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.

Biotransformation-coupled mutasynthesis for the generation of novel pristinamycin derivatives by engineering the phenylglycine residue

“…Assays were carried out and evaluated as reported before. 51 Based on previous studies, iv TT fluorescence values of 0–20% indicate strong inhibition of the assay, 0–40% are considered to represent specific inhibition, whereas values above 40% do not indicate inhibition of the iv TT assay. In iv TT assays with pristinamycin derivatives, it was found that pristinamycin II resulted in strong inhibition of the iv TT assay, whereas neither pristinamycin I ( 1 ) nor the halogenated pristinamycin I derivatives 3 and 4 led to an inhibition (Fig.…”

“…13 In vitro transcription and translation (ivTT) assay with pristinamycin I derivatives Semi-purified HPLC fractions of 1, 3, and 4 were tested in ivTT assays. Assays were carried out as reported before 51 and were conducted in triplicates. Each assay had an end concentration of approximately 0.5 mg mL À1 of 1, 3, 4, or PI standard (Sanofi-Aventis) with or without the addition of 0.5 mg mL À1 pristinamycin II standard (Sanofi-Aventis), respectively.…”

Challenging old microbiological treasures for natural compound biosynthesis capacity

“…The strains DSM 40484 T , DSM 40713 T , DSM 40907 T , DSM 40971 T , and DSM 40976 T were cultivated by inoculation from a GYM plate in 50 mL medium R5 (103 g L −1 sucrose, 10 g L −1 glucose, 0.25 g L −1 K 2 SO 4 , 10.12 g L −1 MgCl 2 x 6 H 2 O, 0.1 g L −1 casamino acids, 5 g L −1 yeast extract, 5.73 g L −1 TES (pH 7.2), 2.94 g L −1 CaCl 2 x 2H 2 O, 0.05 g L −1 KH 2 PO 4 , 3 g L −1 L-proline, and 2 mL trace element solution, according to the work of Handel et al (2022) ; pH 7.4) at 28°C in 250 mL Erlenmeyer flasks on an orbital shaker (180 rpm). After 2 days of cultivation, 5 mL of the preculture was used to inoculate 250 mL Erlenmeyer flasks with 50 mL of production media R5 or NL 800 (5 g L −1 glucose, 10 g L −1 glycerol, 10 g L −1 soluble starch, 58 g L −1 oatmeal, 2 g L −1 yeast extract, 1 g L −1 NaCl, and 1 g L −1 CaCO 3 ; pH 7.2) as production cultures, which were incubated for 3 days at 28°C on an orbital shaker at 180 rpm.…”

“…DSM 40976 T by conjugation, as described by Kieser et al (2000) , resulting in the strain DSM 40976 T pGM1190/ papR2-tipA p. Apramycin (50 μg/mL) was used for selection when appropriate. To test for the effect of papR2 expression on secondary metabolite production, the strain DSM 40976 T pGM1190/ papR2-tipA p and the WT strain were each cultivated in OM medium (20 g L −1 oat meal, 5 mL trace element solution, pH 7.3; according to Handel et al (2022) ) at 28°C; 20 mL of culture was harvested after 72 h of cultivation. For extraction of organic compounds, cultures were treated as described above.…”

Modern Trends in Natural Antibiotic Discovery

“…A specialized platform for the search for such resistance determinants (Antibiotic-Resistant Target Seeker (ARTS)) [ 124 , 125 ] and a database with pre-computed ARTS results for >70,000 genomes were developed by Prof. Ziemert’s group [ 126 ]. This approach, called Ψ-footprinting, was also recently adapted to the search for protein synthesis inhibitors (PSI = Ψ) [ 127 ].…”