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Supplementary Table 1. Annotation of the divamide A biosynthetic cluster. CDSs of the divA biosynthetic gene cluster (GenBank: KY115608) from the E11-036 metagenome listed alongside their most similar BLAST 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 BLAST hit % ID Annotated function
(Organism) Conserved protein
domains (accession) Functional
characterization
-2 ND 369 WP_007353413.1 54 hypothetical protein (Kamptonema) none not characterized
-1 ND 321 XP_010382366.1 32
uncharacterized protein
LOC104678724 (Rhinopithecus
roxellana)
none not characterized
divA ARD09202 276 WP_017718744.1 63 hypothetical protein
(Oscillatoria sp. PCC 10802)
none precursor gene
divM ARD09203 3255 WP_017718743.1 70 hypothetical protein
(Oscillatoria sp. PCC 10802)
LanM-like (cd04792) class II
lanthionine synthetase
divX ARD09204 918 WP_017718741.1 58 hypothetical protein
(Oscillatoria sp. PCC 10802)
Nif11 (pfam07862) putative Asp ß-hydroxylase (16)
divY ARD09205 780 WP_038094010.1 45 hypothetical protein
(Tolypothrix bouteillei)
Het-C (pfam07217) not characterized
divMT ARD09206 798 WP_015192250.1 50
SAM-dependent methyltransferase
(Stanieria cyanosphaera)
Methyltransf_18 (pfam12847) methyltransferase
divT ARD09207 1941 WP_008178015.1 64 ABC transporter
permease and ATPase (Moorea producens)
ABC_membrane_2 (pfam06472),
ABC_tran (pfam00005)
not characterized
divU ARD09208 858 WP_069349017.1 63 hypothetical protein (Scytonema millei)
FGE-sulfatase (pfam03781) not characterized
divN ARD09209 351 WP_008178005.1 53 hypothetical protein
(Oscillatoria sp. PCC 10802)
none putative role in Lal formation
(14-16)
+1 ND 1500 WP_015145144.1 60 phosphopeptide-binding protein
(Pleurocapsa minor)
CHAT (pfam12770), FHA (pfam00498) not characterized
+2 ND 1905 WP_015185269.1 54
serine/threonine protein phosphatase
(Microcoleus sp. PCC 7113)
PP2Cc (cd00143) not characterized
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary Table 2. Relationship of divamide B cluster genes to divamide A cluster. CDSs of the partial divB biosynthetic gene cluster (GenBank: KY115609) from the E11-037 metagenome compared with their respective divA homologues. Individual accession numbers are listed for gene products (ND=data not deposited).
Gene ID Protein accession number Length (bp) Coverage with
corresponding divA gene % ID to corresponding
divA gene -1 ND 286 (partial) 88 100
divA ARD09210 276 100 93 divM ARD09211 111 (partial) 3 100 divX ARD09212 604 (partial) 66 100 divY ARD09213 272 37 86
divMT ARD09214 603 (partial) 76 99
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary Table 3. Synthetic DNA used in this study. DNA gBlocks purchased from Integrated DNA Technologies used to construct pDiv and pDiv-3.
DNA Sequence (5’-3’) Length
(bp)
1a
ctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacaATGCCGACCACCCTGGAAAAACCGAGCGTGGCCTATCTGGAGAAACTGTTTCACCAGACCGCCATCGACAGCGAATTTCGTAGCGAACTGCAGAGCCATCCGGAAGCCTTTGGTATTAGCGCCGATCTGGAACTGCCGCAGAGCGTGGAGAAACAGGACGAAAGCTTCGTGGAACTGCTGAACAACGCCCTGGGCGAAATCGATATTGCCGCCGAATGCGCCAGCACCTGTAGCTTTGGCATCGTGACCATCGTGTGCGATGGTACCACCAAATAAGTTGTTTTACTAGCATATTATATTATTAATATGTCTGGGCTAGCACCACATTGTGAAGCTAGCCTCATTAAATTGATTGTTGGGCATGGTTAGAAAGGAACTAAGTACAAGACAAAACTTGGATGAGAAGCGTAGTAAAATGTCCGAAACTATTGGCTCATAAAGTTTCGAGCAATATTTTCCATCTCTCTTGCTCCATATGAGTTTCAGTTGTTTCAGCTG
549
1b
AATCCGGAGACCAAGGAACTGGTGACCCTGGCCGAGAAACCGAAAATCGTGCGCTTCTTCAAAATCGAAAGCGAGCCGAGCAAAATTCTGTGGCACTGCATTGTTAACACCACCAAGCTGAATCGCTGGAGCTTCGGTTTCAACGTGAGCAGCAAGTGGATGGACAAACTGCTGACAGTGTAAGGAGAATAAAATGAAAATTTTCCTGACATGCCTGCTGGCCCTGGCCCTGCTGTTAGGTATGCCGAGCAGTGCCTTTGCCTTCAAAGTGCCGATCCACGAAGAGATCACCCGCGAAGTGTTTGAGGATTTCCAGGTGGTGGTGGAAGGCGAGACCTTTAAGTTCACCGACTATGCCATCGACCAGATCGTGAAGGCCAACAAGGATACCGATGACCTGCCGAACCAGTTCAATACCGAGATGCACTTCGACGGCGAAGACTTTAGCGGTGGCAGCAATCGTGTGATGTTCCTGAAGGAGCGCACCATTACCAAAGTGACCGACCCTCAGGATCCGCAGGGCACAAGCGCACGTAATGATCTGGGTACCGCCCTGCATACCGTGCAGGACTTCTATGCCCATAGCAATTGGGTGGAACTGGGTCACAGCAGCAGCGATATCAACACCAAGATCGGCCGCGAGGTGTTCAGCGGCGCCGATAAAAACACCGCCACCTGCCCGAACGATCCGGGCATTCTGGGTGGTGCCGGCCTGACCGAACTGACCAGTGGCTACTTCACCTTCATCGGCGTTGTGCCTAGCTGCGATGTTCCTGAGGGTAAATGCCGCCATGGCGTTCCGATTGTGTGCCCGGATGGCCTGAACAAGGACGATAATAGTCGCCCGGGTTTTCCGACCGCCCGTGCATTAGCCGTTAAAGCCACCGAGGACTTCCTGAATCAGATCTTCAGCGACAGCCGTATGGACGGCAACGTGGATGCCATCAAACTGCTGATGCGCATCCGTAATTAATCCCTTTAACAAGTAAATTGTACCTCGCCTAAACCATACCCGACAGGATTGGTTTAGGCTTATCTGCTGCTTTATAAAAGACAGATAGAGATTAGAATTATCGCAATGGAAAATAATAATTATCCGTTCGAACTGAAAGCCTATGACTTTAGCTTTAAAATCTTCAAGGAGATCATTTTCGCCCTGAACGCCTTCTTCATTGGCTTTTGGCTGGGTGTGCTGAAACGCGAACATTACCACCTGGTGGATAGCATTTACTACAACCAGACCGAAATGTACCGCGACGAGAACTACAACAAACGCGGCCTGTGGGACTGGGAAGAGAAAGTTCTGGCCCAGTACTTCCAGCAGTGCCATAATCTGCTGGTGGTTGCAGCAGGCGGTGGTCGTGAGGTTCTGGCCTTATGCAAACGCGGCTATGAAGTGGACGGCTTTGAGTGCAATGCCAACCTGCTGAAATTCGCCAACAACCTGATCAAGCAAGAGGAGTTTGCCAGCCATATCAAACTGGCACCGCGTGATCAGTGCCCGGATAGCCAGAAGGAGTATGATGGCCTGATCGTTGGCTGGGGTGCCTATATGCTGATCCAGGGTAAGGAACGCCGCATTGAATTCCTGCGTCAGCTGCGTACCCAGGCCAAGAAGAACAGTCCGGTGCTGCTGAGCTTCTTCTGCTACAGCGAGACCACCGGCGGCCGTGATTTCAAAGCCATCGCCATGATCGGCAATGCCTTTCGTCGCCTGCTGGGTCGTGAATGCCTGGAAGTTGGCGATAATCTGGCCCCGAACTACGTGCACTACTTCACCAAGGACGAGATCGCAAGCGAACTGCAGGCAGGCGGCTTTGAACTGAAGATCTACTGCACCAATCAGTATGGCCACGCCGTGGGTATCGCAGTGTAAcacctttaagcctatagtctaggaaaaagaggttgaattagagccaacttggtcatctgaaaccctcatgctgtcgtttccccctgaacagttacgaaactacgaagatagagaagctttATGTATAAAGGCCTGGATCGCGACGAAATTCGCCAGATTCAGGTGCTGATGCTGCTGTGCCTGTGCCTGAGCCCGCAGAGCAAACTGCGCCAGCTGCTGGAAATTGCACTGGCCGCCAGCGAAACCCAGATCATGACCCGCATGACACCGTGCGATGATGTGAATGTGGACGGCCTGTTTACCTGGGTGCAGAGCCTGTTTGCCCAGGGTGGTCTGACCGAAGAAGAGAAACGCCTGCTGAAGTGGCAGAACGAAAGCCGTAACATGCTGCCGGCCATTGACGAACTGAAAACCATTGAGAAGAAGCTGGGCTTCAAGATCCGCATTCAGAAGCTGCAGAGCCACAATTAAttattggggtgcggtcaaaaacagttgaattttaagtcgcggcttacccg
2397
2
ATTTTCCATCTCTCTTGCTCCATATGAGTTTCAGTTGTTTCAGCTGCACCGAGCGCAAGGAGAATAACGAACCTTGTTTATTTTTACTGTTAAAGCAAAACACCAATTTCACACTGCGCACCAGCACCATGCTGGACGACTTCAGCCTGTTAAAGTTAGCCAGTCGCGCAAGCAACCTGTGTGAGCAAACCCTGATTGTGAAAGAGCTGGCAAAGAGCAAAGCACCGATCGCCAGCACAACACAACTGAGTCCTGTTGACAGCTGGAAAATTAAGAAACTGACAGGCAAACTGGCCGTGCAGCCGTTCAAAGAGAGCTACGAGCAGGGCACAATCAGTCAGAGCGTGATTGAGGACCTGCGTAAACTGCTGATCGATTACAAACTGTACGAACTGAACCTGGCAAACTTAAGCGAGAGCGACCGCCTGGAATTTATTAAGCCTCACAGCCAGTGGTTAAAGGCATACCAGGCCGCCATGGCAACCCTGGATCTGCCGCGTGAAAAGTTCAGCGGTAGTTGTTGGGGCGAACCGGATATTTACTACGGTAAATTCGCCAAAGTGTGTGAACCGTTTCTGCGCCTGCTGCACCAAACCCTGCGCGGTACCGGCGACGCAATTAACGCCACCGCCGACAATTACCGCATTAATCCGCAGGTGGCCATCGACATCGAGCTGCATCTGCTGAACCGCTTCGAATTAGCCCTGGCCTGGGCCTTAGAGGCCAACATCAATGTTTATTGTAGTCAGAAAGCAATCGCCAAGAGCGAGGATGATAGCGAAGCCTACATCGCATACCTGGAGGAGACCTTCGACCGCAAGCAGAATTATCATGATTTTTATTGTCGTTTCCCGGTGCTGGCCCGTTGGTTAGCCCAAGTGACCTATTTCCTGTGCAATTTTGGCGAGGAAACCCTGCAACGCCTGACCAGCGATCGTGAGCAGATTGGCGCCACCTTCTTCGGCAGCAAGCCGATCAGTCAAATCAAAAGTTTTAAACTGGGTAAAAGCGACTACCATGCCGGTGCCAAGAGCGTGGTGATCGTGGAGCTGGAGCTGGCCAACAGCGAACCTGCCACCCTGGTGTATAAGCCTCGCAGCATTCAGAGCGAAGCCGGCATGCAGGGCCTGCTGGCACAGCTGAACCAAGATAAGGTGGTTCGCTTTGCCCACTATCAGGTGCTGTGTCGCGATGGTTATGGCTATGCAGAGTTCATCCCGAGCGGCAAAAACCAGGTTCAGAATAAAGAAGATCTGAAGAAATTCTACCAACAGCTGGGCGGCTTTTTAAGCATCTTCCATATCCTGGGTGGCGGCGATCTGCACCATGAAAACATCCTGGTTGCAGATGGTAACGCCTTCATCTGTGACTGCGAGACCGTGCTGGAGGTGCTGCCGCAAGGCATGGATAAACTGCCTGGTACAGTTTTAGATAGTGTGTTTAAAACAGCCATGCTGGACTGGCCTCGTGATAGCGCAAGCCCGGAGAACAGCGAGATGATGAGCATTAGTGGTTACAGCGGTGGCGAAAGCTATGAGGTTGCATTCACCGTGCCGCGCGTTAAGGAGCACCGTATGAGCCTGGATCAGGGTGTTGAGTACAAGACAGGTATCACCGTGGAACTGGAAGGTACAAATCGCATTTACTACAACGGTGAGATTGTGGATCCGCAGGACTATAAGGATAGCATTGTGGACGGTTTTAACCAGGTTTATACATGGTTTCAGCAGCACCCGACCAAGGCAATTACCCGTATTAAGGAGCTGTTTAGCAGTAGTTTAGTGCGCTTTATTAATTGGGGCACCCAAGCCTACGCCAAGAGCATTGTGGCCGTTCGTCACCCTAAGTGCCTGGCCGACCCTCTGGAGGTGGACCTGATCTTTAATAGCCTGAAAGAGCATAAACGCCAGTGGGACAAAAAGGGCGAACTGGCAGAGTTAGAGCTGGGCAGCCTGTGGCAACTGGATATCCCGATTTTCACCGCCTTAGCCGCCGAAAGCAAAGACTTAATCTTCAATTATCAGTATAGCGTTAGTGATACCTTAGCCATCAGCCCGTTAGACAATGCCAAACGCCGTCTCGAGCAGCTGAGCACCGAGAACCG
2104
3
