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emi412015-sup-0004-si.doc1315K

Fig. S1. Organization of the pyoverdine genes located upstream of the second lipopeptide biosynthetic gene cluster in P. putida RW10S2, P. ‘reactans’ LMG 5329 and P. fluorescens SBW25. For the encoded NRPS enzymes the predicted domains (labelled circles) are indicated: C, condensation; A, adenylation; T, thiolation; E, epimerization; TE, thioesterase. For P. fluorescens SBW25 the peptide chain that is synthesized by PvdI and PvdJ is indicated (Moon et al., 2008, BMC Microbiol 8: 7). Amino acids are identified by the standard three-letter code (FoOHOrn = N5-formyl-N5-hydroxyornithine). The amino acid specificity of the modules inferred from A-domain analysis (NRPSpredictor2; Röttig et al., 2011, Nucleic Acids Res 39: W362–W367) is shown for the putative pyoverdine synthetases (Orf6, Orf7, Orf8) of strain LMG 5329. Three amino acids predicted for the LMG 5329 pyoverdine that are absent in the SBW25 pyoverdine are shown in dashed boxes. No other pyoverdine genes are located downstream of fpvA in strain RW10S2.

Fig. S2. 16S rRNA phylogenetic relationship of WLIP-producing strains RW10S2 and LMG 5329 to representative Pseudomonas species. The bacterial labels are as indicated in Fig. 3. E. coli K-12 substr. MG1655 16S rRNA was used an out-group. Bootstrap values (%) and substitutions per site (scale bar) are indicated.

Fig. S3. Cladogram of maximum-likelihood tree inferred from amino acid alignment of A-domains extracted from representative functionally characterized Pseudomonas NRPSs. NRPS enzymes are designated with lipopeptide-specific codes: Arf (arthrofactin, Pseudomonas sp. MIS38); Etl (entolysin, P. entomophila L48); Mass (massetolide, P. fluorescens SS101); Ofa (orfamide, P. fluorescens Pf-5); Pso (putisolvin, P. putida PCL1445); Syf (syringafactin, P. syringae DC3000); Visc (viscosin, P. fluorescens SBW25); Wip (WLIP, P. ‘reactans’ LMG 5329) and Wlp (WLIP, P. putida RW10S2). For each domain the substrate specificity is indicated in parentheses using the standard three-letter code. The tree was rooted with the divergent SyrB1 domain (syringomycin, P. syringae pv. syringae strain B301D). Clusters highlighted in red or green colour contain domains derived from Wip (in bold) or Wlp (in bold) respectively.

Fig. S4. Phylogenetic tree of Mass/Visc/Wip/Wlp A-domains corresponding to cladogram representation of Fig. S3. NRPS designation as in Fig. S3. The corresponding matrix with pairwise patristic distance values (Table S3) was used to obtain the differential colour coding for the branches. As an indicator of evolutionary distance, two domains with a patristic distance (summed branches lengths) above 0.45 are represented in a different colour.

Fig. S5. Unrooted maximum-likelihood tree constructed from alignment of TE domains extracted from representative functionally characterized Pseudomonas NRPSs. NRPS designation as in Fig. S3. Clusters highlighted in red or green colour encompass domains derived from Wip (in bold) or Wlp (in bold) respectively. Bootstrap values (%) and substitutions per site (scale bar) are indicated.

Fig. S6. Phylogenetic analysis of proteins associated with representative functionally characterized Pseudomonas NRPS systems: LuxR-type regulators (A) and homologues of MacA (B), MacB (C) and OprM (D). NRPS designation as in Fig. S3. The OprM equivalents of the Syf and Arf systems are not yet known. Clusters highlighted in red or green colour encompass domains derived from Wip (in bold) or Wlp (in bold) respectively. Bootstrap values (%) and substitutions per site (scale bar) are indicated.

Fig. S7. Maximum-likelihood tree inferred from alignment of C-domains extracted from representative functionally characterized Pseudomonas NRPSs. NRPS enzymes are designated with lipopeptide-specific codes: Arf (arthrofactin, Pseudomonas sp. MIS38); Etl (entolysin, P. entomophila L48); Mass (massetolide, P. fluorescens SS101); Ofa (orfamide, P. fluorescens Pf-5); Pso (putisolvin, P. putida PCL1445); Syf (syringafactin, P. syringae pv. tomato DC3000); Syp (syringopeptin, P. syringae pv. syringae B301D); Syr (P. syringae pv. syringae strain B301D); Visc (viscosin, P. fluorescens SBW25); Wip [WLIP, P. fluorescens (P. ‘reactans’) LMG 5329; in red] and Wlp (WLIP, P. putida RW10S2; in green). The tree was rooted with the divergent SyrE-C1 domain. The clusters corresponding to lipoinitiation domains (C1), conventional condensation domains (C) and dual condensation/epimerization domains (C/E) are labelled. C-domains deviating from the ‘Balibar rule’ with respect to the stereochemistry of the amino acid incorporated by the previous module (Balibar et al., 2005, Chem Biol 12: 1189–1200) are indicated with {L} in the C/E cluster and with {D} in the C cluster. Sequences used for alignment in Fig. S8 are marked in bold.

Fig. S8. Multiple amino acid sequence alignment of the C-domains extracted from the sixth module of the NRPSs synthesizing arthrofactin (ArfB), massetolide (MassB), orfamide (OfaB), viscosin (ViscB) and WLIP (WipB, WlpB). The stereochemistry (D or L) of the fifth amino acid present in the respective lipopeptide products is indicated. The positions of a histidine and aspartate residue (red) required for catalytic activity (Bergendahl et al., 2002, Eur J Biochem 269: 620–629) and the additional N-terminal extended His-motif (H-H-I/L-X4-G-D; blue) identified by Balibar and colleagues (2005, Chem Biol 12: 1189–1200) in C/E-type domains are marked.

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Table S1. Pairwise amino acid sequence identities of the NRPS enzymes and auxiliary proteins (LuxR-type regulator, homologues of MacA, MacB and OprM) encoded by the gene clusters mass (massetolide), visc (viscosin), wlp (WLIP, strain RW10S2) and wip (WLIP, strain LMG 5329).

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Table S2. Homology of the pyoverdine genes located upstream of the second lipopeptide biosynthetic gene cluster in P. putida RW10S2, P. ‘reactans’ LMG 5329 and P. fluorescens SBW25. The respective gene organizations are shown in Fig. S1.

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Table S3. Patristic distance matrix for the maximum-likelihood tree of selected Pseudomonas NRPS A-domains. The corresponding tree is shown in Fig. S3.

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