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Bacteria employ small regulatory RNAs (sRNA) and/or RNA binding proteins (RBPs) to respond to environmental cues. In Enterobacteriaceae , the FinO-domain containing RBP ProQ associates with numerous sRNAs and mRNAs, impacts sRNA-mediated riboregulation or mRNA stability by binding to 5'- or 3'-untranslated regions as well as to internal stem loop structures. Global RNA-protein interaction studies and sequence comparisons identified a ProQ-like homolog (PA2582/ProQ Pae ) in Pseudomonas aeruginosa ( Pae ). To address the function of ProQ Pae , at first a comparative transcriptome analysis of the Pae strains PAO1 and PAO1Δ proQ was performed. This study revealed more than 100 differentially abundant transcripts, affecting a variety of cellular functions. Among these transcripts were pprA and pprB , encoding the PprA/PprB two component system, psrA , encoding a transcriptional activator of pprB , and oprI , encoding the outer membrane protein OprI. RNA co-purification experiments with Strep-tagged Pae ProQ protein corroborated an association of ProQ Pae with these transcripts. In accordance with the up-regulation of the psrA , pprA , and pprB genes in strain PAO1Δ proQ a phenotypic analysis revealed an increased susceptibility toward the aminoglycosides tobramycin and gentamicin in biofilms. Conversely, the observed down-regulation of the oprI gene in PAO1Δ proQ could be reconciled with a decreased susceptibility toward the synthetic cationic antimicrobial peptide GW-Q6. Taken together, these studies revealed that ProQ Pae is an RBP that impacts antimicrobial resistance in Pae ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cianciulli Sesso, Resch, Moll, Bläsi and Sonnleitner.)

In the opportunistic human pathogen Pseudomonas aeruginosa ( Pae ), c arbon c atabolite r epression (CCR) orchestrates the hierarchical utilization of N and C sources, and impacts virulence, antibiotic resistance and biofilm development. During CCR, the RNA chaperone Hfq and the c atabolite r epression c ontrol protein Crc form assemblies on target mRNAs that impede translation of proteins involved in uptake and catabolism of less preferred C sources. After exhaustion of the preferred C-source, translational repression of target genes is relieved by the regulatory RNA CrcZ, which binds to and acts as a decoy for Hfq. Here, we asked whether Crc action can be modulated to relieve CCR after exhaustion of a preferred carbon source. As Crc does not bind to RNA per se , we endeavored to identify an interacting protein. In vivo co-purification studies, co-immunoprecipitation and biophysical assays revealed that Crc binds to Pae strain O1 protein PA1677. Our structural studies support bioinformatics analyzes showing that PA1677 belongs to the isochorismatase-like superfamily. Ectopic expression of PA 1677 resulted in de-repression of Hfq/Crc controlled target genes, while in the absence of the protein, an extended lag phase is observed during diauxic growth on a preferred and a non-preferred carbon source. This observations indicate that PA1677 acts as an antagonist of Crc that favors synthesis of proteins required to metabolize non-preferred carbon sources. We present a working model wherein PA1677 diminishes the formation of productive Hfq/Crc repressive complexes on target mRNAs by titrating Crc. Accordingly, we propose the name CrcA ( c atabolite r epression c ontrol protein a ntagonist) for PA1677., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sonnleitner, Bassani, Cianciulli-Sesso, Brear, Lilic, Davidovski, Resch, Luisi, Moll and Bläsi.)

The widely occurring bacterial RNA chaperone Hfq is a key factor in the post-transcriptional control of hundreds of genes in Pseudomonas aeruginosa. How this broadly acting protein can contribute to the regulatory requirements of many different genes remains puzzling. Here, we describe cryo-EM structures of higher order assemblies formed by Hfq and its partner protein Crc on control regions of different P. aeruginosa target mRNAs. Our results show that these assemblies have mRNA-specific quaternary architectures resulting from the combination of multivalent protein-protein interfaces and recognition of patterns in the RNA sequence. The structural polymorphism of these ribonucleoprotein assemblies enables selective translational repression of many different target mRNAs. This system elucidates how highly complex regulatory pathways can evolve with a minimal economy of proteinogenic components in combination with RNA sequence and fold., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

