Lacisediminihabitans profunda gen. nov., sp. nov., a member of the family Microbacteriaceae isolated from freshwater sediment

A novel Gram-stain-positive bacterial strain, CHu50b-6-2T, was isolated from a 67-cm-long sediment core collected from the Daechung Reservoir at a water depth of 17 m, Daejeon, Republic of Korea. The cells of strain CHu50b-6-2T were aerobic non-motile and formed yellow colonies on R2A agar. The phylogenetic analysis based on 16S rRNA gene sequencing indicated that the strain formed a separate lineage within the family Microbacteriaceae, exhibiting 98.0%, 97.7% and 97.6% 16S rRNA gene sequence similarities to Glaciihabitans tibetensis KCTC 29148T, Frigoribacterium faeni KACC 20509T and Lysinibacter cavernae DSM 27960T, respectively. The phylogenetic trees revealed that strain CHu50b-6-2T did not show a clear affiliation to any genus within the family Microbacteriaceae. The chemotaxonomic results showed B1α type peptidoglacan containg 2, 4-diaminobutyric acid (DAB) as the diagnostic diamino acid, MK-10 as the predominant respiratory menaquinone, diphosphatidylglycerol, phosphatidylglycerol, and an unidentified glycolipid as the major polar lipids, 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 combined genotypic and phenotypic data showed that strain CHu50b-6-2T could be distinguished from all genera within the family Microbacteriaceae and represents a novel genus, Lacisediminihabitans gen. nov., with the name Lacisediminihabitans profunda sp. nov., in the family Microbacteriaceae. The type strain is CHu50b-6-2T (= KCTC 49081T = JCM 32673T). Electronic supplementary material The online version of this article (10.1007/s10482-019-01347-8) contains supplementary material, which is available to authorized users.


Introduction
Since Park et al. (1993) proposed the family Microbacteriaceae, 56 genera have been described validly in this family at the time of writing (http:// www.bacterio.net/; Parte 2018). Members of the family Microbacteriaceae are widely distributed in nature including soil, freshwater, groundwater, cyanobacterial mats, the rhizosphere and phyllosphere of plants, air and ice samples, ponds in Antarctica, sludge, seawater, sediment, seaweed, and seafood (Dias and Bhat 1962;Männistö et al. 2000;Reddy et al. 2003;Lee 2007;Kim et al. 2008;Kim and Lee 2011;Shin et al. 2011;Jang et al. 2012;Park et al. 2012;Schumann et al. 2012;Jin et al. 2013;Lai et al. 2015). During an investigation on iron and sulfur oxidizing microbial diversity in the sediment of a eutrophic freshwater reservoir (Jin et al. 2017), a strain designated CHu50b-6-2 T was isolated from the freshwater sediment of the Daechung Reservoir. Herein, we describe the phylogenetic, genetic, phenotypic and chemotaxonomic characteristics of this novel strain, which is proposed to represent a new genus within the family Microbacteriaceae by using a polyphasic approach.

Isolation, morphological and physiological characterization
Strain CHu50b-6-2 T was recovered from a 67-cm-long sediment core (36°22 0 30 00 N, 127°33 0 58 00 E) collected from the Daechung Reservoir at a water depth of 17 m in Daejeon, South Korea. 1 g sediment sample was applied to serial dilution method. A 100 ll sub-sample (10 -6 or 10 -7 ) of the suspended material was spread onto modified 1/10 R2A agar (L -1 : 0.05 g peptone, 0.05 g yeast extract, 0.05 g casamino acid, 0.05 g dextrose, 0.05 g soluble starch, 0.03 g K 2 HPO 4 , 0.005 g MgSO 4 , 0.03 g sodium pyruvate, and 15 g agar) and incubated at room temperature (25°C) for 4 weeks. One yellow colony, designated as CHu50b-6-2 T , was isolated and subcultivated on R2A agar at 30°C for further analysis. The colony characteristics were determined after growing for 5 days at 30°C on R2A agar. Gram staining was performed using a Gram stain kit (Becton-Dickinson) and 3% KOH solution. The cell morphology and motility were examined under a phase-contrast microscope (Nikon Eclipse 80i microscope, 1000 9 magnification) and a transmission electron microscope (CM20, Philips; Netherlands) after negative staining with 2% (w/v) uranyl acetate using cells grown for 48 h on R2A agar.

