title: Functional genomics with a comprehensive library of transposon mutants for the sulfate-reducing bacterium Desulfovibrio alaskensis G20.
abstract: The genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. To demonstrate the utility of the individual mutants, we profiled gene expression in mutants of six regulatory genes and used these data, together with 1,313 high-confidence transcription start sites identified by tiling microarrays and transcriptome sequencing (5' RNA-Seq), to update the regulons of Fur and Rex and to confirm the predicted regulons of LysX, PhnF, PerR, and Dde_3000, a histidine kinase. In addition to enabling single mutant investigations, the D. alaskensis G20 transposon mutants also contain DNA bar codes, which enables the pooling and analysis of mutant fitness for thousands of strains simultaneously. Using two pools of mutants that represent insertions in 2,369 unique protein-coding genes, we demonstrate that the hypothetical gene Dde_3007 is required for methionine biosynthesis. Using comparative genomics, we propose that Dde_3007 performs a missing step in methionine biosynthesis by transferring a sulfur group to O-phosphohomoserine to form homocysteine. Additionally, we show that the entire choline utilization cluster is important for fitness in choline sulfate medium, which confirms that a functional microcompartment is required for choline oxidation. Finally, we demonstrate that Dde_3291, a MerR-like transcription factor, is a choline-dependent activator of the choline utilization cluster. Taken together, our data set and genetic resources provide a foundation for systems-level investigation of a poorly studied group of bacteria of environmental and industrial importance.
  
  
  OBJECTIVE
  Sulfate-reducing bacteria contribute to global nutrient cycles and are a nuisance for the petroleum industry. Despite their environmental and industrial significance, the genomes of sulfate-reducing bacteria remain poorly characterized. Here, we describe a genetic approach to fill gaps in our knowledge of sulfate-reducing bacteria. We generated a large collection of archived, transposon mutants in Desulfovibrio alaskensis G20 and used the phenotypes of these mutant strains to infer the function of genes involved in gene regulation, methionine biosynthesis, and choline utilization. Our findings and mutant resources will enable systematic investigations into gene function, energy generation, stress response, and metabolism for this important group of bacteria.
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Jennifer V
authors.initials: JV
authors.lastname: Kuehl
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: Jayashree
authors.initials: J
authors.lastname: Ray
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Wetmore
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Zuelma
authors.initials: Z
authors.lastname: Esquivel
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Alexey E
authors.initials: AE
authors.lastname: Kazakov
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, California, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Nguyen
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, California, USA.
authors.forename: Raquel
authors.initials: R
authors.lastname: Kuehn
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, California, USA.
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.forename: Terry C
authors.initials: TC
authors.lastname: Hazen
authors.forename: Adam P
authors.initials: AP
authors.lastname: Arkin
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA AMDeutschbauer@lbl.gov.
authors.forename: Adam
authors.initials: A
authors.lastname: Deutschbauer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D003901:Desulfovibrio / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial; D023281:Genomics; D008239:Lysine / Q000378:metabolism; D008715:Methionine / Q000096:biosynthesis; D009154:Mutation; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D013431:Sulfates / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D059467:Transcriptome
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D013431:Sulfates; D008715:Methionine; D008239:Lysine; D002794:Choline