Supporting Information Table of contents Supplementary Methods Assay of the antifungal activities against mycelium growth of F. oxysporum. Supplementary Tables Table S1. Selectivity-conferring code of the amino acid-specific adenylation domains (A domains) of the surfactins (srf), bacillomycin L (bmy), locillomycins (loc) and fengycins gene clusters in B. subtilis 916 genome Table S2. Four family LPs- surfactins, bacillomycin Ls, locillomycins and fengycins production by B. subtilis 916 and its mutant strains at 72 h grown in LB medium Supplementary Figures Fig. S1. MS analysis of four family LPs produced by B. subtilis 916 Fig. S2. The locations of five PKS gene clusters and four NRPS gene clusters in the B. subtilis 916 genome Fig. S3. Phylogenic analysis of the adenylation (A) domains of surfactins, bacillomycin Ls, locillomycins and fengycins in B. subtilis 916 Fig. S4 Sequences alignment between the fourth adenylation domain of bam (for activation of Pro) and the fourth adenylation domain of bmy (for activation of Ser) Fig. S5. HPLC-MS analysis of purified surfactins from culture broth of mutant strain BSBFM Fig. S6. HPLC-MS analysis of purified bacillomycin Ls from culture broth of mutant BSFM Fig. S7. HPLC-MS analysis of purified surfactins from culture broth of mutant BSBFM
Fig. S8. HPLC-MS analysis of purified fengycins from culture broth of mutant BSSM Fig. S9. HPLC spectrograms for bacillomycin L produced by B. subtilis 916 and its mutant strains Fig. S10. HPLC spectrograms for fengycins produced by B.subtilis 916 and its mutants Fig. S11. HPLC spectrograms for locillomycins produced by B. subtilis 916 and its mutant strains Fig. S12. HPLC spectrograms for surfactins produced by B.subtilis 916 and its mutant strains Fig. S13. Antifungal activities of wild-type B. subtilis 916 and its mutant strains against mycelium growth of F. oxysporum.
Supplementary Method Assay of the antifungal activities against mycelium growth of F. oxysporum. The wild-type and mutant strains antifungal activities against mycelium growth of F. oxysporum was performed as follows. Mycelia plugs (5 mm) of F. oxysporum were deposited in the center of the plates at equal distances from the wells. 5-µl portions of culture strains were deposited on both sides at a distance 2.5 cm from the rims of mycelia plugs. The plates were inoculated at 28, and inhibition zones were measured after 3 to 7 days.
Supplementary Tables Table S1. Selectivity-conferring code of the amino acid-specific adenylation domains (A domains) of the surfactins (srf), bacillomycin Ls (bmy), locillomycins (loc) and fengycins (fen) gene clusters in B. subtilis 916 genome A domain Amino acid b Selectivity-conferring amino acid a at position 235 236 239 278 299 301 322 330 331 517 bmy-a1-asn Asn(1) D L T K I G E V G K bmy-a3-asn Asn(3) D L T K I G E V G K loc-a2-gln-asn Gln(2)/Asn(5) D A V Q M D C V D K bmy-a2-tyr Tyr(2) D A L S V G E V V K fen-a3-tyr Tyr(3) D G T L T A E V A K fen-a9-tyr Tyr(9) D G T I T A E V A K loc-a5-tyr Tyr(8) D G L F T V R V E K bmy-a5-glu Glu(5) D A K D L G V V D K srf-a1-glu Glu(1) D A K T L G V V D K fen-a1-glu Glu(1) D A W H F G G V D K fen-a5-glu Glu(5) D A W H F G G V D K bmy-a4-ser Ser(4) D V W H F S L V G K bmy-a6-ser Ser(6) D V W H F S L I D K fen-a6-ala-val Ala(6)/Val(6) D V F W I G G T F K srf-a4-val Val(4) D A F M I G G T F K loc-a6-val Val(9) D A F W I G A T F K srf-a2-leu Leu(2) D A F L M G M V F K srf-a7-leu Leu(7) D A F M L G C V F K srf-a3-leu Leu(3) D A W T K G N V V K srf-a6-leu Leu(6) D A W T L G N V V K fen-a2-orn Orn(2) D V G E V G S I D K fen-a7-pro Pro(7) D V Q F I A H V V K bmy-a7-thr Thr(7) D F W N I G M V H K loc-a1-thr Thr(1) D F W N I G M V H K fen-a4-thr Thr(4) D F W N I G M V H K loc-a4-gly Gly(4)/Gly(7) D I L Q Y G M I W K loc-a3-asp Asp(5)/ Asp(6) D L T K I G H I G K srf-a5-asp Asp(5) D L T M V G H I G K fen-a8-gln Gln(8) D A W H F G S V E K fen-a10-ile Ile(10) D A F F Y G I T F K a Conserved residues locating the substrate-binding pockets of adenylation domains of assigned functions are indicated in bold face. b The positions of the activated amino acid within the respective LPs are given in parentheses.
