A 5′-UTR <i>cis</i> -acting RNA element targeted by RNase III is essential for DNA simple sequence repeat-dependent phase variation in <i>Haemophilus influenzae</i>

Simple sequence repeats (SSRs) are insertion-deletion mutational hotspots causing phase variation in bacterial genomes. When located in intergenic regions, SSR variation impacts transcription and may change post-transcriptional regulatory element targeting. Here, we show that transcription, 5'-UTR structure, and messenger RNA (mRNA) processing are key to bacterial SSR phase variation. Expression of the Haemophilus influenzae HMW1A adhesin is inversely proportional to the (5'-ATCTTTC)n SSR length upstream of the hmw1A gene. This repeat region is transcribed as part of the hmw 5'-UTR, which acts as a cis-acting regulatory element via a long hairpin structure involving 10 repeats on its left arm and a G-rich region on its right arm. The double strand-specific endoribonuclease RNase III processes hmw1 mRNA at this 5'-UTR hairpin increasing HMW1A expression. Deep-sequencing-based SSR quantification in bacterial populations showed that RNase III is also required for changes in SSR count. Moreover, mutations that disrupt the 5'-UTR hairpin in the repeat sequence or in the G-rich region impair HMW1A expression and repeat length changes. This supports hmw 5'-UTR and RNase III contribution to the mechanism of SSR number expansion and contraction. Overall, we present novel regulatory roles for the hmw 5'-UTR in bacterial adaptation, where its transcription, folding, and RNase III processing are essential to HMW1A expression and phase-variable changes.