Small RNAs direct attack and defense mechanisms in a quorum sensing phage and its host.

Many, if not all, bacteria use quorum sensing (QS) to control collective behaviors, and more recently, QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or "listen in" on the host's communication processes, to switch between lytic and lysogenic modes of infection. Here, we study the interaction of Vibrio cholerae with the lysogenic phage VP882, which is activated by the QS molecule DPO. We discover that induction of VP882 results in the binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompetes and downregulates host-encoded small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs, and we demonstrate that one of these sRNAs, named VpdS, promotes phage replication by regulating host and phage mRNA levels. We further show that host-encoded sRNAs can antagonize phage replication by downregulating phage mRNA expression and thus might be part of the host's phage defense arsenal.

Gene expression regulation by modulating Hfq expression in coordination with tailor-made sRNA-based knockdown in Escherichia coli.

The tailor-made synthetic sRNA-based gene expression knockdown system has demonstrated its efficacy in achieving pathway balancing in microbes, facilitating precise target gene repression and fine-tuned control of gene expression. This system operates under a competitive mode of gene regulation, wherein the tailor-made synthetic sRNA shares the intrinsic intracellular Hfq protein with other RNAs. The limited intracellular Hfq amount has the potential to become a constraining factor in the post-transcription regulation of sRNAs. To enhance the efficiency of the tailor-made sRNA gene expression regulation platform, we introduced an Hfq expression level modulation-coordinated sRNA-based gene knockdown system. This system comprises tailor-made sRNA expression cassettes that produce varying Hfq expression levels using different strength promoters. Modulating the expression levels of Hfq significantly improved the repressing capacity of sRNA, as evidenced by evaluations with four fluorescence proteins. In order to validate the practical application of this system, we applied the Hfq-modulated sRNA-based gene knockdown cassette to Escherichia coli strains producing 5-aminolevulinic acid and L-tyrosine. Diversifying the expression levels of metabolic enzymes through this cassette resulted in substantial increases of 74.6% in 5-aminolevulinic acid and 144% in L-tyrosine production. Tailor-made synthetic sRNA-based gene expression knockdown system, coupled with Hfq copy modulation, exhibits potential for optimizing metabolic fluxes through biosynthetic pathways, thereby enhancing the production yields of bioproducts.

RNA compaction and iterative scanning for small RNA targets by the Hfq chaperone.

RNA-guided enzymes must quickly search a vast sequence space for their targets. This search is aided by chaperones such as Hfq, a protein that mediates regulation by bacterial small RNAs (sRNAs). How RNA binding proteins enhance this search is little known. Using single-molecule Förster resonance energy transfer, we show that E. coli Hfq performs a one-dimensional scan in which compaction of the target RNA delivers sRNAs to sites distant from the location of Hfq recruitment. We also show that Hfq can transfer an sRNA between different target sites in a single mRNA, favoring the most stable duplex. We propose that compaction and segmental transfer, combined with repeated cycles of base pairing, enable the kinetic selection of optimal sRNA targets. Finally, we show that RNA compaction and sRNA transfer require conserved arginine patches. We suggest that arginine patches are a widespread strategy for enabling the movement of RNA across protein surfaces.

Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus.

The alphaproteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limited.IMPORTANCEThe conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotroph Caulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy in C. crescentus has been lacking. Here, we identified sRNAs and mRNAs associated with Hfq in vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters in C. crescentus.

The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division.

The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq-dependent small regulatory RNA (sRNA), DinR, which is processed from the 3' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled division inhibition during the SOS response.

The Amyloid Assembly of the Bacterial Hfq Is Lipid-Driven and Lipid-Specific.

Under specific conditions, some proteins can self-assemble into fibrillar structures called amyloids. Initially, these proteins were associated with neurodegenerative diseases in eucaryotes. Nevertheless, they have now been identified in the three domains of life. In bacteria, they are involved in diverse biological processes and are usually useful for the cell. For this reason, they are classified as "functional amyloids". In this work, we focus our analysis on a bacterial functional amyloid called Hfq. Hfq is a pleiotropic regulator that mediates several aspects of genetic expression, mainly via the use of small noncoding RNAs. Our previous work showed that Hfq amyloid-fibrils interact with membranes. This interaction influences Hfq amyloid structure formation and stability, but the specifics of the lipid on the dynamics of this process is unknown. Here, we show, using spectroscopic methods, how lipids specifically drive and modulate Hfq amyloid assembly or, conversely, its disassembly. The reported effects are discussed in light of the consequences for bacterial cell life.

Circular and Linear Dichroism for the Analysis of Small Noncoding RNA Properties.

