S1

Germicidins H-J with Antibacterial Activities from Streptomyces sp. CB00361

Ming Ma1, Mostafa E. Rateb1, Dong Yang1, Jeffrey D. Rudolf1, Xiangcheng Zhu2,3, Yong Huang2, Li-Xing Zhao4, Yi Jiang4, Yanwen Duan2,3, and Ben Shen*1,5,6

1Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458, United States;

2Xiangya International Academy of Translational Medicine, Central South University, Changsha, Hunan 410013, China; 3Hunan Engineering Research Center of Combinatorial Biosynthesis and

Natural Product Drug Discovery, Changsha, Hunan 410329, China; 4Yunan Institute of Microbiology, Yunnan University, Kunming, Yunnan 650091, China, and 5Department of Molecular Therapeutics and 6Natural Products Library Initiative at The Scripps Research

Institute, The Scripps Research Institute, Jupiter, FL 33458, United States.

*Correspondence to: E-mail: shenb@scripps.edu; Tel: (561) 228-2456; Fax: (561) 228-2472

Supplementary Information

General procedures S2 Phylogenetic analysis procedures S2 Fermentation and isolation S2 Antibacterial activity assay S3 Table S1. 1H (400 MHz) and 13C (100 MHz) NMR data of germicidin A (1), C (2), D (3) S4 Figure S1. Structures of selected members of germicidin family of natural products S5 Figure S2. The phylogenetic analysis of S. sp. CB00361 S6 Figures S3-S7. The NMR and HR-ESI-MS spectra of 4 S7-9 Figures S8-S12. The NMR and HR-ESI-MS spectra of 5 S10-12 Figures S13-S17. The NMR and HR-ESI-MS spectra of 6 S13-15 Figures S18-S22. The NMR and HR-ESI-MS spectra of 7 S16-18 References S18

