1

Supporting Information

Appendix:

Media compositions

Seed media (2% glucose, 0.5% yeast extract, 0.5% meat extract, 0.5% peptone, 0.3% hydrolyzed casein, 0.15% NaCl). AF/MS media (2% glucose or 3% dextrin, 0.2% yeast extract, 0.8% organic soybean flour, 0.1% NaCL, 0.4% CaCO3) (1). Minimal MG Base (for 1L: 50 g maltose monohydrate, 0.2 g MgSO4 heptahydrate, 9 mg FeSO4 heptahydrate, 1 g CaCl2 monohydrate, 1 g NaCL, 21 g 3(N-morphilino)propanesulphonic acid, 11.23 g monosodium glutamate monohydrate, 15 mL 1M potassium phosphate buffer pH = 6.5, 4.5 mL of trace mineral solution consisting of 39 mg CuSO4, 5.7 mg H3BO3, 3.7 mg (NH4)6Mo7O24 tetrahydrate, 6.1 mg MnSO4 monohydrate, 880 mg ZnSO4 heptahydrate in 1 L water) (2). Minimal Media C (for 1L: 5 g L-glutamate, 1 g arginine, 1 g aspartate, 0.5 g K2HPO4 heptahydrate, 1 g MgSO4 heptahydrate, 2 g Na2SO4, 0.01 g ZnSO4 heptahydrate, 0.02 g FeSO4 heptahydrate, 3 g CaCO3, 40 g glucose) (3). J Media (10% sucrose, 3% tryptone soya, 1% yeast extract, 1% MgCl2 hexahydrate) (2). SFM (soya flour mannitol) agar plates (2 % organic soya flour, 2 % mannitol, 2% agar, tap water) (2).

General Methods, Materials, and Instrumentation:

All DNA manipulations (sub-cloning) were carried out in DH5α E. coli (Zymo Research). Streptomyces ATCC 55365 was obtained from American Type Culture Collection (Manassas, VA) and E. coli BW25113, E. coli BT340, plasmid pKD46, and plasmid pKD3 were obtained from the E. coli Genetic Stock Center (CGCS, Yale University). S. lividans TK24 and the plasmid pSET152 were generously provided by Suzanne Walker. Methods for the growth, media composition, and DNA isolation of Streptomyces were performed following protocols from Practical Streptomyces Genetics (The John

Innes Foundation, Norwood, UK) (2). Antibiotic concentrations were used as follows: 50 µg/ml apramycin, 100 µg/ml

ampicillin, 50 µg/ml kanamycin, and 25 µg/ml chloramphenicol, 25 µg/mL nalidixic acid. DNA oligos were purchased from Integrated DNA technologies (IDT) and are listed in Table S1. Restriction and PCR enzymes were purchased from New England Biolabs. High resolution LCMS analysis was performed on an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer using an electrospray (ESI) ionization source.

2

Supplementary Figures

GE37468 MGNNEEYFIDVNDLSIDVFDVVEQGGAVTALTADHGMPEVGASTNCFCYICCSCSS-N Thiomuracin MDLSDLPMDVFELADDGVAVESLTAGHGMTEVGASCNCFCYICCSCSS-A

GE2270 MSELESKLNLSDLPMDVFEMADSGMEVESLTAGHGMPEVGASCNCVCGFCCSCSPSA Figure S1 – Structures of the related thiazolyl peptides GE37468, thiomuracin A, and GE2270A. Preprotein sequences are shown below structures with amino acids that become part of the mature antibiotic highlighted in bold. Green residues indicate structural differences between GE37468 and thiomuracinA, blue residues indicate structural differences between GE37468 and GE2270A, and red residues show structural differences from GE37468 that are shared by thiomuracin and GE2270A.