TAGACAATGCCAAACGCCGTCTCGAGCAGCTGAGCACCGAGAACCGTATCCGTCAGAATCAATACATCTATACCAGCCTGAGCACCGACGAGATCAACAGTCCGTACTTTATCGCCGCAGCCGTTAACTATGCCCAGCAGATCGGCTGGCAGCTGTGTGAACAGCTGAGTAGCGATAGTAGCAAAGCACCTTGGCAGACATGGGACTACACCGCAACCGGCAAGCGCTTAGTGGATATCAGCGGCGACCTGTATGATGGCAGCGCCGGCATTTGTCTGTTCCTGGCCTACCTGGATGCCATCAAACCGCAAGTGGAATTCCGTCAGGCCGCCGAACGCGCCTTAGAATACAGTATCGAGAAACGTAACACAACCCTGATCGGTGCATTCCAGGGCGAAACAGGTCTGATTTATCTGCTGACCCATCTGGCACAGTTATGGGACAAACCGGCATTACTGGACCTGGCAGTTGACCTGAGCGACGAGCTGCTGCCGCGCATCAAACAGGACATCTACTTCGATATCCTGCATGGCGTTGCCGGTATCATTCCGGTTATGCTGGGCCTGGCCGAAGCAACCGGTGGTAAAGGCATTGATTGCGCATTACAGTGTGCCGAGCACCTGCTGGAGCAGGGTATTTACCAGGATAACACCCTGAGCTGGCCTCCGGGTCGTCCTGACCTGGTGCGCGGCAATTTTACAGGCTTCAGCCACGGTGCAAGCGGCATCGGTTGGGCCTTAATTATGCTGGGCTGCCACAGCAATAAGAGCGAGTATATTGAGGCAGGCCGTCAGGGCTTTGCCTACGAAGCCACACAGTTTGATGAGGAACAGCGCGATTGGTACGACTTACGCAAGAGCGTTACCACCGCAGATAGCAACGAACCTCACTTTGCCAACGCATGGTGCAATGGTGCCGCCGGTATTGGCCTGAGTCGCATCATTAGTTGGGCCGCCCTGGGCAAAACAGACGACGACATTCTGCGCGATGCCTACACCGCACTGAATGCAACCTTACGCAATTTCAACAAGCTGGGCAACGATAGCCTGTGTCATGGCAAGAGTGGTAACGCCGAGTTATTCCTGCGTTTTGCACAGCTGCGCGATACCCCGTATCTGCAAATGGAGGCAAACGTGCAGGCCCAGGCACAATGGCGCAACTTTGAAAAGGCACGCCGTTGGATGTGCGGTAGCACAGGCAACGATGTTTTCCCGGATTTAATGCTGGGCCTGGCCGGCATTGGCATGCACTTCCTGCGCCTGGCCTACCGTGAACGCGTTCCGAGCCCGTTATTATTAGATCCGCCTCCGCGCGCCATCGACTAAGTTTTGAGTTTTTTAATTCAATATTCAACACCCAAGAGTAATATAGTTTAGTTTGTTTATTAGGATCGAGATGAGCAAAGAAACCGTGATTGAATTTTATGAAGCAATTTTTGAAAGTCCGGAATTCATTCAGGAAATTAAAGCCATTACCCGCCAAGAGGAACTGATCAAACTGGGTGCCCGCAACGGCTACCATTTTACCATGGAAGACCTGGCCCAGGCCGACGCCAGCTATATCCCGAAGAATAACCAGCCGCTGATCAGCATTGATAGCGACGACCGTGCCCGCGAAGAACTGCCGCGCCCGTATCACTATGAATTCGAGTTCAGTGAGATCCCGGGCTTCGAAGAGATCGATCGTGAACTGAAAAAACTGCAGATCAAACCGAACACCGTGGATCTGGACCTGTACGAGAAGAGTTTCCGCGAAGAAGATTTTAAATTTAACTATATTAGCCCGACCGTGCCGGGCTTCCGCCAGTATTACTACAAGAGCCTGAAAAGCTATCTGGATCTGCCTAGCCCTCAGCCGGAATATGCCTGGCGTCCGTTTCACCTGATCAATCTGGATTGCCACGTGGAGGATCCGCTGTACGAGGATTATTTCCAGACCAAGGTGCGTCTGCTGAAGCTGCTGGAGAATTGCCTGGAGACAGAGCTGCGCTTTAGTGGCAGCCTGTGGTATCCGCCGAACGCCTATCGCCTGTGGCACACCAATGAAACCCAGCCGGGCTGGCGCATGTACCTGGTGGATTTCGACAATTTTGACGACAACCAGGAAGGCGAGGTGTTCTTCCGCTACATGAATCCGGAGACCAAGGAACTGGTGACCCTGGCCGAGAAACCGAAAAT
2177
divA-divamide
B
GTGGAACTGCTGAACAACGCCCTGGGCGAAATCGATATTGCCGCCGAATGCGCGTCGACCTGTAGCAGCGGCCCGATCACCGCGATCTGCGATGGTACCACCAAATAAGTTGTTTTACTAGCATATTATATTATTAATATGTCTGGGCTACCACCACATTGTGAAGCTAGCCTCATTAAATTGATTGTTGGGCATGGTTAG
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary Table 4. DNA primers used in this study. Primers used in the construction and sequencing of pDiv, pDiv-2, and pRSFDuet-divMT.
# Primer name Sequence (5’-3’) 1 div1a-fwd CTCGTATGTTGTGTGGAATTGTG 2 div2-rev CGGTTCTCGGTGCTCAG 3 div3-fwd TAGACAATGCCAAACGCC 4 div1b-rev CGGGTAAGCCGCGAC 5 divM-203-fwd CGATCGCCAGCACAAC 6 divM-1424-rev CCAGTCCAGCATGGCTG 7 divM-2818-fwd GCAGGCCGTCAGGG 8 divX-650-rev AGCCCGGCTGGGTTTC 9 divX-580-fwd GAGACAGAGCTGCGCTTTAG 10 divMT-79-rev CAATGAAGAAGGCGTTCAG 11 divMT-20-fwd CGTTCGAACTGAAAGCCTATG 12 divM-203-fwd CGATCGCCAGCACAAC 13 divM-1977-f CTTAGCCGCCGAAAGC 14 divX-461-f TGCCTAGCCCTCAGCC 15 divHypo-780-f CGCATCCGTAATTAATCCC
16 intergenic-divM-native-fwd GAGCAATATTTTCCATCTCTCTTGCTCCATATG
17 intergenic-divM-native-rvs
GGGTGTTGAATATTGAATTAAAAAACTCAAAACTCAATC
18 divMp-fwd GTGGAACTGCTGAACAAC
19 divM-non-optimized-pET16b-Gibson-fwd cgaaggtcgtcatATGCTTGATGATTTTTCTCTCC
20 divM-native-fwd-1 CTTTAGCCTGGGCTCTTG 21 divM-native-fwd-2 GGACAGTGCTTGATTCAG 22 divM-native-rvs-4 CTTCATCAAATTGGGTCG
23 divMT-MCS1-fwd TTCGAGCTCGATGGAAAATAATAATTATCCGTTCG
24 divMT-MCS1-rvs TATGCGGCCGCTTACACTGCGATACCCACG
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary Table 5. Summary of quantification of divamide material by NMR. Divamides were quantified using linear equations shown in Supplementary 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
Sample MW (g/mol) mg/ml mg/ml SD mM SD yield (µg)
L-Tyr 181.19 0.326 0.351 0.00950 1.94 0.0524 42.1
E11-036 divamide A (1) 2020.92 –
0.692 0.167 0.342 0.0824 77.8
0.605 0.120 0.299 0.0596
E. coli divamide A (1) 2020.92 –
1.34 0.251 0.664 0.124 157
1.27 0.198 0.630 0.0980
desmethyl divamide A (9) 1977.92 –
3.85 2.48 1.95 1.25 326
1.59 0.0881 0.802 0.0445
divamide B (2) 1930.83 – 0.983 0.328 0.509 0.170
100 0.691 0.0779 0.358 0.0403
divamide C (3) 2077.95 – 0.272 0.0736 0.131 0.0354
31.7 0.256 0.0797 0.123 0.0383
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary Table 6. Summary of quantification of divamide material by LC/MS. Divamides were quantified using linear equations shown in Supplementary 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/MS was in agreement with the NMR concentration (∆ -5.92%). The solution of 4 used in flow cytometry based assays was quantified using curve ii (*).
