SI for T7A paper 10182011 - Royal Society of Chemistry · 2011-10-26 · Phenomenex Luna C18(2)...

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Supplementary Information

For

Mutagenesis of the thiostrepton precursor peptide at Thr7 impacts both

biosynthesis and function

Chaoxuan Li, Feifei Zhang, and Wendy L. Kelly*

Table of Contents Materials and Methods…………………………….……..……………………………………S2 Figure S1. HPLC-MS analysis of culture extracts from S. laurentii tsrA mutant strains………S5 Figure S2. HPLC and UV-Vis absorption spectra of thiostrepton Thr7Ala 3 and SL105-1 4…S10 Figure S3. Structures of thiostrepton Thr7Ala 3 and SL105-1 4………………………………S11 Figure S4. HPLC-MS analysis of thiostrepton Thr7Ala 3……………………………………S12 Figure S5. MALDI MS and MS/MS analysis of thiostrepton Thr7Ala 3………………………S13 Figure S6. 1H NMR spectrum of thiostrepton Thr7Ala 3……….......…………………………S15 Figure S7. 13C NMR spectrum of thiostrepton Thr7Ala 3……………………………………S16 Figure S8. DEPT-135 NMR spectrum of thiostrepton Thr7Ala 3……….……………………S17 Figure S9. gHMBC spectrum of thiostrepton Thr7Ala 3………………………………………S18 Figure S10. gCOSY spectrum of thiostrepton Thr7Ala 3………………………………………S19 Figure S11. gHSQC spectrum of thiostrepton Thr7Ala 3……….……………………………S20 Figure S12. HPLC-MS analysis of SL105-1 4…………………………………………………S21 Figure S13. MALDI MS and MS/MS analysis of SL105-1 4…………………………………S22 Figure S14. 1H NMR spectrum of SL105-1 4…………………………………………………S24 Figure S15. 13C NMR spectrum of SL105-1 4…………………………………………………S25 Figure S16. DEPT-135 NMR spectrum of SL105-1 4…………………………………………S26 Figure S17. gHMBC spectrum of SL105-1 4…………………………………………………S27 Figure S18. gCOSY spectrum of SL105-1 4………….………………………………………S28 Figure S19. gHSQC spectrum of SL105-1 4….………………………………………………S29 Figure S20. NOESY cross peaks of the thiazoline region of SL105-1 4………………………S30 Figure S21 HPLC-MS analysis of thiostrepton Thr7Val 5……………………………………S31 Figure S22. MALDI MS and MS/MS analysis of thiostrepton Thr7Val 5……………………S32 Figure S23. HPLC-MS analysis of SL106-1 6…………………………………………………S34 Figure S24. MALDI MS and MS/MS analysis of SL106-1 6…………………………………S35 Table S1. 1H and 13C NMR assignments of thiostrepton Thr7Ala 3……………………………S37 Table S2. 1H and 13C NMR assignments of SL105-1 4…………………….…………………S39 Table S3. Antibacterial activity of thiostrepton A 1 and its analogs……………………………S41 Table S4. Strains and plasmids used in this study………………………………………………S42 Table S5. Primers and ultramer used in this study….………………………………….……..S43 References……………………………………………………………………………………...S43

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2011

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Materials and Methods General. Unless specified, common chemicals, solvents, restriction enzymes, DNA ligase

and other materials were purchased from standard commercial sources and used as provided. Oligonucleotides and ultramer DNA were purchased from Integrated DNA Technologies. All DNA sequence analysis was performed at Eurofins MWG Operon. The QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA) was used for the isolation of plasmids and fosmids from E. coli strains. Streptomyces genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI) according to the manufacturer’s recommendations. High performance liquid chromatography (HPLC) analysis was performed on a Beckman Coulter System Gold instrument. HPLC-MS was performed at the Georgia Institute of Technology Bioanalytical Mass Spectrometry Facility with a Phenomenex Syngeri RP column (250 mm × 2 mm, 4 µm) (Torrance, CA) and developed with 20% Buffer B in Buffer A for 8 min followed by a gradient from 20-100% Buffer B over 35 min at 0.25 mL min-1 (Buffer A: 5% acetonitrile and 0.1% formic acid; Buffer B: 95% acetonitrile and 0.1% formic acid). High-resolution matrix-assisted laser desorption/ionization mass spectrometry (HR-MALDI-MS), and MALDI-MS/MS were also performed at the Georgia Institute of Technology Bioanalytical Mass Spectrometry Facility. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed at the Emory University Mass Spectrometry Center. Proton and carbon NMR spectra were recorded on a Bruker 500 MHz spectrometer. All NMR experiments were performed according to standard pulse sequences supplied with the instrument.

Bacterial strains plasmids and growth medium. Streptomyces laurentii ATCC 31255

was obtained from American Type Culture Collection (ATCC). S. laurentii NDS1 was previously generated in our lab.1 All strains and plasmids are listed in Table S4, and primers are listed in Table S5. All Escherichia coli (E. coli) strains were grown in Luria-Bertani liquid or solid medium with the appropriate antibiotic(s). For the selective growth of E. coli or Streptomyces, the following antibiotics and concentrations were used: kanamycin (50 μg mL-1), apramycin (50 μg mL-1), ampicillin (100 μg mL-1), nalidixic acid (25 μg mL-1), and chloramphenicol (30 μg mL-1). MS agar was used for the intergeneric conjugation of S. laurentii NDS1.2

Site-directed mutagenesis of tsrA. Site-directed mutagenesis of TsrA to yield Thr7Ser

and Thr7Ala followed the procedure described previously.1 Two pairs of primers were used: T7S-F and T7S-R for the Thr7Ser mutation and T7A-F and T7A-R for the Thr7Ala mutation (Table S5). Mutant plasmids were amplified from pJP11 by adapting the QuikChange site-directed mutagenesis strategy (Agilent Technologies, La Jolla, CA). DNA sequence analysis confirmed successful mutagenesis in each case, including the absence of any undesired mutations. The resulting plasmids were designated as pCL64 for TsrA Thr7Ser and pCL65 for TsrA Thr7Ala (Table S4).

The following procedure was used to generate the Thr7Val mutant of TsrA. The single-strand DNA ultramer T7V was amplified by polymerase chain reaction using the primers T7V-F and T7V-R (Table S5). The PCR product was cloned into pSC-B-amp/kan using the StrataClone Blunt PCR Cloning Kit (Agilent Technologies, La Jolla, CA). The plasmid encoding the expected Thr7Val mutation was confirmed by DNA sequencing and designated as pCL66 (Table S4).