Useful structural information about the conformation of nucleic acids can be quickly acquired by circular and linear dichroism (CD/LD) spectroscopy. These techniques, rely on the differential absorption of polarised light and are indeed extremely sensitive to subtle changes in the structure of chiral biomolecules. Many CD analyses of DNA or DNA:protein complexes have been conducted with substantial data acquisitions. Conversely, CD RNA analysis are still scarce, despite the fact that RNA plays a wide cellular function. This chapter seeks to introduce the reader to the use of circular, linear dichroism and in particular the use of Synchrotron Radiation for such samples. The use of these techniques on small noncoding RNA (sRNA) will be exemplified by analyzing changes in base stacking and/or helical parameters for the understanding of sRNA structure and function, especially by translating the dynamics of RNA:RNA annealing but also to access RNA stability or RNA:RNA alignment. The effect of RNA remodeling proteins will also be addressed. These analyses are especially useful to decipher the mechanisms by which sRNA will adopt the proper conformation thanks to the action of proteins such as Hfq or ProQ in the regulation of the expression of their target mRNAs.

Global Hfq-mediated RNA interactome of nitrogen starved Escherichia coli uncovers a conserved post-transcriptional regulatory axis required for optimal growth recovery.

The RNA binding protein Hfq has a central role in the post-transcription control of gene expression in many bacteria. Numerous studies have mapped the transcriptome-wide Hfq-mediated RNA-RNA interactions in growing bacteria or bacteria that have entered short-term growth-arrest. To what extent post-transcriptional regulation underpins gene expression in growth-arrested bacteria remains unknown. Here, we used nitrogen (N) starvation as a model to study the Hfq-mediated RNA interactome as Escherichia coli enter, experience, and exit long-term growth arrest. We observe that the Hfq-mediated RNA interactome undergoes extensive changes during N starvation, with the conserved SdsR sRNA making the most interactions with different mRNA targets exclusively in long-term N-starved E. coli. Taking a proteomics approach, we reveal that in growth-arrested cells SdsR influences gene expression far beyond its direct mRNA targets. We demonstrate that the absence of SdsR significantly compromises the ability of the mutant bacteria to recover growth competitively from the long-term N-starved state and uncover a conserved post-transcriptional regulatory axis which underpins this process.

In vivo RNA interactome profiling reveals 3'UTR-processed small RNA targeting a central regulatory hub.

Small noncoding RNAs (sRNAs) are crucial regulators of gene expression in bacteria. Acting in concert with major RNA chaperones such as Hfq or ProQ, sRNAs base-pair with multiple target mRNAs and form large RNA-RNA interaction networks. To systematically investigate the RNA-RNA interactome in living cells, we have developed a streamlined in vivo approach iRIL-seq (intracellular RIL-seq). This generic approach is highly robust, illustrating the dynamic sRNA interactomes in Salmonella enterica across multiple stages of growth. We have identified the OmpD porin mRNA as a central regulatory hub that is targeted by a dozen sRNAs, including FadZ cleaved from the conserved 3'UTR of fadBA mRNA. Both ompD and FadZ are activated by CRP, constituting a type I incoherent feed-forward loop in the fatty acid metabolism pathway. Altogether, we have established an approach to profile RNA-RNA interactomes in live cells, highlighting the complexity of RNA regulatory hubs and RNA networks.

Models of Hfq interactions with small non-coding RNA in Gram-negative and Gram-positive bacteria.

Hfq is required by many Gram-negative bacteria to chaperone the interaction between small non-coding RNA (sRNA) and mRNA to facilitate annealing. Conversely and despite the presence of Hfq in many Gram-positive bacteria, sRNAs in Gram-positive bacteria bind the mRNA target independent of Hfq. Details provided by the Hfq structures from both Gram-negative and Gram-positive bacteria have demonstrated that despite a conserved global structure of the protein, variations of residues on the binding surfaces of Hfq results in the recognition of different RNA sequences as well as the ability of Hfq to facilitate the annealing of the sRNA to the mRNA target. Additionally, a subset of Gram-negative bacteria has an extended C-terminal Domain (CTD) that has been shown to affect the stability of the Hfq hexamer and increase the rate of release of the annealed sRNA-mRNA product. Here we review the structures of Hfq and biochemical data that have defined the interactions of the Gram-negative and Gram-positive homologues to highlight the similarities and differences in the interactions with RNA. These interactions provided a deeper understanding of the how Hfq functions to facilitate the annealing of sRNA-mRNA, the selectivity of the interactions with RNA, and the role of the CTD of Hfq in the interactions with sRNA.

An acetyltranferase moonlights as a regulator of the RNA binding repertoire of the RNA chaperone Hfq in Escherichia coli.

Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, a mediator for sRNA-mRNA annealing. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was previously identified as a silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and investigated its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppressed all tested Hfq-dependent sRNA signaling. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked the interaction interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, and HqbA interacted with Hfq at a site far from the active site. This suggests that HqbA may be bifunctional, with separate roles for regulating via Hfq interaction and for acetylation of undefined substrates. Gel shift assays revealed that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs but not high-affinity RNAs. Comparative RNA immunoprecipitation of Hfq and sequencing showed enrichment of two tRNA precursors, metZWV and proM, by Hfq in mutants that lost the HqbA-Hfq interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.

