Glycomyces salinus sp. nov., an actinomycete isolated from a hypersaline habitat

A Gram-stain-positive, aerobic, nonmotile actinobacterium, designated strain YIM 93776T, was isolated from a saline sediment sample collected from Aiding Lake in Xinjiang Uygur Autonomous Region, Northwest China. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain YIM 93776T was affiliated to the genus Glycomyces and was closely related to Glycomyces albus TRM 49136T (97.6% sequence similarity), Glycomyces lacisalsi XHU 5089T (97.0%) and Glycomyces anabasis EGI 6500139T (96.2%). The cell wall contained meso-diaminopimelic acid and the whole-cell hydrolysates sugars were galactose, mannose, arabinase, glucose and ribose. The predominant menaquinones were MK-9 (H4) and MK-10 (H4). Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, two phosphatidylglyceride, two unidentified phospholipids and two unidentified polar lipids were detected in the polar lipid extracts. Major fatty acids were anteiso-C17:0, iso-C15:0, iso-C16:0, anteiso-C15:0 and anteiso C17:1 A. The draft genome sequence of strain YIM 93776T was 5.37 Mbp in size with 69.5% DNA G + C content. The dDDH and ANI values between strain YIM 93776T and related neighbours were 25.0–34.3% and 77.3–79.8%, respectively. On the basis of morphological, chemotaxonomic and phylogenetic evidence, strain YIM 93776T; therefore, represents a novel species, for which the name Glycomyces salinus sp. nov. is proposed. The type strain is YIM 93776T (= KCTC 49430T = CGMCC 4.7685T).


Introduction
The genus Glycomyces was initially proposed by Labeda et al. (1985) with Glycomyces harbinensis as the type species, and the description was later emended by Labeda and Kroppenstedt (2004). Strains belonging to the genus 1 3 Glycomyces are Gram-stain-positive actinobacteria that form an extensively branched vegetative mycelium and aerial hyphae on certain growth media. At the time of writing, there are 26 validly published names in this genus (https:// lpsn. dsmz. de/ genus/ glyco myces). Most members of Glycomyces strains are isolated from soil, saline environments and traditional Chinese medicinal plants tissues. In the present study, we isolated a halophilic actinobacteria designated as strain YIM 93776 T that was associated with genus Glycomyces. Based on the results of phylogenetic, chemotaxonomic and physiological characterization of strain YIM 93776 T , we propose the strain YIM 93776 T represents a novel Glycomyces species.

Bacterial isolation and cultivation
Strain YIM 93776 T was isolated using standard serial dilution plating technique from a hyper-saline sediment sample obtained from Aiding Lake (GPS,42 68′ 66" N,89 33′ 07" E) in Xinjiang Uygur Autonomous Region, Northwest China. The isolation plates (modified ISP 2 medium with 10% (w/v) NaCl, Shirling and Gottlieb, 1966) were incubated at 37 °C for 2 weeks. Presumptive actinomycete colonies were picked and re-streaked > 3 times to obtain axenic cultures. Strain YIM 93776 T was selected among other strains for further characterization using polyphasic taxonomy based on the phylogenetic profiles of the 16S rRNA gene sequence. Pure cultures of strain YIM 93776 T were routinely cultured and maintained on ISP 2 medium containing 10% (w/v) NaCl at 4 °C and as glycerol suspensions (20%, v/v)

