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Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides

Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are...

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Autores principales: Burger, Brian T., Imam, Saheed, Scarborough, Matthew J., Noguera, Daniel R., Donohue, Timothy J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513736/
https://www.ncbi.nlm.nih.gov/pubmed/28744485
http://dx.doi.org/10.1128/mSystems.00015-17
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author Burger, Brian T.
Imam, Saheed
Scarborough, Matthew J.
Noguera, Daniel R.
Donohue, Timothy J.
author_facet Burger, Brian T.
Imam, Saheed
Scarborough, Matthew J.
Noguera, Daniel R.
Donohue, Timothy J.
author_sort Burger, Brian T.
collection PubMed
description Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosynthetic growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species. IMPORTANCE Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in R. sphaeroides, and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival. Author Video: An author video summary of this article is available.
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spelling pubmed-55137362017-07-25 Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides Burger, Brian T. Imam, Saheed Scarborough, Matthew J. Noguera, Daniel R. Donohue, Timothy J. mSystems Research Article Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosynthetic growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species. IMPORTANCE Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in R. sphaeroides, and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival. Author Video: An author video summary of this article is available. American Society for Microbiology 2017-06-06 /pmc/articles/PMC5513736/ /pubmed/28744485 http://dx.doi.org/10.1128/mSystems.00015-17 Text en Copyright © 2017 Burger et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (http://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Burger, Brian T.
Imam, Saheed
Scarborough, Matthew J.
Noguera, Daniel R.
Donohue, Timothy J.
Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides
title Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides
title_full Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides
title_fullStr Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides
title_full_unstemmed Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides
title_short Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides
title_sort combining genome-scale experimental and computational methods to identify essential genes in rhodobacter sphaeroides
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513736/
https://www.ncbi.nlm.nih.gov/pubmed/28744485
http://dx.doi.org/10.1128/mSystems.00015-17
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