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 from Pseudomonas stutzeri strain 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. 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Vaccaro
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Thorgersen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
authors.forename: W Andrew
authors.initials: WA
authors.lastname: Lancaster
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Morgan N
authors.initials: MN
authors.lastname: Price
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Wetmore
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
authors.forename: Farris L
authors.initials: FL
authors.lastname: Poole
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Deutschbauer
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Arkin
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA adams@bmb.uga.edu.
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D058440:Denitrification; D009154:Mutation; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D044223:Pseudomonas stutzeri / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009566:Nitrates; D009573:Nitrites; D009569:Nitric Oxide; D010088:Oxidoreductases; C034747:nitric-oxide reductase