Discovery of the Cyclic Lipopeptide Gacamide A by Genome Mining

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Discovery of the Cyclic Lipopeptide Gacamide A by Genome Mining and Repair of the Defective GacA Regulator in Pseudomonas fluorescens Pf0‑1 Gahzaleh Jahanshah,†,‡ Qing Yan,§ Heike Gerhardt,∥,# Zoltán Pataj,∥,# Michael Lämmerhofer,∥,# Isabelle Pianet,⊥ Michaele Josten,○,@ Hans-Georg Sahl,○,@ Mark W. Silby,∇ Joyce E. Loper,§,● and Harald Gross*,†,‡ Downloaded via MIDWESTERN UNIV on January 24, 2019 at 09:03:21 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Pharmaceutical Institute, Department of Pharmaceutical Biology, University of Tübingen, 72076 Tübingen, Germany German Centre for Infection Research (DZIF), partner site Tübingen, 72076 Tübingen, Germany § Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, United States ∥ Pharmaceutical Institute, Department of Pharmaceutical Analysis and Bioanalysis, University of Tübingen, 72076 Tübingen, Germany ⊥ CESAMO-ISM, UMR 5255, CNRS, Université Bordeaux I, 351 Cours de la Libération, F-33405 Talence, France # UMR 5060, IRAMAT-CRP2A, Esplanade des Antilles, F-33600 Pessac, France ○ Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), Pharmaceutical Microbiology Unit, University of Bonn, 53115 Bonn, Germany @ German Centre for Infection Research (DZIF), partner site Bonn-Cologne, 53115 Bonn, Germany ∇ Department of Biology, University of Massachusetts Dartmouth, North Dartmouth, Massachusetts 02747, United States ● Agricultural Research Service, U.S. Department of Agriculture, Corvallis, Oregon 97331, United States ‡

S Supporting Information *

ABSTRACT: Genome mining of the Gram-negative bacterium Pseudomonas f luorescens Pf0-1 showed that the strain possesses a silent NRPS-based biosynthetic gene cluster encoding a new lipopeptide; its activation required the repair of the global regulator system. In this paper, we describe the genomics-driven discovery and characterization of the associated secondary metabolite gacamide A, a lipodepsipeptide that forms a new family of Pseudomonas lipopeptides. The compound has a moderate, narrow-spectrum antibiotic activity and facilitates bacterial surface motility.

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for the production of many natural products.8 Briefly, it comprises the membrane-bound sensor kinase GacS, which is activated by environmental signals and phosphorylates the cytoplasmic response regulator GacA. The activated GacA protein binds to the promoter region of sRNA genes rsmX, rsmY, and rsmZ and promotes their transcription.9 The resultant sRNA molecules then sequester translational repressors, termed RsmA and RsmE, which bind to the consensus sequence 5′-A/UA/U CANGGANGU/A-3′ located in the Shine-Dalgarno sequence of target mRNAs. This contact leads to a conformational change (i.e., the formation of an ANGGAN stem loop), which hinders the access of the 30S

enome mining has become a powerful approach for the discovery of microbial natural products and understanding the active principles of bacteria.1 One of the advantages of this approach is that it unveils the full biosynthetic capacity of a microorganism. However, often up to 50% of the detected biosynthetic gene clusters (BGCs) are not transcribed under standard laboratory conditions and are therefore classified as “silent”, or alternatively as “cryptic” or “orphan”2 gene clusters. Among the several strategies for activating silent gene clusters,3 manipulation of the transcriptional regulators has been proposed.4 The deletion of repressors5 or the overexpression of pathway-specific6 and, in rare cases, global7 positive regulators enabled the identification of the corresponding products of silent BGCs. In Pseudomonas spp., the GacS/GacA two-component system is a highly conserved global regulatory system required © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 2, 2018

A

DOI: 10.1021/acs.jnatprod.8b00747 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Organization of the gene cluster encoding gacamide and predicted amino acid composition of the CLP peptide backbone derived from the Pf0-1 genome.