The fosmid int-3A10 is an integrative fosmid harboring the entire tsr biosynthetic gene cluster.1 The fosmid int-3A100 is a derivative of int-3A10, in which the region of tsrA encoding the 17 amino acid structural peptide of TsrA is replaced by a dual-marker disruption cassette


S3

consisting of chlR, a chloramphenicol resistance gene, and a levansucrase-encoding sacB gene.1 The tsrA T7S and T7A mutants were amplified from pCL64 and pCL65 by PCR with primers SD2-F and SD2-R. The tsrA T7V mutant was amplified from pCL66 with primers T7V-F and T7V-R. The resulting PCR products were then used to replace the dual-marker cassette in int-3A100 by PCR-targeted gene replacement,2,3 yielding three sucrose-tolerant fosmids: int-3A104, int-3A105 and int-3A106 (Table S4). The incorporated mutations in int-3A104 to int-3A106 were confirmed by DNA sequence analysis. Fosmids int-3A10 (encoding wild-type TsrA), int-3A104 (for TsrA Thr7Ser), int-3A105 (for TsrA Thr7Ala) and int-3A106 (for TsrA Thr7Val) were first transformed into E. coli ET12567/pUZ8002, and then introduced into S. laurentii NDS1 through intergeneric conjugation. Colonies resistant to apramycin were selected for fermentation analysis.

Evaluation of thiostrepton analog production by S. laurentii NDS1/int-3A104, S. laurentii NDS1/int-3A105 and S. laurentii NDS1/int-3A106. Growth of S. laurentii was performed as described previously.4 After fermentation, the whole cell culture was extracted twice with an equal volume of chloroform. The chloroform layers were pooled and the solvent removed in vacuo. The solid residue from 100 mL of culture was dissolved in 4 mL of chloroform. Samples were analyzed by HPLC with a Phenomenex Luna C18(2) column (250 × 4.6 mm, 5 μm). The column was developed using a gradient of 0-100% acetonitrile in water over 30 min at a flow rate of 1 mL min-1. Absorbance was monitored at 254 nm. Under these conditions, thiostrepton A elutes with a tR of about 23 min.

Purification and structural determination of thiostrepton Thr7Ala 3 and SL105-1 4.

Crude extract from S. laurentii NDS1/int-3A105 (10 L) culture was purified by HPLC with a Phenomenex Luna C18(2) RP-Aqueous semi-preparative column (250 x 10 mm, 5 μm) while monitoring absorbance at 254 nm. The column was first developed using a gradient of 0 to 70% acetonitrile in water over 30 min at a flow rate of 4.7 mL min-1, and fractions containing thiostrepton Thr7Ala 3 and SL105-1 4 were collected separately for each fraction. Solvent was removed in vacuo for the two fractions. The solid residues were dissolved in chloroform for further HPLC purification. For purification of thiostrepton Thr7Ala 3, the column was developed with a gradient of acetonitrile in water at a flow rate of 4.7 mL min-1 as follows: 10 to 40% acetonitrile over 5 min, 40% acetonitrile for 5 min, 40 to 48% acetonitrile over 5 min, 48% acetonitrile for 5 min, 48 to 100% acetonitrile over 5 min, and finally 100% acetonitrile for 5 min. For the purification of SL105-1 4, the column was developed with a gradient of acetonitrile in water at a flow rate of 4.7 mL min-1 as follows: 10 to 35% acetonitrile over 5 min, 35% acetonitrile for 21 min, 35 to 100% acetonitrile over 2 min, and finally 100% acetonitrile for 5 min. Purified samples were analyzed by HPLC-MS, HR-MALDI-MS, MALDI-MS/MS and NMR. HR-MALDI-MS of thiostrepton Thr7Ala 3: C71H84N19O17S5, m/z 1634.4906 [M+H]+ (calculated 1634.4896). HR-MALDI-MS of SL105-1 4: C62H67N16O15S5, m/z 1435.3618 [M+H]+ (calculated 1435.3575). NMR data is included in supplementary figures and tables.

Purification of characterization of thiostrepton Thr7Val 5 and SL106-1 6. Crude

extract from S. laurentii NDS1/int-3A106 (4 L) culture was purified by HPLC with a Phenomenex Luna C18(2) RP-Aqueous semi-preparative column (250 x 10 mm, 5 μm) while monitoring absorbance at 254 nm. The column was first developed using a gradient of 0 to 60% acetonitrile in water over 30 min at a flow rate of 4 mL min-1, and fractions containing thiostrepton Thr7Val 5 and SL106-1 6 were collected separately. Solvent was removed in vacuo


S4

for each fraction. The solid residues were dissolved in chloroform for further HPLC purification. The next column was developed with a gradient of acetonitrile in water at a flow rate of 4.3 mL min-1 as follows: 0 to 100% acetonitrile over 30 min, and finally 100% acetonitrile for 5 min. Purified samples were analyzed by HPLC-MS, HR-MALDI-MS/HR-ESI-MS, and MALDI-MS/MS. HR-ESI-MS of thiostrepton Thr7Val 5: C73H87N19O17S5Na, m/z 1684.5087 [M+Na]+ (calculated 1684.5029). HR-MALDI-MS of SL106-1 6: C64H71N16O15S5, m/z 1463.3890 [M+H]+ (calculated 1463.3888).

Antibacterial activity of thiostrepton analogs. Minimum inhibitory concentrations

(MICs) of thiostrepton analogs against indicator strains were determined following the liquid microdilution method described previously.1 The indicator strains used in this study were methicillin-resistant Staphylococcus aureus ATCC 10537 (MRSA), vancomycin-resistant Enterococcus faecium ATCC 12952 (VRE), Bacillus sp. ATCC 27859 (Bacillus) and Escherichia coli ATCC 27856 (E. coli). Thiostrepton A 1, thiostrepton Thr7Ala 3 and thiostrepton Thr7Val 5 were prepared in DMSO and quantified by UV spectroscopy using an extinction coefficient at 280 nm of 0.027 cm-1 μM-1.5 SL105-1 4 was first quantified by NMR against a tetramethylsilane internal standard and then used to generate a calibration curve by HPLC, and the stock solution was finally prepared in DMSO. SL106-1 6 was quantified by HPLC against the SL105-1 5 standard and prepared in DMSO. The positive antibiotic control used for Bacillus, and VRE was chloramphenicol, whereas the positive control for MRSA was vancomycin. DMSO was used as the negative control in all assays. Cell growth was monitored by comparing the optical density at 600 nm at the time of treatment and after 18 h incubation at 37 °C. A difference in OD600 was considered growth and the lowest concentration causing complete suppression of visible bacterial growth defined the MIC.


S5

Figure S1. HPLC-MS analysis of culture extracts from S. laurentii tsrA mutant strains.

(A) HPLC-MS chromatogram of the S. laurentii NDS1 int-3A104 (TsrA Thr7Ser) culture extract. (1) Chromatogram extracted for m/z 826. (2) Chromatogram extracted for m/z 833 (the calculated [M+2H]2+ m/z 832.7 for wild-type thiostrepton A 1, which ordinarily elutes at about 28 min.1). (3) Total ion chromatogram. (4) Mass spectrum of thiostrepton Thr7Ser 2 from S. laurentii NDS1 int-3A104 extract eluting at tR = 23.6 min (calculated m/z 825.8 [M+2H]2+, observed m/z 825.9 [M+2H]2+).