Table S2. Four family LPs- surfactins, bacillomycin Ls, locillomycins and fengycins production by B. subtilis 916 and its mutant strains at 72 h grown in LB medium a LPs productions (mg liter 1 ) LPs BSBF BSFL BSBL BSBFL WT BSBM BSFM BSLM BSSM M M M M surfactins 21.2(2.9) 30.3(3.6) 25.1(3.3) 20.8(3.1) 0 32.6(4.0) 23.1(3.1) 30.2(3.8) 32.0(4.5) bacillomycin Ls 16.3(1.8) 0 16.8(2.0) 17.9(2.0) 0 0 17.6(2.8) 0 0 locillomycins 3.4(0.5) 3.5(0.6) 3.3(0.5) 0 3.4(0.5) 3.1(0.4) 0 0 0 fengycins 4.2(0.5) 5.3(0.7) 0 4.0(0.6) 1.8(0.4) 0 0 5.8(0.9) 0 a The values are means from three experiments, and the values in parentheses are standard deviations.
Supplementary Figures Fig. S1. MS analysis of four family LPs produced by B. subtilis 916. (a~d) Detection of mass peaks of surfactins, bacillomycin Ls, locillomycins and fengycins in methanol extracts from the acid precipitates of the culture supernatant of B. subtilis 916 grown in the LB medium respectively.
Fig. S2. The locations of five PKS gene clusters and four NRPS gene clusters in the B. subtilis 916 genome. The five PKS gene clusters mln, bae, dfn, dhb and bac are respobsible for the biosynthesis of macrolactin, bacillaene, difficidin, bacillibactin and bacilysin respectively. The four NRPS gene cluster srf, bmy, loc and fen are responsible for biosynthesis of four family LPs-surfactins, bacillomycin Ls, locillomycins and fengycins respectively.
Fig. S3. Phylogenic analysis of the adenylation (A) domains of surfactins (srf), bacillomycin Ls (bmy), locillomycins (loc) and fengycins (fen) in B. subtilis 916. The adenylation sequence comprises the highly conserved core motifs A1 A10. Each adenylation is designed by the peptide synthetase, relative position and corresponding binding amino acid (for example BmyA1Asn correspond to the asparagine binding domain of bmy).
Fig. S4. Sequences alignment between the fourth adenylation domain of bam (for activation of Pro) and the fourth adenylation domain of bmy (for activation of Ser).
Fig. S5. HPLC-MS analysis of purified surfactins from culture broth of mutant strain BSBFM (Δbac:: Nm r ΔfenA::Cm r ). (a) HPLC spectrograms for purified surfactins and the six peaks were used to quantify surfactins production in wild-type B. subtilis 916 and its mutant strains. (b) Mass spectra of surfactin isoform A. (c) Mass spectra of surfactin isoform B. (d) Mass spectra of surfactin isoform C.
Fig. S6. HPLC-MS analysis of purified bacillomycin Ls from culture broth of mutant BSFM (ΔfenA::Cm r ). (a) HPLC spectrograms for purified bacillomycin Ls and the three peaks were used to quantify bacillomycin Ls production in wild-type B. subtilis 916 and its mutant strains. (b) Mass spectra of bacillomycin L isoform A. (c) Mass spectra of bacillomycin L isoform B.
