Supplementary Information

Transcription

1 Supplementary Information Assembly and Clustering of Natural Antibiotics Guides Target Identification Chad W. Johnston 1,2, Michael A. Skinnider 1,2, Chris A. Dejong 1,2, Philip N. Rees 1,2, Gregory M. Chen 1,2, Chelsea Walker 1,2, Shawn French 1, Eric D. Brown 1, Janos Berdy 3, Dennis Y. Liu 1,2 & Nathan A. Magarvey 1,2 * 1 Department of Biochemistry & Biomedical Sciences; M. G. DeGroote Institute for Infectious Disease Research; 2 Department of Chemistry & Chemical Biology; McMaster University, Hamilton, Canada L8S 4K1 3 Eötvös Loránd University, Budapest, Hungary * Corresponding author, address: magarv@mcmaster.ca

2 Supplementary Results Supplementary Figure 1. Antibioticome web application. The antibioticome search web application (accessible at provides a user-friendly mechanism to query the targeted antibioticome, in order to define the putative molecular target of a user-submitted molecule. A single molecular structure or database of molecular structures are submitted in SMILES format, and undergo in silico retrobiosynthesis followed by hierarchical clustering. The single highest-scoring targeted antibiotic match identified by computational retrobiosynthesis is reported for each submitted molecule, as well as the family of natural products to which that targeted antibiotic belongs, the mechanism of action of that family, and the confidence score assigned to the match.

3 Supplementary Figure 2. PRISM a web application for identifying biosynthetic gene clusters. Screenshot of the PRISM user interface. Supplementary Figure 3. PRISM results for the cephalosporin biosynthetic gene cluster. (a) Detection of the cephalosporin biosynthetic gene cluster. Red indicates nonribosomal peptide synthetase genes, brown indicates resistance genes, and green indicates tailoring enzymes. (b) Detection of an embedded resistance determinant related to the cephalosporin mode of action Class C beta lactamase.

4 Supplementary Figure 4. PRISM results for the daptomycin biosynthetic gene cluster. (a) Detection of the daptomycin biosynthetic gene cluster. Red indicates nonribosomal peptide synthetase genes, brown indicates resistance genes, green indicates tailoring enzymes, and gray represents other biosynthetic enzymes. (b) Detection of an embedded resistance determinant related to the daptomycin chemical scaffold, a daptomycin ABC transporter. Supplementary Figure 5. PRISM results for the erythromycin biosynthetic gene cluster. (a) Detection of the erythromycin biosynthetic gene cluster. Blue indicates polyketide synthase genes, brown indicates resistance genes, purple indicates sugar biosynthesis genes, green indicates tailoring enzymes, and gray represents other biosynthetic enzymes. (b) Detection of an embedded resistance determinant related to the erythromycin mode of action, the ribosome modifying rrna methyltransferase.

5 Supplementary Figure 6. PRISM results for the friulimicin biosynthetic gene cluster. (a) Detection of the friulimicin biosynthetic gene cluster. Red indicates nonribosomal peptide synthetase genes, brown indicates resistance genes, and gray represents other biosynthetic enzymes. (b) Detection of an embedded resistance determinant related to the friulimicin chemical scaffold, a friulimicin ABC transporter. Supplementary Figure 7. PRISM results for the mupirocin biosynthetic gene cluster. (a) Detection of the mupirocin biosynthetic gene cluster. Blue indicates polyketide synthase genes, brown indicates resistance genes, and gray represents other biosynthetic enzymes. (b) Detection of an embedded resistance determinant related to the mupirocin mode of action, a mupirocin resistant Ile-tRNA synthetase.

6 Supplementary Figure 8. PRISM results for the albomycin biosynthetic gene cluster. (a) Detection of the albomycin biosynthetic gene cluster. Red indicates nonribosomal peptide synthetase genes, brown indicates resistance genes, and green indicates tailoring enzymes. (b) Detection of an embedded resistance determinant related to the albomycin mode of action, an albomycin resistant Ser-tRNA synthetase. Supplementary Figure 9. PRISM results for the albicidin biosynthetic gene cluster. (a) Detection of the albicidin biosynthetic gene cluster. Red indicates nonribosomal peptide synthetase genes, blue represents polyketide synthetase genes, brown indicates resistance genes, green indicates tailoring enzymes, and gray represents other biosynthetic enzymes. (b) Detection of an embedded resistance determinant related to the albicidin chemical scaffold, an albicidin ABC transporter.

7 Supplementary Figure 10. PRISM results for the teicoplanin biosynthetic gene cluster. (a) Detection of the teicoplanin biosynthetic gene cluster. Red indicates nonribosomal peptide synthetase genes, brown indicates resistance genes, green indicates tailoring enzymes, and gray represents other biosynthetic enzymes. (b) Detection of an embedded resistance determinant related to the teicoplanin mode of action the D-ala-D-lac glycopeptide-resistant ligase. Supplementary Figure 11. PRISM results for the capuramycin biosynthetic gene cluster. (a) Detection of the capuramycin biosynthetic gene cluster. Red indicates nonribosomal peptide synthetase genes, brown indicates resistance genes, purple indicates sugar biosynthesis enzymes, and gray represents other biosynthetic enzymes. (b) Detection of an embedded resistance determinant specific to the capuramycin chemical scaffold A phosphotransferase.