(1)

(2)

(3)

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

Li_091202_08 318 (23.638) Sm (SG, 2x0.20); Cm (310:321-328:349) Scan ES+ 8.38e4766.6

391.4

283.3

319.4

320.4

325.2

737.7

591.5392.4

574.8

418.4495.5

683.4613.5

646.4

825.9

767.6

826.9

827.5

829.0

1033.4867.1919.5 985.5

1940.41292.81135.6 1217.5

1784.31389.31489.1 1651.61539.9

1717.21859.3 1982.6

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00Time0

100

%

0

100

%

0

100

%

Li_091202_08 Scan ES+ 826

2.20e623.56

21.71

20.895.233.15 17.259.687.97 26.76 50.2244.6542.94

32.9227.80 31.73 37.3736.41 38.78

49.47 50.74 53.7858.24

Li_091202_08 Scan ES+ 833

4.09e518.89

14.88

11.310.10

8.493.743.96 6.8613.76 15.32

19.4152.5920.82 43.4634.48

31.9527.5723.5626.83 31.3642.1236.26

46.28 49.47 50.14 52.96 57.20 59.50 59.94

Li_091202_08 Scan ES+ TIC

3.16e842.49

4.633.67

26.2416.366.34 7.5312.95

18.1423.64 37.0028.39

51.9247.84

(4)[M+2H]2+


S6

(B) HPLC-MS chromatogram of the S. laurentii NDS1 int-3A105 (TsrA Thr7Ala) culture extract. (1) Chromatogram extracted for m/z 718. (2) Chromatogram extracted for m/z 818. (3) Chromatogram extracted for m/z 833. (4) Total ion chromatogram. (5) Mass spectrum of thiostrepton Thr7Ala 3 from S. laurentii NDS1 int-3A105 extract eluting at tR = 25.7 min (calculated m/z 817.8 [M+2H]2+, observed m/z 817.9 [M+2H]2+; calculated m/z 1634.5 [M+H]+, observed m/z 1634.6 [M+H]+). (6) Mass spectrum of SL105-1 4 from S. laurentii NDS1 int-3A105 extract eluting at tR = 23.4 min (calculated m/z 718.2 [M+2H]2+, observed m/z 718.4 [M+2H]2+; calculated m/z 1435.4 [M+H]+, observed m/z 1435.5 [M+H]+).

(1)

(2)

(3)

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00Time0

100

%

0

100

%

0

100

%

0

100

%

Li_091202_12 Scan ES+ 718

3.08e623.41

23.04

22.60

21.8511.395.82 9.83 18.66

23.56

38.3326.31 29.50 49.1047.1044.9444.57

39.30 53.4851.9255.48

Li_091202_12 Scan ES+ 818

7.77e625.71

20.07

Li_091202_12 Scan ES+ 833

2.59e545.76

19.7819.2616.81

3.96

3.591.597.606.04 13.549.31 13.02

16.6618.07 38.1927.7926.9020.15

23.04 35.9631.6530.24 42.7939.45 44.79

51.7747.91 49.32

53.55 55.8659.57

59.79


3.16e842.57

23.413.66 4.7020.6718.4416.36

6.347.60

26.2326.83 37.07

47.84 51.55(4)


S7

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

Li_091202_12 346 (25.714) Cm (342:349) Scan ES+ 4.29e5817.9

774.8387.4

316.4319.4

352.4

418.5

699.9559.0450.3

658.4

818.5

818.9

819.4

819.7

1634.61346.5980.4938.71067.7 1218.41184.4

1425.01461.1 1722.3 1962.6

(5) [M+2H]2+

[M+H]+

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

Li_091202_12 315 (23.412) Cm (310:318) Scan ES+ 3.80e5566.9

283.5 553.0

391.5

321.3541.0

395.8526.8

718.4

601.4

675.4

609.9

610.4

610.9

718.9

719.4

1435.5766.6

767.61218.4

1132.3

768.5820.5975.4936.9 1085.5

1133.5

1134.3

1221.4

1222.41366.3

1436.3

1438.5

1639.91439.51717.5 1998.41920.8

(6)

[M+2H]2+

[M+H]+


S8

(C) HPLC-MS chromatogram of the S. laurentii NDS1 int-3A106 (Tsr Thr7Val) culture extract. (1) Chromatogram extracted for m/z 732.2. (2) Chromatogram extracted for m/z 831.8. (3) Chromatogram extracted for m/z 833. (4) Total ion chromatogram. (5) Mass spectrum of thiostrepton Thr7Val 5 from S. laurentii NDS1 int-3A106 extract eluting at tR = 23.5 min (calculated m/z 831.8 [M+2H]2+, observed m/z 832.0 [M+2H]2+). (6) Mass spectrum of SL106-1 6 from S. laurentii NDS1 int-3A106 extract eluting at tR = 25.1 min (calculated m/z 732.2 [M+2H]2+, observed m/z 732.5 [M+2H]2+)

(1)

(2)

(3)

(4)

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00Time0

100

%

0

100

%

0

100

%

0

100

%

Li_110713_12 Scan ES+ 732.2

1.75e725.06

14.2315.62 23.23

Li_110713_12 Scan ES+ 831.8

3.15e723.45

17.89

Li_110713_12 Scan ES+ 833

3.85e723.52

17.89


5.65e818.84

15.77

5.223.542.74

2.306.62 8.37 10.79

17.3020.38

23.5222.20

25.0637.1436.2626.96

31.72 53.7540.5838.60


S9

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

Li_110713_12 342 (25.060) Cm (342) Scan ES+ 2.18e6732.5

623.9

580.9

396.9327.3 523.5488.1

624.5

689.7625.4

732.7

733.4

733.8

734.3

766.4

831.4 1464.5907.8 1209.7

1095.21246.3 1505.3

1652.9 1870.61700.3

y

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

Li_110713_12 321 (23.523) Cm (319:321) Scan ES+ 3.38e6832.0

754.6

737.7

377.9594.3

755.6

756.6

832.5

832.9

833.3

833.9

(5)[M+2H]2+

(6) [M+2H]2+


S10

Figure S2. HPLC and UV-Vis absorption spectra from the TsrA T7A-expressing fermentation extract. (A) HPLC analysis of S. laurentii NDS1 int-3A105 (TsrA Thr7Ala) culture extract. (B) UV-Vis absorption spectrum of SL105-1 4 (tR = 16.9 min). (C) UV-Vis absorption spectrum of thiostrepton Thr7Ala 3 (tR = 18.3 min).

nm200 225 250 275 300 325 350 375 400

0

250

500

750

t/min8 10 12 14 16 18 20 22 24

3

4

A25

4/m

Au

(A)

(B)

(C)

mA

um

Au

nm200 225 250 275 300 325 350 375 400

0

500

1000


S11

Figure S3. Structures of and numbering systems used for (A) thiostrepton Thr7Ala 3 and (B) SL105-1 4. (A) Thiostrepton Thr7Ala 3

(B) SL105-1 4


S12

Figure S4. HPLC-MS analysis of thiostrepton Thr7Ala 3 isolated from S. laurentii NDS1 int-3A105 (TsrA Thr7Ala). (A) Total ion chromatogram. (B) Chromatogram extracted for m/z 818 of thiostrepton Thr7Ala 3. (C) Mass spectrum of thiostrepton Thr7Ala 3 eluting at tR = 26.3 min.

