The major RNA-binding protein Hfq interacts with mRNAs, either alone or together with regulatory small noncoding RNAs (sRNAs), affecting mRNA translation and degradation in bacteria. However, studies tend to focus on single reference strains and assume that the findings may apply to the entire species, despite the important intra-species genetic diversity known to exist. Here, we use RIP-seq to identify Hfq-interacting RNAs in three strains representing the major phylogenetic lineages of Pseudomonas aeruginosa . We find that most interactions are in fact not conserved among the different strains. We identify growth phase-specific and strain-specific Hfq targets, including previously undescribed sRNAs. Strain-specific interactions are due to different accessory gene sets, RNA abundances, or potential context- or sequence- dependent regulatory mechanisms. The accessory Hfq interactome includes most mRNAs encoding Type III Secretion System (T3SS) components and secreted toxins in two strains, as well as a cluster of CRISPR guide RNAs in one strain. Conserved Hfq targets include the global virulence regulator Vfr and metabolic pathways involved in the transition from fast to slow growth. Furthermore, we use rGRIL-seq to show that RhlS, a quorum sensing sRNA, activates Vfr translation, thus revealing a link between quorum sensing and virulence regulation. Overall, our work highlights the important intra-species diversity in post-transcriptional regulatory networks in Pseudomonas aeruginosa .

With the recognition of the importance of posttranscriptional regulation mediated by bacterial small RNAs (sRNAs), their contribution to global gene expression regulatory networks needs to be addressed in a truly comprehensive manner. While a single sRNA targets multiple RNAs, an mRNA can be regulated by multiple sRNAs that can be either transcribed individually or derived by processing of mRNAs.

Toward the end of August 2000, the 6.3 Mbp whole genome sequence of Pseudomonas aeruginosa strain PAO1 was published. With 5570 open reading frames (ORFs), PAO1 had the largest microbial genome sequenced up to that point in time—including a large proportion of metabolic, transport and antimicrobial resistance genes supporting its ability to colonize diverse environments. A remarkable 9% of its ORFs were predicted to encode proteins with regulatory functions, providing new insight into bacterial network complexity as a function of network size. In this celebratory article, we fast forward 20 years, and examine how access to this resource has transformed our understanding of P. aeruginosa. What follows is more than a simple review or commentary; we have specifically asked some of the leaders in the field to provide personal reflections on how the PAO1 genome sequence, along with the Pseudomonas Community Annotation Project (PseudoCAP) and Pseudomonas Genome Database (pseudomonas.com), have contributed to the many exciting discoveries in this field. In addition to bringing us all up to date with the latest developments, we also ask our contributors to speculate on how the next 20 years of Pseudomonas research might pan out.

ABSTRACT The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [ K eq ] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ , resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers. IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

During environmental adaptation bacteria use small regulatory RNAs (sRNAs) to repress or activate expression of a large fraction of their proteome. We extended the use of the in vivo RNA proximity ligation method toward probing global sRNA interactions with their targets in Pseudomonas aeruginosa and verified the method with a known regulon controlled by the PrrF1 sRNA. We also identified two sRNAs (Sr0161 and ErsA) that interact with the mRNA encoding the major porin OprD responsible for the uptake of carbapenem antibiotics. These two sRNAs base pair with the 5 UTR of oprD leading to increase in resistance of the bacteria to meropenem. Additional proximity ligation experiments and enrichment for Sr0161 targets identified the mRNA for the regulator of type III secretion system. Interaction between the exsA mRNA and Sr0161 leads to a block in the synthesis of a component of the T3SS apparatus and an effector. Another sRNA, Sr006, positively regulates, without Hfq, the expression of PagL, an enzyme responsible for deacylation of lipid A, reducing its pro-inflammatory property and resulting in polymyxin resistance. Therefore, an analysis of global sRNA-mRNA interactions can lead to discoveries of novel pathways controlling gene expression that are likely integrated into larger regulatory networks.

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique, referred to as global small non-coding RNA target identification by ligation and sequencing (GRIL-seq), is based on preferential ligation of sRNAs to the ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimaeras. In addition to the RNA chaperone Hfq, the GRIL-seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa , we demonstrated that direct regulatory targets of this sRNA can readily be identified. Therefore, GRIL-seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but also for uncovering novel roles for sRNAs and their targets in complex regulatory networks.

