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The inducible chemical-genetic fluorescent marker FAST outperforms classical fluorescent proteins in the quantitative reporting of bacterial biofilm dynamics.

ABSTRACT: To increase our understanding of bacterial biofilm complexity, real- time quantitative analyses of the living community functions are required. To reach this goal, accurate fluorescent reporters are needed. In this paper, we used the classical fluorescent genetic reporters of the GFP family and demonstrated their limits in the context of a living biofilm. We showed that fluorescence signal saturated after only a few hours of growth and related this saturation to the reduction of oxygen concentration induced by bacterial consumption. This behaviour prevents the use of GFP-like fluorescent proteins for quantitative measurement in living biofilms. To overcome this limitation, we propose the use of a recently introduced small protein tag, FAST, which is fluorescent in the presence of an exogenously applied fluorogenic dye, enabling to avoid the oxygen sensitivity issue. We compared the ability of FAST to report on biofilm growth with that of GFP and mCherry, and demonstrated the superiority of the FAST:fluorogen probes for investigating dynamics in the complex environment of a living biofilm.

SUBMITTER: Monmeyran A

PROVIDER: S-EPMC6037777 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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The inducible chemical-genetic fluorescent marker FAST outperforms classical fluorescent proteins in the quantitative reporting of bacterial biofilm dynamics.

Monmeyran Amaury A Thomen Philippe P Jonquière Hugo H Sureau Franck F Li Chenge C Plamont Marie-Aude MA Douarche Carine C Casella Jean-François JF Gautier Arnaud A Henry Nelly N

Scientific reports 20180709 1

To increase our understanding of bacterial biofilm complexity, real- time quantitative analyses of the living community functions are required. To reach this goal, accurate fluorescent reporters are needed. In this paper, we used the classical fluorescent genetic reporters of the GFP family and demonstrated their limits in the context of a living biofilm. We showed that fluorescence signal saturated after only a few hours of growth and related this saturation to the reduction of oxygen concentra ...[more]

PMID: 29985417

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Project description:Decades of environmental DNA (eDNA) method application, spanning a wide variety of taxa and habitats, has advanced our understanding of eDNA and underlined its value as a tool for conservation practitioners. The general consensus is that eDNA methods are more accurate and cost-effective than traditional survey methods. However, they are formally approved for just a few species globally (e.g., Bighead Carp, Silver Carp, Great Crested Newt). We conducted a meta-analysis of studies that directly compare eDNA with traditional surveys to evaluate the assertion that eDNA methods are consistently "better."Environmental DNA publications for multiple species or single macro-organism detection were identified using the Web of Science, by searching "eDNA" and "environmental DNA" across papers published between 1970 and 2020. The methods used, focal taxa, habitats surveyed, and quantitative and categorical results were collated and analyzed to determine whether and under what circumstances eDNA outperforms traditional surveys.Results show that eDNA methods are cheaper, more sensitive, and detect more species than traditional methods. This is, however, taxa-dependent, with amphibians having the highest potential for detection by eDNA survey. Perhaps most strikingly, of the 535 papers reviewed just 49 quantified the probability of detection for both eDNA and traditional survey methods and studies were three times more likely to give qualitative statements of performance. Synthesis and applications: The results of this meta-analysis demonstrate that where there is a direct comparison, eDNA surveys of macro-organisms are more accurate and efficient than traditional surveys. This conclusion, however, is based on just a fraction of available eDNA papers as most do not offer this granularity. We recommend that conclusions are substantiated with comparable and quantitative data. Where a direct comparison has not been made, we caution against the use of qualitative statements about relative performance. This consistency and rigor will simplify how the eDNA research community tracks methods-based advances and will also provide greater clarity for conservation practitioners. To this end suggest reporting standards for eDNA studies.

Dataset Information

The inducible chemical-genetic fluorescent marker FAST outperforms classical fluorescent proteins in the quantitative reporting of bacterial biofilm dynamics.

Publications

The inducible chemical-genetic fluorescent marker FAST outperforms classical fluorescent proteins in the quantitative reporting of bacterial biofilm dynamics.

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