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

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Many bacterial and archaeal genomes contain a high percentage of hypothetical genes with unknown functions.
• Phylogenetically deep-branching microbes, like methanogens, have up to 50% of genes with unknown functions, hindering a complete understanding of their biology.

Purpose of the Study:

• To develop high-throughput anaerobic techniques for mutagenizing, screening, and selecting mutant strains of *Methanosarcina acetivorans*.
• To identify gene functions by associating genetic mutations with reproducible physiological phenotypes in a strict anaerobe.

Main Methods:

• Development of high-throughput anaerobic techniques for UV mutagenesis, screening, and selection in 96-well plates.
• Isolation and characterization of mutant strains exhibiting altered growth rates on methanol and/or acetate.
• Generation of mutations in known methanogenesis genes, including corrinoid methyltransferases and proton-translocating F₄₂₀H₂:methanophenazine oxidoreductase (Fpo).

Main Results:

• Successfully isolated 10 mutant strains of *Methanosarcina acetivorans* with diverse physiological changes.
• Observed variations in growth rates (increase or decrease) relative to the parent strain when utilizing methanol and/or acetate.
• Generated mutations in essential methanogenesis genes, enabling future in vivo functional complementation studies.

Conclusions:

• The developed high-throughput anaerobic mutagenesis and screening method is effective for associating genetic mutations with reproducible phenotypes.
• This approach is valuable for discovering the functions of hypothetical genes in prokaryotes, particularly those resistant to chemical mutagens.
• The study provides a foundation for systems-level gene function discovery in axenically cultured prokaryotes.