(A)

(B)

(C)

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

Li_110125_04 358 (26.225) Cm (353:364) Scan ES+ 9.88e5818.0

774.9

818.9

819.5

1634.7

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00Time0

100

%


1.87e848.3347.01

26.23

55.6450.15 51.11

0

100

%

Li_110125_04 Scan ES+ 818

2.21e726.30

[M+2H]2+

[M+H]+


S13

Figure S5. MALDI MS and MS/MS analysis of thiostrepton Thr7Ala 3. (A) Full spectrum of MALDI MS of thiostrepton Thr7Ala 3. (B) MALDI MS/MS of parent ion m/z 1634.5. (C) Table and structure showing fragments in MALDI MS/MS of thiostrepton Thr7Ala 3.

7 9 9 .0 1 0 4 0 .8 1 2 8 2 .6 1 5 2 4 .4 1 7 6 6 .2 2 0 0 8 .0M a s s (m /z )

1 .0 E +

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

% I

nten

sity

4700 R eflector S pec #1 M C [B P = 1634.5 , 10147]

Li TS R T7A (C H C A )

1 6 3 4 .5

1 6 8 8 .4

1 6 1 8 .4 1 6 9 0 .41 6 1 7 .4 1 6 9 1 .41 6 5 6 .41 6 1 6 .4

1 2 0 1 .0 1 8 2 3 .51 5 3 9 .9 1 6 9 3 .41 6 0 3 .31 4 0 8 . 9 1 7 7 9 .51 5 4 8 .4 1 8 2 2 .5

1(A)

6 9 . 0 4 0 0 . 2 7 3 1 .4 1 0 6 2 .6 1 3 9 3 . 8 1 7 2 5 .0M a s s (m /z )

4 .5 E +

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

% In

tens

ity

Li TS R T7A M S /M S (C H C A )

1 2 1 7 . 1

1 2 1 9 .2

1 4 0 1 .21 0 7 7 .1

1 1 4 8 .11 4 0 3 .27 8 0 .1 1 0 7 9 .1

1 2 0 0 .11 0 6 0 .1

1 1 5 0 .11 1 0 4 .21 1 8 9 .2 1 4 5 0 .25 4 7 . 1

1 1 3 1 .1 1 4 5 2 .21 0 6 2 .11 2 0 2 .7

1 1 3 3 .1 1 3 1 0 .17 8 2 .1 1 3 9 5 .21 0 8 1 .2 1 5 3 7 .51 1 7 2 .2 1 4 2 3 .2 1 5 3 9 .44 1 8 .1 1 2 2 4 . 2 1 3 1 4 .21 0 5 8 .05 4 9 . 12 5 7 .1 1 3 7 4 .2 1 4 6 8 .3 1 6 0 1 .19 9 1 .18 6 5 .2 1 0 3 4 .23 7 4 . 2 5 1 4 . 1 1 5 5 0 .53 1 9 . 1 1 2 6 0 .21 9 0 . 0 5 5 8 . 2 7 8 4 . 12 7 5 . 1 9 3 9 . 16 2 9 . 1 6 9 2 . 14 5 1 . 1 7 4 0 .19 1 . 0 4 1 4 .11 4 3 . 0

2

3

45

(B)


S14

Fragment Expected Observed

1. M+H+ (Parent ion) 1634.5 1634.5

2. M-QA+H+ 1401.4 1401.2

3. M-QA-Ile1-Ala2+H+ 1217.3 1217.1

4. M-QA-Ile1-Ala2-Dha3+H+ 1148.3 1148.1

5. M-QA-Ile1-Ala2-Dha3-Ala4+H+ 1077.2 1077.1

2

345

(C)


S15

Figure S6. 1H NMR spectrum of thiostrepton Thr7Ala 3 (500 MHz, CDCl3-CD3OD 4:1, 25 °C).


S16

Figure S7. 13C NMR spectrum of thiostrepton Thr7Ala 3 (125 MHz, CDCl3-CD3OD 4:1, 25 °C).


S17

Figure S8. DEPT-135 NMR spectrum of thiostrepton Thr7Ala 3 (125 MHz, CDCl3-CD3OD 4:1, 25 °C).


S18

Figure S9. gHMBC spectrum of thiostrepton Thr7Ala 3 (500 MHz, CDCl3-CD3OD 4:1, 25 °C).


S19

Figure S10. gCOSY spectrum of thiostrepton Thr7Ala 3 (500 MHz, CDCl3-CD3OD 4:1, 25 °C).


S20

Figure S11. gHSQC spectrum of thiostrepton Thr7Ala 3 (500 MHz, CDCl3-CD3OD 4:1, 25 °C).


S21

Figure S12. HPLC-MS analysis of SL105-1 4 isolated from S. laurentii NDS1 int-3A105 (TsrA Thr7Ala). (A) Total ion chromatogram. (B) Chromatogram extracted for m/z 718 of SL105-1 4. (C) Mass spectrum of SL105-1 4 eluting at tR = 23.7 min.

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

Li_100623_07 324 (23.741) Cm (320:326) Scan ES+ 6.90e5718.5

566.9

553.0

518.5369.1

601.5675.5

609.9

610.4

611.0

611.4

719.0

719.5

719.9

1435.6720.51218.5

1132.4745.2

1219.51437.6

1438.61505.5

(A)

(B)

(C)[M+2H]2+

[M+H]+

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00Time0

100

%


1.45e823.74

49.2144.89 55.72

0

100

%

Li_100623_07 Scan ES+ 718

4.79e623.67

23.45

23.23

53.75


S22

Figure S13. MALDI MS and MS/MS analysis of SL105-1 4. (A) Full spectrum of MALDI MS of SL105-1 4. (B) MALDI MS/MS of parent ion m/z 1435.4. (C) Table and structure showing fragments in MALDI MS/MS of SL105-1 4.

7 9 9 . 0 1 0 4 0 . 8 1 2 8 2 . 6 1 5 2 4 . 4 1 7 6 6 . 2 2 0 0 8 . 0M a s s ( m / z )

2 4 2 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

% I

nte

ns

ity

1 4 3 5 . 4

1 2 2 5 . 1

1 4 3 7 . 4

1 2 1 8 . 3 1 4 1 9 . 4

1 2 2 8 . 1

1 4 1 8 . 4

1 2 2 0 . 31 4 3 3 . 3

1 4 5 7 . 31 3 6 6 . 3 1 6 2 4 . 41 4 4 9 . 3

1(A)

2

3

6 9 .0 3 5 8 . 2 6 4 7 .4 9 3 6 . 6 1 2 2 5 .8 1 5 1 5 .0M a s s (m /z )

2 .8 E +

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

% In

tens

ity

L i S L105-1 M S /M S (C H C A )

1 2 1 8 .1

1 2 2 0 .1

1 2 2 2 .0

1 1 9 0 .17 6 4 . 1

1 1 3 2 .01 2 1 6 .0

1 1 3 0 .0 1 2 4 0 .01 1 8 6 .17 6 6 . 1 1 0 6 0 .0 1 4 0 4 .95 4 7 .1 1 3 2 4 .2 1 4 4 6 .54 5 1 .1

4(B)


S23


1. M+H+ (parent ion) 1435.3 1435.4

2. M-NH2-QA+Na+ 1225.3 1225.1

3. M-NH2+H+ 1419.3 1419.4

4. M-QA+H+ 1218.3 1218.1

4

(C) 23


S24

Figure S14. 1H NMR spectrum of SL105-1 4 (500 MHz, CDCl3-CD3OD 4:1, 25 °C).


