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Title: Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

Abstract

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite as an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. Finally, the dominant HemN paralog involved in hememore » biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.« less

Authors:
 [1];  [1];  [1];  [2];  [2];  [1];  [2];  [2];  [1]
  1. Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Biosciences Division
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1378750
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 82; Journal Issue: 1; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES

Citation Formats

Vaccaro, Brian J., Thorgersen, Michael P., Lancaster, W. Andrew, Price, Morgan N., Wetmore, Kelly M., Poole, Farris L., Deutschbauer, Adam, Arkin, Adam P., and Adams, Michael W. W. Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses. United States: N. p., 2015. Web. doi:10.1128/AEM.02602-15.
Vaccaro, Brian J., Thorgersen, Michael P., Lancaster, W. Andrew, Price, Morgan N., Wetmore, Kelly M., Poole, Farris L., Deutschbauer, Adam, Arkin, Adam P., & Adams, Michael W. W. Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses. United States. doi:10.1128/AEM.02602-15.
Vaccaro, Brian J., Thorgersen, Michael P., Lancaster, W. Andrew, Price, Morgan N., Wetmore, Kelly M., Poole, Farris L., Deutschbauer, Adam, Arkin, Adam P., and Adams, Michael W. W. Fri . "Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses". United States. doi:10.1128/AEM.02602-15. https://www.osti.gov/servlets/purl/1378750.
@article{osti_1378750,
title = {Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses},
author = {Vaccaro, Brian J. and Thorgersen, Michael P. and Lancaster, W. Andrew and Price, Morgan N. and Wetmore, Kelly M. and Poole, Farris L. and Deutschbauer, Adam and Arkin, Adam P. and Adams, Michael W. W.},
abstractNote = {Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite as an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. Finally, the dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.},
doi = {10.1128/AEM.02602-15},
journal = {Applied and Environmental Microbiology},
number = 1,
volume = 82,
place = {United States},
year = {2015},
month = {10}
}

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Works referenced in this record:

Protein Crystallography Reveals a Role for the FS0 Cluster of Escherichia coli Nitrate Reductase A (NarGHI) in Enzyme Maturation
journal, January 2010

  • Rothery, Richard A.; Bertero, Michela G.; Spreter, Thomas
  • Journal of Biological Chemistry, Vol. 285, Issue 12
  • DOI: 10.1074/jbc.M109.066027

Structures of protein–protein complexes involved in electron transfer
journal, March 2013

  • Antonyuk, Svetlana V.; Han, Cong; Eady, Robert R.
  • Nature, Vol. 496, Issue 7443
  • DOI: 10.1038/nature11996

Structural basis of inter-protein electron transfer for nitrite reduction in denitrification
journal, November 2009

  • Nojiri, Masaki; Koteishi, Hiroyasu; Nakagami, Takuya
  • Nature, Vol. 462, Issue 7269
  • DOI: 10.1038/nature08507

Structural Basis of Biological N2O Generation by Bacterial Nitric Oxide Reductase
journal, November 2010


N2O binding at a [4Cu:2S] copper–sulphur cluster in nitrous oxide reductase
journal, August 2011

  • Pomowski, Anja; Zumft, Walter G.; Kroneck, Peter M. H.
  • Nature, Vol. 477, Issue 7363
  • DOI: 10.1038/nature10332

Physiological and Transcriptional Studies of Cr(VI) Reduction under Aerobic and Denitrifying Conditions by an Aquifer-Derived Pseudomonad
journal, October 2010

  • Han, Ruyang; Geller, Jil T.; Yang, Li
  • Environmental Science & Technology, Vol. 44, Issue 19
  • DOI: 10.1021/es101152r

Nitrate reductases: Structure, functions, and effect of stress factors
journal, October 2007


Biogenesis of a Respiratory Complex Is Orchestrated by a Single Accessory Protein
journal, April 2007

  • Lanciano, Pascal; Vergnes, Alexandra; Grimaldi, Stéphane
  • Journal of Biological Chemistry, Vol. 282, Issue 24
  • DOI: 10.1074/jbc.M700994200

Phylogenetic Analysis of Nitrite, Nitric Oxide, and Nitrous Oxide Respiratory Enzymes Reveal a Complex Evolutionary History for Denitrification
journal, July 2008

  • Jones, Christopher M.; Stres, Blaž; Rosenquist, Magnus
  • Molecular Biology and Evolution, Vol. 25, Issue 9
  • DOI: 10.1093/molbev/msn146

The Crystal Structure of Siroheme Decarboxylase in Complex with Iron-Uroporphyrin III Reveals Two Essential Histidine Residues
journal, September 2014

  • Haufschildt, Kristin; Schmelz, Stefan; Kriegler, Theresa M.
  • Journal of Molecular Biology, Vol. 426, Issue 19
  • DOI: 10.1016/j.jmb.2014.07.021

