Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

ABSTRACT A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid est...

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Autores principales:	Neissa M. Pinzon, Kelly G. Aukema, Jeffrey A. Gralnick, Lawrence P. Wackett
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2011
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/7b79567c35e14aa3987be92cfb554072
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spelling	oai:doaj.org-article:7b79567c35e14aa3987be92cfb5540722021-11-15T15:38:45ZNile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format10.1128/mBio.00109-112150-7511https://doaj.org/article/7b79567c35e14aa3987be92cfb5540722011-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00109-11https://doaj.org/toc/2150-7511ABSTRACT A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone production as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. IMPORTANCE In recent years, there has been renewed interest in advanced biofuel sources such as bacterial hydrocarbon production. Previous studies used solvent extraction of bacterial cultures followed by gas chromatography-mass spectrometry (GC-MS) to detect and quantify ketones and hydrocarbons (Beller HR, Goh EB, Keasling JD, Appl. Environ. Microbiol. 76:1212–1223, 2010; Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP, Appl. Environ. Microbiol. 76:3850–3862, 2010). While these analyses are powerful and accurate, their labor-intensive nature makes them intractable to high-throughput screening; therefore, methods for rapid identification of bacterial strains that are overproducing hydrocarbons are needed. The use of high-throughput evaluation of bacterial and algal hydrophobic molecule production via Nile red fluorescence from lipids and esters was extended in this study to include hydrocarbons and ketones. This work demonstrated accurate, high-throughput detection of high-level bacterial long-chain ketone and hydrocarbon production by screening for increased fluorescence of the hydrophobic dye Nile red.Neissa M. PinzonKelly G. AukemaJeffrey A. GralnickLawrence P. WackettAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 2, Iss 4 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Neissa M. Pinzon Kelly G. Aukema Jeffrey A. Gralnick Lawrence P. Wackett Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format
description	ABSTRACT A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone production as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. IMPORTANCE In recent years, there has been renewed interest in advanced biofuel sources such as bacterial hydrocarbon production. Previous studies used solvent extraction of bacterial cultures followed by gas chromatography-mass spectrometry (GC-MS) to detect and quantify ketones and hydrocarbons (Beller HR, Goh EB, Keasling JD, Appl. Environ. Microbiol. 76:1212–1223, 2010; Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP, Appl. Environ. Microbiol. 76:3850–3862, 2010). While these analyses are powerful and accurate, their labor-intensive nature makes them intractable to high-throughput screening; therefore, methods for rapid identification of bacterial strains that are overproducing hydrocarbons are needed. The use of high-throughput evaluation of bacterial and algal hydrophobic molecule production via Nile red fluorescence from lipids and esters was extended in this study to include hydrocarbons and ketones. This work demonstrated accurate, high-throughput detection of high-level bacterial long-chain ketone and hydrocarbon production by screening for increased fluorescence of the hydrophobic dye Nile red.
format	article
author	Neissa M. Pinzon Kelly G. Aukema Jeffrey A. Gralnick Lawrence P. Wackett
author_facet	Neissa M. Pinzon Kelly G. Aukema Jeffrey A. Gralnick Lawrence P. Wackett
author_sort	Neissa M. Pinzon
title	Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format
title_short	Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format
title_full	Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format
title_fullStr	Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format
title_full_unstemmed	Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format
title_sort	nile red detection of bacterial hydrocarbons and ketones in a high-throughput format
publisher	American Society for Microbiology
publishDate	2011
url	https://doaj.org/article/7b79567c35e14aa3987be92cfb554072
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Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

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