In Pseudomonas aeruginosa , the RNA chaperone Hfq and the catabolite repression protein Crc act in concert to regulate numerous genes during carbon catabolite repression (CCR). After alleviation of CCR, the RNA CrcZ sequesters Hfq/Crc, which leads to a rewiring of gene expression to ensure the consumption of less preferred carbon and nitrogen sources. Here, we performed a multiomics approach by assessing the transcriptome, translatome, and proteome in parallel in P. aeruginosa strain O1 during and after relief of CCR. As Hfq function is impeded by the RNA CrcZ upon relief of CCR, and Hfq is known to impact antibiotic susceptibility in P. aeruginosa , emphasis was laid on links between CCR and antibiotic susceptibility. To this end, we show that the mexGHI-opmD operon encoding an efflux pump for the antibiotic norfloxacin and the virulence factor 5-Methyl-phenazine is upregulated after alleviation of CCR, resulting in a decreased susceptibility to the antibiotic norfloxacin. A model for indirect regulation of the mexGHI-opmD operon by Hfq is presented., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rozner, Nukarinen, Wolfinger, Amman, Weckwerth, Bläsi and Sonnleitner.)

Pseudomonas aeruginosa PAO1 membrane vesicles (MVs) are known to play a role in cell-to-cell communication. Several studies have shown that the MV composition and physicochemical properties vary according to the bacterial growth stage, but the impact this might have on the externalization of RNA via MVs has not been addressed. Therefore, a study to characterize the RNA content from MVs retrieved at different growth phases was conducted. First, the transcriptome analyses revealed a higher abundance of around 300 RNA species in MVs when compared with the cells. The vesiculation rate along the growth curve was determined, showing that the release of MVs increased during the transition to the stationary phase, whereas it decreased in the late stationary phase. RNA-seq of MVs retrieved along the transition to the stationary phase demonstrated that the RNA cargo of vesicles did not vary. However, the amount of smaller RNAs (<200 nt) inside MVs retrieved in the late exponential phase was higher than in the stationary phase MVs. These results indicate that the externalization of RNA via MVs occurs during late exponential phase and implies the secretion of different types of MVs during growth., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Pseudomonas aeruginosa ( Pae ) is an opportunistic pathogen showing a high intrinsic resistance to a wide variety of antibiotics. It causes nosocomial infections that are particularly detrimental to immunocompromised individuals and to patients suffering from cystic fibrosis. We provide a snapshot on regulatory RNAs of Pae that impact on metabolism, pathogenicity and antibiotic susceptibility. Different experimental approaches such as in silico predictions, co-purification with the RNA chaperone Hfq as well as high-throughput RNA sequencing identified several hundreds of regulatory RNA candidates in Pae . Notwithstanding, using in vitro and in vivo assays, the function of only a few has been revealed. Here, we focus on well-characterized small base-pairing RNAs, regulating specific target genes as well as on larger protein-binding RNAs that sequester and thereby modulate the activity of translational repressors. As the latter impact large gene networks governing metabolism, acute or chronic infections, these protein-binding RNAs in conjunction with their cognate proteins are regarded as global post-transcriptional regulators.

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Here, Crc was shown to enhance Hfq-mediated translational repression of several mRNAs. We have developed a single-molecule fluorescence assay to quantitatively assess the cooperation of Hfq and Crc to form a repressive complex on a RNA, encompassing the translation initiation region and the proximal coding sequence of the P. aeruginosa amiE gene. The presence of Crc did not change the amiE RNA-Hfq interaction lifetimes, whereas it changed the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of the repressive Hfq/Crc/RNA assemblies. These biophysical studies revealed how Crc protein kinetically stabilizes Hfq/RNA complexes, and how the two proteins together fold a large segment of the mRNA into a more compact translationally repressive structure. In fact, the presence of Crc resulted in stronger translational repression in vitro and in a significantly reduced half-life of the target amiE mRNA in vivo. Although Hfq is well-known to act with small regulatory RNAs, this study shows how Hfq can collaborate with another protein to down-regulate translation of mRNAs that become targets for the degradative machinery., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pseudomonas aeruginosa ( Pae ) is notorious for its high-level resistance toward clinically used antibiotics. In fact, Pae has rendered most antimicrobials ineffective, leaving polymyxins and aminoglycosides as last resort antibiotics. Although several resistance mechanisms of Pae are known toward these drugs, a profounder knowledge of hitherto unidentified factors and pathways appears crucial to develop novel strategies to increase their efficacy. Here, we have performed for the first time transcriptome analyses and ribosome profiling in parallel with strain PA14 grown in synthetic cystic fibrosis medium upon exposure to polymyxin E (colistin) and tobramycin. This approach did not only confirm known mechanisms involved in colistin and tobramycin susceptibility but revealed also as yet unknown functions/pathways. Colistin treatment resulted primarily in an anti-oxidative stress response and in the de-regulation of the MexT and AlgU regulons, whereas exposure to tobramycin led predominantly to a rewiring of the expression of multiple amino acid catabolic genes, lower tricarboxylic acid (TCA) cycle genes, type II and VI secretion system genes and genes involved in bacterial motility and attachment, which could potentially lead to a decrease in drug uptake. Moreover, we report that the adverse effects of tobramycin on translation are countered with enhanced expression of genes involved in stalled ribosome rescue, tRNA methylation and type II toxin-antitoxin (TA) systems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cianciulli Sesso, Lilić, Amman, Wolfinger, Sonnleitner and Bläsi.)