S2

General procedures: Optical rotations were measured with an AUTOPOL IV automatic polarimeter (Rudolph Research Analytical). UV spectra were collected with a NanoDrop 2000C spectrophotometer (Thermo Scientific). NMR data was collected on a Bruker 400 Ultra Shield (400 MHz) instruement. HR-ESI-MS data was collected on a 6230 TOF LC/MS spectrometer (Agilent Technologies). MPLC separation was conducted on Biotage Isolera One using a KP-C18-HS (30 g) column. HPLC was carried out on Varian semipreparative HPLC system (Woburn, MA) using a Prostar 330 detector, and a GRACE Apollo C18 column ( 250 × 4.6 mm, 5 μm) for analysis and a Alltima C18 column (250 × 10.0 mm, 5 μm) for purification. All the fermentations were carried out in New Brunswick Scientific Innova 44 incubator shaker or New Brunswick BioFlo/celliGen 115 fermentor. Phylogenetic analysis for S. sp. CB00361: S. sp. CB00361 was grown on ISP4 agar medium at 28 °C for 7 days to harvest the spores. Spores were inoculated into TSB medium and cultured at 28 °C for 2 days to isolate the genomic DNA, following previous protocols.1 Four housekeeping genes, 16S rRNA (accession number: KT819267), recA (encoding recombinase A, accession number: KT819268), rpoB (encoding RNA polymerase B subunit, accession number: KT819269), and trpB (encoding tryptophan synthase subunit B, accession number: KT819270), were amplified by PCR and sequenced. PCR was carried out according to previous protocols.1 The phylogenetic tree was generated from the alignment of concatenated partial sequences2, 3 of the four housekeeping genes 16S rRNA, recA, rpoB, and trpB (3412 bp total) using the Tamura-Nei evolutionary distance method4 and the neighbor-joining algorithm. Bootstrap5 values >60% (based on 1000 resampled trials) are given at nodes. Bar, 0.02 substitutions per nucleotide position. Sequence alignment and phylogenetic tree construction were conducted with MEGA 5.05.6 S. coelicolor A3(2) (AL645882.2), S. avermitilis MA-4680 (BA000030.3), S. rimosus ATCC 10970 (ANSJ00000000.1), S. griseus NBRC 13350 (AP009493), S. scabies 87.22 (NC_013929.1), S. venezuelae ATCC 10712 (FR845719.1), S. clavuligerus ATCC 27064 (ADWJ00000000.1), S. collinus Tu 365 (CP006259.1), S. hygroscopicus subsp. Jinggangensis TL01 (CP003720.1), S. davawensis JCM 4913 (HE971709.1), S. flavogriseus ATCC 33331 (CP002475.1), S. sp. PAMC26508 (CP003990.1) and S. fulvissimus DSM 40593 (CP005080.1) were obtained from the NCBI database and used for representative Streptomyces spp. Mycobacterium tuberculosis H37Rv (AL123456.3) was used as an outgroup. Fermentation of S. sp. CB00361 and isolation of germicidins: S. sp. CB00361 was grown on ISP4 agar plates for sporulation. One week later the spores were harvested and 0.5 mL of spore suspension in 20% glycerol were inoculated into 400 mL of seed medium (Tryptic Soy Broth) and incubated on a rotary shaker at 250 rpm, 28 °C for 2 days. The resultant seed culture (400 mL) was then inoculated into 6 L of production medium [sucrose 100 g, glucose 10 g, casamino acids 0.1 g, yeast extract 5 g, MOPS 21 g, K2SO4 0.25 g, MgCl2·6H2O 10 g, trace elements 1 mL (trace element components: ZnCl2 40 mg, FeCl3·6H2O 200 mg, CuCl2·2H2O 10 mg, MnCl2·4H2O 10 mg, Na2B4O7·10H2O 10 mg, (NH4)6Mo7O24·4H2O 10 mg, in 1.0 L of deionized water), in 1.0 L of deionized water, pH 7.0], and the fermentation in fermentor was continued at 250 rpm, 28 °C for 6 days. After the fermentation, 200 g XAD-16 resins were added into the culture and stirred for 4 h. The resins and cell mass were harvested by centrifugation and washed by deionized water and extracted with MeOH. The MeOH extract was concentrated and loaded onto MPLC, using a Biotage SNAP-HP 100 g silica gel column with an increasing gradient of MeOH in CH2Cl2 as mobile phase. Flowrate was 100 mL/min and every 35 mL was collected as a fraction. The CH2Cl2/MeOH (20/1) elution gave 71 fractions, of which the fractions 55-70 were combined based on the HPLC analysis. These combined fractions were loaded onto preparative HPLC, using CH3CN/H2O (30/70) as the mobile phase, with a flowrate of 3 mL/min and detection at 210 and 290 nm, affording germicidin A (1) (5.3 mg),

S3

germicidin C (2) (65.3 mg), germicidin D (3) (28.9 mg), germicidin H (4) (14.3 mg), germicidin I (5) (10.2 mg), germicidin J (6) (5.9 mg), and keto acid (7) (35.1 mg).

Germicidin A (1): white powder; []25D +31 (c 0.08, DMSO); UV (DMSO) λmax (log ε) 292 (3.90)

nm; 1H NMR (acetone-d6, 400 MHz) data, Table S1; 13C NMR (acetone-d6, 100 MHz) data, Table S1; HR-ESI-MS m/z 197.1176 [M + H]+ (calcd for C11H16O3, 197.1177).