Sample MW (g/mol) mM SD mg/ml SD yield (µg)
1 (E. coli) 2020.92 0.624 0.0363 1.26 0.0733 151.4
10-12
1977.86 0.193 0.114 0.381 0.226 45.7
2048.9 0.0212 0.00269 0.0433 0.00550 5.2
2119.94 0.0232 0.00798 0.0491 0.0169 5.9
2233.02 0.369 0.132 0.825 0.295 99.0
mix 0.606 0.257 1.30 0.544 155.7
9
1977.86 0.440 0.0848 0.871 0.168 104.5
2048.9 0.173 0.0373 0.355 0.0764 42.6
2119.94 0.0701 0.0199 0.149 0.0422 17.8
mix 0.684 0.148 1.37 0.286 164.9
21
2020.92 0.567 0.0343 1.15 0.0694 137.4
2034.9 0.409 0.0666 0.832 0.136 99.8
mix 0.976 0.0324 1.98 0.205 237.3
4
2040.85 0.448 0.0666 0.914 0.136 109.7
2056.85 0.450 0.0624 0.925 0.128 111.1
2072.84 0.148 0.0253 0.306 0.0526 36.7
mix 1.05 0.129 2.15 0.317 257.5
biotinylated divamide A
2430.11 0.103 0.0354 0.250 0.0861 30.0
2684.26 0.115 0.0384 0.309 0.103 37.1
mix 0.218 0.0737 0.559 0.189 67.1
biotinylated cinnamycin
2491.08 0.435 0.0491 1.08 0.122 129.9
2507.08 0.203 0.0290 0.508 0.0726 61.0
mix 0.637 0.0781 1.59 0.195 190.9
4* 2040.85 1.02 0.107 2.08 0.217 624.0
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary Table 7. Summary 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)
Sample Plate Replicate ng/ml nM Total events Gate 1 events gate1/total % Gate 2 events gate2/gate1
% 1 (E11-036) 1 1 4.03 3.73 25709 20303 79 48 0.24 1 (E11-036) 1 1 3.56 3.25 34357 24825 72.3 1339 5.39 1 (E11-036) 1 1 3.08 2.77 30644 20629 67.3 13704 66.4 1 (E11-036) 1 1 2.60 2.30 22842 14034 61.4 10648 75.9 1 (E11-036) 1 1 2.13 1.82 23688 14969 63.2 10620 70.9 1 (E11-036) 1 1 1.65 1.34 25474 17804 69.9 12862 72.2 1 (E11-036) 1 1 1.17 0.87 28341 18654 65.8 12884 69.1 1 (E11-036) 1 1 0.69 0.39 24722 16009 64.8 11878 74.2 1 (E11-036) 1 2 4.03 3.73 32054 23720 74 29 0.12 1 (E11-036) 1 2 3.56 3.25 30221 21169 70 1773 8.38 1 (E11-036) 1 2 3.08 2.77 30127 20105 66.7 12221 60.8 1 (E11-036) 1 2 2.60 2.30 23970 16117 67.2 10611 65.8 1 (E11-036) 1 2 2.13 1.82 26978 17324 64.2 11358 65.6 1 (E11-036) 1 2 1.65 1.34 29093 20576 70.7 14223 69.1 1 (E11-036) 1 2 1.17 0.87 27025 17963 66.5 12971 72.2 1 (E11-036) 1 2 0.69 0.39 19552 11686 59.8 8175 70 1 (E11-036) 1 3 4.03 3.73 35673 28266 79.2 42 0.15 1 (E11-036) 1 3 3.56 3.25 40420 30701 76 3291 10.7 1 (E11-036) 1 3 3.08 2.77 22372 14386 64.3 8911 61.9 1 (E11-036) 1 3 2.60 2.30 28059 16233 57.9 12437 76.6 1 (E11-036) 1 3 2.13 1.82 25145 15337 61 10980 71.6 1 (E11-036) 1 3 1.65 1.34 33887 20245 59.7 14458 71.4 1 (E11-036) 1 3 1.17 0.87 27307 18491 67.7 12590 68.1 1 (E11-036) 1 3 0.69 0.39 26508 16868 63.6 12030 71.3
10-12 1 1 4.43 4.10 20727 4617 22.3 52 1.13 10-12 1 1 3.95 3.62 36096 26327 72.9 128 0.49 10-12 1 1 3.48 3.15 35109 25472 72.6 3776 14.8 10-12 1 1 3.00 2.67 28106 18495 65.8 11101 60 10-12 1 1 2.52 2.19 25192 15286 60.7 9340 61.1 10-12 1 1 2.05 1.72 22842 15781 69.1 9131 57.9 10-12 1 1 1.57 1.24 31067 21842 70.3 14215 65.1 10-12 1 1 1.09 0.76 25192 16078 63.8 11735 73 10-12 1 2 4.43 4.10 19740 5140 26 83 1.61 10-12 1 2 3.95 3.62 33417 24191 72.4 169 0.7 10-12 1 2 3.48 3.15 43569 30048 69 4816 16 10-12 1 2 3.00 2.67 31913 21534 67.5 10323 47.9 10-12 1 2 2.52 2.19 22654 14891 65.7 10407 69.9 10-12 1 2 2.05 1.72 25662 17295 67.4 12095 69.9 10-12 1 2 1.57 1.24 20351 13575 66.7 9337 68.8 10-12 1 2 1.09 0.76 26649 18505 69.4 12651 68.4 10-12 1 3 4.43 4.10 17437 3500 20.1 89 2.54 10-12 1 3 3.95 3.62 37647 27358 72.7 295 1.08 10-12 1 3 3.48 3.15 30456 22565 74.1 2719 12 10-12 1 3 3.00 2.67 32665 21716 66.5 13721 63.2 10-12 1 3 2.52 2.19 28952 17632 60.9 12318 69.9 10-12 1 3 2.05 1.72 27777 18509 66.6 12504 67.6 10-12 1 3 1.57 1.24 31584 22211 70.3 11545 52 10-12 1 3 1.09 0.76 25145 17181 68.3 12257 71.3
uninfected 1 1 0 0 28999 22803 78.6 78 0.34 uninfected 1 2 0 0 35814 29264 81.7 59 0.2 uninfected 1 3 0 0 42300 34471 81.5 109 0.32
HIV 1 1 0 0 28012 18509 66.1 12496 67.5 HIV 1 2 0 0 31866 19590 61.5 13496 68.9
9 2 1 4.43 4.14 24863 5348 21.5 35 0.65 9 2 1 3.96 3.66 50000 38095 76.2 1582 4.15 9 2 1 3.48 3.18 50000 39400 78.8 8268 21 9 2 1 3.00 2.71 49256 33383 67.8 28461 85.3 9 2 1 2.52 2.23 48739 31424 64.5 26468 84.2 9 2 1 2.05 1.75 45167 22766 50.4 19718 86.6 9 2 1 1.57 1.27 46342 25591 55.2 21787 85.1 9 2 1 1.09 0.80 47376 28926 61.1 25343 87.6 9 2 2 4.43 4.14 25380 7430 29.3 109 1.47 9 2 2 3.96 3.66 50000 40576 81.2 1155 2.85