Immune systems respond to "non-self" molecules termed microbe-associated molecular patterns (MAMPs). Microbial genes encoding MAMPs have adaptive functions and are thus evolutionarily conserved. In the presence of a host, these genes are maladaptive and drive antagonistic pleiotropy (AP) because they promote microbe elimination by activating immune responses. The role AP plays in balancing the functionality of MAMP-coding genes against their immunogenicity is unknown. To address this, we focused on an epitope of flagellin that triggers antibacterial immunity in plants. Flagellin is conserved because it enables motility. Here, we decode the immunogenic and motility profiles of this flagellin epitope and determine the spectrum of amino acid mutations that drives AP. We discover two synthetic mutational tracks that undermine the detection activities of a plant flagellin receptor. These tracks generate epitopes with either antagonist or weaker agonist activities. Finally, we find signatures of these tracks layered atop each other in natural Pseudomonads., Competing Interests: Declaration of interests J.L.D. is a co-founder of, and shareholder in, AgBiome LLC, a corporation whose goal is to use plant-associated microbes to improve plant productivity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific response to invading nucleic acids. Therefore, these systems can be harnessed for gene knockdown technologies even in hyperthermophilic archaea to study essential genes. We show here the broader usability of this posttranscriptional silencing technology by expanding the application to further essential genes and systematically analysing and comparing silencing thresholds and escape mutants. Synthetic guide RNAs expressed from miniCRISPR cassettes were used to silence genes involved in cell division ( cdvA ), transcription ( rpo8 ), and RNA metabolism ( smAP2 ) of the two crenarchaeal model organisms Saccharolobus solfataricus and Sulfolobus acidocaldarius . Results were systematically analysed together with those obtained from earlier experiments of cell wall biogenesis ( slaB ) and translation ( aif5A ). Comparison of over 100 individual transformants revealed gene-specific silencing maxima ranging between 40 and 75%, which induced specific knockdown phenotypes leading to growth retardation. Exceedance of this threshold by strong miniCRISPR constructs was not tolerated and led to specific mutation of the silencing miniCRISPR array and phenotypical reversion of cultures. In two thirds of sequenced reverted cultures, the targeting spacers were found to be precisely excised from the miniCRISPR array, indicating a still hypothetical, but highly active recombination system acting on the dynamics of CRISPR spacer arrays. Our results indicate that CRISPR type III - based silencing is a broadly applicable tool to study in vivo functions of essential genes in Sulfolobales which underlies a specific mechanism to avoid malignant silencing overdose.

Carbapenems are often the antibiotics of choice to combat life threatening infections caused by the opportunistic human pathogen Pseudomonas aeruginosa . The outer membrane porins OprD and OpdP serve as entry ports for carbapenems. Here, we report that the RNA chaperone Hfq governs post-transcriptional regulation of the oprD and opdP genes in a distinctive manner. Hfq together with the recently described small regulatory RNAs (sRNAs) ErsA and Sr0161 is shown to mediate translational repression of oprD , whereas opdP appears not to be regulated by sRNAs. At variance, our data indicate that opdP is translationally repressed by a regulatory complex consisting of Hfq and the catabolite repression protein Crc, an assembly known to be key to carbon catabolite repression in P. aeruginosa . The regulatory RNA CrcZ, which is up-regulated during growth of P. aeruginosa on less preferred carbon sources, is known to sequester Hfq, which relieves Hfq-mediated translational repression of genes. The differential carbapenem susceptibility during growth on different carbon sources can thus be understood in light of Hfq-dependent oprD / opdP regulation and of the antagonizing function of the CrcZ RNA on Hfq regulatory complexes., (Copyright © 2020 Sonnleitner, Pusic, Wolfinger and Bläsi.)

