10802) hypothetical protein
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- Paul Whitehead
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1 upplementary Table 1. Annotation of the divamide A biosynthetic cluster. CDs of the div A biosynthetic gene cluster (GenBank: KY11568) from the E11-36 metagenome listed alongside their most similar BLAT search results. Conserved protein domains and biochemical characterizations are included. Individual accession numbers are listed for gene products (ND=data not deposited). Gene Protein accession number Length (bp) Closest BLAT hit ID -2 ND 369 WP_ ND 321 XP_ diva ARD WP_ divm ARD WP_ divx ARD WP_ divy ARD WP_ divmt ARD WP_ divt ARD WP_ divu ARD WP_ divn ARD WP_ ND 15 WP_ ND 195 WP_ Annotated function (Organism) hypothetical protein (Kamptonema) uncharacterized protein LOC (Rhinopithecus roxellana) hypothetical protein (Oscillatoria sp. PCC 182) hypothetical protein (Oscillatoria sp. PCC 182) hypothetical protein (Oscillatoria sp. PCC 182) hypothetical protein (Tolypothrix bouteillei) AM-dependent methyltransferase (tanieria cyanosphaera) ABC transporter permease and ATPase (Moorea producens) hypothetical protein (cytonema millei) hypothetical protein (Oscillatoria sp. PCC 182) phosphopeptidebinding protein (Pleurocapsa minor) serine/threonine protein phosphatase (Microcoleus sp. PCC 7113) Conserved protein domains (accession) none none none LanM-like (cd4792) Nif11 (pfam7862) Het-C (pfam7217) Methyltransf_18 (pfam12847) ABC_membrane_2 (pfam6472), ABC_tran (pfam5) FGE-sulfatase (pfam3781) none CHAT (pfam1277), FHA (pfam498) PP2Cc (cd143) Functional characterization not characterized not characterized precursor gene class II lanthionine synthetase putative Asp ß- hydroxylase (16) not characterized methyltransferase not characterized not characterized putative role in Lal formation (14-16) not characterized not characterized Nature Chemical Biology: doi:1.138/nchembio.2537
2 upplementary Table 2. Relationship of divamide B cluster genes to divamide A cluster. CDs of the partial div B biosynthetic gene cluster (GenBank: KY11569) from the E11-37 metagenome compared with their respective div A homologues. Individual accession numbers are listed for gene products (ND=data not deposited). Gene ID Protein accession Coverage with ID to corresponding Length (bp) number corresponding div A gene div A gene -1 ND 286 (partial) 88 1 diva ARD divm ARD (partial) 3 1 divx ARD (partial) 66 1 divy ARD divmt ARD (partial) Nature Chemical Biology: doi:1.138/nchembio.2537
3 upplementary Table 3. ynthetic DNA used in this study. DNA gblocks purchased from Integrated DNA Technologies used to construct pdiv and pdiv-3. DNA equence (5-3 ) 1a 1b 2 3 divadivamide B ctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacaatgccgaccaccctggaaaaaccgagcgtggcctatctggagaaact GTTTCACCAGACCGCCATCGACAGCGAATTTCGTAGCGAACTGCAGAGCCATCCGGAAGCCTTTGGTATTAGCGCCGATCTGGAACTGCCGCAGAGCG TGGAGAAACAGGACGAAAGCTTCGTGGAACTGCTGAACAACGCCCTGGGCGAAATCGATATTGCCGCCGAATGCGCCAGCACCTGTAGCTTTGGCATC GTGACCATCGTGTGCGATGGTACCACCAAATAAGTTGTTTTACTAGCATATTATATTATTAATATGTCTGGGCTAGCACCACATTGTGAAGCTAGCCT CATTAAATTGATTGTTGGGCATGGTTAGAAAGGAACTAAGTACAAGACAAAACTTGGATGAGAAGCGTAGTAAAATGTCCGAAACTATTGGCTCATAA AGTTTCGAGCAATATTTTCCATCTCTCTTGCTCCATATGAGTTTCAGTTGTTTCAGCTG AATCCGGAGACCAAGGAACTGGTGACCCTGGCCGAGAAACCGAAAATCGTGCGCTTCTTCAAAATCGAAAGCGAGCCGAGCAAAATTCTGTGGCACTG CATTGTTAACACCACCAAGCTGAATCGCTGGAGCTTCGGTTTCAACGTGAGCAGCAAGTGGATGGACAAACTGCTGACAGTGTAAGGAGAATAAAATG AAAATTTTCCTGACATGCCTGCTGGCCCTGGCCCTGCTGTTAGGTATGCCGAGCAGTGCCTTTGCCTTCAAAGTGCCGATCCACGAAGAGATCACCCG