upstream of gamA in the regions flanking the structural NRPS genes. These genes are well conserved in comparison with the ones flanking the viscosin, 19 massetolide, 19 orfamide,19 bananamide,20 or putisolvin21 BGCs of Pseudomonas spp. Each of the NRPS modules consists of a condensation (C), adenylation (A), and thiolation (T) domain, and a terminal TE-TE domain22 was located at the carboxy terminus of GamC. An in silico analysis of the A domain substrate specificity of the 11 modules, employing automated web-based software tools,17,18,23 was conducted to predict the amino acid composition of the peptide moiety. Because genus-dependent deviations of the A domain specificity code are empirically given,24 we complemented the analysis with a phylogenetic approach in which all A domains were compared with those of functionally characterized NRPS systems from pseudomonads encoding CLPs (Figure S1). Application of both approaches suggested that the NRPS-derived peptide moiety of gacamide possesses the Leu1-Asp2-Gln3-Ile4-Leu5-Gln6-Ser7-Leu8Leu9-Ser10-Ile11 amino acid sequence. A combined manual (Figure S2) and automated17,25 analysis of the C domains of each module identified a lipo-initiation (CStarter)26 domain in module 1 of GamA, suggesting that a fatty acid is attached to Leu1. However, because the biosynthesis genes of the fatty acid moiety are not genetically encoded in the gam BGC but rather provided by the primary metabolism in the form of a 3OH fatty acid,27 the exact length and saturation degree could not be predicted. Further analysis of the C domains showed that those of modules 9 and 10 are predicted to be conventional LCL domains, while the remaining C domains are all classified as combined C/E domains.28 This suggests that Leu8, Leu9, and Ile11 are in the L configuration, while all other amino acids are in the D configuration. However, because a number of deviations from sequence-based prediction of epimerization from C/E domains have been reported in the literature,2,29 it is currently not possible to accurately predict the resultant absolute configuration of C/E domains. Therefore, the predicted absolute configuration of all putative Damino acids needs to be validated by chiral amino acid analysis (vide infra). The termination module of GamC consists of a tandem TE domain, which is typical for most of the Pseudomonas lipopeptides.30 A phylogenetic analysis (Figure S3) showed that the first TE domain clusters with type I TE domains of Pseudomonas CLP BGCs that catalyze the intramolecular cyclization between the C-terminal amino acid and a serine or threonine residue.31,32 The second TE of GamC forms a clade with type II TE domains that fulfill functions in the repair and support of the assembly line. Overall, this bioinformatic analysis strongly suggests the formation of a lipodepsipeptide with the following sequence: 3OH-FA-D/L-Leu1-D/L-Asp2-D/L-Gln3-D/L-Ile4-D/L-Leu5-D/L-

ribosomal unit and ultimately represses the initiation of translation of target genes.9 In summary, the activation of the GacS/GacA system prevents the post-transcriptional blocking of all gene clusters that show a certain GGA motif within the ribosome-binding site. The unleashing of the translation of such gene clusters significantly influences many phenotypes, including carbon storage, swarming motility, biofilm formation, and the production of secretion systems, exoproteins, and secondary metabolites.10 Pseudomonas f luorescens Pf0-1 is a soil bacterium11 that has been extensively investigated in an ecological context due to its environmental persistence.12 Its genome was sequenced13 and showed the presence of several orphan gene clusters that encoded secondary metabolites. One gene cluster attracted our attention, because it was putatively responsible for the biosynthesis of a new cyclic lipopeptide (CLP). CLPs are of particular interest due to their various biological properties, which include predominantly surfactant, antibacterial, and antifungal activities.14 It was previously shown that strain Pf0-1 carries a point mutation in the gacA gene that leads to a nonfunctional GacS/ GacA system. 15 Addition of a repaired gacA + gene complemented the gacA mutation present in Pf0-1, resulting in strain Pf0-1-gacA+ that expresses the many phenotypes requiring the GacS/GacA regulatory system. Among them, the repaired strain Pf0-1-gacA+ exhibits enhanced swarming, is hemolytic, and shows activity in a droplet collapse assay.15 All of these observations pointed toward the production of an amphiphilic compound. Considering that several pseudomonads have been shown to produce surface-active rhamnolipids, a bioinformatics search for the corresponding biosynthetic loci rhlABC16 was performed. It revealed that rhlA (42% identity) is present, while the essential genes rhlB and rhlC are absent. Thus, the possibility that the observed effects were caused by rhamnolipids was excluded. In this work, we present the isolation, structure elucidation, and biological evaluation of gacamide A (1), the product of the formerly silent CLP biosynthetic gene cluster of Pf0-1, employing the complemented strain Pf0-1-gacA+

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RESULTS AND DISCUSSION In Silico Analysis of the Gacamide Gene Cluster. A combined manual and automated bioinformatics analysis using antiSMASH 4.017 and PRISM 318 of the Pf0-1 genome predicted seven putative secondary metabolite BGCs (Table S1). One silent NRPS gene cluster thereof, comprised of three genes, namely, gamA (two modules, 6.4 kb), gamB (five modules, 16.3 kb), and gamC (four modules, 14.6 kb), was further analyzed (Figure 1). Putative LuxR-type regulator and transporter genes are present downstream of gamC and B