During translation initiation, the heterotrimeric archaeal translation initiation factor 2 (aIF2) recruits the initiator tRNA i to the small ribosomal subunit. In the stationary growth phase and/or during nutrient stress, Sulfolobus solfataricus aIF2 has a second function: It protects leaderless mRNAs against degradation by binding to their 5'-ends. The S. solfataricus protein Sso2509 is a translation recovery factor (Trf) that interacts with aIF2 and is responsible for the release of aIF2 from bound mRNAs, thereby enabling translation re-initiation. It is a member of the domain of unknown function 35 (DUF35) protein family and is conserved in Sulfolobales as well as in other archaea. Here, we present the X-ray structure of S. solfataricus Trf solved to a resolution of 1.65 Å. Trf is composed of an N-terminal rubredoxin-like domain containing a bound zinc ion and a C-terminal oligosaccharide/oligonucleotide binding fold domain. The Trf structure reveals putative mRNA binding sites in both domains. Surprisingly, the Trf protein is structurally but not sequentially very similar to proteins linked to acyl-CoA utilization-for example, the Sso2064 protein from S. solfataricus-as well as to scaffold proteins found in the acetoacetyl-CoA thiolase/high-mobility group-CoA synthase complex of the archaeon Methanothermococcus thermolithotrophicus and in a steroid side-chain-cleaving aldolase complex from the bacterium Thermomonospora curvata. This suggests that members of the DUF35 protein family are able to act as scaffolding and binding proteins in a wide variety of biological processes., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Translation factor a/eIF5A is highly conserved in Eukarya and Archaea. The eukaryal eIF5A protein is required for transit of ribosomes across consecutive proline codons, whereas the function of the archaeal orthologue remains unknown. Here, we provide a first hint for an involvement of Sulfolobus solfataricus (Sso) aIF5A in translation. CRISPR-mediated knock down of the aif5A gene resulted in strong growth retardation, underlining a pivotal function. Moreover, in vitro studies revealed that Sso aIF5A is endowed with endoribonucleolytic activity. Thus, aIF5A appears to be a moonlighting protein that might be involved in protein synthesis as well as in RNA metabolism.

In diverse bacterial species, the global regulator Hfq contributes to post-transcriptional networks that control expression of numerous genes. Hfq of the opportunistic pathogen Pseudomonas aeruginosa inhibits translation of target transcripts by forming a regulatory complex with the catabolite repression protein Crc. This repressive complex acts as part of an intricate mechanism of preferred nutrient utilisation. We describe high-resolution cryo-EM structures of the assembly of Hfq and Crc bound to the translation initiation site of a target mRNA. The core of the assembly is formed through interactions of two cognate RNAs, two Hfq hexamers and a Crc pair. Additional Crc protomers are recruited to the core to generate higher-order assemblies with demonstrated regulatory activity in vivo. This study reveals how Hfq cooperates with a partner protein to regulate translation, and provides a structural basis for an RNA code that guides global regulators to interact cooperatively and regulate different RNA targets., Competing Interests: XP, TD, ES, SC, UB, BL No competing interests declared, (© 2019, Pei et al.)

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for ~ 10% of hospital-acquired infections worldwide. It is notorious for its high level resistance toward many antibiotics, and the number of multi-drug resistant clinical isolates is steadily increasing. A better understanding of the molecular mechanisms underlying drug resistance is crucial for the development of novel antimicrobials and alternative strategies such as enhanced sensitization of bacteria to antibiotics in use. In P. aeruginosa several uptake channels for amino-acids and carbon sources can serve simultaneously as entry ports for antibiotics. The respective genes are often controlled by carbon catabolite repression (CCR). We have recently shown that Hfq in concert with Crc acts as a translational repressor during CCR. This function is counteracted by the regulatory RNA CrcZ, which functions as a decoy to abrogate Hfq-mediated translational repression of catabolic genes. Here, we report an increased susceptibility of P. aeruginosa hfq deletion strains to different classes of antibiotics. Transcriptome analyses indicated that Hfq impacts on different mechanisms known to be involved in antibiotic susceptibility, viz import and efflux, energy metabolism, cell wall and LPS composition as well as on the c-di-GMP levels. Furthermore, we show that sequestration of Hfq by CrcZ, which was over-produced or induced by non-preferred carbon-sources, enhances the sensitivity toward antibiotics. Thus, controlled synthesis of CrcZ could provide a means to (re)sensitize P. aeruginosa to different classes of antibiotics.

Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen, able to resist host defense mechanisms and antibiotic treatment. In Pae, the master regulator of stress responses RpoS (σ S ) is involved in the regulation of quorum sensing and several virulence genes. Here, we report that the sRNA ReaL translationally silences rpoS mRNA, which results in a decrease of the RpoS levels. Our studies indicated that ReaL base-pairs with the Shine-Dalgarno region of rpoS mRNA. These studies are underlined by a highly similar transcription profile of a rpoS deletion mutant and a reaL over-expressing strain.

