Genomics-driven discovery of taiwachelin, a lipopeptide siderophore from Cupriavidus taiwanensis

Transcription

1 Supporting information Genomics-driven discovery of taiwachelin, a lipopeptide siderophore from Cupriavidus taiwanensis Martin F. Kreutzer and Markus Nett * Table of contents SI-2, SI-3 SI-4 SI-5 SI-6 SI-7 SI-8 SI-9 SI-10 SI-11 SI-12 SI-13 SI-14 SI-15 SI-16 SI-17 SI-18 SI-19 Table S1 Table S2 Figure S1 Figure S2 Figure S3 Table S3 Figure S4 Figure S5 Figure S6 Figure S7 Figure S8 Figure S9 Figure S10 Figure S11 Figure S12 Table S4 Figure S13 Environmental bacteria affiliated with the Burkholderiaceae and their siderophore biosynthentic potential Annotation of the tai biosynthesis genes MALDI-TOF/TOF fragmentation of taiwachelin HR-ESI-MS spectrum of taiwachelin HR-ESI-MS spectrum of Fe(III)-taiwachelin NMR spectroscopic data of taiwachelin in DMSO-d 6 COSY and HMBC correlations in taiwachelin 1 H NMR spectrum of taiwachelin in DMSO-d 6 13 C NMR spectrum of taiwachelin in DMSO-d 6 DEPT135 spectrum of taiwachelin in DMSO-d 6 1 H, 1 H COSY spectrum of taiwachelin in DMSO-d 6 HSQC spectrum of taiwachelin in DMSO-d 6 HMBC spectrum of taiwachelin in DMSO-d 6 1 H, 15 N HMBC spectrum of a crude extract from C. taiwanensis LMG19424 in DMSO-d 6 FT-IR spectrum of taiwachelin Marfey s analysis of the amino acid moieties in taiwachelin Chiral GC-MS analysis of the Hbu moiety in taiwachelin SI-1

2 Table S1. Environmental bacteria affiliated with the Burkholderiaceae and their siderophore biosynthetic potential. Organism Genome size [Mbp] # of identified NRPS clusters 1 # of identified NIS clusters 1 genomic location of siderophore biosynthetic loci and their products 2 Burkholderia gladioli BSR3 Burkholderia glumae BGR1 Burkholderia phymatum STM815 Burkholderia phytofirmans PsJN Burkholderia thailandensis E264 Burkholderia xenovorans LB400 Burkholderia sp. YI (2) 0 bgla_2g16840 bgla_2g16980 (unknown) bgla_4p2050 bgla_4p2140 (micacocidin) (1) 0 bglu_2g09700 bglu_2g09840 (unknown) (1) 0 Bphy_4035 Bphy_4047 (ornibactin) (1) 0 Bphyt_4063 Bphyt_4079 (ornibactin) (2) 0 BTH_I2414 BTH_I2427 (ornibactin) BTH_II1820 BTH_II1833 (pyochelin) (1) 0 Bxe_B0517 Bxe_B0531 (ornibactin) (2) 0 BYI23_C BYI23_C (unknown) BYI23_D BYI23_D (ornibactin) Cupriavidus metallidurans CH34 Cupriavidus necator H16 Cupriavidus necator JMP134 Cupriavidus necator N-1 Cupriavidus taiwanensis LMG (1) Rmet_1110 Rmet_1118 (staphyloferrin B) (1) 1 (1) H16_B1676 H16_B1692 (cupriachelin) PHG120 PHG126 (vibrioferrin) (1) Reut_B3681 Reut_B3687 (unknown) (1) 2c c14900 (staphyloferrin B) (1) 0 RALTA_B1222 RALTA_B1239 (taiwachelin) 3 SI-2

3 Table S1. continued. Organism Genome size [Mbp] # of identified NRPS clusters 1 # of identified NIS clusters 1 genomic location of siderophore biosynthetic loci and their products Ralstonia pickettii 12D Ralstonia pickettii 12J Ralstonia solanacearum CFBP2957 Ralstonia solanacearum GMI1000 Ralstonia solanacearum Po82 Ralstonia solanacearum PSI (1) Rpic12D_0180 Rpic12D_0181 (unknown) (1) Rpic_0173 Rpic_0174 (unknown) (1) 1 (1) (1) 1 (1) (1) 1 (1) (1) 1 (1) RCFBP_mp10439 RCFBP_mp10449 (staphyloferrin B) RCFBP_mp20475 RCFBP_mp20485 (yersiniabactin) RSc1804 RSc1813 (micacocidin) RSp0414 RSp0424 (staphyloferrin B) RSPO_m01293 RSPO_m01304 (yersiniabactin) RSPO_m01404 RSPO_m01415 (staphyloferrin B) RPSI07_1865 RPSI07_1874 (micacocidin) RPSI07_mp0375 RPSI07_mp0385 (staphyloferrin B) Polynucleobacter necessarius subsp. asymbioticus QLW Polynucleobacter necessarius subsp. necessarius STIR The number of gene clusters that are putatively involved in siderophore biosynthesis is set in parentheses. NRPS, nonribosomal peptide synthetase; NIS, NRPSindependent siderophore synthetase. 2 Predicted products are set in parentheses. Compounds that have actually been observed are highlighted in bold. 3 Taiwachelin is first described in this study. SI-3