8 Supplementary Figure 12. PRISM results for the telomycin biosynthetic gene cluster. (a) Detection of the telomycin biosynthetic gene cluster. Red indicates nonribosomal peptide synthetase genes, brown indicates resistance genes, green indicates tailoring enzymes, and gray represents other biosynthetic enzymes. (b) Detection of a generic ABC transporter as the sole known resistance determinant detected in the telomycin gene cluster. Supplementary Figure 13. Telomycin does not cause hemolysis. A solution of 0.25% sheep red blood cell suspension in phosphate-buffered saline was incubated for 1 h at 37 C with either a serial dilution of telomycin, 1% Triton X-100 (positive lysis control), DMSO alone (1%; negative lysis control), or without added content (negative lysis control). After 1 h RBCs were pelleted and lysis was assessed by measuring absorbance of the supernatant at 540 nm.

9 Supplementary Figure 14. Summary of observed spontaneous telomycin-resistance mutations. Sequenced genomes of telomycin-resistant S. aureus Newman and B. subtilis 168 were compared to sensitive parent strains, revealing mutations in the dominant, house-keeping cardiolipin synthase genes cls2 and clsa, respectively. Mutations occurred in the catalytic domains of cardiolipin synthase, and either resulted in truncations, or missense mutations near the active site HKD motifs. Supplementary Figure 15. Fluorescence microscopy of anionic-lipid dye NAO. (a) Structure of the cardiolipin dye 10-N-nonyl-acridine orange (NAO). (b) Fluorescence microscopy images of B. subtilis with the standard membrane dye FM4-64 and the standard cardiolipin dye NAO. Summed pixel intensities over the length of a selected bacteria are depicted below.

10 Supplementary Tables Gene Predicted Function Strand Amino Acids tlo1 Alpha-mannosidase tlo2 Hydrolase tlo3 ABC transporter permease tlo4 ABC transporter permease tlo5 Extracellular binding domain tlo6 Hypothetical protein tlo7 Putative glucokinase tlo8 LacI-family transcription regulator tlo9 Hypothetical protein tlo10 Hydrolase tlo11 Transcriptional regulator tlo12 Tryptophan dehydrogenase tlo13 Antitermination regulator tlo14 GntR-family transcription regulator tlo15 Anthranilate phosphoribosyltransferase tlo16 Phospholipase tlo17 HTH regulatory protein tlo18 Fatty acyl adenylate ligase tlo19 Acyl carrier protein + 90 tlo20 NRPS tlo21 NRPS tlo22 NRPS tlo23 Cytochrome P tlo24 MbtH protein + 75 tlo25 Acylase Transposase Transposase tlo26 / Hydrolase tlo27 Aminotransferase-methyltransferase tlo28 Ferritin-like tlo29 Cytochrome P tlo30 ABC transporter membrane protein tlo31 ABC transporter ATPase tlo32 Proline hydroxylase tlo33 Hypothetical protein tlo34 Hypothetical protein Supplementary Table 1. Telomycin gene cluster analysis.

11 Bacterial strain Telomycin MIC % Cardiolipin Staphylococcus aureus Newman 8 g/ml 100% S. aureus Newman ClsA Q148stop 128 g/ml 1.7% S. aureus Newman ClsA A388stop 128 g/ml 3.8% Bacillus subtilis g/ml 100% B. subtilis 168 ClsA P442L 16 g/ml 10.2% Supplementary Table 2. Cardiolipin levels present in spontaneously telomycin-resistant mutant strains. [NaCl] (M) S. aureus wild type S. aureus Telo R Supplementary Table 3. Increasing osmotic stress increases telomycin potency. MICs of sensitive and telomycin-resistant (cls2 A388stop) S. aureus cultured in CAMHB containing 0, 0.5, 1, or 1.5 M NaCl. MICs were determined after 16 h growth, and are shown in μg ml -1. B. subtilis 168 wildtype B. subtilis 168 Telo R S. aureus Newman wildtype S. aureus Newman Telo R (1) (2) (3) (4) (5) (6) (7) >128 (8) di-5-hydroxytryptophan telomycin (9) di-5-methoxytryptophan telomycin (10) >128 >128 >128 > >128 Supplementary Table 4. MICs of telomycin antibiotics (1-10). Sensitive and resistant strains were exposed to telomycins and MICs were measured by microdilution in CAMHB. MICs were determined after 16 h growth, and are shown in μg ml -1.

12 Supplementary Datasets Supplementary Dataset 1. Results from retrobiosynthetic analysis of microbial modular natural product antibacterials. A retrobiosynthetic algorithm was used to cluster specific antibacterial natural products by their chemical scaffolds. Clustering analysis yielded proposed classes based on conserved chemical scaffold, with the number of structures in each class is listed along with a representative chemical structure and statement on the mechanism of action or known cross resistance. Molecules which are not present in this dataset were not accepted as inputs by our retrobiosynthetic algorithm or were not specific antibacterial agents. Supplementary Dataset 2. Hidden Markov models used by PRISM to detect antibiotic resistance genes.