supplementary table 1. annotation of the divamide …...supplementary table 1. annotation of the...

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


LanM-like (cd04792) class II

lanthionine synthetase

divX ARD09204 918 WP_017718741.1 58 hypothetical protein


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


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


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


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


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


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


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


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


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


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


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 ?


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


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


y13y12y10

y9y8y7y6

y5y4y2y3


OH!

S!S!


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



H!

NiCl2,NaBH455˚C,30min


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


y13y12y10

y9y8y7y6

y5y4y2y3


OH!

S!S!


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


NH

OH+ (Me3)N

SSS


NH

OH

SSS


NH

OH+

(Me3)N

SSS

ECASTCSSGPITAICDGTTK

NH

OH

SSS


NH

OH+ (Me3)N

SSS


NH

OH

SSS


NH

OH+


NH

OH

ECASTCSFGIVTIVCDGTTKOH

SSS



NH

OH


SSS



NH

OH


SSS


NiCl2,NaBH4,55˚C,30min

m/z


y13y12

y10y9y8y7y6y5y4

y2y3


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.


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)


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)


A


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


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


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


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 (*).


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


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


supplementary table 1. annotation of the divamide …...supplementary table 1. annotation of the...

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