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Figure 2. Key 2D NMR correlations for gacamide A (1).

carbons were aliphatic methylenes (Figures S12 and S13). These features, taken together with the molecular formula, suggested that compound 1 is peptidic, consisting of at least 11 amino acids. Preliminary analysis of cross-peaks in the NH amide region of the two-dimensional (2D) 1 H−13C HSQC-TOCSY spectrum corroborated the presence of 11 amino acids (Figure S14). The complete spin system of each amino acid was subsequently established from 2D 1H−1H COSY and 1H−1H TOCSY spectra (Figures S15 and S16). These results, combined with those from 2D 1H−13C HSQC and HMBC analyses (Figures S17 and S18), led to the identification of the following moieties: 4 Leu, 2 Ile, 2 Ser, 2 Gln, and 1 Asp (Figure 2). Subtraction of the C, N, and O atoms, accounted for by the 11 identified amino acid residues from the molecular formula of gacamide A, showed that the remaining fragment of the molecule had to contain 10 carbons and two oxygen atoms. The 13C and 1H−13C HSQC NMR data revealed that the 10 remaining carbon atoms consist of one methyl (δC 13.9), one carbinol methine (δC 69.1), one ester carbonyl (δC 174.1), and seven aliphatic methylene carbons (δC 43.6, 37.9, 25.6, 32.0, 29.5, 29.5, and 27.8), suggesting a linear hydroxy-decanoic acid (Table 1). Correlations in the 1H−1H COSY spectrum delineated a connected spin system for protons at δH 2.50 (H2-2′) and δH 4.10 (H-3′). The observed long-range 1H−13C coupling between H2-2′ and C-1′ subsequently identified the fatty acid fragment as 3-hydroxy-decanoic acid (HDA) (Figure 2). MS/MS analysis of 1 of the [M + Na]+ ion at m/z 1417 produced b- and y-series fragment ions that corroborated the presence of the residues mentioned above and provided the sequence of the amino acids and the fatty acid in gacamide A (Table S2 and Figures S21 and S22): 3-HDA-Xle1-Asp2-Gln3Xle4-Xle5-Gln6-Ser7-Xle8-Xle9-Ser10-Xle11 (Xle = Leu or Ile). The exact planar structure of the lipoundecapeptide with fully assigned leucine and isoleucine residues was determined by further NMR experiments using 1 H− 13 C HMBC correlations from the NH resonances to their corresponding amide carbonyl atoms and observed Hα(i) → HN(i+1) and Hβ(i) → HN(i+1) cross-peaks in 1H−1H ROESY and 1H−1H NOESY spectra, respectively (Figure 2 and Figures S19 and

Gln6-D/L-Ser7-L-Leu8-L-Leu9-D/L-Ser10-L-Ile11, which is cyclized between Ile11 and Ser7. Because of the unique peptide sequence and the new cyclization scheme, the resultant CLP does not belong to the amphisin group of CLPs30 but rather defines a completely new Pseudomonas CLP family. Finally, because the production of gacamide was GacA-dependent,15 the gam BGC was scrutinized for typical binding sites for Pseudomonas repressor proteins. This analysis revealed that indeed a putative target site for RsmA is present upstream of gamA (Figure S4). Isolation and Structural Analysis of Gacamide A. To identify the product of the gacamide BGC, acidified ethyl acetate extracts were prepared from gacA mutant strain Pf0-1 and gacA+-complemented strain Pf0-1-gacA+. Comparison of the metabolite profile by liquid chromatography−mass spectrometry (LC−MS) demonstrated that a group of five peaks (one main compound and four minor congeners) were present in the complemented strain and absent from gacA mutant strain Pf0-1 (Figure S5). The major peak was isolated from a 9 L culture, employing LC−MS-guided fractionation, leading to the purification of gacamide A (1). The lowresolution ESI-MS analysis of compound 1 returned a sodium adduct ion [M + Na]+ at m/z 1417 in positive mode and a deprotonated molecular ion [M − H]− at m/z 1393 in negative mode, while HRESI(+)MS analysis showed a doubly charged sodium adduct of molecular ion [M + 2Na]2+ at m/z 719.91059, corresponding to a molecular formula of C66H115N13O19 requiring 16 double-bond equivalents (Figures S6−S8). Compound 1 displayed end absorption in the ultraviolet (UV) spectrum (Figure S9) and absorption bands for various amide (3300, 1650, and 1540 cm−1) and ester carbonyls (1740 cm−1) in the IR spectrum (Figure S10). The 1 H nuclear magnetic resonance (NMR) spectrum of 1 in d7N,N-dimethylformamide (d7-DMF) displayed resonances for exchangeable NH amide protons at δ 7.4−9.0, overlapping multiplets of α-protons ranging from δ 3.8 to 5.0, a large envelope of CH and CH2 protons at δ 1.2−2.5, and several overlapping methyl group signals centered at δ 0.9 (Figure S11). The 13C and DEPT135 NMR spectra revealed the presence of 15 carbonyls, 18 methines (11 α-amino acid methines, one oxymethine, and six side chain methines), two oxy-methylenes, and 13 methyl groups, and the 18 remaining C



