Identification of the lipodepsipeptide selethramide encoded in a giant nonribosomal peptide synthetase from a Burkholderia bacterium

Abstract

Bacterial natural products have found many important industrial applications. Yet traditional discovery pipelines often prioritize individual natural product families despite the presence of multiple natural product biosynthetic gene clusters in each bacterial genome. Systematic characterization of talented strains is a means to expand the known natural product space. Here, we report genomics, epigenomics, and metabolomics studies of Burkholderia sp. FERM BP-3421, a soil isolate and known producer of antitumor spliceostatins. Its genome is composed of two chromosomes and two plasmids encoding at least 29 natural product families. Metabolomics studies showed that FERM BP-3421 also produces antifungal aminopyrrolnitrin and approved anticancer romidepsin. From the orphan metabolome features, we connected a lipopeptide of 1,928 Da to an 18-module nonribosomal peptide synthetase encoded as a single gene in chromosome 1. Isolation and structure elucidation led to the identification of selethramide which contains a repeating pattern of serine and leucine and is cyclized at the side chain oxygen of the one threonine residue at position 13. A (R)-3-hydroxybutyric acid moiety decorates the N-terminal serine. Initial attempts to obtain deletion mutants to probe the role of selethramide failed. After acquiring epigenome (methylome) data for FERM BP-3421, we employed a mimicry by methylation strategy that improved DNA transfer efficiency. Mutants defective in selethramide biosynthesis showed reduced surfactant activity and impaired swarming motility that could be chemically complemented with selethramide. This work unveils a lipopeptide that promotes surface motility, establishes improved DNA transfer efficiency, and sets the stage for continued natural product identification from a prolific strain.

Keywords: bacterial host development; lipopeptide; natural products.

Fig. 1.

Natural products from Burkholderia sp. FERM BP-3421. (A) Known natural products previously reported (representative structures of the spliceostatin family) and detected in this study (romidepsin and aminopyrrolnitrin). Reported bioactivities are indicated. (B) Selethramide identified in this study.

Fig. 2.

Genome and metabolome of Burkholderia sp. FERM BP-3421. (A) Genome map highlighting the type and distribution of BGCs. The genome contains four circular replicons, i.e., two chromosomes (Chr.) and two plasmids (p). Replicons are oriented with respect to replication genes dnaA or parA and parB as indicated. The sizes of the replicons are not to scale. The location of BGCs encoding natural products is indicated and color-coded by biosynthetic class as shown (lane 1 from the outside in). BGCs were numbered clockwise from the replication gene used to orient each replicon. Known BGCs (orb, ornibactin; prn, pyrrolnitrin; cay, caryoynencin; fr9, spliceostatins; dep, romidepsin) and selethramide (sel) reported here are indicated. BGCs for which we detected products are in bold. Predicted open reading frames (ORFs) on the leading (black) and lagging (gray) strands are shown on lanes 2 and 3, respectively. A normalized and skewed plot of guanosine + cytosine (G+C) content (yellow/purple) is depicted in lanes 4 and 5 respectively. PKS, polyketide synthase; NRPS, nonribosomal peptide synthetase; RiPP, ribosomally synthesized and posttranslationally modified peptides; Hserlactone, homoserine lactone. Genome maps were generated using DNAplotter (22). Genome accession codes CP117782 (chr 1), CP117781 (chr 2), CP117780 (p1), CP117779 (p2). (B) Molecular network analysis of metabolites produced by FERM BP-3421 in 2S4G medium. MS/MS spectra were window filtered by choosing only the top six fragment ions in the ±5.0-Da window throughout the spectrum. The precursor ion mass tolerance was set to 0.03 Da and a MS/MS fragment ion mass tolerance of 0.03 Da. A network was then created where edges were filtered to have a cosine score above 0.7 and more than 6 matched peaks.

Fig. 3.