CGAAGTGTTTGAGGATTTCCAGGTGGTGGTGGAAGGCGAGACCTTTAAGTTCACCGACTATGCCATCGACCAGATCGTGAAGGCCAACAAGGATACCG ATGACCTGCCGAACCAGTTCAATACCGAGATGCACTTCGACGGCGAAGACTTTAGCGGTGGCAGCAATCGTGTGATGTTCCTGAAGGAGCGCACCATT ACCAAAGTGACCGACCCTCAGGATCCGCAGGGCACAAGCGCACGTAATGATCTGGGTACCGCCCTGCATACCGTGCAGGACTTCTATGCCCATAGCAA TTGGGTGGAACTGGGTCACAGCAGCAGCGATATCAACACCAAGATCGGCCGCGAGGTGTTCAGCGGCGCCGATAAAAACACCGCCACCTGCCCGAACG ATCCGGGCATTCTGGGTGGTGCCGGCCTGACCGAACTGACCAGTGGCTACTTCACCTTCATCGGCGTTGTGCCTAGCTGCGATGTTCCTGAGGGTAAA TGCCGCCATGGCGTTCCGATTGTGTGCCCGGATGGCCTGAACAAGGACGATAATAGTCGCCCGGGTTTTCCGACCGCCCGTGCATTAGCCGTTAAAGC CACCGAGGACTTCCTGAATCAGATCTTCAGCGACAGCCGTATGGACGGCAACGTGGATGCCATCAAACTGCTGATGCGCATCCGTAATTAATCCCTTT AACAAGTAAATTGTACCTCGCCTAAACCATACCCGACAGGATTGGTTTAGGCTTATCTGCTGCTTTATAAAAGACAGATAGAGATTAGAATTATCGCA ATGGAAAATAATAATTATCCGTTCGAACTGAAAGCCTATGACTTTAGCTTTAAAATCTTCAAGGAGATCATTTTCGCCCTGAACGCCTTCTTCATTGG CTTTTGGCTGGGTGTGCTGAAACGCGAACATTACCACCTGGTGGATAGCATTTACTACAACCAGACCGAAATGTACCGCGACGAGAACTACAACAAAC GCGGCCTGTGGGACTGGGAAGAGAAAGTTCTGGCCCAGTACTTCCAGCAGTGCCATAATCTGCTGGTGGTTGCAGCAGGCGGTGGTCGTGAGGTTCTG GCCTTATGCAAACGCGGCTATGAAGTGGACGGCTTTGAGTGCAATGCCAACCTGCTGAAATTCGCCAACAACCTGATCAAGCAAGAGGAGTTTGCCAG CCATATCAAACTGGCACCGCGTGATCAGTGCCCGGATAGCCAGAAGGAGTATGATGGCCTGATCGTTGGCTGGGGTGCCTATATGCTGATCCAGGGTA AGGAACGCCGCATTGAATTCCTGCGTCAGCTGCGTACCCAGGCCAAGAAGAACAGTCCGGTGCTGCTGAGCTTCTTCTGCTACAGCGAGACCACCGGC GGCCGTGATTTCAAAGCCATCGCCATGATCGGCAATGCCTTTCGTCGCCTGCTGGGTCGTGAATGCCTGGAAGTTGGCGATAATCTGGCCCCGAACTA CGTGCACTACTTCACCAAGGACGAGATCGCAAGCGAACTGCAGGCAGGCGGCTTTGAACTGAAGATCTACTGCACCAATCAGTATGGCCACGCCGTGG GTATCGCAGTGTAAcacctttaagcctatagtctaggaaaaagaggttgaattagagccaacttggtcatctgaaaccctcatgctgtcgtttccccc tgaacagttacgaaactacgaagatagagaagctttatgtataaaggcctggatcgcgacgaaattcgccagattcaggtgctgatgctgctgtgcct GTGCCTGAGCCCGCAGAGCAAACTGCGCCAGCTGCTGGAAATTGCACTGGCCGCCAGCGAAACCCAGATCATGACCCGCATGACACCGTGCGATGATG TGAATGTGGACGGCCTGTTTACCTGGGTGCAGAGCCTGTTTGCCCAGGGTGGTCTGACCGAAGAAGAGAAACGCCTGCTGAAGTGGCAGAACGAAAGC CGTAACATGCTGCCGGCCATTGACGAACTGAAAACCATTGAGAAGAAGCTGGGCTTCAAGATCCGCATTCAGAAGCTGCAGAGCCACAATTAAttatt ggggtgcggtcaaaaacagttgaattttaagtcgcggcttacccg ATTTTCCATCTCTCTTGCTCCATATGAGTTTCAGTTGTTTCAGCTGCACCGAGCGCAAGGAGAATAACGAACCTTGTTTATTTTTACTGTTAAAGCAA AACACCAATTTCACACTGCGCACCAGCACCATGCTGGACGACTTCAGCCTGTTAAAGTTAGCCAGTCGCGCAAGCAACCTGTGTGAGCAAACCCTGAT TGTGAAAGAGCTGGCAAAGAGCAAAGCACCGATCGCCAGCACAACACAACTGAGTCCTGTTGACAGCTGGAAAATTAAGAAACTGACAGGCAAACTGG CCGTGCAGCCGTTCAAAGAGAGCTACGAGCAGGGCACAATCAGTCAGAGCGTGATTGAGGACCTGCGTAAACTGCTGATCGATTACAAACTGTACGAA CTGAACCTGGCAAACTTAAGCGAGAGCGACCGCCTGGAATTTATTAAGCCTCACAGCCAGTGGTTAAAGGCATACCAGGCCGCCATGGCAACCCTGGA TCTGCCGCGTGAAAAGTTCAGCGGTAGTTGTTGGGGCGAACCGGATATTTACTACGGTAAATTCGCCAAAGTGTGTGAACCGTTTCTGCGCCTGCTGC ACCAAACCCTGCGCGGTACCGGCGACGCAATTAACGCCACCGCCGACAATTACCGCATTAATCCGCAGGTGGCCATCGACATCGAGCTGCATCTGCTG AACCGCTTCGAATTAGCCCTGGCCTGGGCCTTAGAGGCCAACATCAATGTTTATTGTAGTCAGAAAGCAATCGCCAAGAGCGAGGATGATAGCGAAGC CTACATCGCATACCTGGAGGAGACCTTCGACCGCAAGCAGAATTATCATGATTTTTATTGTCGTTTCCCGGTGCTGGCCCGTTGGTTAGCCCAAGTGA CCTATTTCCTGTGCAATTTTGGCGAGGAAACCCTGCAACGCCTGACCAGCGATCGTGAGCAGATTGGCGCCACCTTCTTCGGCAGCAAGCCGATCAGT CAAATCAAAAGTTTTAAACTGGGTAAAAGCGACTACCATGCCGGTGCCAAGAGCGTGGTGATCGTGGAGCTGGAGCTGGCCAACAGCGAACCTGCCAC CCTGGTGTATAAGCCTCGCAGCATTCAGAGCGAAGCCGGCATGCAGGGCCTGCTGGCACAGCTGAACCAAGATAAGGTGGTTCGCTTTGCCCACTATC AGGTGCTGTGTCGCGATGGTTATGGCTATGCAGAGTTCATCCCGAGCGGCAAAAACCAGGTTCAGAATAAAGAAGATCTGAAGAAATTCTACCAACAG CTGGGCGGCTTTTTAAGCATCTTCCATATCCTGGGTGGCGGCGATCTGCACCATGAAAACATCCTGGTTGCAGATGGTAACGCCTTCATCTGTGACTG CGAGACCGTGCTGGAGGTGCTGCCGCAAGGCATGGATAAACTGCCTGGTACAGTTTTAGATAGTGTGTTTAAAACAGCCATGCTGGACTGGCCTCGTG