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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data for Gacamide A (1) in d7-DMF δC, mult.

unit

position

Leu1

1 2 3 4 5 6 NH

175.22, 54.49, 40.16, 24.86, 22.29, 21.85,

1 2 3 4 NH

174.17, 173.44, 53.04, 35.50,

C C CH CH2

1 2 3 4 5 NH NH2

174.72, 174.32, 55.99, 32.30, 27.11,

C C CH CH2 CH2

Asp2

C CH CH2 CH CH3 CH3

δH, mult. [J (Hz)]

unit Ser7

4.17, 1.64, 1.77, 0.90, 0.96, 8.65,

m m m m dd (10.2) d (4.9)

Leu8

4.52, m 2.80, m 8.61, d (5.4) Leu9

Gln3

Ile4

Leu5

Gln6

1 2 3 4 5 6 NH

174.13, 60.99, 35.93, 25.98, 15.91, 11.05,

C CH CH CH2 CH3 CH3

1 2 3 4 5 6 NH

173.41, 54.28, 39.83, 24.73, 23.16, 20.83,

C CH CH2 CH CH3 CH3

1 2 3 4 5 NH NH2

174.38, 171.89, 54.60, 32.12, 27.26,

C C CH CH2 CH2

4.20, 2.34, 2.15, 7.98, 7.42, 6.87,

m 2.41, m m d (11.0) brs brs

4.09, 2.16, 1.52, 0.91, 1.07, 7.84,

m m m m d (6.7) d (6.2)

4.17, 1.54, 1.88, 0.92, 0.88, 8.02,

m 1.88, m m m m d

4.17, 2.41, 2.06, 7.64, 7.27, 6.72,

m 2.27, m 2.12, m d (6.7) brs brs

Ser10

Ile11

3-OH-decanoic acid

position

δC, mult.

1 2 3 NH

171.18, C 63.79, CH2 53.54, CH

δH, mult. [J (Hz)]

1 2 3 4 5 6 NH

174.17, 53.55, 40.48, 24.53, 23.48, 21.48,

C CH CH2 CH CH3 CH3

1 2 3 4 5 6 NH

171.80, 53.35, 38.23, 25.26, 23.29, 20.89,

C CH CH2 CH CH3 CH3

1 2 3 NH

170.15, C 62.31, CH2 57.13, CH

1 2 3 4 5 6 NH

170.43, C 56. 83, CH 36. 71, CH 25.03, CH2 15.77, CH3 11.22, CH3

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′

174.12, 43.58, 69.06, 37.93, 25.60, 29.50, 29.50, 32.03, 27.78, 13.94,

4.41, m 4.49, m 7.75, d (7.2)

4.18, 1.50, 1.99, 0.97, 0.92, 7.99,

m 1.77, m m m m d (9.0)

3.91, 1.85, 1.64, 0.92, 0.92, 8.97,

m 1.99, m m m m d (7.0)

3.93, m 4.45, m 7.71, d (8.2)

4.48, m 2.00, m 1.18, m

7.47, d (9.8) C CH2 CH CH2 CH2 CH2 CH2 CH2 CH2 CH3

2.50, 4.10, 1.51, 1.36, 1.29, 1.29, 1.27, 1.28, 0.88,

d (7.0) m m m m m m m t (7.5)

the ring closure is usually provided by diagnostic cross-peaks obtained from 2D 1H−13C HMBC and 1H−1H NOESY spectra, employing different delay or mixing times, or by the application of one-dimensional (1D) HMBC pulse sequences.33 Because of heavy spectral overlap, no unambiguous diagnostic couplings were observed when we subjected 1 to the 1D and 2D NMR experiments mentioned above. Thus, the ring closure issue was solved ultimately by an acetylation strategy.32 Peracetylation of 1 and subsequent MS/MS analysis of O-acylated gacamide A showed that Ser10 and the βhydroxy group of 3-HDA was acetylated while Ser7 was not affected, indicating that the ring closure occurs between Ile11

