Comparative Genomics and Biosynthetic Cluster Analysis of Antifungal Secondary Metabolites of Three Strains of Streptomyces albidoflavus Isolated from Rhizospheric Soils

Abstract

Streptomyces is a genus of Gram-positive bacteria with high GC content. It remains attractive for studying and discovering new antibiotics, antifungals, and chemotherapeutics. Streptomyces genomes can contain more than 30 cryptic and expressed biosynthetic gene clusters (BGC) encoding secondary metabolites. In this study, three Streptomyces strains isolated from jungle rhizospheric soil exhibited supernatants that can inhibit sensitive and fluconazole-resistant Candida spp. The genomes of the strains Streptomyces sp. A1, J25, J29 ori2 were sequenced, assembled de novo, and analyzed. The genome assemblies revealed that the size of the genomes was 6.9 Mb, with linear topology and 73.5% GC. A phylogenomic approach identified the strains with high similitudes between 98.5 and 98.7% with Streptomyces albidoflavus SM254 and R-53649 strains, respectively. Pangenomic analysis of eight genomes of S. albidoflavus strains deposited in the Genomes database recognized 4707 core protein orthogroups and 745 abundant accessory and exclusive protein orthogroups, suggesting an open pangenome in this species. The antiSMASH software detected candicidin and surugamide BGC-encoding polyene and octapeptide antifungal secondary metabolites in other S. albidoflavus. CORASON software was used to compare the synteny, and the abundance of genes harbored in the clusters was used. In conclusion, although the three strains belong to the same species, each possesses a distinct genome, as evidenced by the different phenotypes, including antifungal and extracellular enzymatic activities.

Keywords: BGC; CORASON; Candida; Streptomyces; antiSMASH; antifungal; candicidin; polyene; surugamide; synteny.

Figure 1

Colonial and microscopic morphologies of axenic cultures of Streptomyces sp. strains. (A–C) and (D–F), colonial and microscopic morphologies of Streptomyces sp. 102, 116-B, and J22, respectively; (G–I) and (J–L), colonial and microscopic morphologies of Streptomyces sp. A1, J25 and J29 ori2, respectively. Conventional Gram staining was used for description of microscopic morphology and observed at 1000×. Bars in panels (A–C) and (G–I) represent colony size in mm. Bars in panels (D–F,J–L) represent the mycelial diameter in μm.

Figure 2

Bioassay of growth inhibition after 24 h at 37 °C of Candida spp. by metabolites produced by Streptomyces sp. strains. Streptomyces sp. 102, 116-B, and J22 did not exhibit antifungal activities on Candida spp. Streptomyces sp. A1 displayed 10–13 mm inhibition halos, Streptomyces sp. J25 displayed 10–12 mm inhibition halos and Streptomyces sp. J29 ori2 displayed 10–13 mm inhibition halos. GAE fresh medium was used as a negative control. Inhibition halos were measured after 24 h of incubation at 37 °C. The symbol * represents the statistically significant difference. Fluconazole sensitive (S) and resistant yeast (R).

Figure 3

Dose–response effect of lyophilized supernatants of Streptomyces spp. and determination of minimum inhibitory concentrations on Candida spp. by the microdilution plate method. Panel (A), graph, and table of MICs obtained for the supernatant of Streptomyces sp. A1; (B), graph, and table of MICs obtained for the supernatant of Streptomyces sp. J25, and (C), graph, and table of MICs obtained for the supernatant of Streptomyces sp. J29 ori2. Figures and line in blue represent growth of C. albicans ATCC 10231; in green, C. krusei ATCC 14423; in purple, C. glabrata CBS 138; and in pink, C. glabrata 43. The symbol on the bar (*) represents statistically significant difference by two-way ANOVA analysis (p < 0.001). Fluconazole sensitive (S) and resistant yeast (R).