Chemotaxonomic characterisation
For fatty acid profiling, strain CHu50b-6-2 T was cultured on R2A agar for 48 h to the late exponential phase. Harvesting of the cell mass was standardized in the instruction of MIDI (http://www.microbialid.com/ PDF/TechNote_101.pdf). Separation and identification of the fatty acids were performed by GC (Hewlett Packard 6890), and the TSBA 6 database provided the Sherlock software 6.1. Extraction of isoprenoid quinone was carried out following the method described by Komagata and Suzuki (1987), and the analysis was done by HPLC (Shimadzu) with an YMC-Pack ODS-A column. Extraction and identification of polar lipids were done using two-dimensional TLC following the method described by Tindall (1990). The isomer of diaminopimelic acid (DAP) in the cell wall was analyzed using the method described by Hasegawa et al. (1983). The cell-wall peptidoglycan was extracted and identified using TLC after hydrolysis with 6 M HCl at 100°C for 18 h (Komagata and Suzuki 1987). Genomic DNA was extracted using a commercial genomic DNA-extraction kit (FastDNA TM SPIN kit). The purity of the extracted DNA was then examined on a ND2000 spectrometer (Nanodrop Technologies, Inc.). DNA G ? C contents (mol%) were analyzed by HPLC after hydrolysis as described by Tamaoka and Komagata (1984). Three reference strains were used: Glaciihabitans tibetensis KCTC 29148 T was obtained from the KCTC (Korean Collection for Type Cultures), Frigoribacterium faeni KACC 20509 T from the KACC (Korean Agricultural Culture Collection), and Lysinibacter cavernae DSM 27960 T from the DSMZ (German Collection of Microorganisms and Cell Cultures).
The bootstrap values were based on 1000 replicates (Felsenstein 1985). The housekeeping gene, recA gene encoding DNA recombinase A, was applied do delineate our strain more clearly from its close species. Housekeeping genes are useful for species identification as phylogenetic markers. The primer sets, recA-F (5 0 -GTT CTC YTT RCC CTG NCC-3 0 ) and recA-R (5 0 -GAR TCS TCS GGW AAG ACB AC-3 0 ), were used for amplifying and sequencing (Katayama et al. 2009). The PCR amplification conditions were as following: 95°C for 5 min, 30 cycles of 95°C for 1.5 min, 55°C for 1 min and 72°C for 1 min and final extension for 10 min at 72°C. To determine genomic relatedness, DNA-DNA hybridisation experiment was carried out between strain CHu50b-6-2 T and type strains of G. tibetensis, F. faeni and L. cavernae, which showed over 97% of 16S rRNA gene similarities to novel strain. The hybridisation test was carried out as described by Ezaki et al. (1989), and salmon sperm DNA (Sigma; D7656) was used as a control.
The draft genome sequence of strain CHu50b-6-2 T was deposited at DDBJ/EMBL/GenBank with the accession number PRJNA559971. The draft genome of strain CHu50b-6-2 T was of 4,022,930 bp, containing 175 contigs, of which the largest was of 845,903 bp. The genome encoded 3975 genes, including 48 tRNAs and 7 rRNAs. The N50 value was 413,391 and the sequencing depth of coverage was 570X. The DNA G ? C content calculated from the draft genome sequence was 67.3 mol% (Table S1).
On the basis of the phylogenetic position and genotypic, chemotaxonomic, and physiological differences, we propose that strain CHu50b-6-2 T should be assigned as a novel species within a new genus, Lacisediminihabitans gen. nov., with the name Lacisediminihabitans profunda sp. nov. within the family Microbacteriaceae.
Description of Lacisediminihabitans profunda sp. nov.
Lacisediminihabitans profunda (pro.fun'da. L. fem. adj. profunda from the deep). In addition to the characteristics described above, the novel species has the following properties. Colonies on R2A are convex, circular with entire edges and yellow color. The cells are observed to be oxidase-negative but catalase-positive. Growth occurs on R2A at temperatures from 4 to 30°C (optimum temperature 25-30°C), but not at 37°C. The pH range for growth is from pH 6-10 (optimum pH 7); however, there is no growth at pH 5 and 11. No growth was observed on TSA, LB, and NA media. The cells are positive for nitrate reduction and b-galactosidase but negative for aesculin hydrolysis, indole production, glucose fermentation, urease, arginine dihydrolase or gelatin hydrolysis (API 20NE test strip). The G ? C content of the genomic DNA is 67.3 mol%.

Compliance with ethical standards
Conflict of interest The authors declare that the study was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.
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