ATAGCGCAAGCCCGGAGAACAGCGAGATGATGAGCATTAGTGGTTACAGCGGTGGCGAAAGCTATGAGGTTGCATTCACCGTGCCGCGCGTTAAGGAG CACCGTATGAGCCTGGATCAGGGTGTTGAGTACAAGACAGGTATCACCGTGGAACTGGAAGGTACAAATCGCATTTACTACAACGGTGAGATTGTGGA TCCGCAGGACTATAAGGATAGCATTGTGGACGGTTTTAACCAGGTTTATACATGGTTTCAGCAGCACCCGACCAAGGCAATTACCCGTATTAAGGAGC TGTTTAGCAGTAGTTTAGTGCGCTTTATTAATTGGGGCACCCAAGCCTACGCCAAGAGCATTGTGGCCGTTCGTCACCCTAAGTGCCTGGCCGACCCT CTGGAGGTGGACCTGATCTTTAATAGCCTGAAAGAGCATAAACGCCAGTGGGACAAAAAGGGCGAACTGGCAGAGTTAGAGCTGGGCAGCCTGTGGCA ACTGGATATCCCGATTTTCACCGCCTTAGCCGCCGAAAGCAAAGACTTAATCTTCAATTATCAGTATAGCGTTAGTGATACCTTAGCCATCAGCCCGT TAGACAATGCCAAACGCCGTCTCGAGCAGCTGAGCACCGAGAACCG TAGACAATGCCAAACGCCGTCTCGAGCAGCTGAGCACCGAGAACCGTATCCGTCAGAATCAATACATCTATACCAGCCTGAGCACCGACGAGATCAAC AGTCCGTACTTTATCGCCGCAGCCGTTAACTATGCCCAGCAGATCGGCTGGCAGCTGTGTGAACAGCTGAGTAGCGATAGTAGCAAAGCACCTTGGCA GACATGGGACTACACCGCAACCGGCAAGCGCTTAGTGGATATCAGCGGCGACCTGTATGATGGCAGCGCCGGCATTTGTCTGTTCCTGGCCTACCTGG ATGCCATCAAACCGCAAGTGGAATTCCGTCAGGCCGCCGAACGCGCCTTAGAATACAGTATCGAGAAACGTAACACAACCCTGATCGGTGCATTCCAG GGCGAAACAGGTCTGATTTATCTGCTGACCCATCTGGCACAGTTATGGGACAAACCGGCATTACTGGACCTGGCAGTTGACCTGAGCGACGAGCTGCT GCCGCGCATCAAACAGGACATCTACTTCGATATCCTGCATGGCGTTGCCGGTATCATTCCGGTTATGCTGGGCCTGGCCGAAGCAACCGGTGGTAAAG GCATTGATTGCGCATTACAGTGTGCCGAGCACCTGCTGGAGCAGGGTATTTACCAGGATAACACCCTGAGCTGGCCTCCGGGTCGTCCTGACCTGGTG CGCGGCAATTTTACAGGCTTCAGCCACGGTGCAAGCGGCATCGGTTGGGCCTTAATTATGCTGGGCTGCCACAGCAATAAGAGCGAGTATATTGAGGC AGGCCGTCAGGGCTTTGCCTACGAAGCCACACAGTTTGATGAGGAACAGCGCGATTGGTACGACTTACGCAAGAGCGTTACCACCGCAGATAGCAACG AACCTCACTTTGCCAACGCATGGTGCAATGGTGCCGCCGGTATTGGCCTGAGTCGCATCATTAGTTGGGCCGCCCTGGGCAAAACAGACGACGACATT CTGCGCGATGCCTACACCGCACTGAATGCAACCTTACGCAATTTCAACAAGCTGGGCAACGATAGCCTGTGTCATGGCAAGAGTGGTAACGCCGAGTT ATTCCTGCGTTTTGCACAGCTGCGCGATACCCCGTATCTGCAAATGGAGGCAAACGTGCAGGCCCAGGCACAATGGCGCAACTTTGAAAAGGCACGCC GTTGGATGTGCGGTAGCACAGGCAACGATGTTTTCCCGGATTTAATGCTGGGCCTGGCCGGCATTGGCATGCACTTCCTGCGCCTGGCCTACCGTGAA CGCGTTCCGAGCCCGTTATTATTAGATCCGCCTCCGCGCGCCATCGACTAAGTTTTGAGTTTTTTAATTCAATATTCAACACCCAAGAGTAATATAGT TTAGTTTGTTTATTAGGATCGAGATGAGCAAAGAAACCGTGATTGAATTTTATGAAGCAATTTTTGAAAGTCCGGAATTCATTCAGGAAATTAAAGCC ATTACCCGCCAAGAGGAACTGATCAAACTGGGTGCCCGCAACGGCTACCATTTTACCATGGAAGACCTGGCCCAGGCCGACGCCAGCTATATCCCGAA GAATAACCAGCCGCTGATCAGCATTGATAGCGACGACCGTGCCCGCGAAGAACTGCCGCGCCCGTATCACTATGAATTCGAGTTCAGTGAGATCCCGG GCTTCGAAGAGATCGATCGTGAACTGAAAAAACTGCAGATCAAACCGAACACCGTGGATCTGGACCTGTACGAGAAGAGTTTCCGCGAAGAAGATTTT AAATTTAACTATATTAGCCCGACCGTGCCGGGCTTCCGCCAGTATTACTACAAGAGCCTGAAAAGCTATCTGGATCTGCCTAGCCCTCAGCCGGAATA TGCCTGGCGTCCGTTTCACCTGATCAATCTGGATTGCCACGTGGAGGATCCGCTGTACGAGGATTATTTCCAGACCAAGGTGCGTCTGCTGAAGCTGC TGGAGAATTGCCTGGAGACAGAGCTGCGCTTTAGTGGCAGCCTGTGGTATCCGCCGAACGCCTATCGCCTGTGGCACACCAATGAAACCCAGCCGGGC TGGCGCATGTACCTGGTGGATTTCGACAATTTTGACGACAACCAGGAAGGCGAGGTGTTCTTCCGCTACATGAATCCGGAGACCAAGGAACTGGTGAC CCTGGCCGAGAAACCGAAAAT GTGGAACTGCTGAACAACGCCCTGGGCGAAATCGATATTGCCGCCGAATGCGCGTCGACCTGTAGCAGCGGCCCGATCACCGCGATCTGCGATGGTAC CACCAAATAAGTTGTTTTACTAGCATATTATATTATTAATATGTCTGGGCTACCACCACATTGTGAAGCTAGCCTCATTAAATTGATTGTTGGGCATG GTTAG Length (bp) Nature Chemical Biology: doi:1.138/nchembio.2537