3

Figure S2 – Fosmid library screening and cluster assembly via next generation Illumnia sequencing. The fosmid library was created as described in the methods and following in protocols in the “CopyControlTM Fosmid Library Production Kit” (Epicentre Biotechnology). (a) Representative gel of a 96-well pooled PCR. Each lane contains the colony PCR product from one pool of approximately 10-12 colonies. Briefly, 10-12 colonies were picked using wooden toothpicks

into 100 µL of elution buffer (5 mM TrisHCl, pH = 8.0). 2 µL of this solution was used as the template in a 25 µL PCR using Platinum taq PCR Supermix (Invitrogen) with primers CycloVNTI F and CycloTSY R (Table S1). Pools which yielded a PCR product at approximately 500 bp (labeled parent pool) were struck out again on appropriate antibiotic plates,

individual colonies picked into 10 µL of elution buffer, and the PCR screen repeated to find unique clones. (b) Unique clones were identified by mapping isolated fosmids with restriction enzymes BamHI and XhoI. Clones A1:A5 and A1:A8 were identified as indistinguishable via this method and were treated as one fosmid for sequencing purposes. The nine unique fosmids were combined and sequenced via next generation Illumina sequencing on an Illumina HiSeq2000 sequencer (Nextera Read 1 primer using a 36 cycle Solexa single end read and analyzed using Illumina OLB Firecrest 1.8.0) at the Biopolymers Facility, Harvard Medical School. The sequencing yielded 13,144,474 reads which were

cleaned and assembled into 526 nodes ≥ 60 bp using GenomeQuest web based software based on the Velvet algorithm with k value set to 31. This resulted in 526 nodes >60 bp which were further assembled into 74 contigs (18,873 bp – 183 bp) using Lasergene Seqman (DNAstar). Four contigs were greater than 10,000 bp and one 18,873 bp contig was found to contain the cyclodehydratase gene PCR product. Extension of this contig to 28,171 bp (Fig. S3) was accomplished by joining contigs using PCR.

a

b

4

Figure S3 – (a) Portion of the Streptomyces ATCC 55365 genome identified by fosmid library panning and next generation Illumina sequencing described in Fig. S2. The flanking open reading frames are shown for genomic context. The genomic sequence was found to have a GC content characteristic of actinomycetes genomic DNA (71.17% G+C). (b) Map of the pSETGE1 plasmid. pSETGE1 was created by ligating the NheI to BglII restriction fragment from fosmid G8:G12 (Fig. S2) to a modified pSET152 vector which was devoid of NheI restriction sites (described in methods). NheI and BglII restriction sites were added to the pSET152 outside the lacZalpha gene by PCR with primers pSET BglII F and pSET NheI R (Table S1). The NheI restriction site was naturally occurring following the getM gene and the BglII restriction site was naturally occurring 2.5 open reading frames upstream of the getA gene in the GE37468 cluster.

a

b

5

Figure S4 – LCMS analysis of pSETGE1 expression in E. coli DH5α. The top chromatogram trace (red) shows the methanolic extract from the wild type producer Streptomyces ATCC 55365 as a reference. The peak corresponding to

GE37468 as confirmed by MS analysis is indicated with a star. After 2 days of growth in LB media (30 °C, 250 rpm) in baffled flasks, methanolic extracts from E. coli harboring pSETGE1 (middle, maroon trace) or pSET152 (negative control, bottom, black trace) failed to show peaks in the UV350 trace indicating lack of GE37468 expression. Additionally, no masses corresponding to GE37468 were identified in the LCMS analysis of the methanolic extracts (data not shown).

6

Figure S5 – Mass spec analysis of GE37468 and mutants. Masses and corresponding errors are tabulated in Fig. S10f. Native GE37468 ionizes under described conditions as [M+OH]+ due to loss of –OH from the mhP residue as previously reported (4). Mutants lacking the mhP residue ionize as [M+H]+. (a) Mass of GE37468 from Streptomyces ATCC 55365 under peak 1 in Fig. 3a. (b) Mass of GE37468 from S. lividans + pSETGE1 under peak 2 in Fig. 3b. (c) Mass of GE37468mhP8I from S. lividans + pSETGE-getJ::FRT under peak 4 in Fig. 3c. (d) Mass of GE37468mhP8A from S. lividans + pSETGE-getAI8A under peak 5 in Fig. 3d. MS conditions: Parameters on an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer using an electrospray (ESI) ionization source were set to: capillary voltage, 3500 kV; fragmentor voltage,

250 V; drying gas temperature, 350 °C. A Gemini-NX C18 reverse phase (5 µm, 110 Å, 2 x 50 mm, Phenomenex) was used to separate analyte with a water/acetonitrile (0.1% formic acid) gradient.