S25

Figure S15. 13C NMR spectrum of SL105-1 4 (125 MHz, CDCl3-CD3OD 4:1, 25 °C).


S26

Figure S16. DEPT-135 NMR spectrum of SL105-1 4 (125 MHz, CDCl3-CD3OD 4:1, 25 °C).


S27

Figure S17. gHMBC spectrum of SL105-1 4 (500 MHz, CDCl3-CD3OD 4:1, 25 °C).


S28

Figure S18. gCOSY spectrum of SL105-1 4 (500 MHz, CDCl3-CD3OD 4:1, 25 °C).


S29

Figure S19. gHSQC spectrum of SL105-1 4 (500 MHz, CDCl3-CD3OD 4:1, 25 °C).


S30

Figure S20. NOESY cross peaks of the thiazoline region of SL105-1 4 (500 MHz, CDCl3-CD3OD 4:1, 25 °C). The cross peaks of Tzn9-2 and Tzn9-3-HA protons are circled in red. The cross peaks of Tzn9-2 and Tzn9-3-HB protons are circled in blue. Schoof and Arndt observed that the NOE signals between the Tzn9-2 and Tzn9-3-HA protons in 1 were of greater intensity than the signals for the Tzn9-2 and Tzn9-3-HB protons.7

Tzn9-2 Tzn9-3-HA Tzn9-3-HB


S31

Figure S21. HPLC-MS analysis of thiostrepton Thr7Val 5 isolated from S. laurentii NDS1 int-3A106 (TsrA Thr7Val). (A) Total ion chromatogram. (B) Chromatogram extracted for m/z 831.8 of thiostrepton Thr7Val 5. (C) Mass spectrum of thiostrepton Thr7Val 5 eluting at tR = 23.6 min.

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00Time0

100

%

Zhang_111005_03 Scan ES+ TIC

1.88e823.52

22.9418.6212.981.35 6.323.40 8.23 10.28 16.72

51.2649.1446.7246.07

44.1628.6526.38 41.8939.6329.52 37.3635.45

52.14 54.70

58.58

0

100

%

Zhang_111005_03 Scan ES+ 831.8

2.14e723.60

(A)

(B)

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

Zhang_111005_03 322 (23.597) Sm (SG, 2x0.20) Scan ES+ 2.50e6832.0

762.9

832.4

832.9

[M+2H]2+(C)


S32

Figure S22. MALDI MS and MS/MS analysis of thiostrepton Thr7Val 5. (A) Full spectrum of MALDI MS of thiostrepton Thr7Val 5. (B) MALDI MS/MS of parent ion m/z 1662.4. (C) Table and structure showing fragments in MALDI MS/MS of thiostrepton Thr7Val 5.

799. 0 1040. 8 1282. 6 1524. 4 1766. 2 2008. 0M ass ( m / z)

1. 9E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsi

ty

1662. 4

1684. 4

1686. 4

1726. 4

1666. 4

1700. 4

1728. 41646. 41645. 4

1634. 31509. 3 1716. 41252. 71088. 2 1680. 41567. 8 1619. 41393. 3 1721. 41595. 41437. 0 1512. 31479. 3 1714. 41319. 2 1851. 51524. 4834. 8 862. 9 1807. 5 1871. 4 1913. 4

1(A)

6 9 . 0 4 0 0 . 2 7 3 1 . 4 1 0 6 2 . 6 1 3 9 3 . 8 1 7 2 5 . 0M a s s ( m / z )

1 . 4 E +

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

% I

nte

ns

ity

1 2 4 5 . 3

1 2 4 8 . 3

1 1 0 5 . 37 8 0 . 1 1 2 2 8 . 3

1 2 1 7 . 35 4 7 . 1

1 2 1 9 . 3 1 4 2 9 . 41 1 0 7 . 2

1 0 8 9 . 2 1 1 7 8 . 3 1 4 3 1 . 47 8 2 . 1 1 5 6 4 . 51 4 7 8 . 31 2 4 2 . 11 0 8 7 . 2 1 1 5 7 . 3 1 3 3 9 . 35 6 4 . 1 1 4 2 4 . 49 6 4 . 3 1 6 4 6 . 91 3 4 1 . 41 0 6 1 . 2 1 5 5 1 . 78 6 6 . 2 1 4 3 5 . 31 2 8 9 . 45 4 9 . 12 8 5 . 1 1 6 4 5 . 19 7 4 . 29 2 0 . 23 7 4 . 2 7 7 9 . 04 1 8 . 1 8 7 0 . 96 9 4 . 15 4 5 . 04 5 6 . 05 0 0 . 2 5 9 8 . 39 3 . 5 7 3 4 . 93 0 1 . 11 8 9 . 8 2 5 1 . 1 6 4 6 . 83 5 4 . 21 5 3 . 7

2

3

4

5

(B)


S33

N

N

NS

NHO O

HN NH2

O

N

S

HN S

O

OHN

NS

HN

O

N

HOOH

S

OHN

O

ON

OH

NH

HN

O

O

HN N

H

O

HN O

OHH


1. M+H+ (Parent ion) 1662.5 1662.4

2. M-QA+H+ 1429.4 1429.4

3. M-QA-Ile1-Ala2+H+ 1245.3 1245.3

4. M-QA-Ile1-Ala2-Dha3+H+ 1176.3 1176.3

5. M-QA-Ile1-Ala2-Dha3-Ala4+H+ 1105.2 1105.3

2

345

(C)


S34

Figure S23. HPLC-MS analysis of SL106-1 6 isolated from S. laurentii NDS1 int-3A106 (TsrA Thr7Val). (A) Total ion chromatogram. (B) Chromatogram extracted for m/z 732.2 of SL106-1 6. (C) Mass spectrum of SL106-1 6 eluting at tR = 25.1 min.

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00Time0

100

%

Zhang_111005_05 Scan ES+ TIC

1.52e847.3847.02

44.9744.0225.13

1.13 24.4822.212.00

4.93 5.3019.79

16.649.54 11.89

36.9934.1427.11 33.7029.74

42.4839.11

53.8252.0049.21 54.41

56.6859.46

0

100

%

Zhang_111005_05 Scan ES+ 732.2

2.01e625.13

22.7210.130.91 1.86 9.183.25 6.69 15.9110.71 21.0316.35

55.5847.4647.0936.4827.26 31.2830.33 32.82 43.3637.65 42.04 54.63

53.0250.60 56.75 59.24

, , y

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

Zhang_111005_05 343 (25.134) Sm (SG, 2x0.20) Scan ES+ 3.08e5732.4

689.5

623.9279.3

538.9441.8357.9

471.3

625.4

732.9

733.2

733.8

1838.5

741.9

1700.61168.1766.61021.8

875.9793.8

903.5965.7

1093.5

1087.7

1536.11484.01409.61248.4

1320.1 1660.1

1752.61850.3

1991.21898.1

(A)

(B)

(C)[M+2H]2+


S35

Figure S24. MALDI MS and MS/MS analysis of SL106-1 6. (A) Full spectrum of MALDI MS of SL106-1 6. (B) MALDI MS/MS of parent ion m/z 1463.5. (C) Table and structure showing fragments in MALDI MS/MS of SL106-1 6.