4 upplementary Table 4. DNA primers used in this study. Primers used in the construction and sequencing of pdiv, pdiv-2, and prfduet-divmt. # Primer name equence (5-3 ) 1 div1a-fwd CTCGTATGTTGTGTGGAATTGTG 2 div2-rev CGGTTCTCGGTGCTCAG 3 div3-fwd TAGACAATGCCAAACGCC 4 div1b-rev CGGGTAAGCCGCGAC 5 divm-23-fwd CGATCGCCAGCACAAC 6 divm-1424-rev CCAGTCCAGCATGGCTG 7 divm-2818-fwd GCAGGCCGTCAGGG 8 divx-65-rev AGCCCGGCTGGGTTTC 9 divx-58-fwd GAGACAGAGCTGCGCTTTAG 1 divmt-79-rev CAATGAAGAAGGCGTTCAG 11 divmt-2-fwd CGTTCGAACTGAAAGCCTATG 12 divm-23-fwd CGATCGCCAGCACAAC 13 divm-1977-f CTTAGCCGCCGAAAGC 14 divx-461-f TGCCTAGCCCTCAGCC 15 divhypo-78-f CGCATCCGTAATTAATCCC 16 intergenic-divmnative-fwd GAGCAATATTTTCCATCTCTCTTGCTCCATATG 17 intergenic-divmnative-rvs ACTCAATC GGGTGTTGAATATTGAATTAAAAAACTCAAA 18 divmp-fwd GTGGAACTGCTGAACAAC 19 divm-non-optimizedpet16b-gibson-fwd cgaaggtcgtcatatgcttgatgatttttctctcc 2 divm-native-fwd-1 CTTTAGCCTGGGCTCTTG 21 divm-native-fwd-2 GGACAGTGCTTGATTCAG 22 divm-native-rvs-4 CTTCATCAAATTGGGTCG 23 divmt-mc1-fwd TTCGAGCTCGATGGAAAATAATAATTATCCGT TCG 24 divmt-mc1-rvs TATGCGGCCGCTTACACTGCGATACCCACG Nature Chemical Biology: doi:1.138/nchembio.2537
5 upplementary Table 5. ummary of quantification of divamide material by NMR. Divamides were quantified using linear equations shown in upplementary Figure 3a. All divamide samples were quantified with (top row) and without (bottom row) inclusion of the methyl region integral because this integral accounts for a large number of protons, which may reduce the accuracy of integration. Compounds 2, 3, and 9 were quantified using integrals for only two proton signals such that numbers obtained with exclusion of methyl integrals represent a single integral. Yields were calculated from the average of the two concentrations. A known concentration of L-Tyr was used to assess the quality of the L-Trp standard curves. A difference of 7.6 was calculated for the NMR-determined concentration relative to the UV-determined concentration. UV NMR ample MW (g/mol) mg/ml mg/ml D mm D yield (µg) L-Tyr E11-36 divamide A (1) E. coli divamide A (1) desmethyl divamide A (9) divamide B (2) divamide C (3) Nature Chemical Biology: doi:1.138/nchembio.2537
6 upplementary Table 6. ummary of quantification of divamide material by LC/M. Divamides were quantified using linear equations shown in upplementary Figure 3b. For mixtures of compounds, the concentrations in mg/ml were calculated based on individual molecular weights before adding together. The concentration of E. coli divamide A determined by LC/M was in agreement with the NMR concentration ( -5.92). The solution of 4 used in flow cytometry based assays was quantified using curve ii (*). ample MW (g/mol) mm D mg/ml D yield (µg) 1 (E. coli) mix mix mix mix biotinylated divamide A mix biotinylated cinnamycin mix * Nature Chemical Biology: doi:1.138/nchembio.2537
7 upplementary Table 7. ummary of flow cytometry experiments. Flow cytometry experiments were performed in triplicate (or duplicate in several cases) for each dose of each sample. A total of four compounds were tested, with eight different doses for dose range 1 and six doses and two infection conditions, uninfected and HIV infected, for dose range 2 (see Figure 4), utilizing four 96-well plates. The total number of events are listed alongside the number of events collected within gate 1, representing live cells, and gate 2, representing living infected cells. Log concentration (viability-dapi) (p24-fitc) ample Plate Replicate ng/ml nm Total events Gate 1 events gate1/total gate2/gate1 Gate 2 events 1 (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) (E11-36) uninfected uninfected uninfected HIV HIV Nature Chemical Biology: doi:1.138/nchembio.2537
8 continued uninfected uninfected uninfected HIV HIV uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected HIV HIV HIV Nature Chemical Biology: doi:1.138/nchembio.2537
9 continued... 9 uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected uninfected HIV HIV HIV Nature Chemical Biology: doi:1.138/nchembio.2537