7

a

b

8

Producer: Streptomyces ATCC 55365 Streptomyces lividans + pSETGE1

Shift1 (ppm) Type J (Hz) (ppm) Shift1 (ppm) Type J (Hz) (ppm) 10.09 s - [10.06 .. 10.11] 10.09 s - [10.01 .. 10.13]

9.59 s - [9.57 .. 9.61] 9.58 s - [9.52 .. 9.65] 9.08 br. s. - [9.05 .. 9.12] 9.08 s - [9.04 .. 9.12] 8.67 m - [8.64 .. 8.71] 8.67 m - [8.63 .. 8.70] 8.59 d 7.04 [8.57 .. 8.61] 8.59 d 7.63 [8.57 .. 8.61] 8.55 d 8.22 [8.52 .. 8.57] 8.55 d 8.22 [8.53 .. 8.56] 8.38 d 7.63 [8.36 .. 8.40] 8.39 d 8.22 [8.36 .. 8.40] 8.18 s - [8.18 .. 8.20] 8.19 s - [8.17 .. 8.20] 7.91 s - [7.90 .. 7.92] 7.90 s - [7.89 .. 7.91] 7.76 s - [7.75 .. 7.77] 7.76 s - [7.75 .. 7.78] 7.29 d 5 [7.27 .. 7.31] 7.28 m 4.7 [7.25 .. 7.31] 7.20 m - [7.18 .. 7.23] 7.19 s - [7.17 .. 7.21] 7.07 d 8.22 [7.04 .. 7.08] 7.11 s - [7.09 .. 7.12] 6.81 br. s. - [6.78 .. 6.83] 6.81 br. s. - [6.78 .. 6.82] 6.59 m - [6.53 .. 6.66] 6.67 br. s. - [6.62 .. 6.71] 6.58 d 8.22 [6.56 .. 6.60] 6.58 d 8.22 [6.56 .. 6.60] 6.45 s - [6.44 .. 6.47] 6.45 br. s. - [6.43 .. 6.47] 6.04 s - [6.03 .. 6.06] 6.04 s - [6.02 .. 6.06] 5.87 d 7 [5.86 .. 5.89] 5.87 d 6.46 [5.84 .. 5.89] 5.78 br. s. - [5.76 .. 5.80] 5.78 br. s - [5.75 .. 5.81] 5.32 t 5.28 [5.30 .. 5.34] 5.32 m - [5.30 .. 5.34] 5.27 dd 13.21, 4.99 [5.25 .. 5.29] 5.26 m - [5.24 .. 5.29] 5.06 td 9.39, 2.93 [5.02 .. 5.09] 5.06 m 7.63 [5.03 .. 5.08] 4.93 td 9.39, 2.35 [4.90 .. 4.96] 4.93 m 11.74 [4.89 .. 4.96] 4.63 d 9.39 [4.60 .. 4.65] 4.63 d 8.8 [4.60 .. 4.65] 3.57 s - [3.55 .. 3.59] 3.50 br. s. - [3.49 .. 3.52] 3.18 s - [3.14 .. 3.21] 3.17 s - [3.15 .. 3.19] 2.94 dd 13.21, 9.10 [2.90 .. 2.99] 2.97 m - [2.91 .. 3.03] 2.72 s - [2.69 .. 2.74] 2.72 s - [2.70 .. 2.73] 2.28 td 7.48, 3.81 [2.24 .. 2.33] 2.28 t 7.34 [2.24 .. 2.30] 2.18 m 7.63 [2.15 .. 2.21] 2.18 t 7.34 [2.15 .. 2.20] 2.11 dd 12.33, 5.87 [2.09 .. 2.14] 2.08 s - [2.07 .. 2.09] 1.99 m - [1.95 .. 2.05] 2.00 m - [1.96 .. 2.04] 1.50 br. s. - [1.43 .. 1.56] 1.51 br. s. - [1.41 .. 1.57] 1.23 s - [1.19 .. 1.31] 1.23 s - [1.19 .. 1.29] 1.14 d 6.46 [1.12 .. 1.16] 1.14 d 6.46 [1.12 .. 1.16] 0.85 m - [0.78 .. 0.90] 0.85 t 7.04 [0.80 .. 0.87]

