High-throughput mutation, selection, and phenotype screening of mutant methanogenic archaea

Mary E. Walter, Alicia Ortiz, Casey Sondgeroth, Nathan M. Sindt, Nikolas Duszenko, Jennie L. Catlett, You Zhou, Shah Valloppilly, Christopher Anderson, Samodha Fernando, Nicole R. Buan

Research output: Contribution to journalArticle

1 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)113-121
Number of pages9
JournalJournal of Microbiological Methods
Volume131
DOIs
StatePublished - Dec 1 2016

Keywords

  • Archaea
  • Methane
  • Methanogen
  • Methanosarcina
  • Struvite

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

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    Walter, M. E., Ortiz, A., Sondgeroth, C., Sindt, N. M., Duszenko, N., Catlett, J. L., Zhou, Y., Valloppilly, S., Anderson, C., Fernando, S., & Buan, N. R. (2016). High-throughput mutation, selection, and phenotype screening of mutant methanogenic archaea. Journal of Microbiological Methods, 131, 113-121. https://doi.org/10.1016/j.mimet.2016.10.010