Ribosome maturation by YbeY stabilises Type III secretion system transcripts in enterohaemorrhagic E. coli. Escherichia coli O157:H7

Enterohaemorrhagic E. coli is a significant human pathogen that utilises a type 3 secretion system (T3S) to colonise the reservoir host, cattle, and humans. Transposon mutagenesis showed the ybeZYX-Int operon to be required for normal T3S levels. Deletion analyses identified the endoribonuclease ybeY, which was previously linked to 16S ribosomal RNA maturation and small RNA (sRNA) function. Loss of ybeY in EHEC had additional pleiotropic effects, including temperature sensitivity and reduced growth. UV-crosslinking revealed specific binding of ybeY to the “neck” and “beak” regions of 16S ribosomal RNA, but identified no significant sRNA association. Sub-inhibitory concentrations of the translation initiation inhibitor kasugamycin provoked degradation of a polycistronic T3S mRNA. We conclude that T3S is particularly sensitive to depletion of initiating ribosomes, explaining the inhibition of T3S in the ∆ybeY strain. Accessory virulence transcripts may be preferentially degraded in cells with reduced translational capacity, potentially reflecting the priorities for protein production. Overall design: UV-crosslinking analysis of RNA-protein interactions was performed as previously described (Tree et al. 2014) in biological duplicate with a control (untagged) sample. PCR amplified cDNA libraries were sequenced using an Illumina MiSeq platform. Data analysis were performed using the pyCRAC software package (Webb et al., 2014) as described in Sy et al. (2017). To identify statistically significant YbeY binding sites pyCalculateFDR was used on mapped reads with settings: 100nt flanking sequence for features (-r 100), p-value threshold of 0.05 (-m 0.05), minimum coverage of 10 reads (--min=10), and 500 iterations (--iterations=500). YbeY binding sites with FDR<0.05, recovered in both HTF tagged replicates, and absent from the untagged control for retained for further analysis.