Nature Chemical Biology: doi:10.1038/nchembio.2537
continued… 9 2 2 3.48 3.18 50000 41132 82.3 11127 27.1 9 2 2 3.00 2.71 45919 31054 67.6 26401 85 9 2 2 2.52 2.23 50000 32579 65.2 28671 88 9 2 2 2.05 1.75 50000 30940 61.9 25388 82.1 9 2 2 1.57 1.27 48692 29871 61.3 27115 90.8 9 2 2 1.09 0.80 44697 27350 61.2 23664 86.5 9 2 3 4.43 4.14 33229 9604 28.9 67 0.7 9 2 3 3.96 3.66 50000 40619 81.2 1369 3.37 9 2 3 3.48 3.18 50000 41092 82.2 12166 29.6 9 2 3 3.00 2.71 50000 33941 67.9 26478 78 9 2 3 2.52 2.23 47141 29983 63.6 25716 85.8 9 2 3 2.05 1.75 50000 31162 62.3 26690 85.6 9 2 3 1.57 1.27 48269 29258 60.6 24387 83.4 9 2 3 1.09 0.80 50000 29807 59.6 25301 84.9 4 2 1 4.72 4.41 25756 351 1.36 87 24.8 4 2 1 4.24 3.93 19787 262 1.32 8 3.05 4 2 1 3.76 3.45 25662 564 2.2 9 1.6 4 2 1 3.28 2.97 50000 40600 81.2 1758 4.33 4 2 1 2.81 2.50 50000 39132 78.3 15413 39.4 4 2 1 2.33 2.02 48880 29916 61.2 23844 79.7 4 2 1 1.85 1.54 50000 28187 56.4 22313 79.2 4 2 1 1.38 1.07 43287 23399 54.1 19711 84.2 4 2 2 4.72 4.41 25051 336 1.34 79 23.5 4 2 2 4.24 3.93 22184 269 1.21 11 4.09 4 2 2 3.76 3.45 20069 462 2.3 10 2.16 4 2 2 3.28 2.97 50000 36501 73 4802 13.2 4 2 2 2.81 2.50 50000 37823 75.6 19498 51.6 4 2 3 2.81 2.50 44039 28139 63.9 16545 58.8 4 2 3 2.33 2.02 47423 22478 47.4 17970 79.9 4 2 3 1.85 1.54 41595 19761 47.5 15915 80.5 4 2 3 1.38 1.07 41830 16014 38.3 13680 85.4
uninfected 2 1 0 0 50000 42500 85 20 0.047 uninfected 2 2 0 0 50000 42514 85 51 0.12 uninfected 2 3 0 0 50000 42584 85.2 48 0.11
HIV 2 1 0 0 44039 23106 52.5 19383 83.9 HIV 2 2 0 0 48222 23674 49.1 19333 81.7
4 uninfected 3 1 5.32 5.01 27354 211 0.77 5 2.37 4 uninfected 3 1 4.84 4.53 24675 200 0.81 9 4.5 4 uninfected 3 1 4.36 4.05 19035 747 3.92 3 0.4 4 uninfected 3 1 3.89 3.58 22748 439 1.93 6 1.37 4 uninfected 3 1 3.41 3.10 47376 34504 72.8 204 0.59 4 uninfected 3 1 2.93 2.62 50000 38324 76.6 207 0.54 4 uninfected 3 2 5.32 5.01 27730 225 0.81 5 2.22 4 uninfected 3 2 4.84 4.53 27871 158 0.57 3 1.9 4 uninfected 3 2 4.36 4.05 3431 204 5.95 10 4.9 4 uninfected 3 2 3.89 3.58 19646 282 1.44 5 1.77 4 uninfected 3 2 3.41 3.10 50000 35255 70.5 111 0.31 4 uninfected 3 2 2.93 2.62 46577 31697 68.1 176 0.56 4 uninfected 3 3 5.32 5.01 26649 240 0.9 5 2.08 4 uninfected 3 3 4.84 4.53 26273 203 0.77 3 1.48 4 uninfected 3 3 4.36 4.05 24017 266 1.11 2 0.75 4 uninfected 3 3 3.89 3.58 20257 186 0.92 3 1.61 4 uninfected 3 3 3.41 3.10 45543 30742 67.5 216 0.7 4 uninfected 3 3 2.93 2.62 50000 37835 75.7 72 0.19
4 3 1 5.32 5.01 20492 217 1.06 5 2.3 4 3 1 4.84 4.53 8742 183 2.09 2 1.09 4 3 1 4.36 4.05 8554 347 4.06 9 2.59 4 3 1 3.89 3.58 7990 189 2.37 4 2.12 4 3 1 3.41 3.10 40044 28271 70.6 4916 17.4 4 3 1 2.93 2.62 32054 18794 58.6 14551 77.4 4 3 2 5.32 5.01 14194 165 1.16 1 0.61 4 3 2 4.84 4.53 9447 161 1.7 7 4.35 4 3 2 4.36 4.05 6110 225 3.68 8 3.56 4 3 2 3.89 3.58 10904 304 2.79 3 0.99 4 3 2 3.41 3.10 41642 29781 71.5 2066 6.94 4 3 2 2.93 2.62 32806 23319 71.1 8731 37.4 4 3 3 5.32 5.01 16638 237 1.42 7 2.95 4 3 3 4.84 4.53 3901 140 3.59 6 4.29 4 3 3 4.36 4.05 7426 327 4.4 10 3.06 4 3 3 3.89 3.58 10857 511 4.71 7 1.37 4 3 3 3.41 3.10 47517 31483 66.3 3081 9.79 4 3 3 2.93 2.62 37694 27434 72.8 7113 25.9
uninfected 3 1 0 0 48504 36417 75.1 111 0.3 uninfected 3 2 0 0 47141 36318 77 68 0.19 uninfected 3 3 0 0 50000 38262 76.5 53 0.14
HIV 3 1 0 0 29422 6308 21.4 5583 88.5 HIV 3 2 0 0 38446 7172 18.7 6605 92.1 HIV 3 3 0 0 28952 5655 19.5 5071 89.7
Nature Chemical Biology: doi:10.1038/nchembio.2537
continued... 9 uninfected 4 1 4.08 3.78 47564 32405 68.1 77 0.24 9 uninfected 4 1 3.60 3.31 50000 34891 69.8 72 0.21 9 uninfected 4 1 3.13 2.83 50000 33779 67.6 108 0.32 9 uninfected 4 1 2.65 2.35 50000 33843 67.7 96 0.28 9 uninfected 4 2 5.03 4.74 6815 169 2.48 6 3.55 9 uninfected 4 2 4.56 4.26 18001 7260 40.3 73 1.01 9 uninfected 4 2 4.08 3.78 50000 35984 72 54 0.15 9 uninfected 4 2 3.60 3.31 50000 35559 71.1 93 0.26 9 uninfected 4 2 3.13 2.83 50000 33542 67.1 55 0.16 9 uninfected 4 2 2.65 2.35 50000 34316 68.6 83 0.24 9 uninfected 4 3 5.03 4.74 9353 308 3.29 6 1.95 9 uninfected 4 3 4.56 4.26 28012 12633 45.1 94 0.74 9 uninfected 4 3 4.08 3.78 47517 30741 64.7 105 0.34 9 uninfected 4 3 3.60 3.31 50000 33967 67.9 72 0.21 9 uninfected 4 3 3.13 2.83 32289 22481 69.6 91 0.4 9 uninfected 4 3 2.65 2.35 50000 32590 65.2 75 0.23
