Antonie van Leeuwenhoek (2020) 113:365–375 https://doi.org/10.1007/s10482-019-01347-8 (0123456789().,-volV)( 0123456789().,-volV) ORIGINAL PAPER Lacisediminihabitans profunda gen. nov., sp. nov., a member of the family Microbacteriaceae isolated from freshwater sediment Ye Zhuo . Chun-Zhi Jin . Feng-Jie Jin . Taihua Li . Dong Hyo Kang . Hee-Mock Oh . Hyung-Gwan Lee . Long Jin Received: 14 August 2019 / Accepted: 2 October 2019 / Published online: 18 October 2019 Ó The Author(s) 2019 Abstract A novel Gram-stain-positive bacterial sequence similarities to Glaciihabitans tibetensis strain, CHu50b-6-2T, was isolated from a 67-cm-long KCTC 29148T, Frigoribacterium faeni KACC sediment core collected from the Daechung Reservoir 20509T and Lysinibacter cavernae DSM 27960T, at a water depth of 17 m, Daejeon, Republic of Korea. respectively. The phylogenetic trees revealed that The cells of strain CHu50b-6-2T were aerobic non- strain CHu50b-6-2T did not show a clear affiliation to motile and formed yellow colonies on R2A agar. The any genus within the family Microbacteriaceae. The phylogenetic analysis based on 16S rRNA gene chemotaxonomic results showed B1a type pepti- sequencing indicated that the strain formed a separate doglacan containg 2, 4-diaminobutyric acid (DAB) lineage within the family Microbacteriaceae, exhibit- as the diagnostic diamino acid, MK-10 as the ing 98.0%, 97.7% and 97.6% 16S rRNA gene predominant respiratory menaquinone, diphos- phatidylglycerol, phosphatidylglycerol, and an unidentified glycolipid as the major polar lipids, Ye Zhuo and Chun-Zhi Jin have contributed equally to this work. anteiso-C15:0, iso-C16:0, and anteiso-C17:0 as the major fatty acids, and a DNA G ? C content of 67.3 mol%. The GenBank/EMBL/DDBJ accession numbers for sequences The combined genotypic and phenotypic data showed T of strain CHu50b-6-2 generated in this study are as follows: that strain CHu50b-6-2T could be distinguished from MF770244 (16S rRNA), MK910353 (recA), and all genera within the family Microbacteriaceae and VRMG00000000 (whole genome sequence). represents a novel genus, Lacisediminihabitans gen. Electronic supplementary material The online version of nov., with the name Lacisediminihabitans profunda this article (https://doi.org/10.1007/s10482-019-01347-8) con- sp. nov., in the family Microbacteriaceae. The type tains supplementary material, which is available to authorized users. Y. Zhuo Á F.-J. Jin Á T. Li Á L. Jin (&) C.-Z. Jin Á D. H. Kang College of Biology and the Environment, Co-Innovation Industrial Biomaterial Research Center, Korea Research Centre for Sustainable Forestry in Southern China, Institute of Bioscience and Biotechnology (KRIBB), Nanjing Forestry University, Nanjing 210037, China Daejeon 34141, Republic of Korea e-mail: [email protected] H.-M. Oh Á H.-G. Lee (&) C.-Z. Jin Á D. H. Kang Cell Factory Research Centre, Korea Research Institute of Department of Bio-Molecular Science, KRIBB School of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Bioscience, Korea University of Science and Technology Republic of Korea (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, Republic e-mail: [email protected] of Korea 123 366 Antonie van Leeuwenhoek (2020) 113:365–375 strain is CHu50b-6-2T (= KCTC 49081T = JCM peptone, 0.05 g yeast extract, 0.05 g casamino acid, 32673T). 0.05 g dextrose, 0.05 g soluble starch, 0.03 g K2HPO4, 0.005 g MgSO4, 0.03 g sodium pyruvate, Keywords Lacisediminihabitans Á and 15 g agar) and incubated at room temperature Lacisediminihabitans profunda Á CHu50b-6-2 Á (25 °C) for 4 weeks. One yellow colony, designated as Sediment CHu50b-6-2T, was isolated and subcultivated on R2A agar at 30 °C for further analysis. The colony char- acteristics were determined after growing for 5 days at 30 °C on R2A agar. Gram staining was performed Introduction using a Gram stain kit (Becton–Dickinson) and 3% KOH solution. The cell morphology and motility were Since Park et al. (1993) proposed the family Mi- examined under a phase-contrast microscope (Nikon crobacteriaceae, 56 genera have been described Eclipse 80i microscope, 1000 9 magnification) and a validly in this family at the time of writing (http:// transmission electron microscope (CM20, Philips; www.bacterio.net/; Parte 2018). Members of the Netherlands) after negative staining with 2% (w/v) family Microbacteriaceae are widely distributed in uranyl acetate using cells grown for 48 h on R2A agar. nature including soil, freshwater, groundwater, The cell growth was checked on R2A agar, cyanobacterial mats, the rhizosphere and phyllosphere trypticase soy agar (TSA; Difco), Luria–Bertani of plants, air and ice samples, ponds in Antarctica, (LB; Difco) medium, and nutrient agar. The growth sludge, seawater, sediment, seaweed, and seafood temperature range was checked by incubating at 4, 