Figure S6 – Characterization of GE37468 from Streptomcyes ATCC 55365 (a) or Streptomyces lividans + pSETGE1 (b) by NMR. (c) Proton shifts tabulated. Small variations in peak shifts and copuling constants between samples are likely attributted to noise in the Streptomyces lividans + pSETGE1 experiment due to low sample amount; however visual inspection indicates the compounds have no significant differences in the proton spectra. Compounds were isolated as described in the methods and purified by HPLC on a Waters 1525EF HPLC equipped with a SunFire C18 Prep column (19 x 250 mm) using a gradient of water (0.1 % formic acid) and acentonitrile (0.1 % formic acid).

c

9

Figure S7 – Analysis of GE37468 expression over time. GE37468 was expressed (200 mL cultures in 500 mL baffled flasks) in AF/MS media (blue data points), Media C (red data points), or MG Base media (green data point) as described in the methods. 10 mL aliquots were removed every 24 hrs, pelleted by centrifugation, and extracted with methanol.

Solvent was removed in vacuo, and the residue was resuspended in 500 µL of 50:50 water:acetonitrile. Ten micoliters were used for LCMS analysis. Peaks in the UV350 chromatogram trace that corresponded to GE37468 (confirmed by mass and retention time) were integrated and relative units shown on the y-axis. The zero hour time point was created by extraction of 10 mL of a common starter culture.

0.00E+00

1.00E+05

2.00E+05

3.00E+05

4.00E+05

5.00E+05

6.00E+05

0hr 24hr 48hr 72hr 96hr 120hr

Inte

grat

ion

of U

V35

0pe

ak

Expression Time (hr)

AF/MS media

Media C

MG Base

10

Figure S8 – Quantification of GE37468 yields. GE37468 was expressed in either Streptomyces ATCC 55365 (top trace, red) or S. lividans + pSETGE1 (bottom trace, orange) in AF/MS media for 72 hours as described. 10 mL was removed from the culture and pelleted by centrifugation. The Streptomyces ATCC 55365 pellet weighed 2.39 g and the S. lividans + pSETGE1 pellet weigh 1.45 g. Pellets were extracted with methanol, solvent removed in vacuo, and residue dissolved

in 500 µL of 50:50 water:acetonitrile. Ten microliters of each sample were analyzed by LCMS and UV350 peaks corresponding to GE37468 (shaded above) were integrated and quantified in relative units. Yields were reported after adjustment for wet cell pellet weight and compared with previously report values (1).

11

12

13

f (a) GE37468 [M-OH]+ Streptomyces ATCC 55365

Expected Observed ppm error

GE37468 1291.2460 1291.2490 2.32 Dihydro 1293.2616 1293.2631 1.16 less one dehydroAla 1221.2405 1221.2434 2.37 less two dehydroAla 1152.2190 1152.2208 1.56 (b) GE37468 [M-OH]+ S. lividans

GE37468 1291.2460 1291.2483 1.78 Dihydro 1293.2616 1293.2575 3.17 less one dehydroAla 1221.2405 1221.2391 1.15 less two dehydroAla 1152.2190 1152.2205 1.30 (c) GE37468 mhP8I [M+H]+ GE37468 mhP8I 1295.2773 1295.2749 1.85 Dihydro 1297.2929 1297.2936 0.54 less one dehydroAla 1225.2718 1225.2657 4.98 less two dehydroAla 1156.2503 1156.2477 2.25 (d) GE37468 mhP8A [M+H]+ GE37468 mhP8A 1253.2303 1253.2292 0.88 Dihydro 1255.2460 1255.2441 1.51 less one dehydroAla 1183.2248 1183.2286 3.21 less two dehydroAla 1114.2033 1114.2049 1.44

Figure S9 – Ion extraction of modified GE37468 products from (a) Streptomyces ATCC 55365, (b) S. lividans + pSETGE1, (c) S. lividans + pSETGE-getJ::FRT, (d) S. lividans + pSET-getAI8A. Peaks are color coded by the type of modification. Expected products are colored black, dihydro products in blue, single dehydroalanine truncation in magenta, and double dehydroalanine truncation in green. Masses extracted to produce each chromatogram are listed above each peak. (e) Structures of modified GE37468 products. The dihydro product is uncharacterized and structure is not shown. (f) Tabulated masses and errors (ppm) from a-d.