Phylogenetic and genomic analyses
Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were carried out as described previously (Feng et al. 2020). The almost-complete 16S rRNA gene sequence (1445 bp) was checked manually and submitted to the GenBank database. Pairwise similarity values between strain YIM 93776 T and its closely related type strains were calculated via the EzBioCloud server (https:// www. ezbio cloud. net) (Yoon et al. 2017). The 16S rRNA gene sequence was aligned with multiple sequences obtained from the GenBank/EMBL/DDBJ databases using CLUSTAL X (Thompson et al. 1997). Phylogenetic trees with gaps completely deleted were reconstructed based on the neighbour-joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981) and maximum-parsimony (MP) (Fitch 1971) algorithms, using the MEGA version 7.0 software package (Kumar et al. 2016). Bootstrap analysis with 1000 replicates was applied to assess confidence levels of the branches (Felsenstein 1985). Kimura's two-parameter model (Kimura 1980) was used to calculate evolutionary distance matrices for the NJ methods. ML was calculated using the general time-reversible model with gamma distribution with invariant sites (G + I).
The draft genome of strain YIM 93776 T was sequenced using PacBio and Illumina Hiseq 2000 sequencers at Shanghai Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China). SOAPdenovo software (version 2.04) was employed to assemble paired-end reads (Li et al. 2010). The G + C contents (mole percent) were calculated from the genome sequences. To assess relationships between the strain YIM 93776 T and sequenced related strains, we performed a phylogenomic analysis and constructed the evolutionary tree based on the orthologous genes using the supermatrix method (Zhi et al. 2017). The digital DNA-DNA hybridization (DDH) similarity between strains was calculated in silico with the Genome-to-Genome distance calculator server version 2.1 (Meier-Kolthoff et al. 2013). Average nucleotide identity (ANI) was calculated with OrthoANI (Richter et al. 2016).

Phenotypic, physiological and biochemical characteristics
Cultural characteristics were determined after 7 and 14 days of incubation at 37 °C on different agar media (ISP 2, ISP 3, ISP 4 and ISP 5, Czapek's agar, PDA and NA media) with 10% (w/v) NaCl concentration. Growth at different temperatures (4, 10, 15, 20, 25, 28, 30, 35, 37, 40, 45, 50 and 55 °C) were determined on ISP 2 medium plates containing 10% (w/v) NaCl concentration and observed after 7 and 14 days. Salt tolerance (0-30%, w/v, NaCl at 5% intervals) was examined at 37 °C on ISP 2 agar medium for 14 days. A series of pH conditions (4.0-11.0, at 0.5 intervals) using the buffer system described by Xu et al. (2005). Growth was assessed by monitoring the turbidity as OD660 using a spectroscopic method (Lambda 35 UV/Vis Spectrometer; PerkinElmer). After incubation on Czapek's agar (containing 10%, w/v NaCl) at 37 °C for 7 days and 28 days, morphological properties and spore motility were examined by means of a light microscope (DM2000, Leica) and a scanning electron microscope (XL30 ESEM-TMP, Philips-FEI). Gram staining was carried out using the standard Gram reaction and was confirmed using the KOH lysis test method (Gregersen 1978). Carbon source utilization for growth was carried out on ISP 9 medium containing 10% (w/v) NaCl as described by Shirling and Gottlieb (1966). Nitrogen source utilization tests were carried out as described by Gordon et al. (1974). Catalase activity was detected based on the bubble formation in 3% (v/v) H 2 O 2 solution. Oxidase activity was detected by the oxidation of tetramethyl-p-phenylenediamine. Hydrolysis of starch, casein, gelatin, cellulose and Tweens 20, 40 and 80, nitrate reduction, urease activity, coagulation and peptonization of milk, melanin and H 2 S production were determined as described by Smibert and Krieg (1994). Other enzymatic activities of strain YIM 93776 T were analyzed using API 20NE and API ZYM kits (bioMérieux, France) according to the manufacturer's instructions. The acid production from carbohydrates was determined using the API 50CH system (bioMérieux, France) according to the manufacturer's instructions.

Chemotaxonomic characteristics
Biomass used for chemotaxonomic studies except fatty acid was obtained from cultures grown on ISP2 plate containing 10% (w/v) NaCl for 14 days at 37 °C. The diagnostic isomers of diaminopimelic acid were determined by TLC method (Hasegawa et al. 1983;Lechevalier and Lechevalier 1970). The whole-cell sugar pattern and peptidoglycan amino acids were detected by HPLC according to methods used by Tang et al. (2009). The respiratory quinones were isolated using the method of Collins et al. (1977) and analyzed by HPLC (Agilent Technologies 1260 Infinity) (Groth et al. 1996). Polar lipids were extracted and then separated using twodimensional TLC and identified using previously described procedures (Minnikin et al. 1984;Hasegawa et al. 1983). Molybdophosphoric acid, molybdenum blue, ninhydrin and α-naphthol were used for the detection of total polar lipids, phospholipids, aminolipids and glycolipids, respectively. For cellular fatty acid analysis, strain YIM 93776 T was grown on tryptic soya agar (TSA; Difco) with 10% (w/v) NaCl at 37 °C and harvested after 7 days. Fatty acid methyl esters were extracted, methylated and analysed using the Microbial Identification System (Sherlock version 6.1; MIDI database: TSBA6) according to the manufacturer's instructions (Sasser 1990).

