title: High-throughput mutation, selection, and phenotype screening of mutant methanogenic archaea.
abstract: Bacterial and archaeal genomes can contain 30% or more hypothetical genes with no predicted function. Phylogenetically deep-branching microbes, such as methane-producing archaea (methanogens), contain up to 50% genes with unknown function. In order to formulate hypotheses about the function of hypothetical gene functions in the strict anaerobe, Methanosarcina acetivorans, we have developed high-throughput anaerobic techniques to UV mutagenize, screen, and select for mutant strains in 96-well plates. Using these approaches we have isolated 10 mutant strains that exhibit a variety of physiological changes including increased or decreased growth rate relative to the parent strain when cells use methanol and/or acetate as carbon and energy sources. This method provides an avenue for the first step in identifying new gene functions: associating a genetic mutation with a reproducible phenotype. Mutations in bona fide methanogenesis genes such as corrinoid methyltransferases and proton-translocating F420H2:methanophenazine oxidoreductase (Fpo) were also generated, opening the door to in vivo functional complementation experiments. Irradiation-based mutagenesis such as from ultraviolet (UV) light, combined with modern genome sequencing, is a useful procedure to discern systems-level gene function in prokaryote taxa that can be axenically cultured but which may be resistant to chemical mutagens.
authors: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, N200 Beadle Center, Lincoln, NE 68588-0664, United States.
authors: Mary E
authors: ME
authors: Walter
authors: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, N200 Beadle Center, Lincoln, NE 68588-0664, United States.
authors: Alicia
authors: A
authors: Ortiz
authors: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, N200 Beadle Center, Lincoln, NE 68588-0664, United States.
authors: Casey
authors: C
authors: Sondgeroth
authors: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, N200 Beadle Center, Lincoln, NE 68588-0664, United States.
authors: Nathan M
authors: NM
authors: Sindt
authors: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, N200 Beadle Center, Lincoln, NE 68588-0664, United States.
authors: Nikolas
authors: N
authors: Duszenko
authors: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, N200 Beadle Center, Lincoln, NE 68588-0664, United States.
authors: Jennie L
authors: JL
authors: Catlett
authors: Morrison Microscopy Core Research Facility, Center for Biotechnology, University of Nebraska-Lincoln, E117 Beadle Center, Lincoln, NE 68588-0664, United States.
authors: You
authors: Y
authors: Zhou
authors: Nebraska Center for Materials and Nanoscience, University of Nebraska-Lincoln, 855 N 16th St, Lincoln, NE 68588, United States.
authors: Shah
authors: S
authors: Valloppilly
authors: School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, United States.
authors: Christopher
authors: C
authors: Anderson
authors: Department of Animal Science, University of Nebraska-Lincoln, C220K Animal Science, Lincoln, NE 68583, United States.
authors: Samodha
authors: S
authors: Fernando
authors: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, N200 Beadle Center, Lincoln, NE 68588-0664, United States. Electronic address: nbuan2@unl.edu.
authors: Nicole R
authors: NR
authors: Buan
mesh_terms: D000085:Acetates / Q000378:metabolism; D001105:Archaea / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000528:radiation effects*; D019641:DNA, Archaeal / Q000235:genetics / Q000528:radiation effects; D019766:Genes, Archaeal; D057166:High-Throughput Screening Assays / Q000379:methods*; D008697:Methane / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D017020:Methanosarcina / Q000235:genetics / Q000254:growth & development / Q000528:radiation effects; D008780:Methyltransferases / Q000235:genetics; D050296:Microbial Viability / Q000528:radiation effects; D016296:Mutagenesis / Q000528:radiation effects; D010641:Phenotype; D017354:Point Mutation / Q000528:radiation effects*; D014466:Ultraviolet Rays
chemical_list: D000085:Acetates; D019641:DNA, Archaeal; D008780:Methyltransferases; D008697:Methane; D000432:Methanol
keywords: Archaea; Methane; Methanogen; Methanosarcina; Struvite