skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

ABSTRACT 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. 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 essentialitymore » 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.« less

Authors:
 [1];  [1];  [2];  [2]; ORCiD logo [1];
  1. Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin, Madison, Wisconsin, USA, Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA
  2. Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin, Madison, Wisconsin, USA, Department of Civil and Environmental Engineering, University of Wisconsin, Madison, Wisconsin, USA
Publication Date:
Research Org.:
Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1617072
Alternate Identifier(s):
OSTI ID: 1427587
Grant/Contract Number:  
FC02-07ER64494
Resource Type:
Published Article
Journal Name:
mSystems
Additional Journal Information:
Journal Name: mSystems Journal Volume: 2 Journal Issue: 3; Journal ID: ISSN 2379-5077
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Rhodobacter sphaeroides; Tn-seq; gene disruption; genomics; metabolic modeling; metabolism; photosynthetic bacteria; proteobacteria

Citation Formats

Burger, Brian T., Imam, Saheed, Scarborough, Matthew J., Noguera, Daniel R., Donohue, Timothy J., and Lindemann, ed., Stephen R. Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides. United States: N. p., 2017. Web. doi:10.1128/mSystems.00015-17.
Burger, Brian T., Imam, Saheed, Scarborough, Matthew J., Noguera, Daniel R., Donohue, Timothy J., & Lindemann, ed., Stephen R. Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides. United States. doi:10.1128/mSystems.00015-17.
Burger, Brian T., Imam, Saheed, Scarborough, Matthew J., Noguera, Daniel R., Donohue, Timothy J., and Lindemann, ed., Stephen R. Tue . "Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides". United States. doi:10.1128/mSystems.00015-17.
@article{osti_1617072,
title = {Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides},
author = {Burger, Brian T. and Imam, Saheed and Scarborough, Matthew J. and Noguera, Daniel R. and Donohue, Timothy J. and Lindemann, ed., Stephen R.},
abstractNote = {ABSTRACT 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. 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.},
doi = {10.1128/mSystems.00015-17},
journal = {mSystems},
number = 3,
volume = 2,
place = {United States},
year = {2017},
month = {6}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1128/mSystems.00015-17

Citation Metrics:
Cited by: 6 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

ARTIST: High-Resolution Genome-Wide Assessment of Fitness Using Transposon-Insertion Sequencing
journal, November 2014


Bayesian analysis of gene essentiality based on sequencing of transposon insertion libraries
journal, January 2013


A Requirement for Sodium in the Growth of Rhodopseudomonas spheroides
journal, June 1960


Multiple Comparisons among Means
journal, March 1961


From Redox Flow to Gene Regulation:  Role of the PrrC Protein of Rhodobacter sphaeroides 2.4.1
journal, February 2000

  • Eraso, Jesus M.; Kaplan, Samuel
  • Biochemistry, Vol. 39, Issue 8
  • DOI: 10.1021/bi9923858

How carotenoids protect bacterial photosynthesis
journal, October 2000

  • Cogdell, Richard J.; Howard, Tina D.; Bittl, Robert
  • Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, Vol. 355, Issue 1402
  • DOI: 10.1098/rstb.2000.0696

The Rhodobacter sphaeroides PufX protein is not required for photosynthetic competence in the absence of a light harvesting system
journal, August 1994


Tn-Seq Explorer: A Tool for Analysis of High-Throughput Sequencing Data of Transposon Mutant Libraries
journal, May 2015


Ultrafast and memory-efficient alignment of short DNA sequences to the human genome
journal, January 2009


KEGG: Kyoto Encyclopedia of Genes and Genomes
journal, January 2000

  • Kanehisa, Minoru; Goto, Susumu
  • Nucleic Acids Research, Vol. 28, Issue 1, p. 27-30
  • DOI: 10.1093/nar/28.1.27

The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases
journal, November 2015

  • Caspi, Ron; Billington, Richard; Ferrer, Luciana
  • Nucleic Acids Research, Vol. 44, Issue D1
  • DOI: 10.1093/nar/gkv1164

Using a Two-Stage Hydrogen-Based Membrane Biofilm Reactor (MBfR) to Achieve Complete Perchlorate Reduction in the Presence of Nitrate and Sulfate
journal, January 2013

  • Zhao, He-Ping; Ontiveros-Valencia, Aura; Tang, Youneng
  • Environmental Science & Technology, Vol. 47, Issue 3
  • DOI: 10.1021/es303823n

A Hidden Markov Model for identifying essential and growth-defect regions in bacterial genomes from transposon insertion sequencing data
journal, October 2013


Unique attributes of cyanobacterial metabolism revealed by improved genome-scale metabolic modeling and essential gene analysis
journal, December 2016

