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.
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DOI：
10.1371/journal.pone.0258934
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Niedermeyer THJ
中科院分区：
文献类型：
--
作者：
Ortlieb N;Klenk E;Kulik A;Niedermeyer THJ
天然产物是开发药物活性成分的先导化合物的重要来源。放线菌对于抗生素的发现尤其具有价值。细菌病原体中抗生素耐药性的日益增加重新激发了人们对放线菌天然产物研究的兴趣。与液体浸没培养相比，放线菌在固体生长培养基上培养时会产生一组不同的天然产物。涉及固体培养基的生物活性测定（例如琼脂块测定）需要对菌株和琼脂块进行手动操作。这对于筛选数百或数千个菌株的较大菌株库来说不太方便。因此，本研究的目的是开发一种基于96孔微孔板的系统，适用于在琼脂块测定中筛选放线菌菌株库。我们开发了一种中等通量的培养和琼脂块测定工作流程，该流程能够方便地用来自菌株库的放线菌孢子悬浮液接种固体琼脂块，并将琼脂块转移到培养皿中进行琼脂块生物活性测定。描述了开发步骤以及开发过程中克服的挑战（例如系统无菌性、琼脂块的处理）。我们展示了一次旨在鉴定抑制基于Agr的群体感应的化合物的示例性筛选活动的结果，其中该工作流程得到了成功应用。我们提出了一种新颖且方便的工作流程，将琼脂扩散测定与基于微量滴定板的培养系统相结合，在该系统中菌株可以在固体表面上生长。这种工作流程促进并加快了在琼脂块测定中针对监测菌株对产生天然产物的放线菌菌株库进行的初始中等通量筛选。
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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影响因子：
5.1
作者：
Crüsemann M;O'Neill EC;Larson CB;Melnik AV;Floros DJ;da Silva RR;Jensen PR;Dorrestein PC;Moore BS
通讯作者：
Moore BS
影响因子：
4.1
作者：
Donadio, S;Monciardini, P;Puglia, AM
通讯作者：
Puglia, AM
DOI：
10.1080/00021369.1989.10869398
发表时间：
1989-04-01
期刊：
AGRICULTURAL AND BIOLOGICAL CHEMISTRY
影响因子：
--
作者：
KAWAI, S;KAWABATA, G;KAWAZU, K
通讯作者：
KAWAZU, K
影响因子：
14.9
作者：
Alanjary, Mohammad;Steinke, Katharina;Ziemert, Nadine
通讯作者：
Ziemert, Nadine
影响因子：
4.4
作者：
Duetz, WA;Rüedi, L;Witholt, B
通讯作者：
Witholt, B