Eukaryotic eIF5A and its bacterial orthologue EF-P are translation elongation factors whose task is to rescue ribosomes from stalling during the synthesis of proteins bearing particular sequences such as polyproline stretches. Both proteins are characterized by unique post-translational modifications, hypusination and lysinylation, respectively, which are essential for their function. An orthologue is present in all Archaea but its function is poorly understood. Here, we show that aIF5A of the crenarchaeum Sulfolobus solfataricus is hypusinated and forms a stable complex with deoxyhypusine synthase, the first enzyme of the hypusination pathway. The recombinant enzyme is able to modify its substrate in vitro resulting in deoxyhypusinated aIF5A. Moreover, with the aim to identify the enzyme involved in the second modification step, i.e. hypusination, a set of proteins interacting with aIF5A was identified.

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources.

Pseudomonas aeruginosa is an opportunistic pathogen that can thrive by anaerobic respiration in the lungs of cystic fibrosis patients using nitrate as terminal electron acceptor. Here, we report the identification and characterization of the small RNA PaiI in the P. aeruginosa strain 14 (PA14). PaiI is a naerobically i nduced in the presence of nitrate and depends on the two-component system NarXL. Our studies revealed that PaiI is required for efficient denitrification affecting the conversion of nitrite to nitric oxide. In the absence of PaiI anaerobic growth was impaired on glucose, which can be reconciled with a decreased uptake of the carbon source under these conditions. The importance of PaiI for anaerobic growth is further underlined by the observation that a paiI deletion mutant was impaired in growth in murine tumors.

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism in Pro- and Eukaryotes. In this study, a collection of 53 mRNAs that co-purified with Sulfolobus solfataricus (Sso) SmAP2 were surveyed for a specific RNA binding motif (RBM). SmAP2 was shown to bind with high affinity to the deduced consensus RNA binding motif (SmAP2-cRBM) in vitro. Residues in SmAP2 interacting with the SmAP2-cRBM were mapped by UV-induced crosslinking in combination with mass-spectrometry, and verified by mutational analyses. The RNA-binding site on SmAP2 includes a modified uracil binding pocket containing a unique threonine (T40) located on the L3 face and a second residue, K25, located in the pore. To study the function of the SmAP2-RBM in vivo, three authentic RBMs were inserted in the 3'UTR of a lacS reporter gene. The presence of the SmAP2-RBM in the reporter-constructs resulted in decreased LacS activity and reduced steady state levels of lacS mRNA. Moreover, the presence of the SmAP2-cRBM in and the replacement of the lacS 3'UTR with that of Sso2194 encompassing a SmAP2-RBM apparently impacted on the stability of the chimeric transcripts. These results are discussed in light of the function(s) of eukaryotic Lsm proteins in RNA turnover., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The conserved Sm and Sm-like proteins are involved in different aspects of RNA metabolism. Here, we explored the interactome of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso) to shed light on their physiological function(s). Both, SmAP1 and SmAP2 co-purified with several proteins involved in RNA-processing/modification, translation and protein turnover as well as with components of the exosome involved in 3΄ to 5΄ degradation of RNA. In follow-up studies a direct interaction with the poly(A) binding and accessory exosomal subunit DnaG was demonstrated. Moreover, elevated levels of both SmAPs resulted in increased abundance of the soluble exosome fraction, suggesting that they affect the subcellular localization of the exosome in the cell. The increased solubility of the exosome was accompanied by augmented levels of RNAs with A-rich tails that were further characterized using RNASeq. Hence, the observation that the Sso SmAPs impact on the activity of the exosome revealed a hitherto unrecognized function of SmAPs in archaea., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The RNA chaperone Hfq regulates virulence and metabolism in the opportunistic pathogen Pseudomonas aeruginosa. During carbon catabolite repression (CCR) Hfq together with the catabolite repression control protein Crc can act as a translational repressor of catabolic genes. Upon relief of CCR, the level of the Hfq-titrating RNA CrcZ is increasing, which in turn abrogates Hfq-mediated translational repression. As the interdependence of Hfq-mediated and RNA based control mechanisms is poorly understood, we explored the possibility whether the regulatory RNA CrcZ can interfere with riboregulation. We first substantiate that the P. aeruginosa Hfq is proficient and required for riboregulation of the transcriptional activator gene antR by the small RNA PrrF1-2. Our studies further revealed that CrcZ can interfere with PrrF1-2/Hfq-mediated regulation of antR. The competition for Hfq can be rationalized by the higher affinity of Hfq for CrcZ than for antR mRNA.