10 upplementary Table 8. ummary table of assay data. IC 5 (anti-hiv), CC 5 (cytotoxicity), and therapeutic index values for all compounds screened are listed. Values from both cytoprotection (Figure 3) and flow cytometry-based (Figure 4) assays are included for comparison. For some samples, the true value of these parameters could not be determined from curve fitting and was estimated based on neighboring data to lie above or below the indicated number (> or <). ome activities were not detected within the concentration range tested (ND). The therapeutic index was not calculated for data that could not be fitted to a curve (NC). Values from repeated experiments were averaged (*). Assay ample IC5 (µm) CC5 (µm) Therapeutic index ND <27. Cytoprotection 1-12 >.35 <8.42 NC ND < ND ND 4 ND >5.35 NC Flow cytometry * <14.4 NC * 6.25 Nature Chemical Biology: doi:1.138/nchembio.2537
11 upplementary Table 9. Divamide-like masses observed from tunicate extracts. Unidentified ions observed from Didemnum molle extracts by LC/M resembled those of characterized divamides in m/z and isotope distribution. ome masses appear to be derivatives of 1-3, but others may reflect alternative amino acid sequences at various stages of posttranslational modification. everal of these display evidence of Hoffmann elimination in negative ionization mode. Thus, additional, uncharacterized divamides are believed to be encoded by E Observed m/z (z = 2) ource ID 11.9 E E11-37? E11-37? 9.5 E11-37? E11-37? E11-37? E x Me E x Me E11-37? E x Me E E x O E11-37? E11-37? 11.5 E11-37? E11-37? E11-37? E11-37? E11-37? E11-37? E E x O E11-37? E11-37? E11-37? E11-37? E11-37? E11-37? E11-37? E11-37? E11-37? Nature Chemical Biology: doi:1.138/nchembio.2537
12 White3 (n=2) White4 (n=4) White1 White2 (n=4) D. psammatode.6 Large1 (n=4) Large3 (n=3) Large4 Large5 Large2 E11-36 mall1 mall2 2.6 substitution/site Brown1 (n=7) Brown3 Gray1 Gray2 (n=3) Gray4 72 Gray3 E11-37 upplementary Figure 1. Ascidian phylogeny. The phylogenetic relationship between ascidians collected in this study (shown in red) to samples collected by Hirose et al. (shown in black) was estimated by maximum likelihood analysis of Didemnum molle cytochrome oxidase subunit I (COI) gene sequences. 49 The tree was generated using RAxML software with 1 bootstrap replicates, with the resulting bootstrap probabilities shown at each node. According to Hirose et al., each of these named morphotypes (such as Large, Brown, etc.) are likely to be different species, even though they are currently all classified as D. molle. Nature Chemical Biology: doi:1.138/nchembio.2537
13 A DNA Length (kbp) i ii i = 88 Da D = 9 Da ladder P L FT W1 W2 T y y H! N!! ECATCFGIVTIVCDGTTK! m/z m/z i y12 y H H! H N! N N! ECATCFGIVTIVCDGTTK!! ECATCFGIVTIVCDGTTK ECATCFGIVTIVCDGTTK!! y12 y9 y7 y5 y2 y13 y1 y8 y6 y4 y3 1! ECATCFGIVTIVCDGTTK! E E1 E2 E3 E4 y6 y y y y9 y ! ECATCFGIVTIVCDGTTK! 1!!! H +H + N (Me3)NN (Me3)N ECATCFGIVTIVCDGTTK ECATCFGIVTIVCDGTTK m/z H! N!! ECATCFGIVTIVCDGTTK! m/z ii kda 27. C m/z DivMT purification Molecular Weight (kda) = 9 Da iii ii iii! ECATCFGIVTIVCDGTTK! i NiCl2, NaBH4 55 C, 3 min = 88 Da!! ECATCFGIVTIVCDGTTK! ECATCFGIVTIVCDGTTK! ECATCFGIVTIVCDGTTK y12 y9 y7 y5 y2!!! y13 y1 y8 y6 y4 y3!! ECATCFGIVTIVCDGTTK! ECATCFGIVTIVCDGTTK NiCl2, NaBH4, y12 y9 y7 y5 y2 y4 NiCl2, NaBH4 55 C, 3 min 1 ECATCFGIVTIVCDGTTK y13 y1 y8 y6 y4 y3 ECATCFGIVTIVCDGTTK C, 3 min! kda ladder P L FT W1 yw2 T E1 E2 E3 E4 ladder 6 y y7 ECATCFGIVTIVCDGTTK!! ECATCFGIVTIVCDGTTK y1 ECATCFGIVTIVCDGTTK y ECATCFGIVTIVCDGTTK! y y9 y1 y13 H N 42.7 H 34.6 ECATCFGIVTIVCDGTTK 86.1 N H kda NH! 27. H! ECATCFGIVTIVCDGTTK N!! N!! ECATCFGIVTIVCDGTTK 2. ECATCFGIVTIVCDGTTK! ECATCFGIVTIVCDGTTK! ii bp ! ECATCFGIVTIVCDGTTK!!!! 