Figure 4

Phylogenomics identification tree by maximum likelihood of Streptomyces albidoflavus strains and related species. The core of 134 protein orthogroups concatenated were defined by Orthofinder. The amino acid substitution model was Q. insect + F + I + I + I + R10. The numbers in the nodes indicate the Ultra-Fast-bootstrap values with 1000 replicates. Leuconostoc mesenteroides strains were used as an outgroup.

Figure 5

Upset plot of the comparative genomics to determine the number of orthogroups shared between S. albidoflavus strains. The colored bars on the left side represent the genomes of each S. albidoflavus strain and parents. The graph represents the number of orthogroups shared between them. The first bar is the number of orthogroups shared among all genomes or core genome of S. albidoflavus. The other columns show the orthogroups shared between groups of strains. For example, in the second column, S. albidoflavus W68 and S. albidoflavus UYFA 156 shared exclusively 288 orthogroups, and in the third column the blue dot represents the orthogroups unique to S. albidoflavus J25. The remaining columns can be interpreted in the same way. The gray dots mean the orthogroups are absent in the indicated genome. Comparative genomics and figure were performed on the OrthoVenn3 online server.

Figure 6

Organization of the candicidin (A) and surugamide (B) BGCs from Streptomyces sp. FR-008 and S. albidoflavus J1074, respectively. The arrows in red indicate the core genes of biosynthesis; in pink, the accessory genes; in green, the regulatory genes; in blue, the genes associated with transport; and in gray, the genes with other functions. Candicidin genes: 1. fscO, putative FAD-dependent monooxygenase (1377 bp); 2. pabC, putative 4-amino-4-deoxychorismate lyase (744 bp); 3. fscRI, putative transcriptional activator (669 bp); 4. fscRII LuxR family transcriptional regulator (2829 bp), 5. fscRIII LuxR family transcriptional regulator (3045 bp); 6. fscRIV LuxR family transcriptional regulator (3018 bp); 7. fscMI glycosyltransferase, MGT family (1377 bp); 8. fscMII, DegT/ DnrJ/ Ery C1/ StrS aminotransferase (1059 bp); 9. fscP cytochrome P450 (1182 bp); 10. fscFE ferredoxin (194 bp); 11. fscTE thioesterase type II (858 bp); 12. pabAB isochorismate synthase (2172 bp); 13. fscA, beta-ketoacyl synthase (5232 bp); 14. fscTI ATP binding protein (transport) (1008); 15. fscTII ABC-2 type transporter (720 bp); 16. fscC acetyl-CoA acetyltransferase (31,878 bp); 17. fscB malonyl CoA-acyl carrier protein transacylase (16,626 bp); 18. fscF beta-ketoacyl synthase (6150 bp); 19. fscE beta-ketoacyl synthase (23,316 bp); 20. fscD beta-ketoacyl synthase (28,653 bp) and 21. fscMIII NAD-dependent epimerase/dehydratase (1209 bp). Surugamide genes: 22. XNR_3438, ABC transporter, permease protein (711 bp); 23. XNR_3439, ABC transporter, permease protein (762 bp); 24. XNR_3440, amino acid ABC transporter amino acid-binding protein (990 bp); 25. XNR_344 1, secreted protein (1221 bp); 26. XNR_3442, major facilitator superfamily permease (1575 bp); 27. XNR_3443, hypothetical protein (309 bp); 28. XNR_3444, TetR family transcriptional regulator (612 bp); 29. XNR_3445, drug resistance transporter EmrB/QacA subfamily protein (1347 bp); 30. surD, non-ribosomal peptide synthetase (12345 bp); 31. surC, non-ribosomal peptide synthetase (23,076 bp); 32. surB, ATP-dependent valine adenylase (12,798 bp); 33. surA, non-ribosomal peptide synthetase (17,202 bp); 34. surE, alpha/beta hydrolase MppK (1356 bp); 35. XNR_3451, putative membrane protein (1098 bp); 36. surR, transcriptional regulator, GntR family (417 bp); 37. XNR_3453, hypothetical protein (372 bp); 38. XNR_3454, ABC transporter ATP-binding protein (942 bp); 39. XNR_3455, ABC transport system membrane protein (798 bp); 40. XNR_3456, MbtH domain-containing protein (258 bp); 41. lipE, abhydrolase_6 biosynthetic-additional (810 bp), and 42. XNR_3458, succinate-semialdehyde dehydrogenase (1428 bp).