Prx1, an AhpF-dependent 2-Cys peroxiredoxin (Prx), was previously identified in Vibrio vulnificus, a facultative aerobic pathogen. In the present study, transcription of the V. vulnificus prx1ahpF genes, which are adjacently located on the chromosome, was evaluated by analyzing the promoter and intergenic region of the two genes. Northern blot analyses revealed that transcription of prx1ahpF results in two transcripts, the prx1 and prx1ahpF transcripts. Primer extension analysis and a point mutational analysis of the promoter region showed that the two transcripts are generated from a single promoter. In addition, the 3' end of the prx1 transcript at the prx1ahpF intergenic region was determined by a 3'RACE assay. These results suggested that the prx1ahpF genes are transcribed as an operon, and the prx1 transcript was produced by transcriptional termination in the intergenic region. RNA secondary structure prediction of the prx1ahpF intergenic region singled out a stem-loop structure without poly(U) tract, and a deletion analysis of the intergenic region showed that the atypical stem-loop structure acts as the transcriptional attenuator to result in the prx1 and prx1ahpF transcripts. The combined results demonstrate that the differential expression of prx1 and ahpF is accomplished by the cis-acting transcriptional attenuator located between the two genes and thereby leads to the production of a high level of Prx1 and a low level of AhpF.

A simple, rapid, and sensitive electrophoretic mobility shift assay (EMSA) can be successfully used to analyze RNA-RNA interactions. The EMSA of RNA-RNA complexes can be further used to evaluate the specificity of interactions using competitor RNAs in combination with their mutated versions or nonspecific RNAs, such as yeast tRNA. RNA is simply prepared by in vitro transcription from PCR product templates. Detailed experimental descriptions for EMSA-based analysis of specific RNA-RNA interactions between Sib RNAs and ibs mRNAs as a representative example are presented.

Escherichia coli and related enteric bacteria can survive under extreme acid stress condition at least for several hours. RpoS is a key factor for acid stress management in many enterobacteria. Although three rpoS-activating sRNAs, DsrA, RprA, and ArcZ, have been identified in E. coli, it remains unclear how these small RNA molecules participate in pathways leading to acid resistance (AR). Here, we showed that overexpression of ArcZ, DsrA, or RprA enhances AR in a RpoS-dependent manner. Mutant strains with deletion of any of three sRNA genes showed lowered AR, and deleting all three sRNA genes led to more severe defects in protecting against acid stress. Overexpression of any of the three sRNAs fully rescued the acid tolerance defects of the mutant strain lacking all three genes, suggesting that all three sRNAs perform the same function in activating RpoS required for AR. Notably, acid stress led to the induction of DsrA and RprA but not ArcZ.

An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism's overall fitness and virulence. Currently it is generally thought that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa, treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD(+) strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.

One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen.