1 B m/z H H N N ECATCGPITAICDGTTK ECATCGPITAICDGTTK H +H + N (Me3)NN (Me 3)N ECATCGPITAICDGTTK ECATCGPITAICDGTTK m/z m/z upplementary Figure 2. Installation and detection of in vitro chemical transformations of pdiv-2 and -3 E. coli products. a, The pdiv expression vector for 1 produced no detectable compounds. The native divm gene was amplified from E11-36 metagenomic DNA by PCR resulting in a faint band (i) that was used as a template for additional PCR amplification (ii). This gene replaced the codon-optimized divm gene of pdiv to yield pdiv-2 (Figure 2a), which produced MeLan-containing products when expressed in E. coli (Figure 2b). b, Desulfurization of 5 (i) results in an 88 Da mass shift (ii) due to the reduction of three MeLan residues and one Dha residue. After base-catalyzed Lal formation, desulfurization yields a 9 Da mass shift (iii), indicating the presence of Lal. c, M/M analysis is hindered by the presence of cyclic residues like MeLan (i), while the linearized desulfurization product is fragmented into y ions (ii). After base treatment, the desulfurization product is resistant to M/M fragmentation due to the Nature Chemical Biology: doi:1.138/nchembio.2537
14 presence of Lal (iii). d, DivMT was purified on Ni-NTA resin from the cell lysate of prfduet- DivMT-expressing E. coli BL21(DE3) and column fractions analyzed by D-PAGE. P = pellet, L = filtered cell lysate (supernatant), FT = Ni-NTA column flowthrough, W = column wash with lysis buffer, T = column wash with transition buffer, E = elutions of increasing imidazole concentration in transition buffer (1 = 5 mm, 2 = 1 mm, 3 = 2 mm, 4 = 5 mm imidazole). A band around 32.6 kda, corresponding to His 6 -DivMT, was observed in E3. e, Methylation was successful for both HPLC-purified 9 (i) and an E. coli C18 fraction containing 17 (ii). Top and bottom spectra show the reaction in absence or presence of DivMT, respectively. A small amount of final product (m/z 965.7) was observed in the C18 fraction prior to DivMT methylation. Nature Chemical Biology: doi:1.138/nchembio.2537
15 A i L-Trp standard curve (! 7.62 ppm) Absolute integral y =.531x +.22 R² = Concentration (mm) ii L-Trp standard curve (! 7.17 ppm) Absolute integral y =.5358x -.13 R² = Concentration (mm) iii L-Trp standard curve (! 3.19 ppm) Absolute integral y =.529x R² = Concentration (mm) upplementary Figure 3. Quantification of divamide material. a, tandard curves for quantification of low abundance materials by NMR were generated using a dilution series of L- Trp in H 2 O, the concentration of which was determined by UV-absorbance (e 28, Trp = nm - 1 ). The absolute integral was plotted as a function of concentration using three different aromatic proton signals: H E3 (i), H Z3 (ii), and methylene H B2 (iii). The linear equations generated were used to determine yields of divamide analytes of unknown concentration in upplementary Table 5. b, tandard curves for quantification by LC/M were generated using a dilution series of synthetic 9, previously quantified by NMR. The resulting linear equations were used to determine concentrations of divamide analytes and estimate relative ratios of compound mixtures, such as those obtained from expression in E. coli, as shown in upplementary Table 6. The majority of analytes were quantified with one curve (i), while a second curve (ii) was used to quantify additional 4. Nature Chemical Biology: doi:1.138/nchembio.2537
