Ψ-Footprinting approach for the identification of protein synthesis inhibitor producers

Abstract

Today, one of the biggest challenges in antibiotic research is a targeted prioritization of natural compound producer strains and an efficient dereplication process to avoid undesired rediscovery of already known substances. Thereby, genome sequence-driven mining strategies are often superior to wet-lab experiments because they are generally faster and less resource-intensive. In the current study, we report on the development of a novel in silico screening approach to evaluate the genetic potential of bacterial strains to produce protein synthesis inhibitors (PSI), which was termed the protein synthesis inhibitor ('psi') target gene footprinting approach = Ψ-footprinting. The strategy is based on the occurrence of protein synthesis associated self-resistance genes in genome sequences of natural compound producers. The screening approach was applied to 406 genome sequences of actinomycetes strains from the DSMZ strain collection, resulting in the prioritization of 15 potential PSI producer strains. For twelve of them, extract samples showed protein synthesis inhibitory properties in in vitro transcription/translation assays. For four strains, namely Saccharopolyspora flava DSM 44771, Micromonospora aurantiaca DSM 43813, Nocardioides albertanoniae DSM 25218, and Geodermatophilus nigrescens DSM 45408, the protein synthesis inhibitory substance amicoumacin was identified by HPLC-MS analysis, which proved the functionality of the in silico screening approach.

Graphical Abstract

Ψ-footprinting approach.

Figure 1.

Agar-based reporter screening with samples from Tü 2108 and bioreporter test strain B. subtilis P_bmrC-lacZ. 1. panel: Tü 2108-grown OM agar plug; 2. panel: 5 μl culture extract from Tü 2108 grown in R5 medium; 3.-4. panel: Pure antibiotics (50 μg berninamycin and 10 μg chloramphenicol (CM)) were prepared on filter discs as controls.

Figure 2.

Whole-genome sequence tree generated with the TYGS web server for strain Tü 2108 and closely related species. Tree inferred with FastME from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d₅. The numbers above branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with an average branch support of 84.9%.

Figure 3.

Cluster comparison between S. bernensis berninamycin A gene cluster and Tü 2108 region 6_3. Black: berninamycin biosynthesis genes. Yellow: ribosomal genes.

Figure 4.

Graphic illustration of the genome sequenced-based screening approach for the identification of PSI producer strains, designated Ψ-footprinting.

Figure 5.

In vitro transcription/translation assay performed with culture extracts of controls. PSI antibiotics, tet₁₅ and apra₅₀ = positive control (orange), medium extracts, extracts of M1146 = negative control (green). For M1146, results are shown exemplary for samples from day 7. Measurements have been performed in triplicate using the same preparation of S12 extract.

Figure 6.

In vitro transcription/translation assay performed with culture extracts in NL800, MS and R5 media from 4, 7 and 10 days (optimal production time of each strain). PSI antibiotics, tet₁₅ and apra₅₀ = positive control (orange), medium extracts, extracts of M1146 = negative control (green), and extracts of DSM cultures (blue). Displayed are the extracts of each DSM strain that resulted in the most decrease of GFP production of the ivTT assay. Measurements have been performed in triplicate using the same preparation of S12 extract.

Figure 7.

In vitro transcription/translation assay performed with R5 culture extract of DSM 43813 from day 7. Displayed are the fractions generated by semi-preparative HPLC (blue). PSI antibiotic apra₅₀ = positive control (orange). Fraction having the greatest inhibition is shown in red. Measurements have been performed in triplicate using the same preparation of S12 extract.

Figure 8.

(A) HPLC chromatogram from fraction F8 of the R5 extract from DSM 25218. Wavelength monitoring was performed at 260 nm. Arrows indicate amicoumacin A and B specific peaks at RT 7.2 min and 7.4 min, respectively. (B, C) Mass spectra from fraction F8 of the R5 extract of the amicoumacin producer strains. Amicoumacin A peaks in positive mode (B) m/z = 424.2 [M + H]⁺ and negative mode (C) m/z = 422.1 [M-H]^– at RT 7.2 min are marked with red ellipses. (D) UV–Vis spectrum of DSM 25218 extract sample of the main peak at RT 7.2 min with amicoumacin B as reference and structure. Here, amicoumacin B was used as a reference since it was the only amicoumacin compound available in the internal database. (E, F) Mass spectra from fraction F8 of the R5 extract of the amicoumacin producer strains. Amicoumacin B peaks in positive mode (E) m/z = 425.1 [M + H]⁺ and negative mode (F) m/z = 423.1 [M-H]^– at RT 7.4 min are marked with red ellipses. (G) UV–Vis spectrum of DSM 25218 extract sample of the shoulder peak at RT 7.4 min with amicoumacin B as reference and structure. The background was subtracted due to the overexposed masses of amicoumacin A for B/C and E/F (from main peak).