Pseudomonas aeruginosa (PA) can thrive in anaerobic biofilms in the lungs of cystic fibrosis (CF) patients. Here, we show that CrcZ is the most abundant PA14 RNA bound to the global regulator Hfq in anoxic biofilms grown in cystic fibrosis sputum medium. Hfq was crucial for anoxic biofilm formation. This observation complied with an RNAseq based transcriptome analysis and follow up studies that implicated Hfq in regulation of a central step preceding denitrification. CrcZ is known to act as a decoy that sequesters Hfq during relief of carbon catabolite repression, which in turn alleviates Hfq-mediated translational repression of catabolic genes. We therefore inferred that CrcZ indirectly impacts on biofilm formation by competing for Hfq. This hypothesis was supported by the findings that over-production of CrcZ mirrored the biofilm phenotype of the hfq deletion mutant, and that deletion of the crcZ gene augmented biofilm formation. To our knowledge, this is the first example where competition for Hfq by CrcZ cross-regulates an Hfq-dependent physiological process unrelated to carbon metabolism.

The opportunistic human pathogen Pseudomonas aeruginosa can thrive under microaerophilic to anaerobic conditions in the lungs of cystic fibrosis patients. RNASeq based comparative RNA profiling of the clinical isolate PA14 cultured in synthetic cystic fibrosis medium was performed after planktonic growth (OD600 = 2.0; P), 30 min after shift to anaerobiosis (A-30) and after anaerobic biofilm growth for 96h (B-96) with the aim to reveal differentially regulated functions impacting on sustained anoxic biofilm formation as well as on tolerance towards different antibiotics. Most notably, functions involved in sulfur metabolism were found to be up-regulated in B-96 cells when compared to A-30 cells. Based on the transcriptome studies a set of transposon mutants were screened, which revealed novel functions involved in anoxic biofilm growth.In addition, these studies revealed a decreased and an increased abundance of the oprD and the mexCD-oprJ operon transcripts, respectively, in B-96 cells, which may explain their increased tolerance towards meropenem and to antibiotics that are expelled by the MexCD-OprD efflux pump. The OprI protein has been implicated as a target for cationic antimicrobial peptides, such as SMAP-29. The transcriptome and subsequent Northern-blot analyses showed that the abundance of the oprI transcript encoding the OprI protein is strongly decreased in B-96 cells. However, follow up studies revealed that the susceptibility of a constructed PA14ΔoprI mutant towards SMAP-29 was indistinguishable from the parental wild-type strain, which questions OprI as a target for this antimicrobial peptide in strain PA14.

The heterotrimeric archaeal IF2 orthologue of eukaryotic translation initiation factor 2 consists of the α-subunit, β-subunit and γ-subunit. Previous studies showed that the γ-subunit of aIF2, besides its central role in Met-tRNAi binding, has an additional function: it binds to the 5'-triphosphorylated end of mRNA and protects its 5'-part from degradation. Competition studies with nucleotides and mRNA, as well as structural and kinetic analyses of aIF2γ mutants, strongly implicate the canonical GTP/GDP-binding pocket in binding to the 5'-triphosphate end of mRNAs. The biological implication of these findings is being discussed., (Copyright © 2015. Published by Elsevier Ltd.)

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism. Sm-based regulation is diverse and can range in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in these processes being mediated by an RNA-associated molecular assembly built on Sm proteins. Despite the availability of several 3D-structures of Sm-like archaeal proteins (SmAPs), their function has remained elusive. The aim of this study was to shed light on the function of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso). Using co-purification followed by RNASeq different classes of non-coding RNAs and mRNAs were identified that co-purified either with both paralogues or solely with Sso-SmAP1 or Sso-SmAP2. The large number of associated intron-containing tRNAs and tRNA/rRNA modifying RNAs may suggest a role of the two Sso-SmAPs in tRNA/rRNA processing. Moreover, the 3D structure of Sso-SmAP2 was elucidated. Like Sso-SmAP1, Sso-SmAP2 forms homoheptamers. The binding of both proteins to distinct RNA substrates is discussed in terms of surface conservation, structural differences in the RNA binding sites and differences in the electrostatic surface potential of the two Sso-SmAP proteins. Taken together, this study may hint to common and different functions of both Sso-SmAPs in Sso RNA metabolism.

