Metabolomic tools for secondary metabolite discovery from marine microbial symbionts

Abstract

Marine invertebrate-associated symbiotic bacteria produce a plethora of novel secondary metabolites which may be structurally unique with interesting pharmacological properties. Selection of strains usually relies on literature searching, genetic screening and bioactivity results, often without considering the chemical novelty and abundance of secondary metabolites being produced by the microorganism until the time-consuming bioassay-guided isolation stages. To fast track the selection process, metabolomic tools were used to aid strain selection by investigating differences in the chemical profiles of 77 bacterial extracts isolated from cold water marine invertebrates from Orkney, Scotland using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Following mass spectrometric analysis and dereplication using an Excel macro developed in-house, principal component analysis (PCA) was employed to differentiate the bacterial strains based on their chemical profiles. NMR 1H and correlation spectroscopy (COSY) were also employed to obtain a chemical fingerprint of each bacterial strain and to confirm the presence of functional groups and spin systems. These results were then combined with taxonomic identification and bioassay screening data to identify three bacterial strains, namely Bacillus sp. 4117, Rhodococcus sp. ZS402 and Vibrio splendidus strain LGP32, to prioritize for scale-up based on their chemically interesting secondary metabolomes, established through dereplication and interesting bioactivities, determined from bioassay screening.

Figure 1

Classification of the isolates by (a) source invertebrate species; (b) cultivation media; (c) phyla and (d) by genera (if known).

Figure 2

Metabolomics and dereplication workflow to aid strain selection.

Figure 3

(a) Principal component score plot analysis of 77 strains clustered according to features (m/z ratios) from mass spectral data (R₂ = 0.4). Bioactivities of outliers are represented using symbols; (*) Anti-trypanosomal activity against Trypanosoma brucei brucei, (•) PTP1B, (Δ) TRPV1, (◊) TRPA1, (□) TRPM8, (○) PPARα, and (▪) Enterococcus faecalis. Rhodococcus sp. ZS402 was also found to be NRPS positive (+); (b) Accompanying PCA loading plot of the 77 strains investigated in this study; (c) Variable intensity plot illustrating two metabolites observed as outliers in (b) (m/z 265.1476 and m/z in 279.1631) in Rhodococcus sp. ZS402.

Figure 4

Heat map based on mass spectrometry data displaying distinct metabolic profiles amongst the 77 bacterial species: (a) dendrogram from multivariate analysis overlaid with heat map; and (b) heat map organized according to species showing differences in the chemical profiles of strains and species. Species observed as outliers from PCA are highlighted and labelled using an asterisk*. (Abbreviations; UBC= uncultured bacterial clone, UGP clone = uncultured gamma proteobacterium, UMB = uncultured marine bacterium).

Figure 5

Positive and negative mode base peak chromatograms from outlying bacterial sample, Bacillus sp. 4115, annotated to indicate metabolites identified in Table 2. NB: several of the metabolites were detected in both positive and negative modes. Positive and negative mode base peak chromatograms from M1 agar medium are shown to indicate that the annotated metabolites are being produced by the bacteria and are not from the medium.

Figure 6

Mass spectrum for Bacillus sp. 4115 in the positive ionization mode showing the presence of a cluster of features within the RT range of 30–39 min. Those annotated with an asterisk * are sodium ion adducts, [M+Na]⁺.

Figure 7

Deconvoluted chromatogram for Bacillus sp. 4115 in the positive ionization mode for extracted ions within the m/z range of 1000–1200 Da.

Figure 8

2D-NMR COSY spectrum of Bacillus sp. 4115, overlaid with spectrum from M1 medium. Signals in orange are from the sample and signals in grey are from the medium.

Figure 9

Positive and negative mode base peak chromatograms from outlying bacterial sample, Vibrio splendidus strain LGP32 annotated to indicate metabolites identified in Table 3. Positive and negative mode base peak chromatograms from M1 agar medium are shown to indicate that the annotated metabolites are being produced by the bacteria and are not from the medium.

Figure 10

2D-NMR COSY spectrum from sample Vibrio splendidus strain LGP32 overlaid with media. Cross signals in orange are from the sample and signals in grey are from the media. Higlighted correlations indicate substructures from oxyplicacetin.

Figure 11

Positive and negative mode base peak chromatograms from outlying bacterial sample, Rhodococcus sp. ZS402, annotated to indicate metabolites identified in Table 4. Positive and negative mode base peak chromatograms from ISP2 agar medium are shown to indicate that the annotated metabolites are being produced by the bacteria and are not from the ISP2 agar medium.

Figure 12

2D-NMR COSY spectrum of Rhodococcus sp. ZS402 overlaid with medium. Signals in orange are from the sample and signals in grey are from the medium.