Kinetics of inter- and intramolecular electron transfer of Pseudomonas nautica cytochrome cd 1 nitrite reductase: regulation of the NO-bound end product
journal, January 2001

  • Lopes, Helder; Besson, Stéphane; Moura, Isabel
  • JBIC Journal of Biological Inorganic Chemistry, Vol. 6, Issue 1
  • DOI: 10.1007/s007750000159

Two c-Type Cytochromes, NirM and NirC, Encoded in the nir Gene Cluster of Pseudomonas aeruginosa Act as Electron Donors for Nitrite Reductase
journal, November 2001

  • Hasegawa, Norio; Arai, Hiroyuki; Igarashi, Yasuo
  • Biochemical and Biophysical Research Communications, Vol. 288, Issue 5
  • DOI: 10.1006/bbrc.2001.5919

Nitric oxide reductases of prokaryotes with emphasis on the respiratory, heme?copper oxidase type
journal, January 2005


Characterization of a member of the NnrR regulon in Rhodobacter sphaeroides 2.4.3 encoding a haem–copper protein
journal, March 2002


A Novel Protein Protects Bacterial Iron-Dependent Metabolism from Nitric Oxide
journal, August 2013

  • Stern, A. M.; Liu, B.; Bakken, L. R.
  • Journal of Bacteriology, Vol. 195, Issue 20
  • DOI: 10.1128/JB.00836-13

Requirement of norD for Brucella suis Virulence in a Murine Model of In Vitro and In Vivo Infection
journal, February 2006


Nitric Oxide Detoxification Systems Enhance Survival of Neisseria meningitidis in Human Macrophages and in Nasopharyngeal Mucosa
journal, May 2005


Nitric oxide and redox mechanisms in the immune response
journal, January 2011

  • Wink, David A.; Hines, Harry B.; Cheng, Robert Y. S.
  • Journal of Leukocyte Biology, Vol. 89, Issue 6
  • DOI: 10.1189/jlb.1010550

Determination of the Active Form of the Tetranuclear Copper Sulfur Cluster in Nitrous Oxide Reductase
journal, January 2014

  • Johnston, Esther M.; Dell’Acqua, Simone; Ramos, Susana
  • Journal of the American Chemical Society, Vol. 136, Issue 2
  • DOI: 10.1021/ja411500p

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration
journal, March 2005


Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons
journal, May 2015

  • Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.
  • mBio, Vol. 6, Issue 3, Article No. e00306-15
  • DOI: 10.1128/mBio.00306-15

Molybdenum Availability Is Key to Nitrate Removal in Contaminated Groundwater Environments
journal, May 2015

  • Thorgersen, Michael P.; Lancaster, W. Andrew; Vaccaro, Brian J.
  • Applied and Environmental Microbiology, Vol. 81, Issue 15
  • DOI: 10.1128/AEM.00917-15

Metallomics of two microorganisms relevant to heavy metal bioremediation reveal fundamental differences in metal assimilation and utilization
journal, January 2014

  • Lancaster, W. Andrew; Menon, Angeli Lal; Scott, Israel
  • Metallomics, Vol. 6, Issue 5
  • DOI: 10.1039/c4mt00050a

Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy
journal, April 2011

  • Letunic, I.; Bork, P.
  • Nucleic Acids Research, Vol. 39, Issue suppl
  • DOI: 10.1093/nar/gkr201

Molybdoproteomes and Evolution of Molybdenum Utilization
journal, June 2008


Bacterial transport of sulfate, molybdate, and related oxyanions
journal, February 2011

  • Aguilar-Barajas, Esther; Díaz-Pérez, César; Ramírez-Díaz, Martha I.
  • BioMetals, Vol. 24, Issue 4
  • DOI: 10.1007/s10534-011-9421-x

A Rhodobacter capsulatus Member of a Universal Permease Family Imports Molybdate and Other Oxyanions
journal, September 2010

  • Gisin, J.; Muller, A.; Pfander, Y.
  • Journal of Bacteriology, Vol. 192, Issue 22
  • DOI: 10.1128/JB.00742-10

STRING 8--a global view on proteins and their functional interactions in 630 organisms
journal, January 2009

  • Jensen, L. J.; Kuhn, M.; Stark, M.
  • Nucleic Acids Research, Vol. 37, Issue Database
  • DOI: 10.1093/nar/gkn760

The InterPro protein families database: the classification resource after 15 years
journal, November 2014

  • Mitchell, Alex; Chang, Hsin-Yu; Daugherty, Louise
  • Nucleic Acids Research, Vol. 43, Issue D1
  • DOI: 10.1093/nar/gku1243

Catalysis of a New Ribose Carbon-Insertion Reaction by the Molybdenum Cofactor Biosynthetic Enzyme MoaA
journal, February 2013

  • Mehta, Angad P.; Hanes, Jeremiah W.; Abdelwahed, Sameh H.
  • Biochemistry, Vol. 52, Issue 7
  • DOI: 10.1021/bi3016026

