A-to-I RNA editing in bacteria increases pathogenicity and tolerance to oxidative stress.

Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes; however, many features remain largely unexplored in prokaryotes. This study focuses on a serine-to-proline recoding event (S128P) that originated in the mRNA offliC, which encodes a flagellar filament protein; the editing event was observed in RNA-seq samples exposed to oxidative stress. Using Sanger sequencing, we show that the S128P editing event is induced by H2O2. To investigate thein vivointeraction between RNAs and TadA, which is the principal enzyme for A-to-I editing, genome-wide RNA immunoprecipitation-coupled high-throughput sequencing (iRIP-Seq) analysis was performed using HA-tagged TadA fromXanthomonas oryzaepv.oryzicola. We found that TadA can bind to the mRNA offliCand the binding motif is identical to that previously reported by Bar-Yaacov and colleagues. This editing event increased motility and enhanced tolerance to oxidative stress due to changes in flagellar filament structure, which was modelled in 3D and measured by TEM. The change in filament structure due to the S128P mutant increased biofilm formation, which was measured by the 3D laser scanning confocal microscopy. RNA-seq revealed that a gene cluster that contributes to siderophore biosynthesis and Fe(3+)uptake was upregulated in S128P compared with WT. Based on intracellular levels of reactive oxygen species and an oxidative stress survival assay, we found that this gene cluster can contribute to the reduction of the Fenton reaction and increases biofilm formation and bacterial virulence. This oxidative stress response was also confirmed inPseudomonas putida. Overall, our work demonstrates that A-to-I RNA editing plays a role in bacterial pathogenicity and adaptation to oxidative stress. Author summary Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes that has only been recently documented in bacteria. In this study, we use multiple 'omic' approaches to show that A-to-I RNA editing can occur infliC, a flagellar filament protein. We show that TadA, which encodes adenosine deaminase, can directly bind to mRNA of target genes through recognition of a GACG motif. This editing event changes a single amino acid residue from serine to proline in FliC, resulting in a structural change in the flagellar filament. This posttranscriptional editing event contributes to virulence and increases tolerance to oxidative stress by enhancing biofilm formation. Our results provide insight into a new mechanism that bacterial pathogens use to adapt to oxidative stress, which can also increase virulence.