8, (Dias and Bhat 1962;Ma¨nnisto¨ et al. 2000; Reddy 15, 20, 30, 37, and 45 °C on R2A agar. Salt tolerance et al. 2003;Lee2007; Kim et al. 2008; Kim and Lee was performed by adding different concentrations of 2011; Shin et al. 2011; Jang et al. 2012; Park et al. NaCl to R2A agar. The pH growth was determined in 2012; Schumann et al. 2012; Jin et al. 2013; Lai et al. R2A broth with a pH range of 5–11 at intervals of 1 pH 2015). During an investigation on iron and sulfur unit. Different biological buffers were applied to oxidizing microbial diversity in the sediment of a adjust the pH values: Na2HPO4/NaH2PO4 buffer for eutrophic freshwater reservoir (Jin et al. 2017), a strain pH 5–7 and Na2CO3/NaHCO3 buffer for pH 8–11 designated CHu50b-6-2T was isolated from the (Bates and Bower 1956; Gomori 1955). The oxidase freshwater sediment of the Daechung Reservoir. activity was checked using 1% tetramethyl-p- Herein, we describe the phylogenetic, genetic, phe- phenylenediamine (Tarrand and Groschel 1982), and notypic and chemotaxonomic characteristics of this the catalase activity was checked using 3% H2O2.We novel strain, which is proposed to represent a new used the API 20NE, ID 32 DN, API ZYM kits genus within the family Microbacteriaceae by using a (bioMe´rieux), and Biolog GN2 MicroPlate to deter- polyphasic approach. mine carbon source utilization and to do enzyme activity assays as well as additional physiological tests following the manufacturer’s instructions. Duplicate Materials and methods antibiotic-susceptibility tests were conducted using filter-paper discs (6 mm) containing the following: Isolation, morphological and physiological amikacin (30 lgml-1), ampicillin/sulbactam characterization (20 lgml-1, 1:1), chloramphenicol (30 lgml-1), erythromycin (30 lgml-1), gentamicin Strain CHu50b-6-2T was recovered from a 67-cm-long (30 lgml-1), kanamycin (30 lgml-1), lincomycin sediment core (36° 220 3000 N, 127° 330 5800 E) (15 lgml-1), nalidixic acid (30 lgml-1), rifampicin collected from the Daechung Reservoir at a water (30 lgml-1), spectinomycin (25 lgml-1), strepto- depth of 17 m in Daejeon, South Korea. 1 g sediment mycin (25 lgml-1), teicoplanin (30 lgml-1), tetra- sample was applied to serial dilution method. A 100 ll cycline (30 lgml-1), and vancomycin (30 lgml-1). sub-sample (10-6 or 10-7) of the suspended material The discs were placed on R2A plates spread with a was spread onto modified 1/10 R2A agar (L-1: 0.05 g culture of strain CHu50b-6-2T and were then incu- 123 Antonie van Leeuwenhoek (2020) 113:365–375 367 bated at 30 °C for 2 days. Susceptibility was recorded then were sequenced with the BigDye Terminator v3.1 as positive at zones with diameters greater than Cycle Sequencing kit (Applied Biosystems). Whole 10 mm. genome was sequenced via the Illumina HiSeq platform. The genome was assembled by the CLC Chemotaxonomic characterisation assembler (CLC-Assembly-Cell-5.1.1), and the gene annotation was performed by the PATRIC 3.5.36 For fatty acid profiling, strain CHu50b-6-2T was (hps://www.patricbrc.org). The average nucleotide cultured on R2A agar for 48 h to the late exponential identity (ANI) was calculated using OrthoANI tool in phase. Harvesting of the cell mass was standardized in the EZBioCloud (Lee et al. 2016). To get the full 16S the instruction of MIDI (http://www.microbialid.com/ rRNA gene, the sequencing primers 27F, 785F (50- PDF/TechNote_101.pdf). Separation and identifica- GGA TTA GAT ACC CTG GTA-30), 800R (50-TAC tion of the fatty acids were performed by GC (Hewlett CAG GGT ATC TAA TCC-30), and 1492R for the Packard 6890), and the TSBA 6 database provided the sequence analysis, were used (Lane 1991). The phy- Sherlock software 6.1. Extraction of isoprenoid qui- logenetic neighbors of strain CHu50b-6-2T were none was carried out following the method described identified, and the pairwise similarities of the 16S by Komagata and Suzuki (1987), and the analysis was rRNA gene sequences were calculated with EzBio- done by HPLC (Shimadzu) with an YMC-Pack ODS- Cloud (Yoon et al. 2017). The retrieved 16S rRNA A column. Extraction and identification of polar lipids gene sequences were aligned using the CLUSTAL X were done using two-dimensional TLC following the program (Thompson et al. 1997). Evolutionary dis- method described by Tindall (1990). The isomer of tances were calculated based on Kimura’s two-pa- diaminopimelic acid (DAP) in the cell wall was ana- rameter model (Kimura 1980). Phylogenetic trees lyzed using the method described by Hasegawa et al. were reconstructed with MEGA version 7.0 (Kumar (1983). The cell-wall peptidoglycan was extracted and et al. 2016) applying the neighbor-joining (Saitou and identified using TLC after hydrolysis with 6 M HCl at Nei 1987), maximum-likelihood (Felsenstein 1981) 100 °C for 18 h (Komagata and Suzuki 1987).