σ28-dependent small RNA regulation of flagella biosynthesis.

Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here, we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3´ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.

Hfq C-terminal region forms a ß-rich amyloid-like motif without perturbing the N-terminal Sm-like structure.

Hfq is a pleitropic actor that serves as stress response and virulence factor in the bacterial cell. To execute its multiple functions, Hfq assembles into symmetric torus-shaped hexamers. Extending outward from the hexameric core, Hfq presents a C-terminal region, described as intrinsically disordered in solution. Many aspects of the role and the structure of this region remain unclear. For instance, in its truncated form it can promote amyloid-like filament assembly. Here, we show that a minimal 11-residue motif at the C-terminal end of Hfq assembles into filaments with amyloid characteristics. Our data suggest that the full-length Hfq in its filamentous state contains a similar molecular fingerprint than that of the short ß-strand peptide, and that the Sm-core structure is not affected by filament formation. Hfq proteins might thus co-exist in two forms in vivo, either as isolated, soluble hexamers or as self-assembled hexamers through amyloid-reminiscent interactions, modulating Hfq cellular functions.

Interactions and Insertion of Escherichia coli Hfq into Outer Membrane Vesicles as Revealed by Infrared and Orientated Circular Dichroism Spectroscopies.

The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs. Moreover, we provide evidence that Hfq interacts with and is inserted into OMV membranes, suggesting a role for this protein in the release of sRNA outside the vesicle. These findings provide clues to the mechanism of host-bacteria interactions which may not be defined solely by protein-protein and protein-outer membrane contacts, but also by the exchange of RNAs, and in particular sRNAs.

Hfq-licensed RNA-RNA interactome in Pseudomonas aeruginosa reveals a keystone sRNA.

The RNA chaperone Hfq plays important regulatory roles in many bacteria by facilitating the base pairing between small RNAs (sRNAs) and their cognate mRNA targets. In the gram-negative opportunistic pathogen Pseudomonas aeruginosa, over a hundred putative sRNAs have been identified but for most, their regulatory targets remained unknown. Using RIL-seq with Hfq in P. aeruginosa, we identified the mRNA targets for dozens of previously known and unknown sRNAs. Strikingly, hundreds of the RNA-RNA interactions we discovered involved PhrS. This sRNA was thought to mediate its effects by pairing with a single target mRNA and regulating the abundance of the transcription regulator MvfR required for the synthesis of the quorum sensing signal PQS. We present evidence that PhrS controls many transcripts by pairing with them directly and employs a two-tiered mechanism for governing PQS synthesis that involves control of an additional transcription regulator called AntR. Our findings in P. aeruginosa expand the repertoire of targets for previously known sRNAs, reveal potential regulatory targets for previously unknown sRNAs, and suggest that PhrS may be a keystone sRNA with the ability to pair with an unusually large number of transcripts in this organism.

Crossing bacterial boundaries: The carbon catabolite repression system Crc-Hfq of Pseudomonas putida KT2440 as a tool to control translation in E. coli.

As a global regulatory mechanism, carbon catabolite repression allows bacteria and eukaryal microbes to preferentially utilize certain substrates from a mixture of carbon sources. The mechanism varies among different species. In Pseudomonas spp., it is mainly mediated by the Crc-Hfq complex which binds to the 5' region of the target mRNAs, thereby inhibiting their translation. This molecular mechanism enables P. putida to rapidly adjust and fine-tune gene expression in changing environments. Hfq is an RNA-binding protein that is ubiquitous and highly conserved in bacterial species. Considering the characteristics of Hfq, and the widespread use and rapid response of Crc-Hfq in P. putida, this complex has the potential to become a general toolbox for post-transcriptional multiplex regulation. In this study, we demonstrate for the first time that transplanting the pseudomonal catabolite repression protein, Crc, into E. coli causes multiplex gene repression. Under the control of Crc, the production of a diester and its precursors was significantly reduced. The effects of Crc introduction on cell growth in both minimal and rich media were evaluated. Two potential factors - off-target effects and Hfq-sequestration - could explain negative effects on cell growth. Simultaneous reduction of off-targeting and increased sequestration of Hfq by the introduction of the small RNA CrcZ, indicated that Hfq sequestration plays a more prominent role in the negative side-effects. This suggests that the negative growth effect can be mitigated by well-controlled expression of Hfq. This study reveals the feasibility of controlling gene expression using heterologous regulation systems.

Small RNAs and Hfq capture unfolded RNA target sites during transcription.

