stallforth si revised 07 18 - pnas · 2013-07-29 · s2 purification and characterization of...

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Supporting Information

Pierre Stallfortha, Debra A. Brockb, Alexandra M. Cantleya, Xiangjun Tianb, David C. Quellerb, Joan E. Strassmannb, and Jon Clardya,1 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115 bDepartment of Biology, Washington University in St. Louis, One Brookings Drive St. Louis, MI 63130

SI Materials and Methods

Fig. S1. Photographs of D. discoideum QS161 grown on lawns of different bacteria. The pictures are taken 72 h after plating the spores. Top left: Klebsiella pneumoniae as a food source. Top right: Pseudomonas fluorescens PfB as a food source. Bottom left: P. fluorescens PfA ΔgacA as a food source. Bottom right: P. fluorescens PfA as a food source. Note: QS161 using P. fluorescens PfA as a food source does not produce fruiting bodies.

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Purification and Characterization of Chromene, Pyrrolnitrin and Pyochelin. Eight 1 L cultures of both P. fluorescens PfA and PfB in SM/5 were grown for 24 h in 4 L Erlenmeyer flasks at 30 °C. The cultures were extracted with the same amount of ethyl acetate and the organic layer was dried over Na2SO4. Solvents were removed in vacuo to give a crude extract, which was dissolved in 2 mL methanol and 11.3 mL water. The suspension was loaded on a C18 (4g Waters Sep-Pak®) column that was pre-conditioned with 15% acetonitrile in water. Fractions with 15%, 50% and 100% acetonitrile in water were collected and solvents were removed in vacuo. The 50% fraction was further subjected to preparative HPLC. Using an Agilent 1200 Series HPLC system equipped with a diode array detector and a Phenomenex Luna C18 column (5 µm, 100 Å 250 x 21.2 mm, flow rate = 12 mL/min). The following gradient was used for HPLC purification of pyrrolnitrin and chromene: 0−3 min, isocratic 40% acetonitrile in water; 3−30 min, linear gradient from 40% acetonitrile in water to 85% acetonitrile in water (retention time chromene: 20.8 min., pyrrolnitrin: 25.5 min.). For the purification of enantio-pyochelin I and II the following gradient was used: 0−3 min, isocratic 40% acetonitrile in water and 0.1% formic acid; 3−30 min, linear gradient from 40% acetonitrile in water to 75% acetonitrile in water and 0.1% formic acid (retention time enantio-pyochelin I: 16.8 min., enantio-pyochelin II: 18.7 min.). NMR spectra were recorded in CD3OD and acetone-d6 on a 400 MHz Varian MR system with the Varian One probe and referenced to the internal solvent peak at δH 3.31 ppm (CD3OD) and 2.05 ppm (acetone-d6), and δC 49.0 ppm (CD3OD) and 29.84 ppm (acetone-d6). High-resolution mass spectrometry (HRMS) was performed at the University of Illinois Urbana−Champaign Mass Spectrometry Facility. A Waters/Micromass Q-tof Ultima (http://scs.illinois.edu/massSpec/instrum/qtof.php) was used for HRES measurements and a Waters/Micromass 70 VSE was used for HREI measurements (http://scs.illinois.edu/massSpec/instrum/vse.php).

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HRMS, IR, 𝛂 𝐃𝟐𝟎 Data and NMR Spectra of Chromene. α !

!" = –6.7 (c = 0.2, MeOH); (+)-HRESIMS calcd. for C14H19O3 [M+H]+: 235.1334 found: 235.1327; IR νmax 3366 (br), 2961, 1622, 1600, 1463, 1144 cm-1.

Fig. S2. HRMS data for chromene.

Table S1. 1H And 13C NMR data (400 MHz, CD3OD) for chromene.

Elemental Composition Report Page 1

Single Mass AnalysisTolerance = 10.0 PPM / DBE: min = -1.5, max = 150.0Element prediction: Off Number of isotope peaks used for i-FIT = 3

Monoisotopic Mass, Even Electron Ions803 formula(e) evaluated with 3 results within limits (all results (up to 1000) for each mass)Elements Used:C: 0-150 H: 0-250 N: 0-10 O: 0-10 S: 0-5

m/z232.00 233.00 234.00 235.00 236.00 237.00 238.00

%0

100

Stallforth, Pierre, PS-Pf2-2 Q-tof UE521University of Illnois, SCS, Mass Spectrometry LabQtof_34838 23 (1.650) AM (Cen,3, 80.00, Ar,15000.0,716.46,0.70,LS 3); Sm (SG, 2x3.00) 1: TOF MS ES+

1.01e+003235.1

233.1232.1232.0 232.7 234.1233.2 234.0 235.0 236.1235.2 236.0235.5 237.1236.9236.3 238.0237.6