Germicidin C (2): white powder; []25D +18.5 (c 0.46, DMSO); UV (DMSO) λmax (log ε) 291

(3.80) nm; 1H NMR (acetone-d6, 400 MHz) data, Table S1; 13C NMR (acetone-d6, 100 MHz) data, Table S1; HR-ESI-MS m/z 183.1017 [M + H]+ (calcd for C10H14O3, 183.1020). Germicidin D (3): white powder; UV (DMSO) λmax (log ε) 291 (3.59) nm; 1H NMR (acetone-d6, 400 MHz) data, Table S1; 13C NMR (acetone-d6, 100 MHz) data, Table S1; HR-ESI-MS m/z 169.0860 [M + H]+ (calcd for C9H12O3, 169.0864). Germicidin E (4): white powder; UV (DMSO) λmax (log ε) 292 (3.80) nm; 1H NMR (acetone-d6, 400 MHz) data, Table 1; 13C NMR (acetone-d6, 100 MHz) data, Table 1; HR-ESI-MS m/z 169.0861 [M + H]+ (calcd for C9H12O3, 169.0864). Germicidin F (5): white powder; UV (DMSO) λmax (log ε) 291 (3.83) nm; 1H NMR (acetone-d6, 400 MHz) data, Table 1; 13C NMR (acetone-d6, 100 MHz) data, Table 1; HR-ESI-MS m/z 183.1016 [M + H]+ (calcd for C10H14O3, 183.1020). Germicidin G (6): white powder; UV (DMSO) λmax (log ε) 292 (3.63) nm; 1H NMR (acetone-d6, 400 MHz) data, Table 1; 13C NMR (acetone-d6, 100 MHz) data, Table 1; HR-ESI-MS m/z 183.1017 [M + H]+ (calcd for C10H14O3, 183.1020). 8-methyl-5-oxo-nonanoic acid (7): colorless oil; 1H NMR (CDCl3, 400 MHz) data, Table 1; 13C NMR (CDCl3,100 MHz) data, Table 1; HRESI-MS m/z 185.1179 [M − H]- (calcd for C10H18O3, 185.1177) and m/z 371.2438 [2M − H]- (calcd for C10H18O3, 371.2432). Antibacterial activity assay: Staphylococcus aureus ATCC 25923, Mycobacterium smegmatis ATCC 607, and Escherichia coli ATCC 25922 were purchased from American Type Culture Collection (ATCC), and Bacillus subtilis NCTC 2116 was purchased from National Collection of Type Cultures (NCTC). The antibacterial activities of compounds 1-7 were initially evaluated against the Gram-positive strains S. aureus ATCC 25923, B. subtilis NCTC 2116, and M. smegmatis ATCC 607, and the Gram-negative strain E. coli ATCC 25922, using the disk diffusion method. Filter paper disks containing ampicillin (10 μg) were used as positive controls. The assay was also performed in a 96-well microtiter plate using Mueller Hinton (MH) broth using the broth dilution method.7 All compounds (1-7) were dissolved in DMSO (Sigma-Aldrich) and added to the cultures, and the effect of different concentrations (0.125-64 μg/mL) on the growth was assessed after 18 h incubation at 37 °C. The MIC values, if any, were determined as the lowest concentration showing no growth compared to the MH broth control.

S4

Table S1. 1H (400 MHz) and 13C (100 MHz) NMR data of germicidin A (1), C (2), D (3) in acetone-d6 positions

germicidin A (1) germicidin C (2) germicidin D (3)

δC, type δH (J in Hz) δC, type δH (J in Hz) δC, type δH (J in Hz)

2 165.4, C 166.9, C 166.2, C

3 104.7, C 96.4, C 98.4, C

4 164.7, C 170.2, C 165.9, C

5 99.2, CH 5.98, s 101.6, CH 6.00, s 98.1, CH 6.03, s

6 167.4, C 165.7, C 168.3, C

7 40.4, CH 2.45, m 40.4, CH 2.37, m 33.1, CH 2.69, m

8 28.0, CH2 1.64, m, 1.51, m 28.1, CH2 1.63, m, 1.49, m 20.3, CH3 1.19, d (6.9)

9 11.8, CH3 0.87, t (7.4) 11.9, CH3 0.87, t (7.4) 20.3, CH3 1.19, d (6.9)