14

Figure S10 – Analysis of cleared media from S. lividans + pSETGE1 expressions in rich AF/MS media (top, green), minimal Media C (second, blue), or minimal MG base (third, orange). After 120 hr of expression as described in the methods,

media was cleared by centrifugation and filtering through Millipore (Billerica, MA) 0.22 µm PFTE filters. 100 mL of cleared media was passed over a Waters (Milford, MA) C18 Sep Pak cartridge, washed with 10 mL of water, and eluted with 2 mL of MeOH. Eluant was dried, and resuspended in 50:50 water:acetonitrile for LCMS analysis. The bottom chromatogram (red) is a methanolic extract of Streptomyces ATCC 55365 mycelia and shown as a control. The location of GE37468 in the bottom chromatogram is indicated with a black star.

15

e

d

c

b

a

16

f

17

S. lividans GE37468

S. lividans 13C6-GE37468

Expected Observed ppm

Expected Observed ppm

M 1308.2493 n/a n/a

1314.2694 n/a n/a

M-OH+ 1291.2460 1291.2470 0.77

1297.2661 1297.2671 0.77

M-OH-CO+ 1263.2511 1263.2521 0.79

1269.2712 1269.2695 1.34

M-OH-CO-NH3+ 1248.2402 1248.2380 1.76

1254.2603 1254.2670 5.34

b6+ 1128.1826 1128.1716 9.75

1134.2027 1134.1834 17.02

b6-NH3+ 1111.1561 n/a n/a

1117.1762 n/a n/a

b4+ 896.1156 896.1099 6.36

902.1357 902.1298 6.54

b2+ 699.0897 699.0711 26.61

705.1098 705.1052 6.52

b2-z13+ 629.0842 629.0842 0.00

635.1043 635.1013 4.72

b2-z12+ 560.0628 560.0675 8.39

566.0829 566.0795 6.01

c2-a8+H+ 496.0315 496.0316 0.20

497.0349 497.0436 17.50

b2-z12-a8+ 479.0048 479.0178 27.14

480.0082 480.0140 12.08

b2-z12-a8-H2O+ 460.9944 461.0100 33.84

461.9978 461.9919 12.77

S. lividans GE37468mhP8I

S. lividans GE37468mhP8A

Expected Observed ppm

Expected Observed ppm

M 1294.2700 n/a n/a

1252.2231 n/a n/a

M+H+ 1295.2773 1295.2749 1.84

1253.2303 1253.2292 0.88

M-CO+ 1278.2507 1278.2479 2.19

1236.2038 1236.2039 0.10

M-CO-NH3+ 1250.2558 1250.2618 4.79

1208.2089 1208.2102 1.11

b6+ 1267.2824 1267.2716 8.52

1225.2354 1225.2350 0.33

b6-NH3+ 1132.2140 1132.2096 3.84

1090.1670 1090.1694 2.20

b4+ 1115.1874 1115.1683 17.13

1073.1405 1073.1368 3.45

b2+ 900.1469 900.1577 11.98

858.1000 858.0952 5.59

b2-z13+ 703.1210 703.1313 14.62

661.0741 661.0723 2.72

b2-z12+ 633.1155 n/a n/a

591.0686 591.0680 1.02

c2-a8+H+ 564.0941 564.0851 15.92

522.0471 522.0393 14.94

b2-z12-a8+ 496.0315 496.0381 13.31

496.0315 496.0381 13.31

b2-z12-a8-H2O+ 479.0048 479.0066 3.76

479.0048 478.9984 13.36

M+ 460.9944 460.9913 6.72

460.9944 461.0028 18.22

S. lividans dihydroGE37468mhP8I

Expected Observed ppm

M 1296.2857 n/a n/a

M+H+ 1297.2929 1297.2936 0.54

M-CO+ 1280.2663 1280.2687 1.86

M-CO-NH3

+ 1252.2714 1252.2670 3.54

b6+ 1269.2980 1269.2998 1.40