Small ribonucleoproteins (sRNPs) target nascent precursor RNAs to guide folding, modification, and splicing during transcription. Yet, rapid co-transcriptional folding of the RNA can mask sRNP sites, impeding target recognition and regulation. To examine how sRNPs target nascent RNAs, we monitored binding of bacterial Hfq⋅DsrA sRNPs to rpoS transcripts using single-molecule co-localization co-transcriptional assembly (smCoCoA). We show that Hfq⋅DsrA recursively samples the mRNA before transcription of the target site to poise it for base pairing with DsrA. We adapted smCoCoA to precisely measure when the target site is synthesized and revealed that Hfq⋅DsrA often binds the mRNA during target site synthesis close to RNA polymerase (RNAP). We suggest that targeting transcripts near RNAP allows an sRNP to capture a site before the transcript folds, providing a kinetic advantage over post-transcriptional targeting. We propose that other sRNPs may also use RNAP-proximal targeting to hasten recognition and regulation.

Hfq protein and GcvB small RNA tailoring of oppA target mRNA to levels allowing translation activation by MicF small RNA in Escherichia coli.

Traffic of molecules across the bacterial membrane mainly relies on porins and transporters, whose expression must adapt to environmental conditions. To ensure bacterial fitness, synthesis and assembly of functional porins and transporters are regulated through a plethora of mechanisms. Among them, small regulatory RNAs (sRNAs) are known to be powerful post-transcriptional regulators. In Escherichia coli, the MicF sRNA is known to regulate only four targets, a very narrow targetome for a sRNA responding to various stresses, such as membrane stress, osmotic shock, or thermal shock. Using an in vivo pull-down assay combined with high-throughput RNA sequencing, we sought to identify new targets of MicF to better understand its role in the maintenance of cellular homoeostasis. Here, we report the first positively regulated target of MicF, the oppA mRNA. The OppA protein is the periplasmic component of the Opp ATP-binding cassette (ABC) oligopeptide transporter and regulates the import of short peptides, some of them bactericides. Mechanistic studies suggest that oppA translation is activated by MicF through a mechanism of action involving facilitated access to a translation-enhancing region in oppA 5'UTR. Intriguingly, MicF activation of oppA translation depends on cross-regulation by negative trans-acting effectors, the GcvB sRNA and the RNA chaperone protein Hfq.

Involvement of RNA chaperone hfq in the regulation of antibiotic resistance and virulence in Shigella sonnei.

The host factor for RNA phage Qß replicase (Hfq) is a crucial post-transcriptional regulator in many bacterial pathogens, facilitating the interaction between small non-coding RNAs (sRNAs) and their target mRNAs. Studies have suggested that Hfq plays a role in antibiotic resistance and virulence in bacteria, although its functions in Shigella are not fully understood. In this study, we investigated the functional roles of Hfq in Shigella sonnei (S. sonnei) by constructing an hfq deletion mutant. Our phenotypic assays showed that the hfq deletion mutant was more sensitivity to antibiotics and had impaired virulence. Transcriptome analyses supported the results concerning the phenotype of the hfq mutant and showed that differentially expressed genes were mainly enriched in the KEGG pathways two-component system, ABC transporters, ribosome, and Escherichia coli biofilm formation. Additionally, we predicted eleven novel Hfq-dependent sRNAs, which were potentially involved in the regulation of antibiotic resistance and/or virulence in S. sonnei. Our findings suggest that Hfq plays a post-transcriptional role in regulating antibiotic resistance and virulence in S. sonnei, and could provide a basis for future studies on Hfq-sRNA-mRNA regulatory networks in this important pathogen.

Role of the 5' end phosphorylation state for small RNA stability and target RNA regulation in bacteria.

In enteric bacteria, several small RNAs (sRNAs) including MicC employ endoribonuclease RNase E to stimulate target RNA decay. A current model proposes that interaction of the sRNA 5' monophosphate (5'P) with the N-terminal sensing pocket of RNase E allosterically activates cleavage of the base-paired target in the active site. In vivo evidence supporting this model is lacking. Here, we engineered a genetic tool allowing us to generate 5' monophosphorylated sRNAs of choice in a controllable manner in the cell. Four sRNAs were tested and none performed better in target destabilization when 5' monophosphorylated. MicC retains full activity even when RNase E is defective in 5'P sensing, whereas regulation is lost upon removal of its scaffolding domain. Interestingly, sRNAs MicC and RyhB that originate with a 5' triphosphate group are dramatically destabilized when 5' monophosphorylated, but stable when in 5' triphosphorylated form. In contrast, the processing-derived sRNAs CpxQ and SroC, which carry 5'P groups naturally, are highly stable. Thus, the 5' phosphorylation state determines stability of naturally triphosphorylated sRNAs, but plays no major role for target RNA destabilization in vivo. In contrast, the RNase E C-terminal half is crucial for MicC-mediated ompD decay, suggesting that interaction with Hfq is mandatory.