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Supporting Information Appendix: Media Compositions 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. 1 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. 2 a b 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. 3 a b 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. 4 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). 5 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. 6 a b 7 c 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 .
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