*3.1. Multi-locus Sequence Analysis and Taxonomy Assignment of the Streptomyces sp. SM17 Isolate*

In order to taxonomically characterise the *Streptomyces* sp. SM17 isolate based on genetic evidence, multi-locus sequence analysis (MLSA) [51] employing the 16S rRNA sequence, in addition to five housekeeping genes, namely *atpD* (ATP synthase subunit beta), *gyrB* (DNA gyrase subunit B), *recA* (recombinase RecA), *rpoB* (DNA-directed RNA polymerase subunit beta), and *trpB* (tryptophan synthase beta chain) was performed, in a similar manner to a previous report [32]. A similarity search was performed in the GenBank database [35], using the NCBI BLASTN tool [36,37], based on the 16S rRNA nucleotide sequence of the SM17 isolate. The top 30 most similar *Streptomyces* species for which complete genome sequences were available in GenBank were selected for further phylogenetic analysis.

The concatenated nucleotide sequences [51,52] of the 16S rRNA and the aforementioned five housekeeping genes, were first aligned using the MAFFT program [38], and the phylogeny analysis was performed using the MrBayes program [39]. The general time reversible (GTR) model of nucleotide substitution with gamma-distributed rates across sites with a proportion of invariable sites was applied [40], with 1 million generations sampled every 100 generations. The final phylogenetic tree was then processed using MEGA X [41], with a posterior probability cut-off of 95% (Figure 1).

**Figure 1.** Phylogenetic tree of the concatenated sequences of the 16S rRNA and the housekeeping genes *atpD*, *gyrB*, *recA*, *rpoB*, and *trpB*, from the *Streptomyces* sp. SM17 together with 30 *Streptomyces* isolates for which complete genome sequences were available in the GenBank database. Analysis was performed using MrBayes, with a posterior probability cut-off of 95%. 1) *albidoflavus* phylogroup. 2) Clade including the neighbour isolate *Streptomyces koyangensis* strain VK-A60T. The strains SM17 and J1074 are indicated with asterisks.

The resulting phylogenetic tree clearly indicates the presence of a clade that includes the isolates *Streptomyces albidoflavus* strain J1074; *Streptomyces* sp. SM17; *Streptomyces albidoflavus* strain SM254; *Streptomyces sampsonii* strain KJ40; *Streptomyces* sp. FR-008; and *Streptomyces koyangensis* strain VK-A60T (clade 2 in Figure 1). In addition, this larger clade contains a sub-clade (clade 1 in Figure 1) that includes *Streptomyces* isolates similar to the strain *Streptomyces albidoflavus* J1074. The J1074 strain is a well-studied *Streptomyces* isolate widely used as a model for the genus and for various biotechnological applications, including the heterologous expression of secondary metabolites biosynthetic gene clusters (BGCs) [25–29]. This isolate was originally classified as "*Streptomyces albus* J1074", but due to recent taxonomy data, it has been reclassified as *Streptomyces albidoflavus* J1074 [30,31]. Hence, in this study, this strain will be referred to as *Streptomyces albidoflavus* J1074, and this clade will from now on be referred to as the *albidoflavus* phylogroup (Figure 1).

Interestingly, members of the *albidoflavus* phylogroup were all isolated from quite different environments. The *Streptomyces albidoflavus* strain J1074 stems from the soil isolate *Streptomyces albus* G [23,24]. The *Streptomyces sampsonii* strain KJ40 was isolated from rhizosphere soil in a poplar plantation [53]. The *Streptomyces* sp. strain FR-008 is a random protoplast fusion derivative of two *Streptomyces hygroscopicus* isolates [54]. On the other hand, two of these strains were isolated from aquatic saline environments, with *Streptomyces* sp. SM17 being isolated from the marine sponge *Haliclona simulans* [13]; while the *Streptomyces albidoflavus* strain SM254 strain was isolated from copper-rich subsurface fluids within an iron mine, following growth on artificial sea water (ASW) [55]. The fact that these isolates, although derived from quite distinct environmental niches, simultaneously share significant genetic similarities is interesting, and raises questions about their potential evolutionary relatedness.
