Microbacterium helvum sp. nov., a novel actinobacterium isolated from cow dung

A Gram-positive, aerobic, non-motile, non-spore-forming, short rod-shaped strain, NEAU-LLCT, was isolated from cow dung in Shangzhi City, Heilongjiang Province, Northeast China and identified by a polyphasic taxonomic study. Colonies was light yellow, round, with entire margin. Strain NEAU-LLCT was grown at 15–45 ℃ and pH 6.0–10.0. NaCl concentration ranged from 0 to 5% (W/V). The 16S rRNA gene sequence of NEAU-LLCT showed the high similarities with Microbacterium kyungheense JCM 18735T (98.5%), Microbacterium trichothecenolyticum JCM 1358T (98.3%) and Microbacterium jejuense JCM 18734T (98.2%). The whole-cell sugars were glucose, rhamnose and ribose. The menaquinones contained MK-12 and MK-13. Ornithine, glutamic acid, lysine and a small amount of alanine and glycine were the amino acids in the hydrolyzed products of the cell wall. The major fatty acids were iso-C16:0, iso-C18:0, anteiso-C15:0 and anteiso-C17:0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The genome of NEAU-LLCT was 4,369,375 bp and G + C content is 70.28 mol%. A combination of DNA–DNA hybridization result and some phenotypic characteristics demonstrated that strain NEAU-LLCT could be distinguished from its closely related strains. Therefore, the strain NEAU-LLCT was considered to represent a novel species, which was named Microbacterium helvum sp. (Type strain NEAU-LLCT = CCTCC AA 2018026T = JCM 32661T).


Introduction
Microbacterium genus was first proposed by Orla-Jensen (1919). Subsequently, the genus was reclassified by Collins et al. (1983) and then Takeuchi and Hatano (1998) revised the description of the genus and tranferred Aureobacterium into Microbacterium. The authors suggested that although the amino acids in the peptidoglycan of the two genera are Communicated by Erko Stackebrandt. different (lysine in members of the genus Microbacterium and ornithine in members of Aureobacterium), the intermixed phenotype of the two genera had similar physiological and biochemical properties. Krishnamurthi et al. (2012), Alves et al. (2014) and Fidalgo et al. (2016) emended the description of the genus Microbacterium. Microbacterium are typically Gram-positive, non-spore-forming and rodshaped (Collins et al. 1983, Takeuchi andHatano 1998). Microbacterium have been isolated from a variety of environmental sources, including soil (Kageyama et al. 2006), water (Torkko et al. 2000), human blood (Clermont et al. 2009), marine environments (Kageyama et al. 2007), biofilms (Kim et al. 2005), plants (Li et al. 2015), faeces (Chen et al. 2016), sediment (Mawlankar et al. 2015) and other environments. Some strains of Microbacterium genus have the ability to degrade crude oil (Schippers et al. 2005) and are halotolerant (Yang et al. 2018) and UV radiation-resistant (Zhang et al. 2010). At the time of writing, the genus containing 122 species effectively released (LPSN, http:// www. bacte rio. cict. fr/ index. html). In this paper, the phenotype and genotype characteristics of a novel were analyzed and its taxonomic place determined by polyphase taxonomic analysis.

Phenotypic characteristics
Morphological characteristics were observed by light microscopy (Nikon ECLIPSE E200) and transmission electron microscope (model JEM1010; JEOL) using cultures grown on ISP 2 medium at 28 ºC for 3 days. Color determination was done with ISCC-NBS colour charts Standard Samples No 2106 (Kelly 1964). Hydrolysis of Tweens (20, 40 and 80) and production of urease were tested as described by Smibert and Krieg (1994). Other physiological and biochemical properties were tested with API 20NE and API ZYM strips (bioMerieux) and acid production was tested using the API 50 CH system (bioMerieux) according to the manufacturers' instructions, using cells grown on ISP 2 medium for 3 days at 28 ºC. The utilization of sole carbon and nitrogen sources, decomposition of cellulose, hydrolysis of starch and aesculin, reduction of nitrate, coagulation and peptonization of milk, liquefaction of gelatin and production of H 2 S were examined as described previously (Gordon et al. 1974;Williams et al. 1989;Yokota et al. 1993a, b). Growth at different temperatures (10, 15, 18, 20, 25, 28, 32, 35, 38, 40, 42 and 45 °C) was determined on glucose-yeast extract (GY) medium (Jia et al. 2013) after incubation for 14 days. The pH range for growth (pH 4-12, at intervals of 1 pH units) was tested in GY broth. The buffer systems were: pH 4.0-5.0, 0.1 M citric acid/0.1 M sodium citrate; pH 6.0-8.0, 0.1 M KH 2 PO 4 /0.1 M NaOH; pH 9.0-10.0, 0.1 M NaHCO 3 /0.1 M Na 2 CO 3 ; pH 11.0-12.0, 0.2 M KH 2 PO 4 /0.1 M NaOH (Cao et al. 2020;Zhao et al. 2019). NaCl tolerance was determined in GY broth supplemented with 0-10% NaCl (w/v, with an interval of 1% w/v) at 28 ºC for 14 days on a rotary shaker. Growth under anaerobic conditions was tested in ISP2 in Hungate tubes filled with oxygen-free N 2 at 28 ºC (Ruan et al. 2014).

