The type 6 secretion system immunity protein RhsFI has been repurposed for small RNA regulation in pathogenic E. coli

RNA-binding proteins (RBPs) play key roles in regulating virulence gene expression in pathogenic, enterohaemorrhagic E. coli (EHEC). Under virulence-inducing conditions total RNA associated protein purification (TRAPP) identified 443 RNA-associated proteins. These included 35 encoded within pathogenicity islands, with RNA-binding confirmed in vitro for a sub-set that included RhsFI. In vivo RNA targets for RhsFI included regulatory small RNAs (sRNAs) encoded in 3' UTRs. The rhsF-rhsFI locus encodes effector and immunity proteins for the type 6 secretion system (T6SS). T6SS expression is repressed in EHEC, but internal initiation generates a transcript encoding a truncated toxin (RhsFint) and downstream RhsFI. Modelling predicted that RhsFint can dimerise with RhsFI. Deletion of rhsFint rhsFI alters the expression of multiple operons in which embedded sRNAs are direct RhsFI targets. Loss of RhsFint:RhsFI dramatically reduced adhesion to bovine cells in vitro, altered cell morphology and increased acid resistance, phenotypes that impact host colonisation. Overall design: RNA seq was performed on biological triplicate cultures of E. coli O157:H7 str. Sakai (stx-) and an isogenic rhsF rhsFI (ECs0605 ECs0604) double deltion strain, and mutant complemented with rhsFint rhsFI (pZE12::rhsFint rhsFI) grown in MEM-HEPES media supplemented with 250nM FeNO3 and 0.1% glucose. In addition, UV-crosslinking and sequencing of RNAs (CRAC) bound to RhsFI was performed on rhsFI-His-TEV-FLAG expressed from pBADmycHis in EHEC. CRAC was performed as previously (Tree et al Molecular Cell 2014) in biological duplicate and the parental EHEC strain was included as a control.