9 4 1 5.03 4.74 28059 148 0.53 5 3.38 9 4 1 4.56 4.26 50000 28362 56.7 846 2.98 9 4 1 4.08 3.78 50000 35477 71 3646 10.3 9 4 1 3.60 3.31 50000 29889 59.8 19621 65.6 9 4 1 3.13 2.83 50000 28213 56.4 19441 68.9 9 4 1 2.65 2.35 50000 20240 40.5 15799 78.1 9 4 2 5.03 4.74 13818 106 0.77 8 7.55 9 4 2 4.56 4.26 50000 29649 59.3 748 2.52 9 4 2 4.08 3.78 50000 37911 75.8 3040 8.02 9 4 2 3.60 3.31 50000 26418 52.8 17202 65.1 9 4 2 3.13 2.83 50000 25183 50.4 16939 67.3 9 4 2 2.65 2.35 50000 15324 30.6 12028 78.5 9 4 3 4.56 4.26 50000 32143 64.3 369 1.15 9 4 3 4.08 3.78 50000 37907 75.8 1151 3.04 9 4 3 3.60 3.31 50000 31510 63 12721 40.4 9 4 3 3.13 2.83 50000 20861 41.7 14394 69
uninfected 4 1 0 0 50000 34860 69.7 122 0.35 uninfected 4 2 0 0 50000 34392 68.8 132 0.38 uninfected 4 3 0 0 50000 32768 65.5 196 0.6 uninfected 4 4 0 0 50000 33084 66.2 104 0.31 uninfected 4 5 0 0 50000 30388 60.8 94 0.31
HIV 4 1 0 0 50000 28937 57.9 15798 54.6 HIV 4 2 0 0 50000 27338 54.7 16680 61 HIV 4 3 0 0 50000 22737 45.5 16926 74.4
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary Table 8. Summary table of assay data. IC50 (anti-HIV), CC50 (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 <). Some 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 Sample IC50 (µM) CC50 (µM) Therapeutic index
Cytoprotection
1 0.225 2.63 11.7 9 0.327 3.38 10.3 5 ND <27.0 –
10-12 >0.035 <8.42 NC 21 0.292 4.43 15.2 2 ND <27.9 – 3 ND ND – 4 ND 0.144 –
Flow cytometry
1 1.03 >5.35 NC 9 1.99* 19 9.55
10-12 0.874 <14.4 NC 4 0.328 2.05* 6.25
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary Table 9. Divamide-like masses observed from tunicate extracts. Unidentified ions observed from Didemnum molle extracts by LC/MS resembled those of characterized divamides in m/z and isotope distribution. Some masses appear to be derivatives of 1-3, but others may reflect alternative amino acid sequences at various stages of post-translational modification. Several of these display evidence of Hoffmann elimination in negative ionization mode. Thus, additional, uncharacterized divamides are believed to be encoded by E11-037.
Observed m/z (z = 2) Source ID 1010.9 E11-036 1 880.5 E11-037 ? 893.5 E11-037 ? 900.5 E11-037 ? 935.9 E11-037 ? 942.9 E11-037 ? 944.9 E11-037 2 - 3 x Me 951.9 E11-037 2 - 2 x Me 953.9 E11-037 ? 958.9 E11-037 2 - 1 x Me 965.9 E11-037 2 973.9 E11-037 2 + 1 x O 974.9 E11-037 ? 982.9 E11-037 ? 1010.5 E11-037 ? 1016.5 E11-037 ? 1018.5 E11-037 ? 1025.5 E11-037 ? 1026.5 E11-037 ? 1027.5 E11-037 ? 1039.5 E11-037 3 1047.5 E11-037 3 + 1 x O 1053.9 E11-037 ? 1089.5 E11-037 ? 1091.9 E11-037 ? 1098.9 E11-037 ? 1099.9 E11-037 ? 1127.6 E11-037 ? 1146.0 E11-037 ? 1185.5 E11-037 ? 1203.6 E11-037 ?
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary 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 100 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.
0.06
Large1(n=4)
Large3(n=3)Large4
Large2E11-036
Small1Small2
Brown1(n=7)Brown3Gray1
Gray4Gray2(n=3)
Gray3E11-037
White3(n=2)White4(n=4)
White2(n=4)
White1
D.psammatode
Large5
0.060.06substitution/site
68
7
2
2
32
100
53
99
72
93
76
46
95
87
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary 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 E11-036 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 90 Da mass shift (iii), indicating the presence of Lal. c, MS/MS 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 MS/MS fragmentation due to the
ladder P L FT W1 W2 T E1 E2 E3 E4 ladder kDa
27.0
66.4
34.642.755.6
20.0
14.3
32.6kDa
99099510001005101010151020102510301035104010451050 m/z
989.8
1010.80
100
%
0
100
%
94094595095596096597097598098599099510001005m/z
944.7965.7
965.7
944.7
0
100
%
0
100
%
OH!N!H!
S!S!S!
ECASTCSFGIVTIVCDGTTK!