IbsC is a small toxin protein in Escherichia coli, whose expression is repressed by a cis-acting small noncoding RNA, SibC (renamed from RygC or QUAD1c). Overexpression of IbsC or the absence of SibC transcription induces the expression of both psp operon (pspABCDE) and pspG gene encoding phage shock proteins, whose expression were known to be induced by multiple environment stresses or agents such as filamentous phage infection, osmotic shock, continued incubation in stationary phase, heat shock, and ethanol treatment. Although exogenous overexpression of IbsC leads to cell death, physiological roles of IbsC remain unknown especially because the strain without ibsC gene in the chromosome shows no growth defects. Considering that only Psp proteins are induced by expression of IbsC, not by other similar small toxin proteins such as LdrD, ShoB, and TisB, the role of IbsC might specifically be related to phage shock proteins. The psp operon is transcribed from pspA promoter by RNA polymerase holoenzyme containing the alternative stress-responsive sigma factor 54 (σ). Transcription from pspA promoter requires PspF as an activator protein that binds to the upstream region of the core promoter elements. Furthermore, PspA inhibits the σ-dependent pspA transcription by interacting with PspF and exerting its negative effects on transcription activation by PspF. Since the IbsC expression causes the induction of pspA transcription, it is attempting to see how the signal of IbsC gets transduced into the transcription activation. The mechanism involved in linking IbsC to pspA transcription is essential for understanding not only the physiological functions of IbsC, but also the regulation mechanism of transcription from σdependent promoters in response to environmental stresses. To understand the molecular mechanism of the pspA induction by IbsC, it is necessary to know which factors are involved in the activation of pspA transcription under conditions of IbsC expression. Although an in vitro transcription system for pspA promoter analysis was previously set up, it is difficult to identify in vitro transcripts because their transcription termination sites are unclear. To overcome the drawback of the previous in vitro transcription system, in this study, we constructed plasmid pPR56 containing a transcription fusion of pspA promoter and rnpB terminator by replacing the rnpB promoter-containing DNA fragment of pLMd23-wt with the pspA promoter-containing fragment. The pspA promoter-containing fragment spanning from −320 through +56 of pspA, which also includes two UAS sequences (UAS I and UAS II) as PspF-binding sites and the IHF binding site, was subcloned into the BamHI/ EcoRI linearlized pLMd23-wt not having the rnpB promotercontaining DNA fragment, to generate the fusion plasmid pPR56 (Fig. 1). The rnpB terminator region in the fusion construct contained the rnpB sequence from +331 to +1286, which includes the three rnpB terminators T1, T2, and T3 leading to transcription termination at +413, +526, and +638, respectively. Therefore, this pPR56 construct was designed to generate pspA-rnpB fusion transcripts of 146 nt terminating at T1 (pspAT1), 259 nt at T2 (pspAT2), and 371 nt at T3 (pspAT3) if transcription starts at the transcription initiation site of pspA. Using supercoiled plasmid pPR56 DNA as a template, in vitro transcription was carried out by adding Eσ and PspF. The pspAT1 transcript of 145 nt was produced as a major band although minor pspAT2 and pspAT3 products were also observed (Fig. 2). The increased abundance of three transcripts was observed with the incremental amount of PspF protein, while RNA I of 108 nt (a transcript transcribed from its own σ specific promoter of the plasmid DNA) was not produced. In contrast, the same transcription reaction performed with Eσ did not produce the pspAT transcripts. Instead, this reaction generated RNA I. All our data confirmed that the pspA transcripts were produced from the σ-specific pspA promoter of pPR56. To

It is well known that 6S RNA, a global regulatory noncoding RNA that modulates gene expression in response to the cellular stresses in Escherichia coli, is generated by processing from primary ssrS (6S RNA) transcripts derived from two different promoters. The 5' processing of 6S RNA from primary transcripts has been well studied; however, it remains unclear how the 3'-end of this RNA is generated although previous studies have suggested that exoribonucleolytic trimming is necessary for 3' processing. Here, we describe several Rho-dependent termination sites located ∼90 bases downstream of the mature 3'-end of 6S RNA. Our data suggest that the 3'-end of 6S RNA is generated via exoribonucleolytic trimming, rather than endoribonucleolytic cleavage, following the transcription termination events. The termination sites identified in this study are within the open reading frame of the downstream ygfA (putative 5-formyl-tetrahydrofolate cyclo-ligase) gene, a part of the highly conserved bacterial operon ssrS-ygfA, which is up-regulated during the biofilm formation. Our findings reveal that ygfA expression, which also aids the formation of multidrug-tolerant persister cells, could be regulated by Rho-dependent termination activity in the cell.

Proteins: Structure, Function, and BioinformaticsVolume 80, Issue 3 p. 963-967 Structure Note Structural analysis of Escherichia coli C5 protein Jae-Sun Shin, Jae-Sun Shin Department of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea Biomedical Proteomics Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, KoreaSearch for more papers by this authorKwang-Sun Kim, Kwang-Sun Kim Systems and Synthetic Biology Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Korea Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, KoreaSearch for more papers by this authorKyoung-Seok Ryu, Kyoung-Seok Ryu Division of Magnetic Resonance Research, Korea Basic Science Institute, Yangcheong-Ri 804-1, Ochang-Eup, Cheongwon-Gun, Chungbuk 363-883, KoreaSearch for more papers by this authorKook Han, Kook Han Department of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, KoreaSearch for more papers by this authorYounghoon Lee, Younghoon Lee Department of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, KoreaSearch for more papers by this authorByong-Seok Choi, Corresponding Author Byong-Seok Choi byongseok.choi@kaist.ac.kr Department of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, KoreaDepartment of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea===Search for more papers by this author Jae-Sun Shin, Jae-Sun Shin Department of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea Biomedical Proteomics Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, KoreaSearch for more papers by this authorKwang-Sun Kim, Kwang-Sun Kim Systems and Synthetic Biology Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Korea Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, KoreaSearch for more papers by this authorKyoung-Seok Ryu, Kyoung-Seok Ryu Division of Magnetic Resonance Research, Korea Basic Science Institute, Yangcheong-Ri 804-1, Ochang-Eup, Cheongwon-Gun, Chungbuk 363-883, KoreaSearch for more papers by this authorKook Han, Kook Han Department of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, KoreaSearch for more papers by this authorYounghoon Lee, Younghoon Lee Department of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, KoreaSearch for more papers by this authorByong-Seok Choi, Corresponding Author Byong-Seok Choi byongseok.choi@kaist.ac.kr Department of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, KoreaDepartment of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea===Search for more papers by this author First published: 16 November 2011 https://doi.org/10.1002/prot.23253Citations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume80, Issue3March 2012Pages 963-967 RelatedInformation

