Networks of Resistance: Small RNA Control of Antibiotic Resistance.

The golden age of antibiotics has passed, and the threat of untreatable antimicrobial resistant infections is now a reality for many individuals. Understanding how bacteria resist antimicrobial treatment and regulate gene expression in response to antibiotics is an important step towards combating resistance. In this review we focus on a ubiquitous class of bacterial gene regulators termed regulatory small RNAs (sRNAs) and how they contribute to antimicrobial resistance and tolerance. Small RNAs have notable roles in modulating the composition of the bacterial envelope, and through these functions control intrinsic antimicrobial resistance in many human pathogens. Recent technical advances that allow profiling of the 'sRNA interactome' have revealed a complex post-transcriptional network of sRNA interactions that can be used to identify network hubs and regulatory bottlenecks. Sequence-specific inhibition of these sRNAs with programmable RNA-targeting therapeutics may present avenues for treating antimicrobial resistant pathogens or resensitizing to our current antibiotics.

Early termination of the Shiga toxin transcript generates a regulatory small RNA.

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.

The Role and Targets of the RNA-Binding Protein ProQ in the Gram-Negative Bacterial Pathogen Pasteurella multocida.

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3' stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3' region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.

Identification of bacteriophage-encoded anti-sRNAs in pathogenic Escherichia coli.

In bacteria, Hfq is a core RNA chaperone that catalyzes the interaction of mRNAs with regulatory small RNAs (sRNAs). To determine in vivo RNA sequence requirements for Hfq interactions, and to study riboregulation in a bacterial pathogen, Hfq was UV crosslinked to RNAs in enterohemorrhagic Escherichia coli (EHEC). Hfq bound repeated trinucleotide motifs of A-R-N (A-A/G-any nucleotide) often associated with the Shine-Dalgarno translation initiation sequence in mRNAs. These motifs overlapped or were adjacent to the mRNA sequences bound by sRNAs. In consequence, sRNA-mRNA duplex formation will displace Hfq, promoting recycling. Fifty-five sRNAs were identified within bacteriophage-derived regions of the EHEC genome, including some of the most abundant Hfq-interacting sRNAs. One of these (AgvB) antagonized the function of the core genome regulatory sRNA, GcvB, by mimicking its mRNA substrate sequence. This bacteriophage-encoded "anti-sRNA" provided EHEC with a growth advantage specifically in bovine rectal mucus recovered from its primary colonization site in cattle.

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E.

RNA sequencing studies have identified hundreds of non-coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high-throughput analysis of RNA-RNA interactions in bacteria. Here we demonstrate that in vivo sRNA-mRNA duplexes can be recovered using UV-crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base-paired sRNA-mRNA duplexes in association with RNase E, allowing proximity-dependent ligation and sequencing of cognate sRNA-mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA-mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co-regulated target mRNAs. We identified multiple mRNA targets for the pathotype-specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli Numerous sRNA interactions were also identified with non-coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.

An RNA-dependent mechanism for transient expression of bacterial translocation filaments.

The prokaryotic RNA chaperone Hfq mediates sRNA-mRNA interactions and plays a significant role in post-transcriptional regulation of the type III secretion (T3S) system produced by a range of Escherichia coli pathotypes. UV-crosslinking was used to map Hfq-binding under conditions that promote T3S and multiple interactions were identified within polycistronic transcripts produced from the locus of enterocyte effacement (LEE) that encodes the T3S system. The majority of Hfq binding was within the LEE5 and LEE4 operons, the latter encoding the translocon apparatus (SepL-EspADB) that is positively regulated by the RNA binding protein, CsrA. Using the identified Hfq-binding sites and a series of sRNA deletions, the sRNA Spot42 was shown to directly repress translation of LEE4 at the sepL 5' UTR. In silico and in vivo analyses of the sepL mRNA secondary structure combined with expression studies of truncates indicated that the unbound sepL mRNA is translationally inactive. Based on expression studies with site-directed mutants, an OFF-ON-OFF toggle model is proposed that results in transient translation of SepL and EspA filament assembly. Under this model, the nascent mRNA is translationally off, before being activated by CsrA, and then repressed by Hfq and Spot42.

Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli.

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.

Methionine biosynthesis and transport are functionally redundant for the growth and virulence of Salmonella Typhimurium.

Methionine (Met) is an amino acid essential for many important cellular and biosynthetic functions, including the initiation of protein synthesis and S-adenosylmethionine-mediated methylation of proteins, RNA, and DNA. The de novo biosynthetic pathway of Met is well conserved across prokaryotes but absent from vertebrates, making it a plausible antimicrobial target. Using a systematic approach, we examined the essentiality of de novo methionine biosynthesis in Salmonella enterica serovar Typhimurium, a bacterial pathogen causing significant gastrointestinal and systemic diseases in humans and agricultural animals. Our data demonstrate that Met biosynthesis is essential for S. Typhimurium to grow in synthetic medium and within cultured epithelial cells where Met is depleted in the environment. During systemic infection of mice, the virulence of S. Typhimurium was not affected when either de novo Met biosynthesis or high-affinity Met transport was disrupted alone, but combined disruption in both led to severe in vivo growth attenuation, demonstrating a functional redundancy between de novo biosynthesis and acquisition as a mechanism of sourcing Met to support growth and virulence for S. Typhimurium during infection. In addition, our LC-MS analysis revealed global changes in the metabolome of S. Typhimurium mutants lacking Met biosynthesis and also uncovered unexpected interactions between Met and peptidoglycan biosynthesis. Together, this study highlights the complexity of the interactions between a single amino acid, Met, and other bacterial processes leading to virulence in the host and indicates that disrupting the de novo biosynthetic pathway alone is likely to be ineffective as an antimicrobial therapy against S. Typhimurium.

VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation.

The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin-Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify the cellular targets of 12 VapCs from M. tuberculosis by applying UV-crosslinking and deep sequencing. Remarkably, these VapCs are all endoribonucleases that cleave RNAs essential for decoding at the ribosomal A-site. Eleven VapCs cleave specific tRNAs while one exhibits SRL cleavage activity. These findings suggest that multiple vapBC modules contribute to the survival of M. tuberculosis in its human host by reducing the level of translation.

Lysogeny with Shiga toxin 2-encoding bacteriophages represses type III secretion in enterohemorrhagic Escherichia coli.

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins.

The 3' UTR of vigR is required for virulence in Staphylococcus aureus and has expanded through STAR sequence repeat insertions.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteremia, and treatment failure is often associated with vancomycin-intermediate S. aureus isolates. The regulatory 3' UTR of the vigR mRNA contributes to vancomycin tolerance and upregulates the autolysin IsaA. Using MS2-affinity purification coupled with RNA sequencing, we find that the vigR 3' UTR also regulates dapE, a succinyl-diaminopimelate desuccinylase required for lysine and peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the 3' UTR increased virulence, while the isaA mutant is completely attenuated in a wax moth larvae model. Sequence and structural analyses of vigR indicated that the 3' UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions that contribute sequence for the isaA interaction seed and may functionalize the 3' UTR.

Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion.

This study has identified horizontally acquired genomic regions of enterohaemorrhagic Escherichia coli O157:H7 that regulate expression of the type III secretion (T3S) system encoded by the locus of enterocyte effacement (LEE). Deletion of O-island 51, a 14.93 kb cryptic prophage (CP-933C), resulted in a reduction in LEE expression and T3S. The deletion also had a reduced capacity to attach to epithelial cells and significantly reduced E. coli O157 excretion levels from sheep. Further characterization of O-island 51 identified a novel positive regulator of the LEE, encoded by ecs1581 in the E. coli O157:H7 strain Sakai genome and present but not annotated in the E. coli strain EDL933 sequence. Functionally important residues of ECs1581 were identified based on phenotypic variants present in sequenced E. coli strains and the regulator was termed RgdR based on a motif demonstrated to be important for stimulation of gene expression. While RgdR activated expression from the LEE1 promoter in the presence or absence of the LEE-encoded regulator (Ler), RgdR stimulation of T3S required ler and Ler autoregulation. RgdR also controlled the expression of other phenotypes, including motility, indicating that this new family of regulators may have a more global role in E. coli gene expression.

Profiling the in vivo RNA interactome associated with the endoribonuclease RNase III in Staphylococcus aureus.

Regulatory small RNA (sRNA) have been extensively studied in model Gram-negative bacteria, but the functional characterisation of these post-transcriptional gene regulators in Gram-positives remains a major challenge. Our previous work in enterohaemorrhagic E. coli utilised the proximity-dependant ligation technique termed CLASH (UV-crosslinking, ligation, and sequencing of hybrids) for direct high-throughput sequencing of the regulatory sRNA-RNA interactions within the cell. Recently, we adapted the CLASH technique and demonstrated that UV-crosslinking and RNA proximity-dependant ligation can be applied to Staphylococcus aureus, which uncovered the first RNA-RNA interaction network in a Gram-positive bacterium. In this chapter, we describe modifications to the CLASH technique that were developed to capture the RNA interactome associated with the double-stranded endoribonuclease RNase III in two clinical isolates of S. aureus. To briefly summarise our CLASH methodology, regulatory RNA-RNA interactions were first UV-crosslinked in vivo to the RNase III protein and protein-RNA complexes were affinity-purified using the His6-TEV-FLAG tags. Linkers were ligated to RNase III-bound RNA during library preparation and duplexed RNA-RNA species were ligated together to form a single contiguous RNA 'hybrid'. The RNase III-RNA binding sites and RNA-RNA interactions occurring on RNase III (RNA hybrids) were then identified by paired-end sequencing technology. RNase III-CLASH represents a step towards a systems-level understanding of regulatory RNA in Gram-positive bacteria.

Detection of Streptococcus pyogenes M1UK in Australia and characterization of the mutation driving enhanced expression of superantigen SpeA.