  • Broddrick, Jared T.; Rubin, Benjamin E.; Welkie, David G.
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 51
  • DOI: 10.1073/pnas.1613446113

DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1T
journal, February 2000


Genome-Scale Identification of Resistance Functions in Pseudomonas aeruginosa Using Tn-seq
journal, January 2011


Metabolically Engineered Rhodobacter sphaeroides RV strains for Improved Biohydrogen Photoproduction Combined with Disposal of Food Wastes
journal, December 2004


Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries
journal, January 2011


IVT-seq reveals extreme bias in RNA sequencing
journal, January 2014


Identifying Genetic Determinants Needed to Establish a Human Gut Symbiont in Its Habitat
journal, September 2009


High yield single stage conversion of glucose to hydrogen by photofermentation with continuous cultures of Rhodobacter capsulatus JP91
journal, January 2013


Photobiotransformation of indole to its value-added derivatives by Rhodobacter sphaeroides OU5
journal, March 1998

  • Rajasekhar, N.; Sasikala, C.; Ramana, C. V.
  • Journal of Industrial Microbiology and Biotechnology, Vol. 20, Issue 3-4
  • DOI: 10.1038/sj.jim.2900501

Genome-scale analysis of gene function in the hydrogenotrophic methanogenic archaeon Methanococcus maripaludis
journal, March 2013

  • Sarmiento, F.; Mrazek, J.; Whitman, W. B.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 12
  • DOI: 10.1073/pnas.1220225110

The essential genome of a bacterium
journal, January 2011

  • Christen, Beat; Abeliuk, Eduardo; Collier, John M.
  • Molecular Systems Biology, Vol. 7, Issue 1
  • DOI: 10.1038/msb.2011.58

Localized transposon Tn5 mutagenesis of the photosynthetic gene cluster of Rhodobacter sphaeroides
journal, June 1990


Using Gene Essentiality and Synthetic Lethality Information to Correct Yeast and CHO Cell Genome-Scale Models
journal, September 2015

  • Chowdhury, Ratul; Chowdhury, Anupam; Maranas, Costas
  • Metabolites, Vol. 5, Issue 4
  • DOI: 10.3390/metabo5040536

The COG database: a tool for genome-scale analysis of protein functions and evolution
journal, January 2000


iRsp1095: A genome-scale reconstruction of the Rhodobacter sphaeroides metabolic network
journal, January 2011


Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants
journal, October 2009

  • Langridge, G. C.; Phan, M. -D.; Turner, D. J.
  • Genome Research, Vol. 19, Issue 12
  • DOI: 10.1101/gr.097097.109

Quantifying the effects of light intensity on bioproduction and maintenance energy during photosynthetic growth of Rhodobacter sphaeroides
journal, November 2014

  • Imam, Saheed; Fitzgerald, Colin M.; Cook, Emily M.
  • Photosynthesis Research, Vol. 123, Issue 2
  • DOI: 10.1007/s11120-014-0061-1

KEGG as a reference resource for gene and protein annotation
journal, October 2015

  • Kanehisa, Minoru; Sato, Yoko; Kawashima, Masayuki
  • Nucleic Acids Research, Vol. 44, Issue D1
  • DOI: 10.1093/nar/gkv1070

COBRApy: COnstraints-Based Reconstruction and Analysis for Python
journal, January 2013

  • Ebrahim, Ali; Lerman, Joshua A.; Palsson, Bernhard O.
  • BMC Systems Biology, Vol. 7, Issue 1
  • DOI: 10.1186/1752-0509-7-74

Essential Genome of the Metabolically Versatile Alphaproteobacterium Rhodopseudomonas palustris
journal, December 2015

  • Pechter, Kieran B.; Gallagher, Larry; Pyles, Harley
  • Journal of Bacteriology, Vol. 198, Issue 5
  • DOI: 10.1128/JB.00771-15

Adjusting for multiple testing when reporting research results: the Bonferroni vs Holm methods.
journal, May 1996


Studies on the expression of the pufX polypeptide and its requirement for photoheterotrophic growth in Rhodobacter sphaeroides.
journal, August 1992


Anaerobic biosynthesis of the lower ligand of vitamin B 12
journal, August 2015

  • Hazra, Amrita B.; Han, Andrew W.; Mehta, Angad P.
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 34
  • DOI: 10.1073/pnas.1509132112

Using Genome-scale Models to Predict Biological Capabilities
journal, May 2015


Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes
journal, February 2016

  • Spero, Melanie A.; Brickner, Joshua R.; Mollet, Jordan T.
  • Journal of Bacteriology, Vol. 198, Issue 8
  • DOI: 10.1128/JB.01025-15

Differential Expression of the CO2 Fixation Operons of Rhodobacter sphaeroides by the Prr/Reg Two-Component System during Chemoautotrophic Growth
journal, December 2002


