# A Modified Methyl Transferase Cofactor to Selectively Silence Gene Expression in Escherichia coli

## TL;DR

A modified cofactor can selectively silence specific genes in E. coli, offering a new way to control bacterial gene expression.

## Contribution

AdoHcy azide selectively silences genes in E. coli by steric inhibition, not seen with other modifications.

## Key findings

- AdoHcy azide disables ampicillin, kanamycin, and eGFP genes in modified plasmids in some E. coli strains.
- Sequencing shows 3–6 nucleotides missing near the modification site, indicating steric inhibition.
- Modified plasmids resist demethylation in the BL21 strain of E. coli.

## Abstract

Artificial control of gene expression in bacteria offers interesting prospects for influencing bacterial pathogenicity and antibiotic resistance. We show that the methyl‐transferase cofactor, AdoHcy azide, can silence gene expression in modified plasmids in some strains of Escherichia coli, where ampicillin and kanamycin resistance as well as eGFP genes were selectively and independently disabled. The disabling of transcription is likely due to steric inhibition during transcription initiation, which is supported by Sanger and nanopore sequencing results. Both sequencing methods showed that 3–6 nucleotides were absent from around the modification site. Postgrowth, extracted AmpR/eGFP plasmid shows evidence of restriction, with sections of the plasmid, including the modification site, missing for the AdoHcy azide modified plasmids. Notably, the AdoHcy azide modification on the DNA appears to be resistant against demethylation in the BL21 strain of E. coli.

Graphical abstract and table of contents entry

A modified methyl transferase cofactor, containing AdoHcy azide, creates a specific single‐site molecular switch near a promoter, on plasmid DNA, preventing the expression of a single gene at the designated site, not observed with other modifications. Likely of steric origin, this observation points to a new way for controlling gene expression in vivo, in conjunction with a wider variety of synthetic methyl transferase cofactors.© 2026 WILEY‐VCH GmbH