4 Table S2. Annotation of the tai biosynthesis genes. Gene designation C. taiwanensis Protein Accession no. Size (aa) Proposed function LMG19424 gene no. (GenBank) taia RALTA_B1222 YP_ Cyclase/dehydrase taib RALTA_B1223 YP_ MbtH-like taic RALTA_B1224 YP_ Type-II thioesterase taid RALTA_B1225 YP_ Loading module: FAAL-ACP-C-TauD taie RALTA_B1226 YP_ NRPS: C-A-PCP taif RALTA_B1227 YP_ NRPS: C-A-PCP-E-C-A-PCP taig RALTA_B1228 YP_ NRPS: C-A-PCP-E-C-A-PCP-C-A-PCP-TE taih RALTA_B1229 YP_ TonB-dependent siderophore receptor taii RALTA_B1230 YP_ Iron-regulated membrane protein taij RALTA_B1231 YP_ Hypothetical protein taik RALTA_B1232 YP_ Siderophore-iron reductase tail RALTA_B1233 n/a 244 Transposase taim RALTA_B1234 YP_ ABC-type siderophore exporter tain RALTA_B1235 YP_ Esterase/lipase taio RALTA_B1236 YP_ N δ -hydroxyornithine O-acyltransferase taip RALTA_B1237 YP_ Ornithine N δ -monooxygenase taiq RALTA_B1238 YP_ Phosphopantetheinyl transferase tair RALTA_B1239 YP_ Iron-regulated transcription activating factor SI-4

5 Figure S1. MALDI-TOF/TOF fragmentation of taiwachelin. SI-5

6 Figure S2. HR-ESI-MS spectrum of taiwachelin. Theoretical mass (m/z): [M+H] + Delta (ppm): 0.17 Composition (calculated): C 41 H 71 O 18 N 8 SI-6

7 Figure S3. HR-ESI-MS spectrum of Fe(III)-taiwachelin. Theoretical mass (m/z): [M-H] - Delta (ppm): Composition (calculated): C 41 H 66 O 18 N 8 Fe SI-7

8 Table S3. NMR spectroscopic data of taiwachelin in dimethylsulfoxide-d 6. residue pos. δ C δ H, M (J in Hz) residue pos. δ C δ H, M (J in Hz) residue pos. δ C δ H, M (J in Hz) Fatty acid C L-Asp NH 8.15, d (7.8) L -N δ - NH 8.09, d (8.4) C , t (7.3) CO OH-Orn CO C , m Cα , ddd (7.8, 7.2, 6.8) (cyclic) Cα , ddd (10.7, 8.4, 5.1) C , m Cβ 36.1 a: 2.70, dd (16.5, 6.8) Cβ 27.3 a: 1.92, m C , m b: 2.53, dd (16.5, 7.2) b: 1.70, m C , m Cγ Cγ , m C-7 C-8 C-9 C-10 C-11 C , m 1.23, m 1.23, m 1.23, m 1.26, m 0.85, t (7.0) D-N δ - OH-Orn NH CO Cα Cβ Cγ Cδ , d (7.8) 4.29, m 1.47, m 1.55, m a: 3.50, m Cδ , m L-threo- NH 7.85, d (9.2) b: 3.42, m β-oh-asp CO Cα Cβ Cγ , dd (9.2, 2.7) 4.48, d (2.7) L-Hbu C-1 C-2 C a: 2.52, dd (15.0, 6.3) b: 2.37, dd (15.0, 6.3) 4.00, sext (6.3) D-allo-Thr NH 7.73, d (8.0) C , d (6.3) CO Cα Cβ Cγ , dd (8.0, 6.4) 3.82, quint (6.4) 1.03, d (6.4) L-Ser NH CO Cα Cβ , d (7.6) 4.31, m 3.59, d (7.2) SI-8

9 Figure S4. NMR-based structure elucidation of taiwachelin: COSY (bold lines) and selected HMBC correlations (arrows) observed in dimethylsulfoxide-d 6. SI-9

10 Figure S MHz 1 H NMR spectrum of taiwachelin in dimethylsulfoxide-d 6. SI-10

11 Figure S MHz 13 C NMR spectrum of taiwachelin in dimethylsulfoxide-d 6. SI-11

12 Figure S7. DEPT135 spectrum of taiwachelin in dimethylsulfoxide-d 6. SI-12

13 Figure S8. 1 H, 1 H COSY spectrum of taiwachelin in dimethylsulfoxide-d 6. SI-13

14 Figure S9. HSQC spectrum of taiwachelin in dimethylsulfoxide-d 6. SI-14

15 Figure S10. HMBC spectrum of taiwachelin in dimethylsulfoxide-d 6. SI-15

16 Figure S11. 1 H, 15 N HMBC spectrum of a crude extract from an iron-deficient culture of C. taiwanensis LMG19424 in dimethylsulfoxide-d 6. putative proton-to-hydroxamate nitrogen correlations SI-16

17 Figure S12. FT-IR spectrum of taiwachelin. SI-17

18 Table S4. Marfey s analysis of the amino acid moieties in taiwachelin. amino acid m/z [M+H] + retention time (min): Marfey product of L-amino acid standard retention time (min): Marfey product of D-amino acid standard retention time (min): Marfey product of amino acid from taiwachelin hydrolysis threo-β-hydroxyaspartic acid allo-threonine aspartic acid ornithine / 9.4 serine* *HPLC separation using 10 mm ammonium formate buffer SI-18

19 Figure S13. Chiral GC-MS analysis of trifluoroacetylated Hbu samples. A: S-Hbu derivative; B: R,S-Hbu derivative; C: taiwachelin derivative after cleavage with 0.3 M HCl; D: EI-MS fragmentation of the Hbu peak in chromatogram C the fragmentation pattern is identical to the derivatised commercial standards of Hbu. D SI-19