16 B i continued Desmethyl divamide A standard curve ii Area Under Curve (AUC) Concentration (µm) Desmethyl divamide A standard curve y = x R² =.9878 Area Under Curve (AUC) y = 25.95x R² = Concentration (µm) Nature Chemical Biology: doi:1.138/nchembio.2537
17 A relative survival B R 2 =.9998 IC 5 =.292 µm CC 5 = 4.43 µm 9 R 2 =.9976 IC 5 =.327 µm CC 5 = 3.38 µm 1 (E11-36) live infected cells (p24-fitc) (E11-36) R 2 =.9987 IC 5 = 1.3 µm CC 5 = >5.35 µm Dose range 1 HIV infected 1-12 log concentration (ng/ml) R 2 =.9935 IC 5 =.874 µm CC 5 = <14.4 µm live cells (viability-dapi) Controls Uninfected AZT Infected Controls Uninfected p24 Infected p24 Uninfected viability Infected viability upplementary Figure 4. Additional cytoprotection and flow-cytometry assay doseresponse curves. a, Cytoprotection assay dose-response curves for divamide species not shown in Figure 3. Experimental controls, shown as dashed lines, include uninfected (blue), infected (red), and AZT (at either.1 or.5 mg/ml) responses. Points represent an average of three assay responses per dose (error bars = ± standard deviation). R 2 values are included for compounds for which curves could be fit with equation 7 (see online methods) or a single fourparameter IC 5 equation. Curves could not be fit for E11-36-derived 1 due to abnormal curve shape. b, Flow-cytometry dose-response curves for divamide species not shown in Figure 4. Cytotoxicity and anti-hiv activities of divamides and 4 were monitored simultaneously via flow cytometry using a fluorescein-labeled antibody to HIV-1 capsid protein p24 (green; live infected cells) and viability stain (purple; live cells) with HIV-infected CEM TART T-cells. The biological marker and corresponding fluorescence channel are shown in parentheses along the vertical axes. Points represent an average of three assay responses per dose (error bars = ± standard deviation). Average uninfected and infected cell control responses are shown as horizontal lines for each biological marker. All plots shown employed dose range 1 with HIVinfected cells, yielding full anti-hiv dose-response curves but only partial cytotoxicity curves. Nature Chemical Biology: doi:1.138/nchembio.2537
18 A 1 [M+H] 2+ = pos i 1 [M-2H] 2- = = 63 Da neg ii 1 1 pos m/z [M-2H] 2- = = neg [M+H] 2+ = = 63 Da iii m/z pos [M-2H] 2-2- = neg [M+H] = = 63 Da m/z upplementary Figure 5. Chemical diversity within the divamide family. a, Hoffman elimination of trimethylamine from a quaternary N-trimethylamine results in a net loss of 6 Da and the elimination of a positive charge. This characteristic was observed for N- trimethylglutamate-containing lanthipeptides 1 (i), 2 (ii), and 3 (iii) by LC/M in negative ionization mode. b, All divamide-like lanthipeptide gene clusters that could be identified by BLAT search of div pathway genes are shown. They occur mostly in cyanobacteria (i) and actinobacteria (ii), with one example from chloroflexi (iii). Cyanobacteria may contain multiple lanthipeptide clusters within a single genome or multiple lana precursor proteins in a single cluster, suggesting multiple products are produced from a single strain. Each pathway maintains the genes responsible for introducing conserved modifications, including Lan/MeLan and Hya. Most also contain divn homologs, but this gene does not appear to be 1 conserved across all pathways. ome cyanobacterial pathways have incorporated unique modifying enzymes. A methyltransferase bearing no resemblance to DivMT can be found in the first pathway from Cylindrospermum stagnale PCC7417 ( orf1 ). The recently reported oscillamycin is the product