*3.1. Polyphasic Taxonomic Characterization of NEAU-SSA 1T*

The morphological characteristics of strain NEAU-SSA 1<sup>T</sup> showed that the strain had the typical characteristics of the genus *Streptomyces*. Observation of 6-week cultures of strain NEAU-SSA 1<sup>T</sup> grown on ISP 3 medium revealed that it formed well-developed, branched substrate hyphae and aerial mycelia. Sporangia consisted of cylindrical, and rough-surfaced spores (0.6–0.8 μm × 0.9–1.6 μm) were produced on aerial mycelia, but spore chains were not observed (Figure 1). Strain NEAU-SSA 1<sup>T</sup> exhibited good growth on ISP 3, ISP 4, ISP 7, and Nutrient agar media; moderate growth on ISP 1, ISP 2, ISP 5, ISP 6, and Czapek's agar media; and poor growth on Bennett's agar medium. The cultural characteristics of strain NEAU-SSA 1T is shown in Table S1. Strain NEAU-SSA 1<sup>T</sup> grew well between pH 6.0 and 11.0, with an optimum pH of 7.0. The range of temperature of the strain was determined to be 15–45 ◦C, with the optimum growth temperature being 28 ◦C. The strain grew in the presence of 0–6% NaCl (*w*/*v*) with an optimal level of 0–1% (*w*/*v*). Detailed physiological characteristics are presented in the species description (Table 2 and Table S1).

**Figure 1.** Scanning electron micrograph of strain NEAU-SSA 1<sup>T</sup> grown on ISP 3 agar for 6 weeks at 28 ◦C; Scale bar represents 1 μm.

Chemotaxonomic analyses revealed that strain NEAU-SSA 1T exhibited characteristics that are typical of representatives of the genus *Streptomyces*. The strain was found to contain *LL*-diaminopimelic acid as diamino acid. The whole-cell hydrolysates of the strain were determined to contain ribose, mannose, and galactose. The menaquinones of strain NEAU-SSA 1<sup>T</sup> were MK-9(H4) (29.5%), MK-9(H6) (41.2%), and MK-9(H8) (29.4%). The cellular fatty acid profile of strain NEAU-SSA 1<sup>T</sup> was composed of *iso*-C17:0 (30.9%), C16:0 (26.4%), C17:1ω9c (19.9%), C15:0 (7.8%), C17:0 (4.4%), C14:0 (3.3%), *iso*-C16:0

(1.7%), *anteiso*-C15:0 (1.7%), C18:1ω9c (1.7%), C16:0 1-OH (1.2%), and *iso*-C18:0 (1.1%). The polar lipids of the strain consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), hydroxy-phosphatidylethanolamine (OH-PE), phosphatidylinositol (PI), two phosphatidylinositol mannosides (PIMs), and an unidentified phospholipid (PL) (Supplementary Figure S1). All the chemotaxonomic data are consistent with the assignment of strain NEAU-SSA 1T to the genus *Streptomyces*.


**Table 2.** Differential characteristics of strain NEAU-SSA 1T, *S. aureoverticillatus* JCM 4347T, *S. vastus* JCM4524T, *S. cinereus* DSM43033T, *S. xiangluensis* NEAU-LA29T, and *S. flaveus* JCM3035T.

Strains: 1—NEAU-SSA 1T; 2—*S. aureoverticillatus* JCM 4347T; 3—*S. vastus* JCM4524T; 4—*S. cinereus* DSM43033T; 5—*S. xiangluensis* NEAU-LA29T; 6—*S. flaveus* JCM3035T. Abbreviation: +, positive; –, negative. All data are from this study except where marked. <sup>a</sup> Data from Michael Goodfellow et al. [45].

Sequence analysis of the 16S rRNA gene showed that strain NEAU-SSA 1<sup>T</sup> were affiliated with the genus *Streptomyces* and most closely related to *S. aureoverticillatus* JCM 4347T (97.9%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain formed a cluster with *S. vastus* JCM4524<sup>T</sup> (97.4%), *S. cinereus* DSM43033<sup>T</sup> (97.2%), *S. xiangluensis* NEAU-LA29<sup>T</sup> (97.1%), and *S. flaveus* JCM3035T (97.1%) in the neighbor-joining tree (Figure 2), a relationship also recovered by the maximum-likelihood algorithm (Figure S2). Phylogenetic trees based on the neighbor-joining and maximum-likelihood algorithms were constructed from the concatenated sequence alignment of the five housekeeping genes (Figure 3 and Figure S3), and had the same topology as the 16S rRNA gene tree. Moreover, pairwise distances calculated for NEAU-SSA 1T and the related species using the concatenated sequences of *atp*D-*gyr*B-*rec*A-*rpo*B-*trp*B were well above 0.007 (Table S2), which is considered to be the threshold for species determination by Rong et al. [46]. DNA–DNA hybridization

