Results and Discussion

This study was designed to acquire a whole genome sequence for strain TFH56, which was isolated from tomato flower, to classify this strain within the genus Streptomyces, and to identify genes in strain TFH56 that are homologous to sequences associated with the biosynthesis of bioactive compounds in other Actinobacteria genomes. The acquired genome sequence was assembled in three contigs totaling 7.67 Mbp with 71.5% G + C content, 7172 CDS, 66 tRNA, and 18 rRNA (Figure 1 and Table 1). In evolutionary terms, TFH56 was closest to S. globisporus in terms of 16S rRNA (Figure 2). ANI calculations performed with MUMmer indicated that the genome of strain TFH56 had 96.11% sequence similarity with that of S. globisporus C-1027, and 99.99% similarity with that of S. globisporus S4-7 (Cha et al. 2016; Kim et al. 2019), which was isolated from strawberry rhizosphere (Figure 3). Unexpectedly, Kitasatospora was closely related to Streptomyces in the analysis of ANI and 16S rRNA, both of which were associated with the former classification of Kitasatospora albolonga as Streptomyces albolongus (Labeda et al. 2017). Moreover, consistent with the linear chromosome and two linear plasmids (pTFSG1 and pTFSG2) identified in this study, linear chromosomes and plasmids have been reported for S. globisporus (Li et al. 2016). In strain TFH56, the plasmids had less G + C content than did the chromosome (Figure 1 and Table 1).

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Figure 1

Genome map of Streptomyces globisporus TFH56. The outermost open ring shows the linear chromosome and linear plasmids. The innermost open ring shows the GC ratio, calculated from 10,000 bp. The remaining two open rings indicate CDS, rRNA, and tRNA on each strand.

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Figure 2

Evolutionary history tree for the 16S rRNA sequence, inferred using the Tamura-Nei mode and maximum likelihood method. The tree involved 24 strains of Actinobacteria, and its sequences are ∼1500 bp. Taxonomically, TFH56 is related most closely to Streptomyces globisporus.

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Figure 3

Average nucleotide identity based on MUMmer (ANIm) identification and alignment of 21 Streptomyces strains and three Actinobacteria members. The lower left heatmap shows ANIm identity. The trees show similarity according to ANIm identity scores. The upper right subplots are dot plots of whole genome alignments. Black dots represent same-direction alignments, and red dots represent reverse-direction alignments.

antiSMASH predicted 27 secondary metabolite gene clusters in the genome of strain TFH56 (Table 2). Caryolan-1-ol synthase was identified in cluster 9, and a thiopeptide sequence associated with conprimycin biosynthesis in S. globisporus S4-7 (Cha et al. 2016) was identified in cluster 15. Lidamycin biosynthesis genes were also detected in cluster 27. S. globisporus S4-7 is known to produce the antifungal compounds conprimycin (Cha et al. 2016) and caryolan-1-ol (Cho et al. 2017), and S. globisporus C-1027 is known to produce the antitumor agent lidamycin (originally called C-1027) (Hu et al. 1988; Liu et al. 2002; Li et al. 2016). Lidamycin biosynthesis gene sequences could not be found in other Streptomyces spp., and sequences associated with caryolan-1-ol and conprimycin biosynthesis could be found only among close relatives of S. globisporus based on the ANI phylogeny.

The sequences of these biosynthetic genes were compared among 10 strains in ANI species-similarity order (Figure 4). The caryolan-1-ol synthase sequence in strain TFH56 comprised 978 bp. This sequence was identical to that in strain S4-7, but differed by 24 bp from the sequence in strain C-1027. Streptomyces griseus NBRC 13350, K. albolonga YIM 101047, and Streptomyces fulvissimus DSM 40593 all had similar caryolan-1-ol synthase sequences (Figure 4C). Based on the antiSMASH analyses of CDS structure (Figure 4, D and E), which produced results consistent with the ANI results, the conprimycin biosynthesis sequence in strain TFH56 was 99–100% similar to the sequence in strain S4-7 (Table S1), and was also similar to the homologous sequences in strain C-1027, S. griseus, K. albolonga, and S. fulvissimus (Figure 4, A and D). The homolog of the S. globisporus lidamycin biosynthetic cluster in strain TFH56 was located in pTFSG1. This finding corresponded to a previous report that this cluster is located in the linear plasmid SGLP1 in strain C-1027 (Li et al. 2016). The lidamycin cluster in strain TFH56 differed from that in strain S4-7 in the 0–25 and >65 kbp regions (Figure 4, B and E). However, the lidamycin CDS structure in strain TFH56 was similar to that in strain C-1027, with the exception of the regulator gene, which differed by roughly 22 kbp in the most similar sequence (Figure 4E and Table S2). These results indicate that genes involved in the biosynthesis of caryolan-1-ol, conprimycin, and lidamycin are unique to S. globisporus and its closest relatives among sequenced Streptomyces strains (Figure 4 and Figure S1). This finding could improve our understanding of the diversity and evolution of biosynthetic gene clusters among plant probiotic Streptomyces spp. strains.

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Figure 4

Dot plots of aligned secondary metabolite related sequences and comparisons of their CDS, performed with antiSMASH. (A and D) C-1027 lidamycin synthetic gene cluster and homologs. (B and E) S4-7 conprimycin synthetic gene cluster and homologs. (C) Caryolan-1-ol synthase. All axes at dot plots are lengths of DNA sequences. Black and red diagonals in the upper right block correspond to forward and reverse strands, respectively. In the lower left block, the gradient from green to blue to magenta indicates alignment from the highest to the lowest score. Strain or species axis order depends on the ANI results. In (D and E), each filled block indicates a CDS.