Fluorescence recovery allows theimplementation of a fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments

Research output: Contribution to journalJournal articlepeer-review

Standard

Fluorescence recovery allows theimplementation of a fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments. / Pinilla-Redondo, Rafael ; Riber, Leise; Sørensen, Søren Johannes.

In: Applied and Environmental Microbiology, Vol. 84, No. 6, e02507-17, 2018.

Research output: Contribution to journalJournal articlepeer-review

Harvard

Pinilla-Redondo, R, Riber, L & Sørensen, SJ 2018, 'Fluorescence recovery allows theimplementation of a fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments', Applied and Environmental Microbiology, vol. 84, no. 6, e02507-17. https://doi.org/10.1128/AEM.02507-17

APA

Pinilla-Redondo, R., Riber, L., & Sørensen, S. J. (2018). Fluorescence recovery allows theimplementation of a fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments. Applied and Environmental Microbiology, 84(6), [e02507-17]. https://doi.org/10.1128/AEM.02507-17

Vancouver

Pinilla-Redondo R, Riber L, Sørensen SJ. Fluorescence recovery allows theimplementation of a fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments. Applied and Environmental Microbiology. 2018;84(6). e02507-17. https://doi.org/10.1128/AEM.02507-17

Author

Pinilla-Redondo, Rafael ; Riber, Leise ; Sørensen, Søren Johannes. / Fluorescence recovery allows theimplementation of a fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments. In: Applied and Environmental Microbiology. 2018 ; Vol. 84, No. 6.

Bibtex

@article{28b97a810da54eb6ad8a1c2853f02df7,
title = "Fluorescence recovery allows theimplementation of a fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments",
abstract = "The study of horizontal gene transfer (HGT) in microbial communities has been revolutionized by significant advances in cultivation-independent methods based on fluorescence reporter gene technologies. Recently, the combination of these novel approaches with flow cytometry has presented itself as one of the most powerful tools to study the spread of mobile genetic elements (MGEs) in the environment. However, the use of fluorescent markers, like green fluorescent protein (GFP) and mCherry, is limited by environmental constraints, such as oxygen availability and pH levels, that affect the correct maturation of their fluorophores. Few studies have characterized the effects of such environmental conditions in a systematic way, and the sheer amount of distinct protein variants requires each system to be examined in an individual fashion. The lack of efficient and reliable markers to monitor HGT in anaerobic environments, coupled to the abundance of ecologically and clinically relevant oxygen-deprived niches in which bacteria thrive, calls for the urgent development of suitable tools that permit its study. In an attempt to devise a process that allows the implementation of the mentioned dual-labeling system to anoxic milieus, the aerobic fluorescence recovery of mCherry and GFPmut3, as well as the effect of pH on their fluorescence intensities, was studied. The findings present a solution to an intrinsic problem that has long hampered the utilization of this system, highlight its pH limitations, and provide experimental tools that will help broaden its horizon of application to other fields.IMPORTANCEMany anaerobic environments, like the gastrointestinal tract, anaerobic digesters, and the interiors of dense biofilms, have been shown to be hotspots for horizontal gene transfer (HGT). Despite the increasing wealth of reports warning about the alarming spread of antibiotic resistance determinants, to date, HGT studies mainly rely on cultivation-based methods. Unfortunately, the relevance of these studies is often questionable, as only a minor fraction of bacteria can be cultivated. A recently developed approach to monitoring the fate of plasmids in microbial communities is based on a fluorescence dual-labeling system and allows the bypassing of cultivation. However, the fluorescent proteins on which it is founded are constrained by pH levels and by their strict dependence on oxygen for the maturation of their fluorophores. This study focused on the development and validation of an appropriate aerobic fluorescence recovery (AFR) method for this platform, as this embodies the missing technical link impeding its implementation in anoxic environments.",
author = "Rafael Pinilla-Redondo and Leise Riber and S{\o}rensen, {S{\o}ren Johannes}",
note = "Copyright {\textcopyright} 2018 American Society for Microbiology.",
year = "2018",
doi = "10.1128/AEM.02507-17",
language = "English",
volume = "84",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "6",

}

RIS

TY - JOUR

T1 - Fluorescence recovery allows theimplementation of a fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments

AU - Pinilla-Redondo, Rafael

AU - Riber, Leise

AU - Sørensen, Søren Johannes

N1 - Copyright © 2018 American Society for Microbiology.