Minimum: -1.5Maximum: 5.0 10.0 150.0

Mass Calc. Mass mDa PPM DBE i-FIT Formula

235.1327 235.1334 -0.7 -3.0 5.5 4.9 C14 H19 O3 235.1341 -1.4 -6.0 1.5 47.2 C7 H19 N6 O S 235.1307 2.0 8.5 6.5 13.7 C10 H15 N6 O

Position δC δH (mult., J in Hz)

2 78.7 4.50 (dd, J = 9.8, 2.6) 3 135.5

4 112.6 6.33 (s) 4a 105.0

5 154.4 6 96.5 5.86 (d, J = 2.3)

7 154.5 8 96.3 5.78 (d, J = 2.3)

8a 158.8 1' 35.7 1.74-1.64 (m), 1.46-1.36 (m)

2' 19.7 1.62-1.50 (m), 1.46-1.36 (m) 3' 14.2 0.92 (t, J = 7.24) 1" 26.8 2.20-2.00 (m) 2" 12.3 1.14 (t, J = 7.42)

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A 1H NMR spectrum of chromene in CD3OD (400MHz)

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B 13C NMR spectrum of chromene in CD3OD (400MHz)

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C gCOSY NMR spectrum of chromene in CD3OD (400MHz)

D HSQC NMR spectrum of chromene in CD3OD (400MHz)

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Fig. S3. NMR spectra of chromene in CD3OD (A: 1H; B: 13C; C: H-H gCOSY; D: HSQC; E: gHMBC).

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E gHMBC NMR spectrum of chromene in CD3OD (400MHz)

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HRMS Data and NMR Spectra of Pyrrolnitrin. HREIMS calcd. for C10H6N2O2

35Cl2 [M]+: 255.9806 found: 255.9804.

Fig. S4. HRMS data for pyrrolnitrin.

Fig. S5. 1H NMR spectra of commercially available pyrrolnitrin (Sigma Aldrich, spectrum 1, bottom) and pyrrolnitrin isolated from PfA (spectrum 2, top) both are recorded in CD3OD.


Single Mass Analysis Tolerance = 5.0 mDa / DBE: min = -1.5, max = 50.0Element prediction: Off

Monoisotopic Mass, Odd and Even Electron Ions2 formula(e) evaluated with 1 results within limits (up to 50 best isotopic matches for each mass)Elements Used:C: 0-500 H: 0-1000 N: 2-2 O: 2-2 Cl: 2-2

m/z253.00 254.00 255.00 256.00 257.00 258.00 259.00 260.00

%0

100

Pierre Stallforth, PN-01 PN-01-HREI 135 (4.950) TOF MS EI+

2.49e+002255.9804

253.2915 255.2056254.1517257.9765

256.2386 256.9803 257.2479259.9660258.9651258.2233 259.2343 260.1500

Minimum: -1.5Maximum: 5.0 10.0 50.0


255.9804 255.9806 -0.2 -0.8 8.0 1.2 C10 H6 N2 O2 Cl2

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HRMS, IR, 𝛂 𝐃𝟐𝟎 Data and NMR Spectra of enantio-Pyochelin I and II. α !

!" = –15 (c = 0.1, acetone) (reported: –14 (1)); (+)-HRESI calcd. for C14H17N2O3S2 [M+H]+: 325.0681 found: 325.0681.

Fig. S6. HRMS data for enantio-pyochelin.

Enantio-Pyochelin I δH (mult., J in Hz)

Enantio-Pyochelin II δH (mult., J in Hz)

Reported (1) observed Reported (1) observed

H4, H6 7.40-7.46 (m) 7.38-7.45 (m) 7.40-7.46 (m) 7.38-7.45 (m) H3, H5 6.93-6.97 (m) 6.90-6.98 (m) 6.93-6.97 (m) 6.90-6.98 (m) H4' 5.20 (td, J=9.0, 5.5) 5.21 (td, J=8.8, 5.3) 5.04 (q, J=8.0) 5.01 (q, J=8.2) H2' 4.65 (d, J=5.5) 4.62 (d, J=5.4) 4.56 (d, J=8.0) 4.56 (d, J=8.2) H4" 3.76 (dd, J=7.0, 8.5) 3.73 (dd, J=6.8, 8.1) 4.27 (t, J=6.5) 4.25 (t, J=6.5) H5' 3.52 (d, J=9.0) 3.50 (d, J=8.9) 3.66 (dd, J=11.5, 8.5) 3.67 (dd, J=11.3, 8.6)

3.45 (dd, J=11.5, 8.0) 3.46 (dd, J=11.3, 7.9)

H5" 3.23-3.25 (m) 3.20-3.26 (m) 3.23-3.25 (m) 3.20-3.26 (m) NCH3 2.66 (s) 2.65 (s) 2.52 (s) 2.51 (s) Table S2. 1H Data (400 MHz, CD3OD) for enantio-pyochelin I and II from PfB and reported data (1).