Fig. S7. HPLC-MS analysis of purified surfactins from culture broth of mutant BSBFM (Δbac:: Nm r ΔfenA::Cm r ). (a) HPLC spectrograms for purified locillomycins and six peaks were used to quantify locillomycins production in wild-type B. subtilis 916 and its mutant strains. (b) Mass spectra of locillomycin isoform A. (c) Mass spectra of locillomycin isoform B. (d) Mass spectra of locillomycin isoform C.
Fig. S8. HPLC-MS analysis of purified fengycins from culture broth of mutant BSSM (ΔfenA::Cm r ). (a) HPLC spectrograms for purified fengycins standards and five peaks were used to quantify fengycins production in wild-type B. subtilis 916 and its mutant strains. (b) Mass spectra of fengycin isoform A. (c) Mass spectra of fengycin isoform B. (d) Mass spectra of fengycin isoform C. (e) Mass spectra of fengycin isoform D. (f) Mass spectra of fengycin isoform E.
Fig. S9. HPLC spectrograms for bacillomycin Ls produced by B. subtilis 916 and its mutant strains. The mutants BSBM (Δbac::Nm r ) and BSSM (ΔsrfA::Spec r ) were deficient in biosynthesis of bacillomycin Ls. The mutants BSFM (ΔfenA::Cm r ) and BSLM (ΔlocA::Em r ) showed no significant difference in the production of bacillomycin Ls compared to wild-type B. subtilis 916.
Fig. S10. HPLC spectrograms for fengycins produced by B.subtilis 916 and its mutants. The mutant BSFM (ΔfenA::Cm r ) was deficient in biosynthesis of fengycins. The mutant BSSM (ΔsrfA::Spec r ) was severely impaired in biosynthesis of fengycins. The mutants BSBM (Δbac::Nm r ) and BSLM (ΔlocA::Em r ) enhanced differently in the production of fengycins compared to wild-type B.subtilis 916.
Fig. S11. HPLC spectrograms for locillomycins produced by B. subtilis 916 and its mutant strains. The mutant BSLM (ΔlocA::Em r ) was deficient in biosynthesis of locillomycins. The mutants BSBM (Δbac::Nm r ), BSFM (ΔfenA::Cm r ), BSLM (ΔlocA::Em r ) and BSSM (ΔsrfA::Spec r ) showed no significant difference in the production of locillomycins compared to wild-type B. subtilis 916.
Fig. S12. HPLC spectrograms for surfactins produced by B.subtilis 916 and its mutant strains. The mutant BSSM was deficient in the biosynthesis of surfactins. The mutant BSBM (Δbac::Nm r ) and BSBFM (Δbac::Nm r, ΔfenA::Cm r ) enhanced differently in the production of surfactins compared to wild-type B. subtilis 916. The mutant BSFM (ΔfenA::Cm r ) showed no significant difference in the production of surfactins when compared to wild-type B. subtilis 916.
Fig. S13. Antifungal activities of wild-type B. subtilis 916 and its mutant strains against mycelium growth of F. oxysporum. (a) wild-type B. subtilis 916 was able to produce all four families lipopeptides: surfactin, bacillomycin L, locillomycin and fengycin. (b) BSBM (ΔbmyD:: Nm r ) was deficient in the production of bacillomycin Ls. (c) BSFM (ΔfenA::Cm r ) was deficient in the production of fengycins. (d) BSLM (ΔlocA::Em r ) was deficient in the production of locillomycins. (e) BSSM (ΔsrfA::Spec r ) was deficient in the production of surfactins and bacillomycin Ls, and also impaired the biosynthesis of locillomycins. (f) BSBFM (Δbac:: Nm r ΔfenA::Cm r ) was deficient in the production of both bacillomycin Ls and fengycins. (g) BSFLM (ΔfenA::Cm r ΔlocA::Em r ) was deficient in the production of both fengycins and locillomycins. (h) BSBLM (Δbac:: Nm r ΔlocA::Em r ) was deficient in the production of both bacillomycin Ls and locillomycins. (i) BSBFLM (Δbac:: Nm r ΔfenA::Cm r ΔlocA::Em r ) was deficient in the production of bacillomycin Ls, fengycins and locillomycins. The bacillomycin Ls and fengycins contributed mainly to the antifungal activities of B. subtilis 916 and its mutant strains.