The type 6 secretion system (T6SS) is a molecular ‘harpoon’ that is used by bacteria to assault neighbouring cells and inject toxic effector proteins. The tip of the harpoon can be loaded with a diverse set of toxins and to prevent self-intoxication, the toxin loci encode cognate immunity proteins. In an effort to better understand post-transcriptional gene regulation in the human pathogen, enterohaemorrhagic E. coli (EHEC), we have applied the UV-crosslinking and organic extraction technique, TRAPP, to identify RNA-binding proteins within the EHEC RBPome. This analysis identified 443 proteins that were statistically enriched by TRAPP and included the T6SS immunity protein RhsFI. Using UV-crosslinking and RNA sequencing (CRAC) we find that RhsFI binds to a range of 3’UTR encoded small RNAs (sRNA) and partially overlaps with known targets of the RNA chaperones ProQ and Hfq. We have confirmed a subset of RhsFI-RNA targets using dot hybridisation assays. Alphafold analysis indicates that RhsFI forms a complex with its cognate toxin, RhsF, and together these proteins have a large positively charged surface that is predicted to bind RNA. Our results suggest that the RhsF-RhsFI toxin-immunity complex may bind RNA within the T6SS-encoding cell itself. In support of this hypothesis, we find that an internal promoter allows sub-genic expression of the toxin domain of RhsF and RhsFI immunity protein. This internal promoter uncouples RhsF-RhsFI expression from the T6SS harpoon complex.
Collectively, our results indicate that the toxin-immunity proteins RhsF-RhsFI are expressed independently of the T6SS harpoon in enterohaemorrhagic E. coli and bind to a subset of 3’UTR-encoded sRNAs known to associate with Hfq and ProQ. These data suggests that RhsF-RhsFI has been repurposed from a toxin used in offense, to an internal gene regulator that modulates Hfq- and ProQ-bound regulatory sRNAs.