S20). The combination of all data suggested the lipopeptide sequence HDA-Leu1-Asp2-Gln3-Ile4-Leu5-Gln6-Ser7-Leu8Leu9-Ser10-Ile11, which is in full agreement with the sequence proposed on the basis of bioinformatics. The assigned residues account for only 15 of the 16 required double-bond equivalents; thus, gacamide A has to be cyclic. The two Ser residues or the β-hydroxy group of HDA could in principle provide the hydroxy group for a C-terminal ring closure via an ester bond. However, residue Ser10 could be excluded from participation in a ring closure, because a COSY correlation between Ser10-Hβ (δH 3.93) and Ser10-OH (δH 4.95) revealed that Ser10 possesses a free hydroxy group. Proof of D



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Chart 1

Certain cyclic lipopeptides interact to form a white precipitate, which can be viewed in a white-line-in-agar assay. Pf0-1-gacA+ showed the characteristic white precipitate when interacting with tolaasin, consistent with its production of a cyclic lipopeptide (Figure S25). Because of its amphiphilic nature, 1 was further evaluated for its surface activity in comparison with the standard surfactant surfactin, using tensiometry. Gacamide A decreased the surface tension of phosphate buffer from 71.5 to 28.6 mN/m at a concentration as low as 23 μM, whereas the same concentration of the reference standard surfactin decreased the surface tension to 27.1 mN/m (Figure S26). Because the surfactant activity of lipopeptides commonly assists swarming, we tested this hypothesis by testing the swarming behavior of Pf0-1 on soft agar with and without supplementation of gacamide A at different concentrations. Notably, in the concentration range of ≥0.1 mg/mL, Pf0-1 shows extensive swarming (Figure S27), thus proving one physiological role of 1.

and Ser7 (Table S3 and Figures S23 and S24). With the planar structure of 1 determined, the absolute configuration of the amino acids and that at C-3′ of the lipid side chain required resolution. Stereoanalysis of the 3-hydroxy-decanoic acid of 1 was accomplished by enantioselective HPLC−ESI-MS analysis with Chiralpak ZWIX (+) as the chiral stationary phase34 and found to be in the R configuration. The configuration of the amino acids was determined by enantioselective gas chromatography−mass spectrometry (GC−MS) analysis using Chirasil Val.35 Prior to GC−MS analysis, samples were subjected to acidic hydrolysis and subsequent derivatization. Configurations were determined by comparison of the retention time of the amino acids in the sample with those of authentic standards (Table S4) and found to be 1 L-Ile, 1 Dallo-Ile, 2 D-Leu, 2 L-Leu, 2 D-Ser, 1 D-Asp, 2 D-Gln (detected as D-Glu). To assign the relative positions of the D- and L-Leu, L-Ile, and D-allo-Ile residues, we turned our attention to the information available from the gacamide biosynthesis gene cluster. As mentioned above in the bioinformatics analyses, we deduced that Leu8, Leu9, and Ile11 are in the L configuration. Thus, Leu1 has to be in the D configuration and Ile4 in the Dallo configuration. It is worth mentioning that, in contrast to many other examples of Pseudomonas lipopeptide BGCs, all C/ E domains appear to be fully functional. Confirmation of the Putative Gacamide Gene Cluster. To confirm the identity of the putative cluster, we performed comparative metabolite profiling using LC−MS of the Pf0-1gacA+ strain and a gamA knockout mutant in a gacA+complemented strain, which was available from a former study.15 In the Pf0-1-gacA+/ΔgamA (syn. gacA+ Δ2211) mutant, the production of gacamides was completely abolished (Figure S5). A former phenotypic analysis using motility assays revealed reduced swarming motility and the absence of surfactant ring formation ahead of the cellular front as the main alterations of the Pf0-1-gacA+/ΔgamA mutant.15 We thus concluded that the gamABC operon indeed represents the gacamide BGC. Biological Activity. Several cyclic lipopetides were reported to possess significant antibacterial activity.11 To investigate if gacamide A has antibacterial activity, 1 was evaluated in two antimicrobial assay panels. One assay investigated the activity toward human pathogens, while the other panel focused on plant pathogens (Tables S5 and S6). However, gacamide A (1) did not convincingly inhibit growth of any of the tested bacteria, with the exception of moderate inhibitory activity toward the bacterium Arthrobacter crystallopoietes DSM 20117 [minimum inhibitory concentration (MIC) of 32 μg/mL].

