Bioreporters for direct mode of action-informed screening of antibiotic producer strains

Wex, Katharina W.; Saur, Julian S.; Handel, Franziska; Ortlieb, Nico; Mokeev, Vladislav; Kulik, Andreas; Niedermeyer, Timo H. J.; Mast, Yvonne; Grond, Stephanie; Berscheid, Anne; Brötz‐Oesterhelt, Heike

doi:10.1016/j.chembiol.2021.02.022

Cited by 15 publications

(17 citation statements)

References 67 publications

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“…Since the development of the system, we have conducted 3 screening campaigns. The results of these campaigns will be reported elsewhere or have already been published [55]. Sterilization of the replicator proved to be the rate-limiting step in the developed workflow, but inoculating multiple plates from the same pore suspension master plate is a rapid process.…”

Section: Discussionmentioning

confidence: 95%

Development of an agar-plug cultivation system for bioactivity assays of actinomycete strain collections

et al. 2021

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Natural products are an important source of lead compounds for the development of drug substances. Actinomycetes have been valuable especially for the discovery of antibiotics. Increasing occurrence of antibiotic resistance among bacterial pathogens has revived the interest in actinomycete natural product research. Actinobacteria produce a different set of natural products when cultivated on solid growth media compared with submersed culture. Bioactivity assays involving solid media (e.g. agar-plug assays) require manual manipulation of the strains and agar plugs. This is less convenient for the screening of larger strain collections of several hundred or thousand strains. Thus, the aim of this study was to develop a 96-well microplate-based system suitable for the screening of actinomycete strain collections in agar-plug assays. We developed a medium-throughput cultivation and agar-plug assay workflow that allows the convenient inoculation of solid agar plugs with actinomycete spore suspensions from a strain collection, and the transfer of the agar plugs to petri dishes to conduct agar-plug bioactivity assays. The development steps as well as the challenges that were overcome during the development (e.g. system sterility, handling of the agar plugs) are described. We present the results from one exemplary screening campaign targeted to identify compounds inhibiting Agr-based quorum sensing where the workflow was used successfully. We present a novel and convenient workflow to combine agar diffusion assays with microtiter-plate-based cultivation systems in which strains can grow on a solid surface. This workflow facilitates and speeds up the initial medium throughput screening of natural product-producing actinomycete strain collections against monitor strains in agar-plug assays.

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Section: Discussionmentioning

confidence: 95%

Development of an agar-plug cultivation system for bioactivity assays of actinomycete strain collections

et al. 2021

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“…Compared to instrumental methods, the whole-cell bioreporter approach could represent the true biological activity of the antibiotics in the natural environment, as well as serving as a promising candidate in understanding the distinct toxic mechanisms of antibiotics. 67,68 Song et al designed TC-specic bioreporters, which successfully responded to TC with the detection limits of 0.0026-0.11 mg mL À1 . 69 Bacillus subtilis bioreporter strains with different promoters fused to the rey luciferase reporter gene were constructed for the classication of distinct toxic mechanisms of antibiotics.…”

Section: Response Of the Whole-cell Bioreporter To Antibiotics In Aqu...mentioning

confidence: 99%

Toxicity assessment and microbial response to soil antibiotic exposure: differences between individual and mixed antibiotics

Jiang

Shen

et al. 2022

Environ. Sci.: Processes Impacts

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“…One such new approach, which we also refer to in the current manuscript, is represented by the mode of action (MoA)-based Bacillus subtilis bioreporter panel, recently reported by Wex et al. ( 5 ), which allows for fast and efficient combined bioactivity and MoA screening of antibiotic producer strains. The bioreporter assay employs selected biomarkers indicative of interference with the main bacterial metabolic pathways and thereby enables to selectively follow up and prioritize metabolic activities of interest ( 5 ).…”

Section: Introductionmentioning

confidence: 99%

“…( 5 ), which allows for fast and efficient combined bioactivity and MoA screening of antibiotic producer strains. The bioreporter assay employs selected biomarkers indicative of interference with the main bacterial metabolic pathways and thereby enables to selectively follow up and prioritize metabolic activities of interest ( 5 ). This MoA-based bioreporter assay was used to screen strains from the Tübingen actinomycetes strain collection for novel natural compounds ( 5 ).…”

Section: Introductionmentioning

confidence: 99%

“…The bioreporter assay employs selected biomarkers indicative of interference with the main bacterial metabolic pathways and thereby enables to selectively follow up and prioritize metabolic activities of interest ( 5 ). This MoA-based bioreporter assay was used to screen strains from the Tübingen actinomycetes strain collection for novel natural compounds ( 5 ). Preceding this study, the Tübingen strain collection had been subjected to a bioactivity test using the newly developed 96-well microplate system, designed to rapidly screen actinomycetes strain collections in agar plug assays ( 6 ).…”

Section: Introductionmentioning

confidence: 99%

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

Handel

Kulik

Wex

et al. 2022

NAR Genomics and Bioinformatics

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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.

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Bioreporters for direct mode of action-informed screening of antibiotic producer strains

Cited by 15 publications

References 67 publications

Development of an agar-plug cultivation system for bioactivity assays of actinomycete strain collections

Development of an agar-plug cultivation system for bioactivity assays of actinomycete strain collections

Toxicity assessment and microbial response to soil antibiotic exposure: differences between individual and mixed antibiotics

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

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