (Wayne et al. 1987). The whole-cell fatty acid compositions T T imum-parsimony and maximum-likelihood algorithms of strain YS-37 , L.