ExcludonFinder: mapping transcriptional overlaps between neighboring genes.

Bacteria regulate neighboring genes via overlapping transcription in untranslated regions (UTRs), forming excludons. This overlap leads to transcriptional interference and RNase III-mediated mRNA degradation, resulting in mutually exclusive gene expression, where the activation of one gene suppresses its neighbor. Although individual examples of excludons have been described in various bacterial species, a comprehensive excludon map of a bacterial genome has yet to be established. In this study, we constructed the excludon map of Escherichia coli and Staphylococcus aureus using publicly available RNA-seq data and a newly developed computational tool, ExcludonFinder (https://excludonfinder-unavarra.com). Our analysis identified 16 divergent and 165 convergent excludons in E. coli, as well as 10 divergent and 28 convergent excludons in S. aureus. To validate these findings, we used four independent datasets: detection of double-stranded RNA capture via the Tombusvirus p19 protein, accumulation of short RNAs from RNase III activity, overlap with predicted transcriptional terminators, and single-cell expression analysis. As a proof of concept, we examined transcriptional changes in E. coli under antibiotic stress, revealing that the relBE-ydfV excludon exhibits opposing expression patterns in response to multiple antibiotics. Our findings reveal the widespread presence of excludons and their broad relevance in bacterial gene regulation.