of the second cluster from Oscillatoria sp. PCC 182 and contains hydroxyproline, presumably introduced by the putative proline hydroxylase located in the first cluster ( orf ) (Yang, J. J., 216, Thesis, University of Helsinki, Finland). Many of the actinobacterial cluster strongly resemble that of cinnamycin, containing cinnamycin regulatory gene homologs, while cinnamycin B, recently reported from Actinomadura atramentaria NRBC (the cluster described here was identified by BLAT search from Actinomadura atramentaria DM 43919), includes only the essential divamide-like biosynthetic genes (Kodani,. et al., 216, J. Ind. Microbiol. Biotechnol. 43, ). c, Alignment of LanA precursor proteins from the divamide extended family, generated using ClustalW2 and then manually adjusted. The Nature Chemical Biology: doi:1.138/nchembio.2537
19 occurrence of multiple cassettes, as is seen for putative proteins 4-6, is a rare feature among RiPPs but common within the diversity-generating cyanobactins. 4 The alignment reveals conservation of Lan/MeLan, Lal, Gly, and Hya positions within the core, as well as hypervariable positions (*). Nature Chemical Biology: doi:1.138/nchembio.2537
20 B i ii iii Cylindrospermumstagnale PCC 7417 Genbank: CP Moorea producens 3L Genbank: GL Oscillatoria sp. PCC 182 Genbank: KB KB cytonemamillei VB Genbank: JTJC Actinomadura atramentaria DM Genbank: KB Actinomadura oligospora ATCC Genbank: JADG111.1 JADG13.1 Actinomadura macra NBRC 1412 Genbank: BCQT13.1 Actinomadura rubrobrunea NBRC Genbank: BCQU11.1 Frankia sp. EUN1f Genbank: ADGX Nocardiopsistrehalosi NBRC 1421 Genbank: BCRK12.1 Nocardiopsis potens DM Genbank: ANBB18.1 Marinaactinospora thermotolerans DM Genbank: NZ_FUW11.1 treptomyces roseoverticillatus strain NRRL B-35 Genbank: JOFL116.1 treptomyces africanus NRRL B Genbank: NZ_MUKA141.1 Anaerolineaceae bacterium 4572_78 Genbank: NBMH continued lana: WP_ lana: WP_ orf1 A orf2 M T orf3 A orf2 M T N X M A-1 A-2 T lana-1: EGJ lana: WP_ oscillamycin lana: WP_ T M orf A X orf M A N T X M A lana: WP_ orf X M A N cinnamycin B lana: WP_ lana: WP_ lana: WP_ cint/h X M A-1 N A-2 cint X M A N lana: WP_ cint X M A N lana: WP_ Y X M A N cinh cint lana: WP_ cinh cint X M A N cinr1 cinorf11 lana: WP_ orf cinh cint X M A N cinr1 cinorf11 lana: WP_ cinh cint X M A N cinr1 cinorf11 lana: WP_ Y cinh cint X M A N cinr cink cinorf1 11 cinr1 lana: WP_ cinh cint X M A N cinr cink cinorf1 11 cinr1 cinorf12 13 lana: WP_ orf M A lana: OQY kb Nature Chemical Biology: doi:1.138/nchembio.2537
21 C continued Prochloron didemni E11-36 (divamidea) 2. Prochloron didemni E11-37 (divamideb) 3. Anaerolineaceae bacterium 4572_78 4. Cylindrospermum stagnale PCC Cylindrospermum stagnale PCC 7417 (2) 6. Oscillatoria sp. PCC cytonema millei VB Moorea producens 3L 9. Moorea producens 3L (2) 1. Oscillatoria sp. PCC 182 (2; oscillamycin) 11. Frankia sp. EUN1f 12. treptomyces roseoverticillatus NRRL B Actinomadura atramentaria DM (cinnamycin B) 14. Actinomadura oligospora ATCC Actinomadura oligospora ATCC (2) 16. Actinomadura macra NRBC Actinomadura rubrobrunea NRBC treptomyces africanus NRRL B Nocardiopsis trehalosi NRBC Marinactinospora thermotolerans DM Nocardiopsis potens DM treptomyces cinnamoneous DM 45 (cinnamycin) 23. treptomyces cinnamoneous ATCC (duramycin) 24. treptomyces cinnamoneous ATCC (2) 25. treptoverticillium sp. R275 (duramycin B) 26. treptoverticillium sp. R217 (duramycin C) 27. treptomyces sp. No. A647P-2 (ancovenin) * * * * * * * * * * * Nature Chemical Biology: doi:1.138/nchembio.2537
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