799. 0 1040. 8 1282. 6 1524. 4 1766. 2 2008. 0M ass ( m / z)

2. 5E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsi

ty

1485. 4

1487. 5

1463. 5

1253. 2

1465. 5

1246. 4

1248. 41224. 7

1501. 41249. 41469. 41219. 7 1503. 51446. 41074. 4 1315. 4 1525. 41479. 5 1686. 61436. 81268. 4 1310. 3928. 3835. 3 1364. 4 1517. 41397. 41124. 3 1674. 51009. 3 1326. 4876. 1 1571. 5 1718. 61631. 5

1

(A)

3

2

6 9 3 6 4 6 5 9 9 5 4 1 2 4 9 1 5 4 4M a s s ( m / z )

2 . 4 E +

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

% I

nte

ns

ity

L i T 7 V f ra c t i o n 3 M S/M S (CHCA)

1 2 4 6 . 3

1 2 4 8 . 3

1 2 1 8 . 3

7 6 4 . 1

1 2 2 1 . 31 1 6 0 . 2

1 2 2 8 . 21 2 2 9 . 9

1 1 5 8 . 2 1 2 4 2 . 21 1 0 8 . 27 6 2 . 1 1 2 0 0 . 31 0 6 3 . 3 1 4 3 8 . 45 4 7 . 1 1 3 5 0 . 28 5 4 . 1 1 2 9 8 . 21 0 2 1 . 2 1 3 9 6 . 77 6 9 . 7 9 4 0 . 07 0 6 . 1 9 7 4 . 74 6 4 . 2 9 0 6 . 88 1 1 . 34 9 7 . 14 2 8 . 11 6 0 . 3 2 9 0 . 69 9 . 7 3 9 0 . 0 6 6 0 . 17 4 . 3 6 0 2 . 53 5 5 . 82 3 7 . 51 9 4 . 41 2 5 . 3

3(B)


S36


1. M+H+ (parent ion) 1463.4 1463.5

2. M+Na+ 1485.4 1485.4

3. M-NH2-QA+Na+ 1253.3 1253.2

4. M-QA+H+ 1246.3 1246.3

4

(C) 3


S37

Table S1. 1H and 13C NMR assignments of thiostrepton Thr7Ala 3

Position δC [ppm]; mult

δH [ppm]; (mult, J in Hz) HMBCa COSYb

Ile1 Ile1-1 173.1; C q Ile1-2 65.2; CH 2.92 (d, 4.0) Ile1-1, Ile1-3, Ile1-4, Ile1-6, Q8 Ile1-3 Ile1-3 38.2; CH 1.88-1.82 (m) Ile1-4-HB, Ile1-6 Ile1-4 23.9; CH2 HA: 1.32-1.26 (m)

HB: 1.04-0.98 (m) Ile1-5, Ile1-6 Ile-5, Ile1-6

Ile1-4-HB, Ile1-5 Ile1-3, Ile1-4-HA, Ile1-5

Ile1-5 11.4; CH3 0.82 (t, 7.3) Ile1-3, Ile1-4 Ile1-4-HA, Ile1-4-HB Ile1-6 15.7; CH3 0.94 (d, 6.9) Ile1-2, Ile1-3, Ile1-4 Ile1-3 Ala2 Ala2-1 169.0; C q Ala2-2 49.4; CH 3.75 (q, 6.8) Ile1-1, Ala2-1, Ala2-3 Ala2-3 Ala2-3 18.9; CH3 1.13 (d, 6.7) Ala2-1, Ala2-2 Ala2-2 Ala2-NHd 7.79 (d, 5.9) Ala2-2 Dha3 Dha3-1 162.8; C q Dha3-2 132.1; C q Dha3-3 103.4; CH2 HA: 5.74 (d, 2.0)

HB: 5.25 (d, 2.0) Dha3-1, Dha3-2 Dha3-1, Dha3-2

Dha3-3-HB

Dha3-3-HA Ala4 Ala4-1 173.7; C q Ala4-2 51.6; CH 4.68 (q, 6.6) Dha3-1Ala4-1, Ala4-3 Ala4-3 Ala4-3 19.5; CH3 1.37 (d, 6.7) Ala4-1, Ala4-2 Ala4-2 Ala4-NHd 6.93 (d, 7.6) Ala4-2 Pip Pip-2 162.2; C q Pip-3 23.9; CH2 HA: 3.63-3.55 (m)

HB: 2.89-2.85 (m) Pip-4-HB

Pip-4 27.8; CH2 HA: 3.95 (m) HB: 2.25 (m)

Pip-2, Pip-3, Pip-5, Pip-6, Thz6-4, Thz13-2 Pip-3, Pip-5, Pip-6

Pip-4-HB

Pip-4-HA, Pip-3-HB Pip-5 56.9; C q Pip-6 64.4; CH 5.05 (s) Ala4-1, Pip-2, Pip-3, Pip-4, Pip-5, Thz13-2,

Thz13-3, Thz15-4

Thz6 Thz6-1 161.4; C q Thz6-2 146.8; C q Thz6-3 124.2; CH 7.99 (s) Thz6-1, Thz6-2, Thz6-4 Thz6-4 169.2; C q Ala7 Ala7-1 169.6; C q Ala7-2 47.0; CH 3.89 (q, 7.0) Thz6-1, Ala7-1, Ala7-3 Ala7-3, Ala7-NH Ala7-3 19.1; CH3 0.70 (d, 7.2) Ala7-1, Ala7-2 Ala7-2 Ala7-NHd 6.61 (d, 9.5) Ala7-2 Dhb8 Dhb8-2 128.2; C q Dhb8-3 135.2; CH 6.20 (q, 7.0) Dhb8-2, Dhb8-4, Tzn9-4 Dhb8-4 Dhb8-4 14.9; CH3 1.58 (d, 7.0) Dhb8-2, Dhb8-3, Tzn9-4 Dhb8-3 Tzn9 Tzn9-1 171.4; C q Tzn9-2 78.1; CH 4.83 (dd, 13.1, 8.9) Dhb8-2, Tzn9-1, Tzn9-3, Tzn9-4 Tzn9-3-HA, Tzn9-3-HBTzn9-3 35.1; CH2 HA: 3.63-3.55 (m)

HB: 3.11 (dd, 13.1, 11.6)Tzn9-2, Tzn9-4 Tzn9-1, Tzn9-2

Tzn9-2, Tzn9-3-HB Tzn9-2, Tzn9-3-HA

Tzn9-4 169.8; C q Ile10 Ile10-2 52.4; CH 5.63 (s) Tzn9-1, Ile10-3, Ile10-4, Thz11-4 Ile10-NH Ile10-3 77.0c Ile10-4 67.6; CH 3.68 (q, 6.4) Ile10-2, Ile10-3, Ile10-5, Ile10-6 Ile10-5 Ile10-5 15.9; CH3 1.24 (d, 6.4) Ile10-3, Ile10-4, Ile10-6 Ile10-4 Ile10-6 18.1; CH3 1.08 (s) Ile10-2, Ile10-3, Ile10-4 Ile10-NHd 7.54 (d, 9.6)