10 17.2, CH3 1.17, d (6.9) 18.4, CH3 1.15, d (6.9) 8.6, CH3 1.86, s

11 18.1, CH2 2.40, q (7.4) 9.0, CH3 1.82, s

12 12.9, CH3 1.02, t (7.4)

S5

Figure S1. Structures of selected members of germicidin family of natural products featuring

the 3,6-disubstituted 4-hydroxy--pyrone core, including germicidin A,8-12 B,8-11 C,9,10 D,9,10 F,11 G,11 isogermicidin A12 and B,12 surugapyrone A,13 the violapyrones,14 the photopyrones,15 the

marine -pyrones,16 the myxopyronins,17 the corallopyronins,18 the dactylfungins,19 the

phytotoxic -pyrones,20 and the csypyrones.21,22 The additional compounds, isolated from the endophytic Streptomyces sp. A00122 together with germicidin A, were named as germicidin D and E (boxed),12 which unfortunately is inconsistent with the germicidin nomenclature.

S6

Figure S2. The phylogenetic analysis identified strain CB00361 (labeled with a red square) as a

Streptomyces species.

S7

Figure S3. The 1H (400 MHz) NMR spectrum of germicidin H (4) in acetone-d6

Figure S4. The 13C (100 MHz) NMR spectrum of germicidin H (4) in acetone-d6

S8

Figure S5. The COSY spectrum of germicidin H (4) in acetone-d6

Figure S6. The HMBC spectrum of germicidin H (4) in acetone-d6

S9

Figure S7. The HR-ESI-MS spectrum of germicidin H (4)

S10

Figure S8. The 1H (400 MHz) NMR spectrum of germicidin I (5) in acetone-d6

Figure S9. The 13C (100 MHz) NMR spectrum of germicidin I (5) in acetone-d6

S11

Figure S10. The COSY spectrum of germicidin I (5) in acetone-d6

Figure S11. The HMBC spectrum of germicidin I (5) in acetone-d6

S12

Figure S12. The HRESIMS spectrum of germicidin I (5)

S13

Figure S13. The 1H (400 MHz) NMR spectrum of germicidin J (6) in acetone-d6

Figure S14. The 13C (100 MHz) NMR spectrum of germicidin J (6) in acetone-d6

S14

Figure S15. The COSY spectrum of germicidin J (6) in acetone-d6

Figure S16. The HMBC spectrum of germicidin J (6) in acetone-d6

S15

Figure S17. The HRESIMS spectrum of germicidin J (6)

S16

Figure S18. The 1H (400 MHz) NMR spectrum of 8-methyl-5-oxo-nonanoic acid (7) in CDCl3

Figure S19. The 13C (100 MHz) NMR spectrum of 8-methyl-5-oxo-nonanoic acid (7) in CDCl3

S17

Figure S20. The COSY spectrum of 8-methyl-5-oxo-nonanoic acid (7) in CDCl3

Figure S21. The HMBC spectrum of 8-methyl-5-oxo-nonanoic acid (7) in CDCl3

S18

Figure S22. The HR-ESI-MS spectrum of 8-methyl-5-oxo-nonanoic acid (7)

References 1. Xie, P. et al. Biosynthetic potential-based strain prioritization for natural product discovery

– a showcase for diterpenoid producing actinomycetes. J. Nat. Prod. 77, 337-387 (2014). 2. Guo, Y., Zheng, W., Rong, X. & Huang, Y. A multilocus phylogeny of the Streptomyces

griseus 16S rRNA gene clade: use of multilocus sequence analysis for streptomycete systematics . Int. J. Syst. Evol. Microbiol. 58, 149-159 (2008).

3. Labeda, D. P. Multilocus sequence analysis of phytopathogenic species of the genus Streptomyces. Int. J. Syst. Evol. Microbiol. 61, 2525-2531 (2011).