Microbial nitrate respiration – Genes, enzymes and environmental distribution
journal, August 2011


Ecological and evolutionary factors underlying global and local assembly of denitrifier communities
journal, January 2010


Novel structural and functional insights into the MoxR family of AAA+ ATPases
journal, August 2012


Mutational Analysis of the Nor Gene Cluster which Encodes Nitric-Oxide Reductase from Paracoccus denitrificans
journal, December 1996


Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes
journal, March 1998

  • Liu, X.; Miller, M. J. S.; Joshi, M. S.
  • Proceedings of the National Academy of Sciences, Vol. 95, Issue 5
  • DOI: 10.1073/pnas.95.5.2175

Distinct Roles of the Salmonella enterica Serovar Typhimurium CyaY and YggX Proteins in the Biosynthesis and Repair of Iron-Sulfur Clusters
journal, January 2014

  • Velayudhan, Jyoti; Karlinsey, Joyce E.; Frawley, Elaine R.
  • Infection and Immunity, Vol. 82, Issue 4
  • DOI: 10.1128/IAI.01022-13

Dynamics of the Heme-binding Bacterial Gas-sensing Dissimilative Nitrate Respiration Regulator (DNR) and Activation Barriers for Ligand Binding and Escape
journal, July 2014

  • Lobato, Laura; Bouzhir-Sima, Latifa; Yamashita, Taku
  • Journal of Biological Chemistry, Vol. 289, Issue 38
  • DOI: 10.1074/jbc.M114.571398

Dissimilatory Metabolism of Nitrogen Oxides in Bacteria: Comparative Reconstruction of Transcriptional Networks
journal, January 2005


Nitric Oxide Signaling and Transcriptional Control of Denitrification Genes in Pseudomonas stutzeri
journal, April 2001


NO sensing in Pseudomonas aeruginosa: Structure of the Transcriptional Regulator DNR
journal, May 2008

  • Giardina, Giorgio; Rinaldo, Serena; Johnson, Kenneth A.
  • Journal of Molecular Biology, Vol. 378, Issue 5
  • DOI: 10.1016/j.jmb.2008.03.013

Regulators of bacterial responses to nitric oxide
journal, March 2007


Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501
journal, May 2008

  • Yan, Y.; Yang, J.; Dou, Y.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 21
  • DOI: 10.1073/pnas.0801093105

Histidine-containing Phosphotransfer Protein-B (HptB) Regulates Swarming Motility through Partner-switching System in Pseudomonas aeruginosa PAO1 Strain
journal, November 2011

  • Bhuwan, Manish; Lee, Hui-Ju; Peng, Hwei-Ling
  • Journal of Biological Chemistry, Vol. 287, Issue 3
  • DOI: 10.1074/jbc.M111.256586

The Chemical Genomic Portrait of Yeast: Uncovering a Phenotype for All Genes
journal, April 2008


Towards an Informative Mutant Phenotype for Every Bacterial Gene
journal, August 2014

  • Deutschbauer, A.; Price, M. N.; Wetmore, K. M.
  • Journal of Bacteriology, Vol. 196, Issue 20
  • DOI: 10.1128/JB.01836-14

Role of the Extracytoplasmic Function Protein Family Sigma Factor RpoE in Metal Resistance of Escherichia coli
journal, March 2005


The Pseudomonas aeruginosa Reference Strain PA14 Displays Increased Virulence Due to a Mutation in ladS
journal, December 2011


SyntTax: a web server linking synteny to prokaryotic taxonomy
journal, January 2013


High Specificity in CheR Methyltransferase Function: CheR2 OF PSEUDOMONAS PUTIDA IS ESSENTIAL FOR CHEMOTAXIS, WHEREAS CheR1 IS INVOLVED IN BIOFILM FORMATION
journal, May 2013

  • García-Fontana, Cristina; Reyes-Darias, José Antonio; Muñoz-Martínez, Francisco
  • Journal of Biological Chemistry, Vol. 288, Issue 26
  • DOI: 10.1074/jbc.M113.472605

Inactivation of gltB Abolishes Expression of the Assimilatory Nitrate Reductase Gene (nasB) in Pseudomonas putida KT2442
journal, June 2000


Pseudomonas aeruginosa PA1006 Is a Persulfide-Modified Protein That Is Critical for Molybdenum Homeostasis
journal, February 2013


Pseudomonas aeruginosa PA1006, Which Plays a Role in Molybdenum Homeostasis, Is Required for Nitrate Utilization, Biofilm Formation, and Virulence
journal, February 2013


Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis
journal, May 2015

  • Hover, Bradley M.; Tonthat, Nam K.; Schumacher, Maria A.
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 20
  • DOI: 10.1073/pnas.1500697112

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    journal, June 2019

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    • eLife, Vol. 8
    • DOI: 10.7554/elife.39733