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EXPERIMENTAL SECTION

General Experimental Procedures. HPLC was performed with a Waters system, consisting of a Waters 600 controller and pump, a Waters 2996 photodiode array detector, a Rheodyne 7725i injector, and a 200 series PerkinElmer vacuum degasser. For LC−MS analysis, a 1100 Series HPLC system (Agilent Technologies) was fitted with a G1322A degasser, a G1312A binary pump, a G1329A autosampler, and a G1315A diode array detector. The Agilent HPLC components were connected with an ABSCIEX 3200 QTRAP LC/MS/MS mass spectrometer (Sciex, Darmstadt, Germany). 1D and 2D NMR spectra were measured either on a Bruker AV-III 600 MHz spectrometer using a 5 mm inverse broadband probe head (located at CESAMOISM, Talence, France) or on a Bruker Avance III 600 MHz spectrometer, equipped with a cryo-platform (located at the HansKnöll-Institute, Jena, Germany). All spectra were recorded in d7-DMF (Deutero, Kastellaun, Germany) and calibrated to the residual solvent signals (resonances at δH 2.92 and δC 34.89). Optical rotation values were measured on a Jasco P-2000 polarimeter, using a 3.5 mm × 10 mm cylindrical quartz cell. UV spectra were recorded on a PerkinElmer Lambda 25 UV/vis spectrometer. Infrared spectra were obtained by employing a Jasco FT/IR 4200 spectrometer, interfaced with a MIRacle ATR device (ZnSe crystal). High-resolution mass spectra were acquired on an HR-ESI-TOF-MS Bruker maXis 4G mass spectrometer. All solvents were purchased as HPLC or LC−MS grade. Cultural Conditions for Isolation of Gacamide A. P. f luorescens Pf0-1-gacA+ is Pf0-1 containing a plasmid-borne gacA+. Seed cultures of P. f luorescens Pf0-1-gacA+ were grown in 12.5 mL of Davis Minimal Broth without dextrose35 but containing 20 mM glycerol (DMBgly) and 10 mg/mL tetracycline (to maintain the plasmid) in 50 mL Falcon tubes for 3 days employing a Gerhardt horizontal shaker at 110 rpm. Six 5 L Erlenmeyer flasks containing 1.5 L of DMBgly were inoculated with 3 mL of seed culture. Cultures E