b6-NH3

+ 1134.2296 1134.2281 1.30

b4+ 1117.2030 1117.1992 3.42

b2

+ 902.1625 902.1643 1.95

b2-z13+ 705.1366 705.1214 21.61

b2-z12

+ 635.1312 n/a n/a

c2-a8+H+ 566.1097 n/a n/a

b2-z12-a8+ 498.0471 n/a n/a

b2-z12-a8-H2O+ 481.0204 n/a n/a

M+ 463.0100 n/a n/a

g

18

Figure S11 – MS/MS analysis of GE37468 analogs. High resolution mass spectrometry analysis was performed on an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer using an electrospray (ESI) ionization source in positive mode. Parameters were set as described in Fig. S5 with the exception that collision induced dissociation (CID) was used for tandem MS/MS analysis. Isolated ions and scan times are listed in chromatogram titles. Identified masses are labeled in red. CID was set to 0, 45, or 70 in 3 independent ion isolations and data combined to create ion spectra. (a) S. lividans GE37468 (b) S. lividans 13C6-GE37468 (c) S. lividans GE37468mhP8I (d) S. lividans GE37468mhP8A (e) S. lividans dihydroGE37468mhP8I (f) Biemann annotation of fragmentation. (g) Tabulated results from a-e with ppm errors.

19

Figure S12 – Preprotein gene replacement. The pSETGE1 vector (Fig. S3) was linearized using the restriction sites (AclI and Scal) as described in the methods. Overlap PCR (primers GetA F new, IA QC 1R for PCR 1; primers IA QC 2F, and AclI R for PCR 2; combined via overlap PCR with primers GetAF new and AclI R) was used to create a 2178 bp fragment harboring an I8A mutation in the preprotein gene (mhP8A mutation in the mature antibiotic). The ends of this fragment contained > 50 bp homology with the AclI and ScaI ends of the cut pSETGE1 vector. Using the enzyme mix reported by Gibson, D.G. and coworkers, the fragment was ligated into the cut vector to create pSETGE-getAmhP8A (5).

20

Name Sequence AclI R new CCGAAAAGGGCCGGTATCCGTCAG Cyclo H-1 F GGTGGTCGAGCGGGAYGCNTTYYT CycloTSY R ATGCCGATGGTCGGGCCGTCGAACAGG CycloVNTI F GGGATGCGTTCCTGTGCACGTGGTA Cylco L-6 R CCGGTTCCGGTGGCCRAANGTCAT GetA F new GCTCTTCGAATCCGGAGTGCGCATCCGGATTCACTTGGGGGTCTGTCTTGCAGCAAAGG GetA hygroR CGAAGCGGGGAATTCCTCCCATCAACTGACGTGGCGTCATGTAGGCTGGAGCTGCTTC GetA prim 2F GGGTCTGTCTTGCAGCAAAGGACGGAAGGAAGTCTCATGATGGGAATTAGCCATGGTCC GetJ alt F CGCGGAGGACGTGCGCACCGTGCTGTCGGACAGCGGGACGTGTAGGCTGGAGCTGCTTC GetJ alt R GGCCTGCCCTGCGAGCACGAGGCGCCGGATGGAGAGGGTATGGGAATTAGCCATGGTCC I-A QC 1R GCTGCAGGAGCAGCAGGCGTAGCAGAAGCAGTTGGTCGACGC I-A QC 2F ACCAACTGCTTCTGCTACGCCTGCTGCTCCTGCAGCTCCAAC pSET BglII F TCGTAAGAGATCTTCGTAATCATGTCATAGCTGTTTCCTGTGT pSET NheI QC F GTCGACTCTAGCAAGCTGGCCAGGGCCCGATACGTCGCGGTGAGT pSET NheI QC R CCCTGGCCAGCTTGCTAGAGTCGACCTGCAGGTCCCCGGGGATCG pSET NheI R ACCTCAAGCTAGCCGATTCTAGACCGATGCAAAGTGCCGATCAACATAAC Table S1 – List of primers.