A new variant of Streptococcus pyogenes serotype M1 (designated 'M1UK') has been reported in the United Kingdom, linked with seasonal scarlet fever surges, marked increase in invasive infections, and exhibiting enhanced expression of the superantigen SpeA. The progenitor S. pyogenes 'M1global' and M1UK clones can be differentiated by 27 SNPs and 4 indels, yet the mechanism for speA upregulation is unknown. Here we investigate the previously unappreciated expansion of M1UK in Australia, now isolated from the majority of serious infections caused by serotype M1 S. pyogenes. M1UK sub-lineages circulating in Australia also contain a novel toxin repertoire associated with epidemic scarlet fever causing S. pyogenes in Asia. A single SNP in the 5' transcriptional leader sequence of the transfer-messenger RNA gene ssrA drives enhanced SpeA superantigen expression as a result of ssrA terminator read-through in the M1UK lineage. This represents a previously unappreciated mechanism of toxin expression and urges enhanced international surveillance.

Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence-blocking compounds.

A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.

Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7.

Type III secretion (T3S) plays a pivotal role in the colonization of ruminant hosts by Enterohemorrhagic Escherichia coli (EHEC). The T3S system translocates effector proteins into host cells to promote bacterial attachment and persistence. The repertoire and variation in prophage regions underpins differences in the pathogenesis and epidemiology of EHEC strains. In this study, we have used a collection of deletions in cryptic prophages and EHEC O157 O-islands to screen for novel regulators of T3S. Using this approach we have identified a family of homologous AraC-like regulators that indirectly repress T3S. These prophage-encoded secretion regulator genes (psr) are found exclusively on prophages and are associated with effector loci and the T3S activating Pch family of regulators. Transcriptional profiling, mutagenesis and DNA binding studies were used to show that these regulators usurp the conserved GAD acid stress resistance system to regulate T3S by increasing the expression of GadE (YhiE) and YhiF and that this regulation follows attachment to bovine epithelial cells. We further demonstrate that PsrA and effectors encoded within cryptic prophage CP933-N are required for persistence in a ruminant model of colonization.

The Small RNA CyaR Activates Translation of the Outer Membrane Haem Receptor chuA in Enterohemorrhagic Escherichia coli.

To sense the transition from environment to host, bacteria use a range of environmental cues to control expression of virulence genes. Iron is tightly sequestered in host tissues and in the human pathogen enterohemorrhagic Escherichia coli (EHEC) iron-limitation induces transcription of the outer membrane haem transporter encoded by chuAS. ChuA expression is post-transcriptionally activated at 37°C by a FourU RNA thermometer ensuring that the haem receptor is only expressed under low iron, high temperature conditions that indicate the host. Here we demonstrate that expression of chuA is also independently regulated by the cAMP-responsive small RNA (sRNA) CyaR and transcriptional terminator Rho. These results indicate that chuAS expression is regulated at the transcription initiation, transcript elongation, and translational level. We speculate that additional sensing of the gluconeogenic environment allows further precision in determining when EHEC is at the gastrointestinal epithelium of the host. With previous studies, it appears that the chuAS transcript is controlled by eight regulatory inputs that control expression through six different transcriptional and post-transcriptional mechanisms. The results highlight the ability of regulatory sRNAs to integrate multiple environmental signals into a layered hierarchy of signal input.

RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.

RNase III-CLASH of multi-drug resistant Staphylococcus aureus reveals a regulatory mRNA 3'UTR required for intermediate vancomycin resistance.

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.

Burning the Candle at Both Ends: Have Exoribonucleases Driven Divergence of Regulatory RNA Mechanisms in Bacteria?

Regulatory RNAs have emerged as ubiquitous gene regulators in all bacterial species studied to date. The combination of sequence-specific RNA interactions and malleable RNA structure has allowed regulatory RNA to adopt different mechanisms of gene regulation in a diversity of genetic backgrounds. In the model Gammaproteobacteria Escherichia coli and Salmonella, the regulatory RNA chaperone Hfq appears to play a global role in gene regulation, directly controlling ∼20 to 25% of the entire transcriptome. While the model Firmicutes Bacillus subtilis and Staphylococcus aureus encode a Hfq homologue, its role has been significantly depreciated. These bacteria also have marked differences in RNA turnover. E. coli and Salmonella degrade RNA through internal endonucleolytic and 3'→5' exonucleolytic cleavage that appears to allow transient accumulation of mRNA 3' UTR cleavage fragments that contain stabilizing 3' structures. In contrast, B. subtilis and S. aureus are able to exonucleolytically attack internally cleaved RNA from both the 5' and 3' ends, efficiently degrading mRNA 3' UTR fragments. Here, we propose that the lack of 5'→3' exoribonuclease activity in Gammaproteobacteria has allowed the accumulation of mRNA 3' UTR ends as the "default" setting. This in turn may have provided a larger pool of unconstrained RNA sequences that has fueled the expansion of Hfq function and small RNA (sRNA) regulation in E. coli and Salmonella. Conversely, the exoribonuclease RNase J may be a significant barrier to the evolution of 3' UTR sRNAs in B. subtilis and S. aureus that has limited the pool of RNA ligands available to Hfq and other sRNA chaperones, depreciating their function in these model Firmicutes.