Unlabelled: Pseudomonas aeruginosa bacteriophage ϕKZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ∼ 280-kb ϕKZ genome to single-nucleotide resolution, we combine 369 ϕKZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the ϕKZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to ϕKZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the ϕKZ genome is performed by the consecutive action of two ϕKZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus., Importance: The data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage ϕKZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial β and β'-like RNAP subunits., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Carbon Catabolite repression (CCR) allows a fast adaptation of Bacteria to changing nutrient supplies. The Pseudomonas aeruginosa (PAO1) catabolite repression control protein (Crc) was deemed to act as a translational regulator, repressing functions involved in uptake and utilization of carbon sources. However, Crc of PAO1 was recently shown to be devoid of RNA binding activity. In this study the RNA chaperone Hfq was identified as the principle post-transcriptional regulator of CCR in PAO1. Hfq is shown to bind to A-rich sequences within the ribosome binding site of the model mRNA amiE, and to repress translation in vitro and in vivo. We further report that Crc plays an unknown ancillary role, as full-fledged repression of amiE and other CCR-regulated mRNAs in vivo required its presence. Moreover, we show that the regulatory RNA CrcZ, transcription of which is augmented when CCR is alleviated, binds to Hfq with high affinity. This study on CCR in PAO1 revealed a novel concept for Hfq function, wherein the regulatory RNA CrcZ acts as a decoy to abrogate Hfq-mediated translational repression of catabolic genes and thus highlights the central role of RNA based regulation in CCR of PAO1.

The RNA chaperone Hfq acts as a central player in post-transcriptional gene regulation in several Gram-negative Bacteria, whereas comparatively little is known about its role in Gram-positive Bacteria. Here, we studied the function of Hfq in Bacillus subtilis, and show that it confers a survival advantage. A comparative transcriptome analysis revealed mRNAs with a differential abundance that are governed by the ResD-ResE system required for aerobic and anaerobic respiration. Expression of resD was found to be up-regulated in the hfq- strain. Furthermore, several genes of the GerE and ComK regulons were de-regulated in the hfq- background. Surprisingly, only six out of >100 known and predicted small RNAs (sRNAs) showed altered abundance in the absence of Hfq. Moreover, Hfq positively affected the transcript abundance of genes encoding type I toxin-antitoxin systems. Taken the moderate effect on sRNA levels and mRNAs together, it seems rather unlikely that Hfq plays a central role in RNA transactions in Bacillus subtilis.

The translation initiation factor aIF2 of the crenarchaeon Sulfolobus solfataricus (Sso) recruits initiator tRNA to the ribosome and stabilizes mRNAs by binding via the γ-subunit to their 5'-triphosphate end. It has been hypothesized that the latter occurs predominantly during unfavorable growth conditions, and that aIF2 or aIF2-γ is released on relief of nutrient stress to enable in particular anew translation of leaderless mRNAs. As leaderless mRNAs are prevalent in Sso and aIF2-γ bound to the 5'-end of a leaderless RNA inhibited ribosome binding in vitro, we aimed at elucidating the mechanism underlying aIF2/aIF2-γ recycling from mRNAs. We have identified a protein termed Trf (translation recovery factor) that co-purified with trimeric aIF2 during outgrowth of cells from prolonged stationary phase. Subsequent in vitro studies revealed that Trf triggers the release of trimeric aIF2 from RNA, and that Trf directly interacts with the aIF2-γ subunit. The importance of Trf is further underscored by an impaired protein synthesis during outgrowth from stationary phase in a Sso trf deletion mutant.

At low temperatures the Escherichia coli rpoS mRNA, encoding the stationary phase sigma factor RpoS, forms an intramolecular secondary structure (iss) that impedes translation initiation. Under these conditions the small RNA DsrA, which is stabilzed by Hfq, forms a duplex with rpoS mRNA sequences opposite of the ribosome-binding site (rbs). Both the DEAD box helicase CsdA and Hfq have been implicated in DsrA·rpoS duplex formation. Hfq binding to A-rich sequences in the rpoS leader has been suggested to restructure the mRNA, and thereby to accelerate DsrA·rpoS duplex formation, which, in turn, was deemed to free the rpoS rbs and to permit ribosome loading on the mRNA. Several experiments designed to elucidate the role of Hfq in DsrA-mediated translational activation of rpoS mRNA have been conducted in vitro. Here, we assessed RpoS synthesis in vivo to further study the role of Hfq in rpoS regulation. We show that RpoS synthesis was reduced when DsrA was ectopically overexpressed at 24 °C in the absence of Hfq despite of DsrA·rpoS duplex formation. This observation indicated that DsrA·rpoS annealing may not be sufficient for efficient ribosome loading on rpoS mRNA. In addition, a HfqG29A mutant protein was employed, which is deficient in binding to A-rich sequences present in the rpoS leader but proficient in DsrA binding. We show that DsrA·rpoS duplex formation occurs in the presence of the HfqG29A mutant protein at low temperature, whereas synthesis of RpoS was greatly diminished. RNase T1 footprinting studies of DsrA·rpoS duplexes in the absence and presence of Hfq or HfqG29A indicated that Hfq is required to resolve a stem-loop structure in the immediate coding region of rpoS mRNA. These in vivo studies corroborate the importance of the A-rich sequences in the rpoS leader and strongly suggest that Hfq, besides stabilizing DsrA and accelerating DsrA·rpoS duplex formation, is also required to convert the rpoS mRNA into a translationally competent form.