It is well known that 6S RNA, a global regulatory noncoding RNA that modulates gene expression in response to the cellular stresses in Escherichia coli, is generated by processing from primary ssrS (6S RNA) transcripts derived from two different promoters. The 5′ processing of 6S RNA from primary transcripts has been well studied; however, it remains unclear how the 3′-end of this RNA is generated although previous studies have suggested that exoribonucleolytic trimming is necessary for 3′ processing. Here, we describe several Rho-dependent termination sites located ∼90 bases downstream of the mature 3′-end of 6S RNA. Our data suggest that the 3′-end of 6S RNA is generated via exoribonucleolytic trimming, rather than endoribonucleolytic cleavage, following the transcription termination events. The termination sites identified in this study are within the open reading frame of the downstream ygfA (putative 5-formyl-tetrahydrofolate cyclo-ligase) gene, a part of the highly conserved bacterial operon ssrS-ygfA, which is up-regulated during the biofilm formation. Our findings reveal that ygfA expression, which also aids the formation of multidrug-tolerant persister cells, could be regulated by Rho-dependent termination activity in the cell.

Five Sib antitoxin RNAs, members of a family of cis-encoded small regulatory RNAs (sRNAs) in Escherichia coli, repress their target mRNAs, which encode Ibs toxins. This target repression occurs only between cognate sRNA-mRNA pairs with an exception of ibsA. We performed co-transformation assays to assess the ability of SibC derivatives to repress ibsC expression, thereby revealing the regions of SibC that are essential for ibsC mRNA recognition. SibC has two target recognition domains, TRD1 and TRD2, which function independently. The target site for TRD1 is located within the ORF of ibsC, whereas the target site for TRD2 is located in the translational initiation region. The TRD1 sequence is sufficient to repress ibsC expression. In contrast, TRD2 requires a specific structure in addition to the recognition sequence. An in vitro structural probing analysis showed that the initial interactions at these two recognition sites allowed base-pairing to progress into the flanking sequences. Displacement of the TRD1 and TRD2 domains of SibC by the corresponding domains of SibD changed the target specificity of SibC from ibsC to ibsD, suggesting that these two elements modulate the cognate target recognition of each Sib RNA by discriminating among non-cognate ibs mRNAs.

RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing of a large fraction of RNAs in the cell. To better understand the molecular mechanisms of RNase E action, we performed a genetic screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that knock down the ability of RNase E to support survival of E. coli. Comparative phylogenetic analysis of RNase E homologs shows that wild-type residues at these mutated positions are nearly invariably conserved. Cells conditionally expressing these N-Rne mutants in the absence of wild-type RNase E show a decrease in copy number of plasmids regulated by the RNase E substrate RNA I, and accumulation of 5S ribosomal RNA, M1 RNA, and tRNAAsn precursors, as has been found in Rne-depleted cells, suggesting that the inability of these mutants to support cellular growth results from loss of ribonucleolytic activity. Purified mutant proteins containing an amino acid substitution in the DNase I subdomain, which is spatially distant from the catalytic site posited from crystallographic studies, showed defective binding to an RNase E substrate, p23 RNA, but still retained RNA cleavage activity—implicating a previously unidentified structural motif in the DNase I subdomain in the binding of RNase E to targeted RNA molecules, demonstrating the role of the DNase I domain in RNase E activity.