4. Tamura, K. & Nei, M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10, 512-526 (1993).

5. Felsenstein, J. Confidence limits on phylogenies: an approach using the bootstrap Evol. 39, 783-791 (1983).

6. Tamura, K., et al. MEGA5: Molecular Evolutionary Genetics Analysis using Likelihood, Distance, and Parsimony methods. Mol. Biol. Evol. 28, 2731-2739 (2011).

7. Wiegand, I., Hilpert, K. & Hancock, R. E. W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3, 163-175 (2008).

8. Petersen, F. & Zahner, H. Germicidin, an autoregulative germination inhibitor of Streptomyces viridochromogenes NRRL B-1551. J. Antibiot. 46, 1126-1138 (1993).

9. Aoki, Y., Matsumoto, D., Kawaide, H. & Natsume, M. Physiological role of germicidins in spore germination and hyphal elongation in Streptomyces coelicolor A3(2). J. Antibiot. 64, 607-611 (2011).

S19

10. Song, L. et al. Type III polyketide synthase -ketoacyl-ACP starter unit and ethylmalonyl-CoA extender unit selectivity discovered by Streptomyces coelicolor genome mining. J. Am. Chem. Soc. 128, 14754 -14755 (2006).

11. Xu, Z., Ding, L. & Hertweck, C. A branched extender unit shared between two orthogonal polyketide pathways in an Endophyte, Angew. Chem. Int. Ed. 50, 4667-4670 (2011).

12. Li, Y., et al. Two new germicidins from the endophytic Streptomyces sp. A00122 of Camptotheca acuminate. Rec. Nat. Prod. 7, 45-48 (2013).

13. Sugiyama, Y., Oya, A., Kudo, T. & Hirota, A. Surugapyrone A from Streptomyces coelicoflavus strain USF-6280 as a new DPPH radical-scavenger. J. Antibiot. 63, 365-369 (2010).

14. Zhang, J. et al. Violapyrones A-G, a-pyrone derivatives from Streptomyces violascens isolated from Hylobates hoolock feces. J. Nat. Prod. 76, 2126-2130 (2013).

15. Brachmann, A. O. et al. Pyrones as bacterial signaling molecules. Nat. Chem. Biol. 9, 573-578 (2013).

16. Zhang, H. et al. -Pyrones with diverse hydroxyl substitutions from three marine-derived Nocardiopsis strains. J. Nat. Prod. ASAP, DOI:10.1021/acs.jnatprod.6b00175.

17. Irschik, H., Gerth, K., Hofle, G., Kohl, W. & Reichenbach, H. The myxopyronins, new inhibitors of bacterial RNA synthesis from Myxococcus fulvus (Myxobacterales). J. Antibiot. 36, 1651-1658 (1983).

18. Irschik, H., Jansen, R., Hofle, G., Gerth, K. & Reichenbach, H. The corallopyronins, new inhibitors of bacterial RNA synthesis from Myxobacteria. J. Antibiot. 38, 145-152 (1985).

19. Xaio, J. Z., Kumazawa, S., Yoshikawa, N. & Mikawa, T. Dactylfungins, novel antifungal antibiotics produced by Dactylaria parvispora. J. Antibiot. 46, 48-55 (1993).

20. Evidente, A. et al. Phytotoxic -pyrones produced by Pestalotiopsis guepinii, the causal agent of hazelnut twig blight. J. Antibiot. 65, 203-206 (2012).

21. Seshime, Y., Juvvadi, P. R., Kitamoto, K., Ebizuka, Y. & Fujii, I. Identification of csypyrone B1 as the novel product of Aspergillus oryzae type III polyketide synthase CsyB. Bioorg. Med. Chem. 18, 4542-4546 (2010).

22. Hashimoto, M. et al. Identification of csypyrone B2 and B3 as the minor products of Aspergillus oryzae type III polyketide synthase CsyB. Bioorg. Med. Chem. Lett. 23, 650-653 (2013).