By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to efficiently adapt to changes in their environment. To better understand how Escherichia coli acclimatizes to nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover Hfq-associated RNA-RNA interactions at specific growth stages. We demonstrate that Hfq CLASH robustly captures bona fide RNA-RNA interactions. We identified hundreds of novel sRNA base-pairing interactions, including many sRNA-sRNA interactions and involving 3’UTR-derived sRNAs. We rediscovered known and identified novel sRNA seed sequences. The sRNA-mRNA interactions identified by CLASH have strong base-pairing potential and are highly enriched for complementary sequence motifs, even those supported by only a few reads. Yet, steady state levels of most mRNA targets were not significantly affected upon over-expression of the sRNA regulator. Our results reinforce the idea that the reproducibility of the interaction, not base-pairing potential, is a stronger predictor for a regulatory outcome.

Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen that colonizes humans and its reservoir host, cattle. Colonization requires the expression of a type 3 secretion (T3S) system that injects a mixture of effector proteins into host cells to promote bacterial attachment and disease progression. The T3S system is tightly regulated by a complex network of transcriptional and post-transcriptional regulators. Using transposon mutagenesis, here we identified the ybeZYX-Int operon as being required for normal T3S levels. Deletion analyses localized the regulation to the endoribonuclease YbeY, previously linked to 16S rRNA maturation and small RNA (sRNA) function. Loss of ybeY in EHEC had pleiotropic effects on EHEC cells, including reduced motility and growth and cold sensitivity. Using UV cross-linking and RNA-Seq (CRAC) analysis, we identified YbeY-binding sites throughout the transcriptome and discovered specific binding of YbeY to the “neck” and “beak” regions of 16S rRNA but identified no significant association of YbeY with sRNA, suggesting that YbeY modulates T3S by depleting mature ribosomes. In E. coli, translation is strongly linked to mRNA stabilization, and subinhibitory concentrations of the translation-initiation inhibitor kasugamycin provoked rapid degradation of a polycistronic mRNA encoding needle filament and needle tip proteins of the T3S system. We conclude that T3S is particularly sensitive to depletion of initiating ribosomes, explaining the inhibition of T3S in the ΔybeY strain. Accessory virulence transcripts may be preferentially degraded in cells with reduced translational capacity, potentially reflecting prioritization in protein production.

Enterohemorrhagic Escherichia coli is a significant human pathogen that causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome. The latter can lead to potentially fatal renal failure and is caused by the release of Shiga toxins that are encoded within lambdoid bacteriophages. The toxins are encoded within the late transcript of the phage and are regulated by antitermination of the PR′ late promoter during lytic induction of the phage. During lysogeny, the late transcript is prematurely terminated at tR′ immediately downstream of PR′, generating a short RNA that is a byproduct of antitermination regulation. We demonstrate that this short transcript binds the small RNA chaperone Hfq, and is processed into a stable 74-nt regulatory small RNA that we have termed StxS. StxS represses expression of Shiga toxin 1 under lysogenic conditions through direct interactions with the stx1AB transcript. StxS acts in trans to activate expression of the general stress response sigma factor, RpoS, through direct interactions with an activating seed sequence within the 5′ UTR. Activation of RpoS promotes high cell density growth under nutrient-limiting conditions. Many phages utilize antitermination to regulate the lytic/lysogenic switch and our results demonstrate that short RNAs generated as a byproduct of this regulation can acquire regulatory small RNA functions that modulate host fitness.

Treatment of methicillin-resistant Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA–RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA–RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA–mRNA interactions are recovered and we find that an mRNA encoding a long 3′ untranslated region (UTR) (termed vigR 3′UTR) functions as a regulatory ‘hub’ within the RNA–RNA interaction network. We demonstrate that the vigR 3′UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA–mRNA base-pairing. Deletion of the vigR 3′UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3′UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.

27By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to very 28 efficiently adapt to constant changes in their environment. To better understand how 29 Escherichia coli acclimatizes to changes in nutrient availability, we performed UV cross-30 linking, ligation and sequencing of hybrids (CLASH) to uncover sRNA-target interactions. 31Strikingly, we uncovered hundreds of novel Hfq-mediated sRNA-target interactions at specific 32 growth stages, involving many novel 3'UTR-derived sRNAs and a plethora of sRNA-sRNA 33 interactions. We discovered sRNA-target interaction networks that play a role in adaptation to 34 changes in nutrient availability. We characterized a novel 3'UTR-derived sRNA (MdoR), which 35 is part of a regulatory cascade that enhances maltose uptake by (a) inactivating repressive 36 pathways that block the accumulation of maltose transporters and (b) by reducing the flux of 37 general porins to the outer membrane. Our work provides striking examples of how bacteria 38 utilize sRNAs to integrate multiple regulatory pathways to enhance nutrient stress adaptation.39 3 Microorganisms are renowned for their ability to adapt to environmental changes by 40 rapidly rewiring their gene expression program. These responses are mediated through 41 integrated transcriptional and post-transcriptional networks. Control at the transcriptional level 42 dictates which genes are expressed (Balleza et al., 2009; Martínez-Antonio et al., 2008) and 43 is well-characterised in Escherichia coli. Post-transcriptional regulation is key for controlling 44 adaptive responses. By using riboregulators and RNA-binding proteins (RBPs), cells can 45 efficiently integrate multiple pathways and incorporate additional signals into regulatory 46 circuits. E. coli employs many post-transcriptional regulators, including small regulatory RNAs 47 (sRNAs (Waters and Storz, 2009)), cis-acting RNAs (Kortmann and Narberhaus, 2012), and 48 RNA binding proteins (RBPs) (Holmqvist and Vogel, 2018). The sRNAs are the largest class 49 of bacterial regulators, which work in tandem with RBPs to regulate their RNA targets (Storz 50 et al., 2011; Waters and Storz, 2009). The base-pairing interactions are often mediated by 51 RNA chaperones such as Hfq and ProQ, which help to anneal or stabilize the sRNA and 52 sRNA-target duplex (Smirnov et al., 2017(Smirnov et al., , 2016 Updegrove et al., 2016). Small RNAs can 53 repress or stimulate translation and transcription, as well as control mRNA stability 54 (Sedlyarova et al., 2016; Updegrove et al., 2016; Vogel and Luisi, 2011; Waters and Storz, 55 2009). 56During growth in rich media, E. coli are exposed to continuously changing conditions, 57 such as fluctuations in nutrient availability, pH and osmolarity. Consequently, E. coli elicit 58 complex responses that result in physiological and behavioural changes such as envelope 59 composition remodelling, quorum sensing, nutrient scavenging, swarming and biofilm 60 formation. Even subtle changes in the growth conditions can trigg...