Ile10-2


S38



Thz11 Thz11-1 162.1; C q Thz11-2 150.0; C q Thz11-3 125.8; CH 8.22 (s) Ile10-2, Thz11-1, Thz11-2, Thz11-4 Thz11-4 166.5; C q Thr12 Thr12-2 55.8; CH 5.72 (s) Thz11-1, Thr12-3, Thr12-4, Thz13-2,

Thz13-3, Thz13-4 Thr12-NH

Thr12-3 71.7; CH 6.38 (q, 6.6) Thr12-4, Thz13-4, Q-1 Thr12-4 Thr12-4 18.9; CH3 1.69 (d, 6.6) Thr12-2, Thr12-3 Thr12-3 Thr12-NHd 9.01 (d, 7.6) Thr12-2 Thz13 Thz13-2 156.8; C q Thz13-3 118.3; CH 7.35 (s) Pip-6, Thz13-2, Thz13-4 Thz13-4 170.8; C q Thz15 Thz15-1 159.5; C q Thz15-2 149.8; C q Thz15-3 127.7; CH 8.25 (s) Thz15-1, Thz15-2, Thz15-4 Thz15-4 168.2; C q Dha16 Dha16-1 162.0; C q Dha16-2 134.0; C q Dha16-3 103.2; CH2 HA: 6.67 (d, 2.3)


Dha16-3-HB

Dha16-3-HA Dha17 Dha17-1 166.0; C q Dha17-2 132.8; C q Dha17-3 104.3; CH2 HA: 6.50 (d, 1.9)


Dha17-3-HB Dha17-3-HA

Q Q-1 160.8; C q Q-2 143.7; C q Q-3 122.5; CH 7.22 (s) Q-1, Q-5, Q-11 Q-4 153.1; C q Q-5 127.0; C q Q-6 123.2; CH 6.86 (d, 10.1) Q-4, Q-5, Q-8, Q-10 Q-7 Q-7 129.9; CH 6.32 (dd, 9.7, 5.5) Q-5, Q-8, Q-9 Q-6, Q-8 Q-8 58.9; CH 3.63-3.55 (m) Q-6, Q-7, Q-9, Q-10 Q-7, Q-9 Q-9 67.4; CH 4.32 (s) Q-5, Q-7, Q-8, Q-10 Q-8 Q-10 155.1; C q Q-11 64.2; CH 5.17 (q, 6.5) Q-3, Q-4, Q-5, Q-12 Q-12 Q-12 22.4; CH3 1.29 (d, 6.6) Q-4, Q-11 Q-11

a HMBC correlations are from the proton to the indicated carbon. b COSY correlations are from the proton to the proton attached to the indicated position. c The δ of this resonance was determined by HMBC due to overlap with the CDCl3 peak. d Only those amide resonances demonstrating COSY correlations to neighboring protons were assigned.


S39

Table S2. 1H and 13C NMR assignments of SL105-1 4



Pyr3 Pyr3-1 159.6; C q Pyr3-2 196.3; C q Pyr3-3 24.0; CH3 2.14 (s) Pyr3-1, Pyr3-2

Ala4 Ala4-1 172.2; C q Ala4-2 49.8; CH 4.48 (q, 7.2) Ala4-1, Ala4-3 Ala4-3 Ala4-3 17.0; CH3 1.49 (d, 7.2) Ala4-1, Ala4-2 Ala4-2 Pip Pip-2 162.7; C q Pip-3 24.8c HA: 3.29 (m)

HB: 2.90 (m) Pip-4-HB Pip-4-HA, Pip-4-HB

Pip-4 27.8; CH2 HA: 3.82-3.77 (m) HB: 2.10-2.03 (m)

Pip-2, Pip-3, Pip-5, Pip-6 Pip-3

Pip-3-HB, Pip-4-HB Pip-3-HA, Pip-3-HB, Pip-4-HA

Pip-5 57.3; C q Pip-6 64.5; CH 5.13 (s) Pip-2, Pip-5, Thz13-2, Thz13-3, Thz15-4 Thz6 Thz6-1 159.6; C q Thz6-2 145.7; C q Thz6-3 125.0; CH 7.60 (s) Thz6-1, Thz6-2, Thz6-4 Thz6-4 170.4; C q Ala7 Ala7-1 169.5; C q Ala7-2 47.2; CH 3.82-3.77 (m) Ala7-1, Ala7-3 Ala7-3 19.8; CH3 1.06 (d, 7.0) Ala7-1, Ala7-2 Ala7-NHd 6.84 (d, 7.5) Dhb8 Dhb8-2 128.3; C q Dhb8-3 135.3; CH 6.18 (q, 6.9) Dhb8-2, Tzn9-4 Dhb8-4 Dhb8-4 14.7; CH3 1.52 (d, 6.9) Dhb8-2, Dhb8-3, Tzn9-4 Dhb8-3 Tzn9 Tzn9-1 171.4; C q Tzn9-2 78.1; CH 4.83 (dd, 12.0, 9.7) Tzn9-1 Tzn9-3-HA, Tzn9-3-HB Tzn9-3 35.1; CH2 HA: 3.57 (dd, 10.7, 9.2)

HB: 3.14 (t, 12.1) Tzn9-2, Tzn9-3-HB

Tzn9-2, Tzn9-3-HA Tzn9-4 169.6; C q Ile10 Ile10-2 52.7; CH 5.60 (s) Tzn9-1, Ile10-3, Thz11-4 Ile10-NH Ile10-3 75.7d; C q Ile10-4 68.2; CH 3.82-3.77 (m) Ile10-2, Ile10-3, Ile10-6 Ile10-5 Ile10-5 16.1; CH3 1.29 (d, 6.3) Ile10-3, Ile10-4 Ile10-4 Ile10-6 18.1; CH3 1.08 (s) Ile10-2, Ile10-3, Ile10-4 Ile10-NHd

Thz11

7.84 (d, 10.3) Ile10-2

Thz11-1 162.5; C q Thz11-2 149.9; C q Thz11-3 125.3; CH 8.15 (s) Thz11-1, Thz11-2, Thz11-4 Thz11-4 167.3; C q Thr12 Thr12-2 55.0; CH 5.73 (brs) Thz11-1, Thz13-4 Thr12-NH, Thr12-3 Thr12-3 72.1; CH 6.41-6.35 (m) Thr12-2, Thr12-4 Thr12-4 16.8; CH3 1.57 (d, 6.6) Thr12-2, Thr12-3 Thr12-3 Thr12-NHd 9.37 (d, 8.9) Thz13 Thz13-2 157.0; C q Thz13-3 119.3; CH 7.46 (s) Pip-5, Thz13-2, Thz13-4 Thz13-4 171.6; C q Thz15 Thz15-1 159.6; C q Thz15-2 149.8; C q Thz15-3 127.1; CH 8.15 (s) Thz15-1, Thz15-2, Thz15-4 Thz15-4 168.3; C q


S40



Dha16 Dha16-1 161.9; C q Dha16-2 134.1; C q Dha16-3 103.2; CH2 HA: 6.55 (d, 1.5)

