Transposon mutagenesis for methylotrophic bacteria using Methylorubrum extorquens AM1 as a model system

Publication Date

1-1-2021

Document Type

Contribution to a Book

Publication Title

Methods in Enzymology

Volume

650

DOI

10.1016/bs.mie.2021.01.015

First Page

159

Last Page

184

Abstract

Transposon mutagenesis utilizes transposable genetic elements that integrate into a recipient genome to generate random insertion mutations which are easily identified. This forward genetic approach has proven powerful in elucidating complex processes, such as various pathways in methylotrophy. In the past decade, many methylotrophic bacteria have been shown to possess alcohol dehydrogenase enzymes that use lanthanides (Lns) as cofactors. Using Methylorubrum extorquens AM1 as a model organism, we discuss the experimental designs, protocols, and results of three transposon mutagenesis studies to identify genes involved in different aspects of Ln-dependent methanol oxidation. These studies include a selection for transposon insertions that prevent toxic intracellular formaldehyde accumulation, a fluorescence-imaging screen to identify regulatory processes for a primary Ln-dependent methanol dehydrogenase, and a phenotypic screen for genes necessary for function of a Ln-dependent ethanol dehydrogenase. We anticipate that the methods described in this chapter can be applied to understand other metabolic systems in diverse bacteria.

Keywords

Alcohol dehydrogenase, Lanthanides, Methanol oxidation, Methylotrophy, Mutagenesis, Transposon

Department

Biological Sciences

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