*2.4. Phylogenetic Analysis of NEAU-SSA 1<sup>T</sup>*

For DNA extraction, strain NEAU-SSA 1T was cultured in GY medium for 3 days to the early stationary phase and harvested using centrifugation. The chromosomal DNA was extracted according to the method of sodium dodecyl sulfate (SDS)-based DNA extraction [28]. PCR amplification of the 16S rRNA gene sequence was carried out using the universal bacterial primers 27F (5 -AGAGTTTGATCCTGGCTCAG-3´) and 1541R (5´-AAGGAGGTGATCCAGCC-3´) under conditions described previously [29,30]. The PCR product was purified and cloned into the vector pMD19-T (Takara) and sequenced using an Applied Biosystems DNA sequencer (model 3730XL, Applied Biosystems Inc., Foster City, California, USA). The almost complete 16S rRNA gene sequence of strain NEAU-SSA 1T (1412bp) was obtained and compared with type strains available at the EzBioCloud server (https://www.ezbiocloud.net/), retrieved using NCBI BLAST (National Center for Biotechnology Information, Basic Local Alignment Search Tool; https://blast.ncbi.nlm.nih.gov/Blast.cgi;) and then submitted to the GenBank database. Phylogenetic trees were constructed based on the 16S rRNA gene sequences of strain NEAU-SSA 1T and related reference species. Sequences were multiply aligned in Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 using the Clustal W algorithm and trimmed manually where necessary. Phylogenetic trees were constructed with neighbor-joining [31] and maximum likelihood [32] algorithms using MEGA [33]. The stability of the topology of the phylogenetic tree was assessed using the bootstrap method with 1000 repetitions [34]. A distance matrix was generated using Kimura's two-parameter model [35]. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). 16S rRNA gene sequence similarities between strains were calculated on the basis of pairwise alignment using the EzBioCloud server [36]. To further clarify the affiliation of strain NEAU-SSA 1T to its closely related strains, phylogenetic relationships of the strain NEAU-SSA 1T were also confirmed using sequences of five individual housekeeping genes (*atp*D, g*yr*B, *rec*A, *rpo*B, and *trp*B) for core-genome analysis. The sequences of NEAU-SSA 1<sup>T</sup> and its related strains were obtained from the genomes or GenBank/EMBL/DDBJ (European Molecular Biology Laboratory/DNA Data Bank of Japan). GenBank accession numbers of the sequences used are given in Table 1. The sequences of each locus were aligned using MEGA 7.0 software and trimmed manually at the same position before being used for further analysis. Trimmed sequences of the five housekeeping genes were concatenated head-to-tail in-frame in the order *atp*D-*gyr*B-*rec*A-*rpo*B-*trp*B. Phylogenetic analysis was performed as described above. Genome mining for bioactive secondary metabolites was performed using "antibiotics and secondary metabolite analysis shell" (antiSMASH) version 4.0 [37].


GenBank Accession Numbers of the Sequences Used in

 MLSA.

**Table 1.**

*Microorganisms* **2019**, *7*, 360


**Table 1.** *Cont.*