Efficient hydrogen production from acetate through isolated Rhodobacter sphaeroides
journal, December 2011

  • Kobayashi, Jyumpei; Yoshimune, Kazuaki; Komoriya, Tomoe
  • Journal of Bioscience and Bioengineering, Vol. 112, Issue 6
  • DOI: 10.1016/j.jbiosc.2011.08.008

Summarizing and correcting the GC content bias in high-throughput sequencing
journal, February 2012

  • Benjamini, Yuval; Speed, Terence P.
  • Nucleic Acids Research, Vol. 40, Issue 10
  • DOI: 10.1093/nar/gks001

DEG 10, an update of the database of essential genes that includes both protein-coding genes and noncoding genomic elements: Table 1.
journal, November 2013

  • Luo, Hao; Lin, Yan; Gao, Feng
  • Nucleic Acids Research, Vol. 42, Issue D1
  • DOI: 10.1093/nar/gkt1131

Comparison of H2H2 accumulation by Rhodobacter sphaeroides KD131 and its uptake hydrogenase and PHB synthase deficient mutant
journal, January 2006


Essential Genes Predicted in the Genome of Rubrivivax gelatinosus
journal, June 2016


Estimation of the Medians for Dependent Variables
journal, March 1959


Coenzyme Q10 production in a 150-l reactor by a mutant strain of Rhodobacter sphaeroides
journal, February 2010

  • Kien, Nguyen Ba; Kong, In-Soo; Lee, Min-Gyu
  • Journal of Industrial Microbiology & Biotechnology, Vol. 37, Issue 5
  • DOI: 10.1007/s10295-010-0699-4

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides
journal, January 2013

  • Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.
  • BMC Systems Biology, Vol. 7, Issue 1
  • DOI: 10.1186/1752-0509-7-89

Global Assessment of Genomic Regions Required for Growth in Mycobacterium tuberculosis
journal, September 2012


In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria
journal, February 1999

  • Rubin, E. J.; Akerley, B. J.; Novik, V. N.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 4
  • DOI: 10.1073/pnas.96.4.1645

Applications of genome‐scale metabolic reconstructions
journal, January 2009

  • Oberhardt, Matthew A.; Palsson, Bernhard Ø; Papin, Jason A.
  • Molecular Systems Biology, Vol. 5, Issue 1
  • DOI: 10.1038/msb.2009.77

(p)ppGpp: Still Magical?
journal, October 2008


Complete DNA sequence, specific Tn5 insertion map, and gene assignment of the carotenoid biosynthesis pathway of Rhodobacter sphaeroides.
journal, April 1995


Capturing Uncertainty by Modeling Local Transposon Insertion Frequencies Improves Discrimination of Essential Genes
journal, January 2015

  • DeJesus, Michael A.; Ioerger, Thomas R.
  • IEEE/ACM Transactions on Computational Biology and Bioinformatics, Vol. 12, Issue 1
  • DOI: 10.1109/TCBB.2014.2326857

The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases
journal, November 2013

  • Caspi, Ron; Altman, Tomer; Billington, Richard
  • Nucleic Acids Research, Vol. 42, Issue D1
  • DOI: 10.1093/nar/gkt1103

Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms
journal, September 2009

  • van Opijnen, Tim; Bodi, Kip L.; Camilli, Andrew
  • Nature Methods, Vol. 6, Issue 10
  • DOI: 10.1038/nmeth.1377

Genetic analysis of the bchC and bchA genes of Rhodobacter sphaeroides
journal, January 1993

  • McGlynn, Peter; Neil Hunter, C.
  • Molecular and General Genetics MGG, Vol. 236-236, Issue 2-3
  • DOI: 10.1007/BF00277117

PrrC, a Sco homologue from Rhodobacter sphaeroides , possesses thiol-disulfide oxidoreductase activity
journal, September 2007


Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung
journal, September 2009

  • Gawronski, J. D.; Wong, S. M. S.; Giannoukos, G.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 38
  • DOI: 10.1073/pnas.0906627106

Genome-scale Reconstruction of Metabolic Network in Bacillus subtilis Based on High-throughput Phenotyping and Gene Essentiality Data
journal, June 2007

  • Oh, You-Kwan; Palsson, Bernhard O.; Park, Sung M.
  • Journal of Biological Chemistry, Vol. 282, Issue 39
  • DOI: 10.1074/jbc.M703759200

The roles of Rhodobacter sphaeroides copper chaperones PCuAC and Sco (PrrC) in the assembly of the copper centers of the aa3-type and the cbb3-type cytochrome c oxidases
journal, June 2012

  • Thompson, Audie K.; Gray, Jimmy; Liu, Aimin
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1817, Issue 6
  • DOI: 10.1016/j.bbabio.2012.01.003