HB: 5.48 (brs) Dha16-1, Dha16-2 Dha16-1

Dha16-3-HB Dha16-3-HA

Dha17 Dha17-1 166.0; C q Dha17-2 132.8; C q Dha17-3 104.4; CH2 HA: 6.40 (s)

HB: 5.55 (s) Dha17-1, Dha17-2 Dha17-1

Dha17-3-HB

Dha17-3-HA Q Q-1 163.0; C q Q-2 f Q-3 116.6; CH 7.57 (s) Q-5, Q-11 Q-4 154.7; C q Q-5 127.0; C q Q-6 123.3; CH 8.05 (d, 8.1) Q-4, Q-8, Q-10 Q-7 Q-7 128.8; CH 7.68-7.61 (m) Q-5, Q-9 Q-6 Q-8 130.5; CH 7.68-7.61 (m) Q-6, Q-10 Q-9 Q-9 129.0; CH 8.24 (d, 8.1) Q-7 Q-8 Q-10 146.1; C q Q-11 64.3; CH 5.51 (q, 6.5) Q-3, Q-12 Q-12 Q-12 23.4; CH3 1.41 (d, 6.5) Q-4, Q-11 Q-11

a HMBC correlations are from the proton to the indicated carbon. b COSY correlations are from the proton to the proton attached to the indicated position. c Multiplicity of this resonance was unassigned due to weak signal. d The δ of this resonance was determined by HMBC due to overlap with the methanol-d6 peak. e Only those amide resonances demonstrating COSY correlations to neighboring protons were assigned. f Unable to assign the resonance for this carbon


S41

Table S3. Antibacterial activity of thiostrepton A 1 and its analogs

Compound MICa (µg/mL)

MRSAb VREc Bacillusd

Thiostrepton A 1 0.012 0.012 0.025 Thiostrepton Thr7Ala 3 >3.4 >3.4 >3.4 SL105-1 4 >3.4 >3.4 >3.4 Thiostrepton Thr7Val 5 >3.4 >3.4 >3.4 SL106-1 6 >3.4 >3.4 >3.4 Vancomycin 0.39 NDe ND Chloramphenicol ND 3.9 0.98

a Minimum inhibitory concentration. b Staphylococcus aureus ATCC 10537. c Enterococcus faecium ATCC 12952. d Bacillus sp. ATCC 27859. e Not determined.


S42

Table S4. Strains and plasmids used in this study

Strain/Plasmid Description Reference or source Streptomyces strains

S. laurentii ATCC 31255 Wild-type, thiostrepton producer ATCC S. laurentii NDS1 S. laurentii containing an in-frame deletion of tsrA 1 S. laurentii NDS1/int-3A104 S. laurentii NDS1 containing int-3A104 This study S. laurentii NDS1/int-3A105 S. laurentii NDS1 containing int-3A105 This study S. laurentii NDS1/int-3A106 S. laurentii NDS1 containing int-3A106 This study

E. coli strains

BW25113/pKD46 Host for PCR-targeted disruption of a gene from a fosmid or plasmid

3

ET12567/pUZ8002 Host for conjugation with Streptomyces species 6 Strains used for antimicrobial assays

Bacillus sp. ATCC 27859 Wild-type ATCC Escherichia coli ATCC 27856 Wild-type ATCC Staphylococcus aureus ATCC 10537 Methicillin-resistant ATCC Enterococcus faecium ATCC 12952 Vancomycin-resistant ATCC

Plasmids

pSC-B-amp/kan A routine vector from StrataClone Blunt PCR Cloning Kit, for cloning blunt-end PCR product

Agilent Technologies

pJP11 pCR4-Blunt vector harboring a 1.8 kb fragment PCR amplified from S. laurentii genomic DNA, which contains the 0.2 kb tsrA gene and its two flanking 0.8 kb regions

4

pCL64 Plasmid containing the tsrA variant encoding the Thr7Ser mutation

This study

pCL65 Plasmid containing the tsrA variant encoding the Thr7Ala mutation

This study

pCL66 Plasmid containing the tsrA variant encoding the Thr7Val mutation

This study

int-3A100 Derived from int-3A10, tsrA is replaced by chlR–sacB cassette

1

int-3A104 Derived from int-3A100. The chlR–sacB cassette is replaced by the tsrA variant encoding the Thr7Ser mutation

This study

int-3A105 Derived from int-3A100. The chlR–sacB cassette is replaced by the tsrA variant encoding the Thr7Ala mutation

This study

int-3A106 Derived from int-3A100. The chlR–sacB cassette is replaced by the tsrA variant encoding the Thr7Val mutation

This study


S43

Table S5. Primers and ultramer used in this study

Primer Sequence Description T7S-F 5’-GTCCGCCTCCTGCTCCACCTGCATCTGC-3’ Primers to generate TsrA Thr7Ser. T7S-R 5’-GCAGATGCAGGTGGAGCAGGAGGCGGAC-3’ T7A-F 5’-GTCCGCC TCCTGCGGCACCTGCATCTGC-3’ Primers to generate TsrA Thr7Ala. T7A-R 5’-GCAGATGCAGGTGCCGCAGGA GGCGGAC-3’ T7V

5’-ACTACATGGACGAGACGCTGCTCGACGGTGA GGACCTGACCGTCACGATGATCGCGTCCGCCTCCTGCGTCACCTGCATCTGCACCTGCAGCTGCAGCTCCTGAGGTAACACCCGGCGCGGAGGACTGTTCCTCCCCGCCCGCCGGACCTG-3’

Chemically synthesized ultramer to generate TsrA Thr7Val.

T7V-F T7V-R

5’-ACTACATGGACGAGACGCTGCTC-3’ 5’-CAGGTCCGGCGGGCGGGGA-3’

Primers for amplification of chemically synthesized ultramer TsrA T7V.

SD2-F 5’-GCGCGATCGACGCGACCGCAG-3’ Primers used in the amplification of tsrA variants from pCL64 and pCL65. SD2-R 5’-GGCGGGGAGGAACAGTCCTCC-3’

SD3-F 5’-ATCGTGTTGGGCTTGACG-3’ Primers used in DNA sequencing to confirm int-3A104, int-3A105, and int-3A106. SD3-R 5’-CGCGGTGCAATAGGACAT-3’

References 1. Li, C.; Zhang, F.; Kelly, W. L. Mol. Biosyst. 2011, 7, 82-90. 2. Gust, B.; Challis, G. L.; Fowler, K.; Kieser, T.; Chater, K. F. Proc. Natl. Acad. Sci. U.S.A.

2003, 100, 1541-1546. 3. Datsenko, K. A.; Wanner, B. L. Proc. Natl. Acad. Sci. U.S.A 2000, 97, 6640-6645. 4. Kelly, W. L.; Pan, L.; Li, C. J. Am. Chem. Soc. 2009, 131, 4327-4334. 5. Ryan, P. C.; Lu, M.; Draper, D. E. J. Mol. Biol. 1991, 221, 1257-1268. 6. Gust, B.; Kieser, T.; Chater, K. F. REDIRECT technology: PCR-targeting system in

Streptomyces coelicolor; John Innes Center, Norwich Research Park: Norwich, UK, 2002. 7. Schoof, S.; Arndt, H. D. Chem. Commun., 2009, 7113-7115.


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