PY - 2018

Y1 - 2018

N2 - The study of horizontal gene transfer (HGT) in microbial communities has been revolutionized by significant advances in cultivation-independent methods based on fluorescence reporter gene technologies. Recently, the combination of these novel approaches with flow cytometry has presented itself as one of the most powerful tools to study the spread of mobile genetic elements (MGEs) in the environment. However, the use of fluorescent markers, like green fluorescent protein (GFP) and mCherry, is limited by environmental constraints, such as oxygen availability and pH levels, that affect the correct maturation of their fluorophores. Few studies have characterized the effects of such environmental conditions in a systematic way, and the sheer amount of distinct protein variants requires each system to be examined in an individual fashion. The lack of efficient and reliable markers to monitor HGT in anaerobic environments, coupled to the abundance of ecologically and clinically relevant oxygen-deprived niches in which bacteria thrive, calls for the urgent development of suitable tools that permit its study. In an attempt to devise a process that allows the implementation of the mentioned dual-labeling system to anoxic milieus, the aerobic fluorescence recovery of mCherry and GFPmut3, as well as the effect of pH on their fluorescence intensities, was studied. The findings present a solution to an intrinsic problem that has long hampered the utilization of this system, highlight its pH limitations, and provide experimental tools that will help broaden its horizon of application to other fields.IMPORTANCEMany anaerobic environments, like the gastrointestinal tract, anaerobic digesters, and the interiors of dense biofilms, have been shown to be hotspots for horizontal gene transfer (HGT). Despite the increasing wealth of reports warning about the alarming spread of antibiotic resistance determinants, to date, HGT studies mainly rely on cultivation-based methods. Unfortunately, the relevance of these studies is often questionable, as only a minor fraction of bacteria can be cultivated. A recently developed approach to monitoring the fate of plasmids in microbial communities is based on a fluorescence dual-labeling system and allows the bypassing of cultivation. However, the fluorescent proteins on which it is founded are constrained by pH levels and by their strict dependence on oxygen for the maturation of their fluorophores. This study focused on the development and validation of an appropriate aerobic fluorescence recovery (AFR) method for this platform, as this embodies the missing technical link impeding its implementation in anoxic environments.

AB - The study of horizontal gene transfer (HGT) in microbial communities has been revolutionized by significant advances in cultivation-independent methods based on fluorescence reporter gene technologies. Recently, the combination of these novel approaches with flow cytometry has presented itself as one of the most powerful tools to study the spread of mobile genetic elements (MGEs) in the environment. However, the use of fluorescent markers, like green fluorescent protein (GFP) and mCherry, is limited by environmental constraints, such as oxygen availability and pH levels, that affect the correct maturation of their fluorophores. Few studies have characterized the effects of such environmental conditions in a systematic way, and the sheer amount of distinct protein variants requires each system to be examined in an individual fashion. The lack of efficient and reliable markers to monitor HGT in anaerobic environments, coupled to the abundance of ecologically and clinically relevant oxygen-deprived niches in which bacteria thrive, calls for the urgent development of suitable tools that permit its study. In an attempt to devise a process that allows the implementation of the mentioned dual-labeling system to anoxic milieus, the aerobic fluorescence recovery of mCherry and GFPmut3, as well as the effect of pH on their fluorescence intensities, was studied. The findings present a solution to an intrinsic problem that has long hampered the utilization of this system, highlight its pH limitations, and provide experimental tools that will help broaden its horizon of application to other fields.IMPORTANCEMany anaerobic environments, like the gastrointestinal tract, anaerobic digesters, and the interiors of dense biofilms, have been shown to be hotspots for horizontal gene transfer (HGT). Despite the increasing wealth of reports warning about the alarming spread of antibiotic resistance determinants, to date, HGT studies mainly rely on cultivation-based methods. Unfortunately, the relevance of these studies is often questionable, as only a minor fraction of bacteria can be cultivated. A recently developed approach to monitoring the fate of plasmids in microbial communities is based on a fluorescence dual-labeling system and allows the bypassing of cultivation. However, the fluorescent proteins on which it is founded are constrained by pH levels and by their strict dependence on oxygen for the maturation of their fluorophores. This study focused on the development and validation of an appropriate aerobic fluorescence recovery (AFR) method for this platform, as this embodies the missing technical link impeding its implementation in anoxic environments.

U2 - 10.1128/AEM.02507-17

DO - 10.1128/AEM.02507-17

M3 - Journal article

C2 - 29330182

VL - 84

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 6

M1 - e02507-17

ER -

ID: 191910907