21

Gene Function Homolog Accession ID Sim Other Thiopeptide Clusters

Superfamily

GetA Structural gene

GetB ABC transporter associated

ABC transporter-like protein [Gordonia bronchialis DSM 43247]

YP_003272700.1 61 74 P-loop NTPase (DrrA)

GetC ABC type II transporter

ABC transporter permease [Corynebacterium kroppenstedtii DSM 44385]

YP_002905681.1 57 74 ABC 2 Membrane

GetD Dehydratase Dehydratase [Nonomuraea sp. WU8817]

ACS83767.1 35 46 TpdB (thiomuracin/GE2270)

Lantibiotic dehydratase, C-term

GetE Dehydratase

Dehydratase [Thermobispora bispora DSM 43833]

YP_003651171.1 35 49 TpdC (thiomuracin/GE2270)

GetF Putative aza-Diels -Alderase

Putative aza-Diels-Alderase [Thermobispora bispora DSM 43833]

YP_003651172.1 36 44 TpdD (thiomuracin/GE2270)

GetG Dehydrogenase

Dehydrogenase [Thermobispora bispora DSM 43833]

YP_003651173.1 50 62 TpdE (thiomuracin/GE2270)

Nitro FMN oxidoreductase (McbC)

GetH Unknown

Hypothetical protein Tbis_0554 [Thermobispora bispora DSM 43833]

YP_003651174.1 35 45 TpdF (thiomuracin/GE2270)

GetI Cyclodehydratase

Cyclodehydratase Tbis_0555 [Thermobispora bispora DSM 43833]

YP_003651175.1 48 60 TpdG (thiomuracin/GE2270)

YcaO

GetJ P450 Monooxygenase

Cytochrome P450 monooxygenase [Thermobispora bispora DSM 43833]

YP_003651166.1 43 57 TpdQ (GE2270)/ TpdJ1 (thiomuracin)

P450 monooxygenase

GetK Dehydrogenase NADH oxidase [Actinomadura melliaura]

ABC02783.1 28 36 CltC (cyclothiazomycin) McbC

22

GetL Transcriptional Regulator

Transcriptional regulator [Micromonospora sp. ATCC 39149]

ZP_04604482.1 35 51 CtlW (Sch 40832) TetR Transcriptional Regulator (AcrR)

GetM Aminohydrolase/ peptidease

Amidohydrolase [Mycobacterium abscessus ATCC 19977]

YP_001705460.1 40 56 M20 dimer (AbgB)

Table S2 – GE37468 cluster proposed genes and nearest homologs. Protein identity (ID) and similarity (Sim) are listed according to a BLAST search.

23

Bacillus subtilisa MRSAb

GE37468c 0.047 0.047

GE37468d 0.047 0.047

GE37468mhP8I 0.37 0.19

GE37468mhP8A 0.093 0.093

Table S3 – Minimal inhibitory concentrations (MIC, µg/mL) for GE37468 and analogs against Bacillus subtilis and MRSA. Assays were carried out as described in Acker et al. and Bowers et al. (6-7) with the exception that serial dilutions were

1:4 or 1:1 in DMSO from 150-0.05 µg/mL (1.5-0.0005 µg/mL final assay concentration). MICs were determined based on the lowest concentration of antibiotic that produced an increase of less than 10% in OD600 over a control well containing erythromycin. aBacillus subtilis MW2 grown in LB media. bMethicillin-resistant Streptomyces aureus MW2 grown in TSB media. cExpressed in Streptomyces ATCC 55365. dExpressed in S. lividans.

24

References for Supporting Information 1. Marinelli F, Gastaldo L, Toppo G, & Quarta C (1996) Antibiotic GE37468A: a new inhibitor of bacterial protein

synthesis. III. Strain and fermentation study. J Antibiot (Tokyo) 49(9):880-885. 2. Kieser T, Bibb MJ, Buttner MJ, Chater KF, & Hopwood DA (2000) Practical Streptomyces Genetics (Crowes,

Norwich, UK) p 613. 3. Houck DR, Chen LC, Keller PJ, Beale JM, & Floss HG (1988) Biosynthesis of the modified peptide antibiotic

nosiheptide in Streptomyces actuosus. J. Am. Chem. Soc. 110(17):5800-5806. 4. Ferrari P, Colombo L, Stella S, Selva E, & Zerilli LF (1995) Antibiotic GE37468 A: a novel inhibitor of bacterial

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