Recent studies identified a 5´ to 3´ exoribonuclease termed Sso-RNase J in the crenarchaeon Sulfolobus solfataricus (Sso), which has been reclassified to the aCPSF2 (archaeal cleavage and polyadenylation specificity factor 2) group of β-CASP proteins. In this study, the Sso-aCPSF2 orthologue of Sulfolobus acidocaldarius (Saci-aCPSF2) was functionally characterized. Like Sso-aCPSF2, Saci-aCPSF2 degrades RNA with 5´ to 3´ directionality in vitro. To address the biological significance of Saci-aCPSF2, a deletion mutant was constructed, and the influence of Saci-aCPSF2 on the transcriptome profile was assessed employing high throughput RNA sequencing. This analysis revealed 560 genes with differential transcript abundance, suggesting a considerable role of this enzyme in RNA metabolism. In addition, bioinformatic analyses revealed several transcripts that are preferentially degraded at the 5´ end. This was exemplarily verified for two transcripts by Northern-blot analyses, showing for the first time that aCPSF2 proteins play a role in 5' to 3' directional mRNA decay in the crenarchaeal clade of Archaea.

Among the five superfamilies of helicases involved in RNA and DNA metabolism, superfamily 2 and superfamily 5 include bacterial RNA-helicases. These enzymes have been shown to be involved in ribosome biogenesis and post-transcriptional gene regulation. Here, we focus on bacterial regulatory mechanisms that are mediated by RNA helicases belonging to superfamily 2, which includes DEAD-box and DEAH-box helicases. Some of these helicases are part of bacterial degradosomes and were shown to unwind RNA duplexes. We will review examples where these enzymes have been implicated in translatability and metabolic stability of bacterial transcripts. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We report the first example of antisense RNA regulation in a hyperthermophilic archaeon. In Sulfolobus solfataricus, the transposon-derived paralogous RNAs, RNA-257(1-4), show extended complementarity to the 3' UTR of the 1183 mRNA, encoding a putative phosphate transporter. Phosphate limitation results in decreased RNA-257(1) and increased 1183 mRNA levels. Correspondingly, the 1183 mRNA is faster degraded in vitro upon duplex formation with RNA-257(1). Insertion of the 1183 3' UTR downstream of the lacS gene results in strongly reduced lacS mRNA levels in transformed cells, indicating that antisense regulation can function in trans.

A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.

The Crc protein has been shown to mediate catabolite repression control in Pseudomonas, leading to a preferential assimilation of carbon sources. It has been suggested that Crc acts as a translational repressor of mRNAs, encoding functions involved in uptake and breakdown of different carbon sources. Moreover, the regulatory RNA CrcZ, the level of which is increased in the presence of less preferred carbon sources, was suggested to bind to and sequester Crc, resulting in a relief of catabolite repression. Here, we determined the crystal structure of Pseudomonas aeruginosa Crc, a member of apurinic/apyrimidinic (AP) endonuclease family, at 1.8 Å. Although Crc displays high sequence similarity with its orthologs, there are amino acid alterations in the area corresponding to the active site in AP proteins. Unlike typical AP endonuclease family proteins, Crc has a reduced overall positive charge and the conserved positively charged amino-acid residues of the DNA-binding surface of AP proteins are partially substituted by negatively charged, polar and hydrophobic residues. Crc protein purified to homogeneity from P. aeruginosa did neither display DNase activity, nor did it bind to previously identified RNA substrates. Rather, the RNA chaperone Hfq was identified as a contaminant in His-tagged Crc preparations purified by one step Ni-affinity chromatography from Escherichia coli, and was shown to account for the RNA binding activity observed with the His-Crc preparations. Taken together, these data challenge a role of Crc as a direct translational repressor in carbon catabolite repression in P. aeruginosa.