0
100
%
945950955960965970975980985990995m/z
944.80
945.83
989.78
∆=88Da
∆=90Da
ECASTCSFGIVTIVCDGTTK!OH!
ECASTCSFGIVTIVCDGTTK!OH!N!
H!
NiCl2,NaBH455˚C,30min
ECASTCSFGIVTIVCDGTTK!S!
OH!
S!S!
0
100
%
0
100
%
100200300400500 6007008009001000110012001300m/z
86.1
1159.2988.4804.2
889.2
691.2592.2
521.1
390.2
147.1y1
y4
y5y6
y7y8 y9 y10 y12 y13
1305.3
ECASTCSFGIVTIVCDGTTK!OH!
y13y12y10
y9y8y7y6
y5y4y2y3
ECASTCSFGIVTIVCDGTTK!S!
OH!
S!S!
ECASTCSFGIVTIVCDGTTK!OH!N!
H!86.1
604.1703.2426.1272.0
199.1
0
100
%
0
100
%
0
100
%
0
100
%
945950955960965970975980985990995m/z
944.80
945.83
989.78
∆=88Da
∆=90Da
ECASTCSFGIVTIVCDGTTK!OH!
ECASTCSFGIVTIVCDGTTK!OH!N!
H!
NiCl2,NaBH455˚C,30min
ECASTCSFGIVTIVCDGTTK!S!
OH!
S!S!
0
100
%
0
100
%
100200300400500 6007008009001000110012001300m/z
86.1
1159.2988.4804.2
889.2
691.2592.2
521.1
390.2
147.1y1
y4
y5y6
y7y8 y9 y10 y12 y13
1305.3
ECASTCSFGIVTIVCDGTTK!OH!
y13y12y10
y9y8y7y6
y5y4y2y3
ECASTCSFGIVTIVCDGTTK!S!
OH!
S!S!
ECASTCSFGIVTIVCDGTTK!OH!N!
H!86.1
604.1703.2426.1272.0
199.1
0
100
%
0
100
%
0
100
%
B C
i
ii
iii
D E
i
ii
i
ii
iii
DivMT purificationladderPLFTW1W2TE1E2E3E4
32.6kDa
14.3
20.0
27.0
34.642.755.666.4
MolecularW
eight(kD
a)A i ii
DNALength(kbp
)
3423bp
10.0
4.03.02.0
1.00.5
10.0
4.03.02.0
1.00.5
5
9
1
17
2
m/z
m/z
(Me3)N
SSS
ECASTCSFGIVTIVCDGTTK
NH
OH
SSS
ECASTCSFGIVTIVCDGTTK
NH
OH+ (Me3)N
SSS
ECASTCSFGIVTIVCDGTTK
NH
OH
SSS
ECASTCSFGIVTIVCDGTTK
NH
OH+
(Me3)N
SSS
ECASTCSSGPITAICDGTTK
NH
OH
SSS
ECASTCSSGPITAICDGTTK
NH
OH+ (Me3)N
SSS
ECASTCSSGPITAICDGTTK
NH
OH
SSS
ECASTCSSGPITAICDGTTK
NH
OH+
ECASTCSFGIVTIVCDGTTK
NH
OH
ECASTCSFGIVTIVCDGTTKOH
SSS
ECASTCSFGIVTIVCDGTTKOH
ECASTCSFGIVTIVCDGTTK
NH
OH
ECASTCSFGIVTIVCDGTTKOH
SSS
ECASTCSFGIVTIVCDGTTKOH
ECASTCSFGIVTIVCDGTTK
NH
OH
ECASTCSFGIVTIVCDGTTKOH
SSS
ECASTCSFGIVTIVCDGTTKOH
NiCl2,NaBH4,55˚C,30min
m/z
ECASTCSFGIVTIVCDGTTKOH
y13y12
y10y9y8y7y6y5y4
y2y3
Nature Chemical Biology: doi:10.1038/nchembio.2537
presence of Lal (iii). d, DivMT was purified on Ni-NTA resin from the cell lysate of pRSFDuet-DivMT-expressing E. coli BL21(DE3) and column fractions analyzed by SDS-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 = 50 mM, 2 = 100 mM, 3 = 200 mM, 4 = 500 mM imidazole). A band around 32.6 kDa, corresponding to His6-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:10.1038/nchembio.2537
Supplementary Figure 3. Quantification of divamide material. a, Standard curves for quantification of low abundance materials by NMR were generated using a dilution series of L-Trp in H2O, the concentration of which was determined by UV-absorbance (e280, Trp = 5170.9 nm-
1). The absolute integral was plotted as a function of concentration using three different aromatic proton signals: HE3 (i), HZ3 (ii), and methylene HB2 (iii). The linear equations generated were used to determine yields of divamide analytes of unknown concentration in Supplementary Table 5. b, Standard curves for quantification by LC/MS 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 Supplementary Table 6. The majority of analytes were quantified with one curve (i), while a second curve (ii) was used to quantify additional 4.
i
ii
iii
y=0.5301x+0.022R²=0.99967
012345678
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Absolutein
tegral
Concentration(mM)
L-Trp standardcurve(! 7.62ppm)
y=0.5358x- 0.0013R²=0.99982
012345678
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Absolutein
tegral
Concentration(mM)
L-Trp standardcurve(! 7.17ppm)
y=0.5209x- 0.0171R²=0.99975
012345678
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Absolutein
tegral
Concentration(mM)
L-Trp standardcurve(! 3.19ppm)
A
Nature Chemical Biology: doi:10.1038/nchembio.2537
continued…
y=231.14xR²=0.98708
0500100015002000250030003500400045005000
0 5 10 15
Area
Und
erCurve(A
UC)
Concentration(µM)
Desmethyl divamide Astandardcurve
y=205.95xR²=0.99289
0
1000
2000
3000
4000
5000
6000
7000
0 5 10 15 20 25 30 35
Area
Und
erCurve(A
UC)
Concentration(µM)
Desmethyl divamide Astandardcurve
i
ii
B
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary Figure 4. Additional cytoprotection and flow-cytometry assay dose-response 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 0.1 or 0.05 mg/ml) responses. Points represent an average of three assay responses per dose (error bars = ± standard deviation). R2 values are included for compounds for which curves could be fit with equation 7 (see online methods) or a single four-parameter IC50 equation. Curves could not be fit for E11-036-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 CEMTART 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 HIV-infected cells, yielding full anti-HIV dose-response curves but only partial cytotoxicity curves.