Molecular phylogenetic analysis
16S rRNA gene sequence analysis showed that strain YIM 93776 T was related most closely to the type strains of Glycomyces albus (97.6% similarity), Glycomyces lacisalsi (97.0% similarity), Glycomyces anabasis (96.2% similarity). Lower levels of 16S rRNA gene sequence similarity (92.0-95.1%) were found with the type strains of all other type species within the genus Glycomyces. These values were below the 98.7% cutoff point recommended for recognition of genomic species which need not to calculate overall genome related index (OGRI) (Stackebrandt and Ebers 2006;Kim et al. 2014). The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences indicated that YIM 93776 T formed a branch distinct from those of their related species (Fig. 1). This relationship was supported by a high level of bootstrap support. The topologies of phylogenetic trees constructed with the maximum-likelihood and maximum-parsimony algorithms were similar to that of the tree reconstructed by neighbour-joining analysis (Supplementary information Fig. S1, S2) Wayne et al. (1987) for assigning strains to the same genomic species. Similarly, the low ANI values between strain YIM 93776 T and above species were ranged 77.3-79.8%, well below the threshold used to delineate prokaryote species (Richter and Rosselló-Móra 2009; Chun and Rainey 2014) (Supplementary information Table S1). The ANI or digital DDH values between strain YIM 93776 T and the most closely related species G. albus, G. lacisalsi and G. anabasis were not calculated due to the genome sequences were not available at the time of writing. The phylogeomic tree also supported that strain YIM 93776 T formed a distinct phylogenetic lineage within the genus Glycomyces (Supplementary information Fig. S3). These results indicated that strain YIM 93776 T should be considered as a separate novel species.

Phenotypic, physiological, and biochemical characteristics
Growth of strain YIM 93776 T was observed at pH 5.5-11 (optimum 8) and in the presence of 5-13% (w/v) NaCl. The temperature range for growth was 25-45 °C, with optimum growth at 37 °C. Strain YIM 93776 T grew well on Czapek's agar and NA and moderately well on ISP 2, ISP 3, ISP 4, ISP 5 and PDA media (10%, w/v NaCl). White substrate mycelia developed well on the above media. Aerial mycelium was white and abundantly produced on Czapek's and NA media, but absent on ISP 2, ISP 3, ISP 4 and ISP 5 media. Diffusible pigments or melanin were not observed on any test media. When strain YIM 93776 T grown on Czapek's agar containing 10% (w/v) NaCl, fragmented substrate was observed for 7 days and aerial hyphae with fragmentation was detected on 28 days. The mobility of spores was not observed (Supplementary information Fig. S4). The detailed physiological characteristics of strain YIM 93776 T are presented in Table 1, Table S2 and in the species description.
The phylogenetic data indicate that strain YIM 93776 T represents a novel species in the genus Glycomyces. In addition, the morphological and chemotaxonomic characteristics clearly differentiate strain YIM 93776 T from other related species of genus Glycomyces (Table 1). Also, strain YIM 93776 T contained glucose, mannose, ribose, arabinose and galactose, but no xylose which Glycomyces albus does contain. In conclusion, on the basis of phylogenetic analysis, chemotaxonomic data and phenotypic traits, strain YIM 93776 T is considered to represent a novel species in the genus Glycomyces, for which the name Glycomyces salinus sp. nov. is proposed.

Accession number
The NCBI GenBank accession number for the 16S rRNA gene sequence of strain YIM 93776 T is MW380665. The draft whole genome sequence for strain YIM 93776 T has been deposited at DDBJ/ENA/GenBank under accession number GCA_016428645.1.