m6A Modification Is Incorporated into Bacterial mRNA Without Specific Functional Benefit

N 6-Methyladenosine (m6A), the most abundant modification in eukaryotic messenger RNAs (mRNAs), has also been found at a low level in bacterial mRNAs. However, enzyme(s) that introduce m6A modification on mRNAs in bacteria remain elusive. In this work, we combine deep-sequencing approaches that identify m6A sites with in vitro biochemical studies to identify putative m6A methyltransferases that would modify Escherichia coli mRNAs. We tested four uncharacterized candidates predicted to encode proteins with putative methyltransferase domains, whose deletion decreased the m6A level. However, in vitro analysis with the purified putative methyltransferases revealed that none of them installs m6A on mRNA. Exposure to heat and oxidative stress also changed the m6A level; however, we found no clear correlation between the m6A change and the specific stress. Considering two deep-sequencing approaches with different resolution, we found that m6A methylation on bacterial mRNAs is very low and appears randomly introduced. These results suggest that, in contrast to eukaryotes, the m6A modification in bacterial mRNA lacks a direct enzymatic recognition mechanism and has no clear biological function. © 2025 The Author(s).