was employed to further clarify the relatedness between the strain and *S. aureoverticillatus* JCM 4347T, *S. vastus* JCM4524T, *S. cinereus* DSM 43033T, and *S. xiangluensis* NEAU-LA29T. Results showed that strain NEAU-SSA 1<sup>T</sup> shared DNA–DNA relatedness of 37.1 <sup>±</sup> 3.4% with *S. aureoverticillatus* JCM <sup>4347</sup>T, 35.4 <sup>±</sup> 4.3% with *S. vastus* JCM 4524T, 33.1 <sup>±</sup> 4.1% with *S. cinereus* DSM 43033T, and 29.0 <sup>±</sup> 4.9% with *S. xiangluensis* NEAU-LA29T. Digital DNA–DNA hybridization was employed to clarify the relatedness between strain NEAU-SSA 1T and *S. flaveus* JCM 3035T. The level of digital DNA–DNA hybridization between them was 24.9 ± 2.4%. These five values are all below the threshold value of 70% recommended by Wayne et al. [47] for assigning strains to the same genomic species. Similarly, a low ANI value of 80.99% was found between strain NEAU-SSA 1T and *S. flaveus* JCM 3035T, a result well below the threshold used to delineate prokaryote species [48,49].

The assembled genome sequence of strain NEAU-SSA 1T was found to be 10,364,704 bp long and composed of 352 contigs with an N50 of 59,982 bp, a DNA G+C content of 69.9 mol % and a coverage of 200x. It was deposited into GenBank under the accession number VCHX00000000. The 16S rRNA gene sequence from the whole genome sequence shared a 100% similarity with that from PCR sequencing, suggesting that the genome sequence was not contaminated. Detailed genomic information is presented in the Table S3.

Comparison of phenotypic characteristics between strain NEAU-SSA 1<sup>T</sup> and its closely related species—*S. aureoverticillatus* JCM 4347T, *S. vastus* JCM4524T, *S. cinereus* DSM 43033T, *S. xiangluensis* NEAU-LA29T, and *S. flaveus* JCM 3035T—was performed to differentiate these strains (Table 2). Differential cultural characteristics included: NaCl tolerance of the strain was up to 5.0%, which is lower than that of *S. aureoverticillatus*JCM 4347<sup>T</sup> (15%) and *S. flaveus*JCM 3035<sup>T</sup> (7%); and the strain could grow at pH 11.0, while *S. vastus* JCM4524T, *S. cinereus* DSM 43033T, and *S. xiangluensis* NEAU-LA29<sup>T</sup> could not. Other phenotypic differences included the production of H2S; decomposition of cellulose; liquefaction of gelatin; growth temperature; hydrolysis of Tweens (20, 40, and 80); and utilization of L-arabinose, D-galactose, D-fructose, D-maltose, lactose, L-rhamnose, D-ribose, D-sorbitol, D-mannose, raffinose, D-xylose, *myo*-inositol, L-glutamine, glycine, L-threonine, L-tyrosine, L-serine, L-proline, L-asparagine, and L-arginine.

On the basis of morphological, physiological, chemotaxonomic, and phylogenetic results, strain NEAU-SSA 1T is considered to represent a novel species within the genus *Streptomyces*, for which the name *Streptomyces sporangiiformans* is proposed.

**Figure 2.** Neighbor-joining tree showing the phylogenetic position of strain NEAU-SSA 1T (1412 bp) and the related species of the genus *Streptomyces* based on 16S rRNA gene sequences. The out-group used was *Kitasatospora setae* LM-6054T. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Asterisks indicate branches also recovered in the maximum-likelihood tree. Scale bar represents 0.005 nucleotide substitutions per site.

**Figure 3.** Neighbor-joining tree based on MLSA analysis of the concatenated partial sequences from five housekeeping genes (*atp*D, *gyr*B, *rec*A, *rpo*B, and *trp*B) of isolate NEAU-SSA 1T (in bold) and related taxa. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. *Kitasatospora setae* LM-6054T was used as an out-group. Asterisks indicate branches also recovered in the maximum-likelihood tree. Scale bar represents 0.02 nucleotide substitutions per site.