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were incubated for 48 h at 23 °C in an INFORS HT Multitron Pro orbital incubator shaker with shaking at 120 rpm. Extraction and Isolation. All of the fermentation broth from a 9 L fermentation of P. f luorescens Pf0-1-gacA+ was acidified with trifluoroacetic acid to pH 2 and subsequently repeatedly extracted with 1:1 EtOAc to yield 8.82 g of crude extract. This extract was fractionated using vacuum liquid chromatography (VLC). The reversed phase (RP) C18 column was eluted stepwise under vacuum with solvents of decreasing polarity, ranging from a 50:50 MeOH/ H2O mixture to pure methanol, followed by 100% dichloromethane (DCM) to give seven fractions (A−G). Fraction F (99.6 mg), eluting with 100% MeOH, was further subfractionated by solid phase extraction (SPE) (Chromabond C18, 2 g) using stepwise gradient elution from a 15:85 H2O/MeOH mixture to 100% MeOH to give five subfractions (Fa−Fe). Purification of subfraction Fb was performed by RP-HPLC using a linear gradient from 50:50 to 100 MeOH/H2O (0.1% TFA) over a period of 30 min, followed by isocratic elution at 100 MeOH for an additional 10 min (Phenomenex C18 Luna, 10 mm × 250 mm, 5 μm; 2 mL/min flow rate; UV monitoring at 215 nm). A second purification was done using the same gradient with a Phenomenex Kinetex XB-C18, 4.6 mm × 250 mm column (1 mL/min flow rate, 215 mm), which yielded pure gacamide A (tR = 30.4 min; 27 mg) and minute amounts of its derivatives. Gacamide A (1). Amorphous, white opaque powder: [α]25D −7 (c 0.33, DMF); UV (MeCN) λmax (log ε) 196 nm (5.3); FT-IR (ATR) νmax 3300, 1740, 1650, 1540 cm−1; see Table 1 for 1H NMR and 13C NMR data; positive HR-ESI-MS m/z 719.91059 [M + 2Na]2+ (calcd for C66H115N13O19Na2, 719.91138; Δ = −1.1 ppm). Chiral HPLC−MS Analysis of the 3-Hydroxy-decanoic Acid Portion of 1. The sample was hydrolyzed using 250 μL of 6 N deuterated hydrochloric acid (DCl/D2O) at 110 °C for 24 h. Afterward, DCl/D2O was removed using a Thermo Savant ISS110 SpeedVac (Thermo Scientific, Holbrook, MA) at 43 °C for 1−1.5 h. To decrease the level of ion suppression in the ESI process, the free 3hydroxy-decanoic acid was isolated by liquid−liquid extraction using 200 μL of a mixture containing water and chloroform in a 1:1 ratio. The chloroform layer was used for the determination of the absolute configuration of the 3-hydroxy-decanoic acid. The chloroform was removed in the Speed Vac, and the residue was dissolved in methanol. Subsequently, analysis was performed using a Chiralpak ZWIX (+) [150 mm × 4 mm (inside diameter)] column from Chiral Technologies Europe (Illkirch, France). The column was thermostated at 10 °C, and a 30 min isocratic run was performed using an acetonitrile/methanol/acetic acid mixture (95:5:0.025) as the mobile phase and a flow rate of 0.3 mL/min. The injection volume was set to 10 μL. HPLC−MS experiments were performed on an Agilent 1100 LC MSD ion trap system equipped with an autosampler, a binary gradient pump, and a column thermostat (Agilent, Waldbronn, Germany). The ESI source was operated in negative ionization mode at 350 °C and 40 psi with a dry gas flow of 7.5 L/min. Analysis was performed in a scan range of m/z 180−200. Analysis of the authentic standards of racemic and 3R-hydroxy-decanoic acid showed that the R enantiomer elutes earlier on the ZWIX (+) column at a retention time of 13.48 min while the S enantiomer elutes at 14.67 min. The retention time of the obtained 3-hydroxy-decanoic acid portion of 1 (tR = 13.21 min) matched that of 3R-hydroxy-decanoic acid. Chiral GC−MS Analysis of the Peptide Moiety of Gacamide A (1). One milligram of gacamide A was dissolved in 500 μL of 6 N DCl/D2O (deuterated hydrochloric acid), and hydrolysis allowed to proceed at 110 °C for 24 h. The solvent was removed in a Speed Vac. Afterward, 250 μL of EtOD/DCl (obtained from reaction of 15% acetyl chloride in deuterated ethanol) was added and the derivatization carried out at 110 °C for 20 min to give the ethyl esters of the amino acids. The solvent was removed by a gentle stream of nitrogen at 110 °C. The residue was treated with 250 μL of a 1:2 (v:v) trifluoroacetic acid anhydride/trifluoroacetic acid ethyl ester mixture at 130 °C for 10 min. The derivatization reagent was removed by nitrogen at room temperature. The derivatized amino acids were dissolved in 100 μL of dichloromethane and injected into the GC−