Chemotaxonomic characterization
Biomass for chemotaxonomic studies was prepared by growing the strain in GY broth in shake flasks at 28 ºC for 7 days. Cells were harvested by centrifugation, washed with distilled water twice and then freeze-dried. The preparation of cell wall peptidoglycan in the cell wall was performed in accordance with the work by Komagata and Suzuki (1987). Cell wall amino acids were identified by TLC (Hasegawa et al. 1983) and High-Speed Amino Acid Analyzer (Hitachi LA8080, Japan). The whole-cell sugars were tested according to the procedures developed by Lechevalier and Lechevalier (1980). The phospholipids in cell were examined by two-dimensional TLC and identified using the method of Minnikin et al. (1984). Menaquinones were extracted from freeze-dried biomass and purified according to Collins (1985) and analyzed by a HPLC-UV method (Wu et al. 1989) using an Agilent Extend-C18 Column (150 × 4.6 mm, i.d. 5 µm), monitored at 270 nm. The mobile phase was acetonitrile/propyl alcohol (60:40, v/v). The strain NEAU-LLC T was incubated in ISP2 broth at 28 ºC in shaking flasks for 7 days to determine the fatty acid composition of cells. Fatty acid methyl esters were extracted from the biomass as described by Gao et al. (2014) and analyzed by GC-MS using the method of Xiang et al. (2011).

DNA preparation, amplification and determination of 16S rRNA gene sequence
Extraction of chromosomal DNA and PCR amplification of the 16S rRNA gene sequence were carried out according to the procedure developed by Kim et al. (2000). The PCR product was purified and cloned into the vector pMD19-T (Takara) and sequenced using an Applied Biosystems DNA sequencer (model 3730XL). The almost full-length 16S rRNA gene sequence of strain NEAU-LLC T (1514 bp) was obtained and aligned with multiple sequences obtained from the GenBank/EMBL/DDBJ databases using Clustal W algorithm. Phylogenetic trees were generated with the neighbour-joining (Saitou and Nei 1987) and maximumlikelihood (Felsenstein 1981) algorithms using Molecular Evolutionary Genetic Analysis (MEGA) software version MEGA7.0 (Kumar et al. 2016). The stability of the topology of the phylogenetic tree was assessed using the bootstrap method with 1000 replicates (Felsenstein 1985). The distance matrix was generated using Kimura's two-parameter model (Kimura 1980). All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). Pairwise alignment analysis of 16S rRNA gene sequence similarities between strains were calculated on the EzBioCloud server (Yoon et al. 2017a).

DNA base composition and DNA-DNA hybridization
Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were employed to further genomically distinguish strain NEAU-LLC T from M. kyungheense JCM 18735 T and M. trichothecenolyticum JCM 1358 T (Meier-Kolthoff et al. 2013;Yoon et al. 2017b). In the present study, ANI and dDDH values were determined from the genomes of these three strains using the ortho-ANIu algorithm from Ezbiocloud (Yoon et al. 2017a(Yoon et al. , 2017b and the genome-to-genome distance calculator (GGDC 2.0) at http:// ggdc. dsmz. de. Because of the lack of the genome sequence of strain M. jejuense JCM 18734 T , DNA-DNA relatedness test between strain NEAU-LLC T and M. jejuense JCM 18734 T was carried out by the thermal renaturation method described by De Ley et al. (1970) under consideration of the modifications described by Huss et al. (1983), using a model Cary 100 Bio UV/VIS spectrophotometer equipped with a Peltier-thermostated 6 × 6 multicell changer and a temperature controller with in situ temperature probe (Varian). The concentration and purity of DNA samples were determined by measuring the optical density at 260, 280 and 230 nm. The DNA samples used for hybridization were diluted to OD260 around 1.0 using 0.1 × SSC (saline sodium citrate buffer (Thomas et al. 2000), then sheared using a JY92-II ultrasonic cell disruptor (ultrasonic time 3 s, interval time 4 s, 90 times). The DNA renaturation rates were detected in 2 × SSC at 70 ℃. The experiments were performed with three replications and the DNA-DNA relatedness value was expressed as mean of the three values.