%re
lativesurvival 21
R2 =0.9998IC50=0.292µMCC50=4.43µM
9
R2 =0.9976IC50=0.327µMCC50=3.38µM
1 (E11-036)150
100
50
0
-50
-100
-150
logconcentration(ng/ml)
%liveinfected
cells
(p24
-FITC)
%livecells
(viability-DAPI)
Doserange1
HIVinfected
10-12
1 2 3 4 50
R2 =0.9935IC50=0.874µMCC50=<14.4µM
1(E11-036)100
80
60
40
20
0
1 2 3 4 50
R2 =0.9987IC50=1.03µMCC50=>5.35µM
A
B100
80
60
40
20
0
1 2 3 4 50 1 2 3 4 50 1 2 3 4 50
Uninfectedp24Infectedp24Uninfectedviability
Controls
Infectedviability
UninfectedAZTInfected
Controls
Nature Chemical Biology: doi:10.1038/nchembio.2537
Supplementary Figure 5. Chemical diversity within the divamide family. a, Hoffman elimination of trimethylamine from a quaternary N-trimethylamine results in a net loss of 60 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/MS in negative ionization mode. b, All divamide-like lanthipeptide gene clusters that could be identified by BLAST 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 100% conserved across all pathways. Some 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 10802 and contains hydroxyproline, presumably introduced by the putative proline hydroxylase located in the first cluster (“orf”) (Yang, J. J., 2016, 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 14695 (the cluster described here was identified by BLAST search from Actinomadura atramentaria DSM 43919), includes only the essential divamide-like biosynthetic genes (Kodani, S. et al., 2016, J. Ind. Microbiol. Biotechnol. 43, 1159-1165). c, Alignment of LanA precursor proteins from the divamide extended family, generated using ClustalW2 and then manually adjusted. The
10051010101510201025103010351040m/z
930935940945950955960965970975980m/z
9809859909951000100510101015 m/
100
%
0
100
%
0
100
%
0100
%
0
100
%
0
100
%
0
[M+H]2+ =1011.1
∆=63Da
∆=63Da
∆=63Da
[M+H]2+ =965.8
[M+H]2+ =1039.4
[M-2H]2- =1007.8
[M-2H]2- =934.3
i
ii
iii
pos
pos
pos
neg
neg
neg
930935940945950955960965970975980m/z
9809859909951000100510101015 m/z
100
%
0
100
%
0
100
%
0100
%
0
100
%
0
100
%
0
[M+H]2+ =1011.0
∆=63Da
∆=63Da
∆=63Da
[M+H]2+ =965.9
[M+H]2+ =1039.5
[M-2H]2- =1008
[M-2H]2- =934.45
[M-2H]2- =979.5i
ii
iii
pos
pos
pos
neg
neg
neg
A
Nature Chemical Biology: doi:10.1038/nchembio.2537
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:10.1038/nchembio.2537
continued…
lanA:WP_015207184.1
Cylindrospermum stagnale PCC7417Genbank:CP003642.1
lanA:WP_015208675.1
lanA-1:EGJ35561.1Mooreaproducens 3LGenbank:GL890819.1
Oscillatoria sp.PCC10802Genbank:KB235949.1
KB235948.1
lanA:WP_017720753.1 oscillamycin lanA:WP_017718744.1
cinnamycin B lanA:WP_019631332.1Actinomadura atramentaria DSM43919Genbank:KB907211.1
Actinomadura oligospora ATCC43269Genbank:JADG01000011.1
JADG01000003.1
lanA:WP_026413721.1 lanA:WP_026411382.1
Frankiasp.EUN1fGenbank:ADGX01000154.1 lanA:WP_006544294.1
Nocardiopsis potens DSM45234Genbank:ANBB01000008.1
lanA:WP_017591584.1
StreptomycesroseoverticillatusstrainNRRLB-3500
Genbank:JOFL01000016.1lanA:WP_030367889.1
orf1Aorf2MTorf3
Scytonema millei VB511283Genbank:JTJC02000234.1
lanA:WP_069351382.1
Aorf2MT
NXMA-1A-2T
TMorf A Xorf MAN
TXMA
orf XMAN
cinT/HXMA-1N A-2
YXMANcinH cinT
orf cinH cinT XMANcinR1cinorf11
YcinH cinT XMANcinR cinK cinorf1011cinR1
2kb
B
ii
cinT XMANActinomadura macra NBRC14102
Genbank:BCQT01000003.1
Actinomadura rubrobrunea NBRC15275Genbank:BCQU01000010.1
cinT XMAN
lanA:WP_067451807.1
lanA:WP_067909139.1
cinH cinT XMANcinR1cinorf11Nocardiopsis trehalosi NBRC14201
Genbank:BCRK01000002.1 lanA:WP_067962807.1
Marinaactinospora thermotoleransDSM45154
Genbank:NZ_FUWS01000010.1lanA:WP_078762901.1
cinH cinT XM ANcinR1 cinorf11
Streptomycesafricanus NRRLB-24243Genbank:NZ_MUKA01000041.1 lanA:WP_086559519.1
cinH cinT XM ANcinR cinK cinorf1011cinR1cinorf1213
Anaerolineaceae bacterium4572_78Genbank:NBMH01000181.1 lanA:OQY43268.1
orfMA
iii
i
Nature Chemical Biology: doi:10.1038/nchembio.2537
continued…
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1.
Proc
hlor
ondi
dem
niE
11-0
36(d
ivam
ideA
)2.
Pr
ochl
oron
dide
mni
E11
-037
(div
amid
eB)
3.
Anae
rolin
eace
aeba
cter
ium
457
2_78
4.
Cyl
indr
ospe
rmum
stag
nale
PC
C 7
417
5.
Cyl
indr
ospe
rmum
stag
nale
PC
C 7
417
(2)
6.
Osc
illato
riasp
. PC
C 1
0802
7.
Scyt
onem
am
illeiV
B51
1283
8.
Moo
rea
prod
ucen
s3L
9.
Moo
rea
prod
ucen
s3L
(2)
10. O
scilla
toria
sp.P
CC
108
02 (2
; osc
illam
ycin
)11
.Fra
nkia
sp.
EU
N1f
12. S
trept
omyc
es ro
seov
ertic
illatu
sN
RR
L B
-350
013
. Act
inom
adur
aat
ram
enta
riaD
SM
439
19 (c
inna
myc
inB
)14
. Act
inom
adur
aol
igos
pora
ATC
C 4
3269
15. A
ctin
omad
ura
olig
ospo
raA
TCC
432
69 (2
)16
. Act
inom
adur
am
acra
NR
BC
141
0217
. Act
inom
adur
aru
brob
rune
aN
RB
C 1
5275
18. S
trept
omyc
es a
frica
nus
NR
RL
B-2
4243
19. N
ocar
diop
sis
treha
losi
NR
BC
142
0120
. Mar
inac
tinos
pora
ther
mot
oler
ans
DS
M 4
5154
21. N
ocar
diop
sis
pote
nsD
SM
452
3422
. Stre
ptom
yces
cin
nam
oneo
usD
SM
400
05(c
inna
myc
in)
23. S
trept
omyc
es c
inna
mon
eous
ATC
C 1
2686
(dur
amyc
in)
24. S
trept
omyc
es c
inna
mon
eous
ATC
C 1
2686
(2)
25. S
trept
over
ticilli
umsp
.R20
75 (d
uram
ycin
B)
26. S
trept
over
ticilli
umsp
. R21
07 (d
uram
ycin
C)
27. S
trept
omyc
es s
p. N
o. A
647P
-2 (a
ncov
enin
)
* *
*
* *
*
*
*
* *
*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27C
Nature Chemical Biology: doi:10.1038/nchembio.2537