MS instrument. GC analysis was performed using an Agilent Technologies 7890 A GC-System with a 5975 C inert MSD with Triple-Axis. The GC−MS instrument was equipped with an LChirasil Val column from C.A.T. (Tübingen, Germany) (20 m × 300 μm × 0.28 μm). The column temperature was held at 50 °C for 0.5 min, increased in a first step from 50 to 77 °C at a heating rate of 50 °C/min and in a second step from 77 to 195 °C at a rate of 4 °C/min, and finally held at 195 °C for an additional 7 min. The flow rate was 3.2 mL/min with an injection temperature of 220 °C and splitless injection with helium as the carrier gas. For MS detection, 70 eV was used for ionization and the SIM mode was used for data acquisition. Standards were analyzed by including all enantiomers of the proteinogenic amino acids, including allo-Ile, which were derivatized as described above. Each peak in the chromatographic trace was identified by comparing the retention time and mass spectra of sample and standard runs. Acetylation of Gacamide A (1). Eight milligrams of pure 1 was incubated with 1 mL of pyridine (water free) and 1 mL of acetic anhydride overnight under light exclusion. The acetylated product was precipitated with 20 mL of ice-cold water. Subsequently, the substance was extracted three times with 20 mL of chloroform. The organic phase was washed twice with distilled water and dried with Na2SO4. After the removal of the solvents under reduced pressure, the product was analyzed by analytical HPLC, and afterward, the acetylated gacamide A was subjected to MS/MS measurements. Antibacterial Assays. The antibacterial activity was determined as previously described36 by agar diffusion assays. In brief, culture plates (5% sheep blood Columbia agar, BD) were overlaid with a 3 mL tryptic soy soft agar, inoculated with the tryptic soy broth (TSB, Oxoid) growth suspension of the bacteria to be tested. Compounds were diluted with dimethyl sulfoxide (DMSO) to a concentration of 1 mg/mL, and 3 μL of this dilution was spotted on the surface of the agar; the diameter of the inhibition zone was measured after incubation at 37 °C for 24 h. The indicator strains represent clinical isolates from distinct patients.37 These strains were maintained on Mueller−Hinton (MH) agar or on blood agar. MIC determinations were carried out in microtiter plates. Arthrobacter crystallopoietes DSM 20117 was grown in tryptic soy broth (Oxoid); all other strains were grown in half-concentrated Mueller−Hinton broth (Oxoid). MICs with 2-fold serial dilution steps were determined (1:2). Bacteria were added to give a final concentration of 105 colony-forming units in a volume of 0.2 mL. After incubation for 24 h at 37 °C, the MIC was read as the lowest compound concentration causing inhibition of visible growth. Results are mean values of three independent determinations. Pure compounds demonstrating an MIC of >50 μg/mL were regarded as inactive samples. The antibacterial activity against plant-associated bacteria was determined as previously described by agar diffusion assays.38 Streptomycin was included as a positive control. White-Line-In-Agar Test. The white-line test was performed according to the method of Wong and Preece.39 Determination of the Decrease in Surface Tension. The surface tension was measured at 23 °C with a Sinterface PAT-1 tensiometer. The reference compound surfactin was purchased from Sigma-Aldrich Chemicals (Taufkirchen, Germany) and used as supplied. Samples were dissolved in 1 mL of a mixture consisting of 950 μL of phosphate buffer (pH 8) and 50 μL of DMSO at different concentrations, and three replicates were measured. The surface tension values for distilled water and the buffer/DMSO mixture were almost the same. Swarming Assay. Gacamide A (1) was dissolved at a final concentration of 5 mg/mL in DMSO and stored at −20 °C. To test whether pure 1 could restore swarming motility to P. f luorescens Pf0-1, gacamide A was diluted to 0.1 and 0.05 mg/mL in a buffer composed of 20 mM Tris and 0.85% NaCl (pH 7). Swarming motility was tested on LB agar plates solidified with 0.6% bacto agar. On these plates we spread 25 μL of diluted gacamide A or equivalently diluted DMSO as a control. The plates were then allowed to dry for 30 min at room temperature, after which 2.5 μL of a 20 h old Pf0-1 culture was F



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spotted in the center. Plates were incubated at 25 °C and examined daily to score motility. Nucleotide Sequence Accession Number. The nucleotide sequence of the whole genome sequence of Pf0-1 can be found in the GenBank under accession number CP000094.2 (for coordinates of the gacamide BGC, see the Supporting Information). Furthermore, the gene cluster for gacamide A has been deposited in the Minimum Information about a Biosynthetic Gene Cluster (MIBiG) repository40 under accession number BGC0001842.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00747. Bioinformatics analyses, spectral data (HR-ESI-MS, IR, UV, and 1D and 2D NMR spectra), chiral GC−MS analysis, and biological assay results of compound 1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +49-7071-2976970. Fax: +49-7071-295250. Email: [email protected]. ORCID

Michael Lämmerhofer: 0000-0002-1318-0974 Harald Gross: 0000-0002-0731-821X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS G.J. gratefully acknowledges the German Academic Exchange Service (DAAD) for a Ph.D. scholarship (A/11/96661). The authors thank Dr. D. Wistuba and her team (Mass Spectrometry Department, Institute for Organic Chemistry, University of Tübingen) for HRMS measurements and Prof. M. Nett (Department of Biochemical and Chemical Engineering, Technical University Dortmund, Dortmund, Germany) for recording NMR spectra. The authors are grateful for the kind help of A. Färber (Department of Pharmaceutical Technology, Pharmaceutical Institute, University of Tübingen) for performing tensiometric measurements and Brenda T. Shaffer (Agricultural Research Service, U.S. Department of Agriculture, Corvallis, OR) for assistance with the biological assays. Funding from the Deutsche Forschungsgemeinschaft (DFG), who supported H. Gross (GR2672/2-1) and H.-G. Sahl (Sa292/13-1) within the frame of the “Research Unit FOR854-Post-Genomic Strategies for New Antibiotic Drugs and Targets”, is gratefully acknowledged. The contribution of H. Gross and H.-G. Sahl was also financially supported by the German Centre for Infection Research (DZIF). M.L. acknowledges support by the “Struktur- und Innovationsfonds BadenWü rttemberg (SI-BW)” and by the German Research Foundation DFG for funding scientific equipment as part of the DFG’s Major Research Instrumentation Program as per Art. 91b GG (INST 37/821-1 FUGG).

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