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 C Fernando, Nicole R Buan

Research output: Contribution to journalArticle

1 Citation (Scopus)

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

Fingerprint

Archaea
Phenotype
Mutation
Genes
Archaeal Genome
Corrinoids
Methanosarcina
Bacterial Genomes
Methane
Mutagens
Methyltransferases
Ultraviolet Rays
Mutagenesis
Methanol
Protons
Oxidoreductases
Acetates
Carbon
Genome
Growth

Keywords

  • Archaea
  • Methane
  • Methanogen
  • Methanosarcina
  • Struvite

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

Cite this

High-throughput mutation, selection, and phenotype screening of mutant methanogenic archaea. / Walter, Mary E.; Ortiz, Alicia; Sondgeroth, Casey; Sindt, Nathan M.; Duszenko, Nikolas; Catlett, Jennie L.; Zhou, You; Valloppilly, Shah; Anderson, Christopher; Fernando, Samodha C; Buan, Nicole R.

In: Journal of Microbiological Methods, Vol. 131, 01.12.2016, p. 113-121.

Research output: Contribution to journalArticle

Walter, ME, Ortiz, A, Sondgeroth, C, Sindt, NM, Duszenko, N, Catlett, JL, Zhou, Y, Valloppilly, S, Anderson, C, Fernando, SC & Buan, NR 2016, 'High-throughput mutation, selection, and phenotype screening of mutant methanogenic archaea', Journal of Microbiological Methods, vol. 131, pp. 113-121. https://doi.org/10.1016/j.mimet.2016.10.010
Walter, Mary E. ; Ortiz, Alicia ; Sondgeroth, Casey ; Sindt, Nathan M. ; Duszenko, Nikolas ; Catlett, Jennie L. ; Zhou, You ; Valloppilly, Shah ; Anderson, Christopher ; Fernando, Samodha C ; Buan, Nicole R. / High-throughput mutation, selection, and phenotype screening of mutant methanogenic archaea. In: Journal of Microbiological Methods. 2016 ; Vol. 131. pp. 113-121.
@article{9c64390b13b74eaa8dd0c8b9e4503dbd,
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.",
keywords = "Archaea, Methane, Methanogen, Methanosarcina, Struvite",
author = "Walter, {Mary E.} and Alicia Ortiz and Casey Sondgeroth and Sindt, {Nathan M.} and Nikolas Duszenko and Catlett, {Jennie L.} and You Zhou and Shah Valloppilly and Christopher Anderson and Fernando, {Samodha C} and Buan, {Nicole R}",
year = "2016",
month = "12",
day = "1",
doi = "10.1016/j.mimet.2016.10.010",
language = "English (US)",
volume = "131",
pages = "113--121",
journal = "Journal of Microbiological Methods",
issn = "0167-7012",
publisher = "Elsevier",

}

TY - JOUR

T1 - High-throughput mutation, selection, and phenotype screening of mutant methanogenic archaea

AU - Walter, Mary E.

AU - Ortiz, Alicia

AU - Sondgeroth, Casey

AU - Sindt, Nathan M.

AU - Duszenko, Nikolas

AU - Catlett, Jennie L.

AU - Zhou, You

AU - Valloppilly, Shah

AU - Anderson, Christopher

AU - Fernando, Samodha C

AU - Buan, Nicole R

PY - 2016/12/1

Y1 - 2016/12/1

N2 - 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.

AB - 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.

KW - Archaea

KW - Methane

KW - Methanogen

KW - Methanosarcina

KW - Struvite

UR - http://www.scopus.com/inward/record.url?scp=84994021813&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84994021813&partnerID=8YFLogxK

U2 - 10.1016/j.mimet.2016.10.010

DO - 10.1016/j.mimet.2016.10.010

M3 - Article

C2 - 27771305

AN - SCOPUS:84994021813

VL - 131

SP - 113

EP - 121

JO - Journal of Microbiological Methods

JF - Journal of Microbiological Methods

SN - 0167-7012

ER -