Single Mass AnalysisTolerance = 100.0 PPM / DBE: min = -1.5, max = 150.0Element prediction: Off Number of isotope peaks used for i-FIT = 3

Monoisotopic Mass, Even Electron Ions44 formula(e) evaluated with 6 results within limits (all results (up to 1000) for each mass)Elements Used:C: 0-150 H: 0-200 N: 0-2 O: 0-3 S: 2-2

m/z324.50 325.00 325.50 326.00 326.50 327.00 327.50

%0

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Stallforth, Pierre, 50-11 Q-tof UE521University of Illnois, SCS, Mass Spectrometry LabQtof_36553 26 (1.864) AM (Cen,3, 80.00, Ar,15000.0,716.46,0.70,LS 3); Sm (SG, 2x3.00); Cm (21:26) 1: TOF MS ES+

4.47e+002325.1

325.0324.7324.5325.2 327.2326.1325.2 326.0325.6 325.6

327.1326.2326.4 326.9326.6 327.2 327.4 327.6

Minimum: -1.5Maximum: 5.0 100.0 150.0


325.0681 325.0681 0.0 0.0 7.5 5.2 C14 H17 N2 O3 S2 325.0721 -4.0 -12.3 11.5 5.5 C19 H17 O S2 325.0833 -15.2 -46.8 11.5 9.3 C18 H17 N2 S2 325.0469 21.2 65.2 12.5 12.0 C17 H13 N2 O S2 325.0932 -25.1 -77.2 6.5 15.0 C16 H21 O3 S2 325.0357 32.4 99.7 12.5 21.3 C18 H13 O2 S2

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Fig. S7. 1H NMR spectrum of enantio-pyochelin I and II in acetone-d6 (400MHz).

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Fig. S8. LC-MS traces of ethyl acetate extract of farmer QS161 co-cultured with PfA/PfB. The LC-MS traces of purified chromene, pyrrolnitrin and pyochelin (3rd, 5th and 7th trace from the top) are added for comparison.

Current Chromatogram(s)

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Print of window 38: Current Chromatogram(s)Data File : D:\DATA\SNAPSHOT.DSample Name : Coculture-Pf-QS161-5uL=====================================================================Acq. Operator : Pierre Seq. Line : 3Acq. Instrument : Instrument 1 Location : Vial 22Injection Date : 6/3/2013 7:23:33 PM Inj : 1 Inj Volume : 5.0 µlAcq. Method : D:\DATA\PIERRE\COCULTURE 2013-06-03 18-21-30\DEFAULT_ACID.MLast changed : 6/3/2013 6:21:28 PM by PierreAnalysis Method : C:\CHEM32\1\METHODS\CHRISTINE\M32_COLUMN_SHORT.MLast changed : 6/9/2013 8:36:20 PM by ki-hyun (modified after loading)

Instrument 1 6/9/2013 8:37:06 PM ki-hyun Page 2 of 2


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UV trace (245 nm) farmer !D. discoideum QS161 co-culture!

Ion-extraction (neg. mode) of farmer !D. discoideum QS161 co-culture!m/z= 255 (pyrrolnitrin)!

UV trace (245 nm) isolated and !purified pyrrolnitrin!

Ion-extraction (neg. mode) of farmer !D. discoideum QS161 co-culture!m/z= 233 (chromene)!

UV trace (245 nm) isolated and !purified chromene!

Ion-extraction (neg. mode) of farmer !D. discoideum QS161 co-culture!m/z= 323 (enantio-pyochelin I and II)!

UV trace (245 nm) isolated and !purified enantio-pyochelin I and II!


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Antibiotic assay. Initial testing of purified isolates from P. fluorescens PfA and PfB was performed using a disc assay against B. subtilis 3610, E. coli K12, and S. cerevisiae. Each organism was grown overnight in either minimal medium (B. subtilis ATCC 3610), LB (E. coli K12), or YPD (S. cervisiae). 200 µL of the overnight culture was added to 3 mL of the respective top agar containing 0.3% agar (which was cooled to approximately 40 °C after melting). The mixture was poured onto an agar plate of the respective medium and left to solidify. Sterile filter paper discs (6mm) were placed on top of the agar surface. The compound of interest was dissolved in methanol to a concentration of 5 mg/mL and 9 µL were added to the discs. After 24 – 48 h the size of the inhibition zone was measured. Bacillus subtilis (ATCC 3610) was grown in 5 mL minimal medium over night at 37 °C. The overnight culture was diluted with additional minimal medium to OD600 = 0.01. Chromene was dissolved in DMSO to give a concentration of 10 mM and a series of dilutions was. Of each dilution, 1 µL was added to the wells of a 96-well plate, followed by the diluted culture of the test organism (99 µL) to give a final compound concentration ranging from 200 µM to 0.4 µM. The cultures were allowed to grow for 24 h at 30 °C before the OD600 was measured using a plate reader. The antibiotic assay was performed in triplicate.