Phylogenetic analysis
Identification using the EzBioCloud server revealed that strain NEAU-LLC T belongs to the genus Microbacterium and shared high sequence similarities with M. kyungheese JCM 18735 T (98.5%), M. trichothecenolyticum JCM 1358 T (98.3%) and M. jejuense JCM 18734 T (98.2%). The phylogenetic tree based on the 16S rRNA gene sequences indicated that strain NEAU-LLC T formed a cluster with M. trichothecenolyticum JCM 1358 T (98.3%) and M. jejuense JCM 18734 T (98.2%) in the neighbor-joining tree (Fig. 1), this relationship also recovered by the maximum likelihood (Fig. S3). Thus, based on the phylogenetic relationship, 16S rRNA sequences similarities, the isolate was mostly related to M. kyungheese JCM 18735 T , M. trichothecenolyticum JCM 1358 T and M. jejuense JCM 18734 T (Fig. 2).

Phenotypic characteristics
Morphological observation of NEAU-LLC T strain cultured on ISP 2 medium revealed that the strain has typical characteristics of members of the genus Microbacterium. Strain NEAU-LLC T had characteristics shared by all members of the genus Microbacterium (Fidalgo et al. 2016). Detailed physiological and biochemical properties, enzyme activity (API 20NE, API ZYM) and the production of acid (API 50CH) are presented in the species description and the differential characteristics of strain NEAU-LLC T and three closely related species of the genus Microbacterium are summarized in Table 1. For example, the NEAU-LLC T strain could grow at pH 6.0, while the closely related strain could not. The tolerance of NEAU-LLC T to NaCl was up to 5%, lower than that of M. kyungheese JCM 18735 T and M. jejuense JCM 18734 T , but higher than M. trichothecenolyticum JCM 1358 T . Other phenotypic differences include the hydrolysis of urea and Tweens (20, 40 and 80) and patterns of carbon and nitrogen utilization. The negative characteristics of nitrogen assimilation tests, the enzyme activities (API 20 NE, API ZYM) and the production of acid (API 50CH) for NEAU-LLC T are summarized in Table S3.

Molecular characteristics
To determine whether strain NEAU-LLC T could be considered to represent a new species, DNA-DNA hybridization by the thermal renaturation was carried out with M. jejuense JCM 18734 T and the DNA-DNA relationship was are shown at branch points. Arthrobacter globiformis DSM 20124 T (M23411) was used as an outgroup. Asterisks indicate branches also recovered in the maximum-likelihood tree; Bar, 0.0100 substitutions 24.9 ± 2.5%. The digital DNA-DNA hybridization levels between NEAU-LLC T and M. kyungheese JCM 18735 T , M. trichothecenolyticum JCM 1358 T were 37.1 ± 2.5% and 25.7 ± 2.5%, respectively. These values are below the 70% threshold recommended by Wayne et al. (1987) for assigning strains to the same genomic species. Similarly, the ANI values of NEAU-LLC T and the two reference strains were 86.36% and 82.41%, respectively, which were lower than the 95%-96% threshold defined by prokaryotic species (Richter and Rossello-Mora 2009;Chun and Rainey 2014). Detailed genomic information and other general features of genome sequences are shown in Table S2.
In conclusion, it is evident from phenotype and genotype data that NEAU-LLC T strain represents a novel species in the genus Microbacterium, for which the name Microbacterium helvum is proposed.
The type strain is NEAU-LLC T (= CCTCC AA 2018026 T = JCM 32661 T ), isolated from cow dung Fig. 2 Whole-genome sequence tree generated with TYGS for strain NEAU-LLC T and closely related species of the genus Microbacterium. Tree inferred with FastME from GBDP distances calculated from genome sequences. Branch lengths are scaled in terms of GBDP distance formula d5; numbers above branches are GBDP pseudo-bootstrap support values from 100 replications  The GenBank accession number for the 16S rRNA gene sequence and the draft genome sequence of the type strain are MW009703 and JACXZS000000000, respectively.