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

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:
Grant/Contract Number:
AC02-05CH11231
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
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)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES
OSTI Identifier:
1378750

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., 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.. 2015. "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}
}