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Fig. S9. Pictures of fruiting bodies of D. discoideum non-host QS160 and host QS161 in buffer (top) and chromene (5 ng/mL, bottom).

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[Pyrrolnitrin] (ng/mL)

%-change in spore production farmer QS161 (SD)

%-change in spore production non-farmer

QS160 (SD) 0.005 11.4 (10.3)

-8.1 (4.2)

0.05 12.2 (8.0)

-2.3 (6.8) 0.5 9.7 (4.3)

-4.3 (7.9)

5 20.5 (6.2)

-4.6 (1.6) 50 13.3 (2.7)

-7.9 (2.6)

500 18.8 (17.0)

-7.7 (1.8) 1000 19.1 (3.2)

-13.0 (7.0)

5000 19.0 (8.8)

-6.9 (4.6) 10000 26.0 (5.9)

-16.8 (7.3)

40000 -1.0 (8.3)

-26.8 (3.5)

[Chromene] (ng/mL)

%-change in spore production farmer QS161 (SD)

%-change in spore production non-farmer

QS160 (SD) 0.005 2.2 (4.5)

-3.0 (3.8)

0.05 11.3 (5.0)

1.7 (10.3) 0.5 20.5 (4.9)

-2.6 (8.6)

5 17.8 (8.8)

-10.1 (5.0) 50 24.1 (9.6)

-20.8 (12.6)

500 15.4 (5.8)

-11.4 (1.8) 5000 22.5 (8.3)

-11.6 (0.9)

Table S3. Effect of pyrrolnitrin and chromene produced by PfA on the spore production of farmer D. discoideum QS161 vs. a non-farmer clone QS160.

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Fig. S10. Photographs of D. discoideum QS161 grown on lawns of different bacteria. The pictures are taken 72 h after plating the spores. Left: PfA as a food source. Middle: PfB as a food source. Right: PfA as a food source complemented with supernatant from PfB. Note: QS161 using P. fluorescens PfA as a food source does not produce fruiting bodies. Filtered supernatant of PfA alone is not sufficient to restore the non-food source properties of PfA.

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Construction of gacA Gene Deletion Mutant. In-frame deletion mutant of gacA locus in P. fluorescens PfA was constructed via homologous recombination using SacB as a counter-selection marker (2, 3). In order to construct the suicide vectors for deletion of gacA, two regions flanking the target gene were amplified using genomic DNA isolated from P. fluorescens PfA as template. A primer (5′-TGTCCCGGGTGGCGGCAAAAGGG -3′) containing an XmaI site and one (5′- ACCCTCGAGGCAGACACCTCGCGATAT -3′) with an XhoI site were used for upstream fragment amplification (restriction sites are underlined). A primer (5′-TGTCTCGAGAATGACCGAACAGTTTGATCC-3′) with an XhoI site and one (5′-AACGAATTCCTCCAGGAACATCACCGAGT-3′) containing a EcoRI site were used for amplification of the downstream fragment of the gacA coding sequence. The two amplicons were mixed, digested with XhoI endonuclease, ligated with T4 ligase and amplified by overlap extension PCR using the two distal primers: (5′-AACGAATTCCTCCAGGAACATCACCGAGT-3′) and (5′- TGTCCCGGGTGGCGGCAAAAGGG -3′). After digesting the resulting amplicon with XmaI and EcoRI endonucleases the DNA fragment was ligated into the XmaI and EcoRI site of vector pEXG2 (4). The construct was confirmed by sequencing and then transformed into the E. coli donor strain SM10λpir that was used for conjugational transfer of the plasmid into P. fluorescens PfA as described previously (2) with some modifications to construct a merodiploid. Mating was carried out at 30 °C. After counter-selection on LB-agar plates containing 15 µg/mL gentamicin and 15 µg/mL nalidixic acid, several clones were plated on LB-plates containing 15 µg/mL gentamicin and 5% sucrose for counter selection. The loss of gacA gene was confirmed by PCR and sequencing.

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Fig. S11. HPLC (254nm) traces of an ethyl acetate extract of PfB (blue) and PfAΔgacA (red) cultures (overlaid).


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LCMS trace (254nm) !PfB!PfA ΔgacA!

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1. Youard ZA, Mislin GLA, Majcherczyk PA, Schalk IJ, Reimmann C (2007) Pseudomonas fluorescens CHA0 Produces Enantio-pyochelin, the Optical Antipode of the Pseudomonas aeruginosa Siderophore Pyochelin. J Biol Chem 282:35546–35553.

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stallforth si revised 07 18 - pnas · 2013-07-29 · s2 purification and characterization of...

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