Identification of selethramide. (A) Predicted biosynthesis of selethramide. Locus 1.2 contains one contiguous NRPS gene selB encoding 18 modules, in addition to a MbtH-like chaperone selA, that together are proposed to represent the sel BGC. Genes surrounding selAB are expected to be involved in primary metabolism, i.e., a glutathione-S-transferase, and a glycine betaine/l-proline transporter ProP upstream of selA, and a malonate-utilizing operon downstream of selB. The predicted domains of SelB are indicated. The stereoconfiguration of the amino acids was predicted based on the C-domain type present at each module. See also SI Appendix, Fig. S17 for a C-domain phylogenetic tree and sequence alignment. (B) MS/MS fragmentation pattern for selethramide. The amino acid sequences with major fragments consistent with the losses of acyl chain-Ser1 (b2 ion, calcd 1755.0873, expt 1755.0752), Ser2 (b3 ion, calcd 1668.0552, expt 1668.0560), Leu3 (b4 ion, calcd 1554.9712, expt 1544.9613), Leu4 (b5 ion, calcd 1441.8871, expt 1441.8886), Ser5 (b6 ion, calcd 1354.8551, expt 1354.8472), Ser6 (b7 ion, calcd 1267.8231, expt 1267.8250), Leu7 (b8 ion, calcd 1154.7390, expt 1154.7352), Leu8 (b9 ion, calcd 1041.6549, expt 1041.6518), Ser9 (b10 ion, calcd 954.6229, expt 954.6245), Leu10 (b11 ion, calcd 841.5388, expt 841.5395), Leu11 (b12 ion, calcd 728.4548, expt 728.4480), and Ser12 (b13 ion, calcd 641.4227, expt 641.4178) are indicated. For the full-length fragmentation pattern of the linear basic methanolysis product, see SI Appendix, Fig. S4. (C) Selected 2D NMR correlations.

Fig. 4.

Harnessing epigenome data to improve DNA transfer efficiency. (A) Methylome and predicted R-M systems of Burkholderia sp. FERM BP-3421. Putative R-M systems predicted using REBASE are shown on the table to the right, where underlined bases represent m6A and m4C. S, G, or C; W, A, or T. To illustrate the density of each methylation site, the distribution of methylated motifs is shown on the left for chromosome 1, in lane 1 (M.BurIV, blue), lane 2 (M.BurII, gray), and lane 3 (M.BurI, green), from the outside in. The CR motif is not shown because it appears to be a sequencing artifact. The normalized and skewed plots of G+C content (yellow/purple) are depicted in lanes 4 and 5 respectively. (B) Schematic representation of the mimicry by methylation strategy used to improve DNA transfer efficiency. (C) Comparison of electroporation efficiency calculated as yellow colony forming units (CFU)/µg plasmid DNA. Burkholderia FERM BP-3421 was electroporated with plasmid pMo168 prepared from E. coli carrying either pACYC184_MBurI_MBurII (+) or the empty vector pACYC184 as negative control (−). The error bars indicate SDs (N = 3, P-value < 0.001 using a two-tailed t test). Representative plate results are shown (LB agar with kanamycin selection and catechol metabolism via the xylE reporter present in pMo168, i.e., yellow indicates pMo168 uptake).

Fig. 5.

Selethramide is required for swarming motility. (A) Liquid chromatography-mass spectrometry analysis of culture extracts from the wild-type FERM BP-3421 (WT) and the selethramide deletion mutant (ΔselB MT). Extracts were obtained after cultivation in 2S4G medium for 5 d. Extracted ion chromatograms at 1928.84. (B) Swarming assay on LB containing Bacto agar at 0.5% w/v. Pictures taken after 72 h. For chemical complementation, phenotypic concentrations of selethramide were used (3 µg/mL added to plate prior to inoculation). (C) Drop collapse assay. To 20-µL droplets of water containing crystal violet at 0.0025% to facilitate visualization, 5 µL of water or 2S4G production medium was added as negative control (4 mm drop diameter observed), whereas the same volume of aqueous Tween 20 at 0.1% w/v served as positive control (5 mm drop diameter observed). WT (7 mm drop diameter observed) and MT (4 mm drop diameter observed) samples consisted of 5 µL of culture normalized to the same OD₆₀₀.