Figure 7

Synteny of BGC of candicidin of S. albidoflavus and related species. The figure was composed with the genetic contexts detected using fscB PKS and pabAB genes as a query in the CORASON software. The blue arrow points to the reference cluster, and the red box points to the S. albidoflavus A1, J25, and J29 ori2 genomes described in this work. Candicidin genes: 1. fscO, putative FAD-dependent monooxygenase (1377 bp); 2. pabC, putative 4-amino-4-deoxychorismate lyase (744 bp); 3. fscRI, putative transcriptional activator (669 bp); 4. fscRII LuxR family transcriptional regulator (2829 bp), 5. fscRIII LuxR family transcriptional regulator (3045 bp); 6. fscRIV LuxR family transcriptional regulator (3018 bp); 7. fscMI glycosyltransferase, MGT family (1377 bp); 8. fscMII, DegT/DnrJ/Ery C1/StrS aminotransferase (1059 bp); 9. fscP cytochrome P450 (1182 bp); 10. fscFE ferredoxin (194 bp); 11. fscTE thioesterase type II (858 bp); 12. pabAB isochorismate synthase (2172 bp); 13. fscA, beta-ketoacyl synthase (5232 bp); 14. fscTI ATP binding protein (transport) (1008); 15. fscTII ABC-2 type transporter (720 bp); 16. fscC acetyl-CoA acetyltransferase (31,878 bp); 17. fscB malonyl CoA-acyl carrier protein transacylase (16,626 bp); 18. fscF beta-ketoacyl synthase (6150 bp); 19. fscE beta-ketoacyl synthase (23,316 bp); 20. fscD beta-ketoacyl synthase (28,653 bp) and 21. fscMIII NAD-dependent epimerase/dehydratase (1209 bp).

Figure 8

Synteny of biosynthetic gene clusters of surugamide A of S. albidoflavus and related species. The figure was composed with the genetic contexts detected using the surB gene as a query in the CORASON software. The data were taken from the antiSMASH comparison of the BGC query with the cluster reported in the MIBiG. The blue arrow points to the reference cluster, and the red box points to the S. albidoflavus A1, J25, and J29 ori2 genomes described in this work. Surugamide genes: 22. XNR_3438, ABC transporter, permease protein (711 bp); 23. XNR_3439, ABC transporter, permease protein (762 bp); 24. XNR_3440, amino acid ABC transporter amino acid-binding protein (990 bp); 25. XNR_3441, secreted protein (1221 bp); 26. XNR_3442, major facilitator superfamily permease (1575 bp); 27. XNR_3443, hypothetical protein (309 bp); 28. XNR_3444, TetR family transcriptional regulator (612 bp); 29. XNR_3445, drug resistance transporter EmrB/QacA subfamily protein (1347 bp); 30. surD, non-ribosomal peptide synthetase (12,345 bp); 31. surC, non-ribosomal peptide synthetase (23,076 bp); 32. surB, ATP-dependent valine adenylase (12,798 bp); 33. surA, non-ribosomal peptide synthetase (17,202 bp); 34. surE, alpha/beta hydrolase MppK (1356 bp); 35. XNR_3451, putative membrane protein (1098 bp); 36. surR, transcriptional regulator, GntR family (417 bp); 37. XNR_3453, hypothetical protein (372 bp); 38. XNR_3454, ABC transporter ATP-binding protein (942 bp); 39. XNR_3455, ABC transport system membrane protein (798 bp); 40. XNR_3456, MbtH domain-containing protein (258 bp); 41. lipE, abhydrolase_6 biosynthetic-additional (810 bp), and 42. XNR_3458, succinate-semialdehyde dehydrogenase (1428 bp).