RESUMO
The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components. Evaluation of substrate accessibility suggests that the paused 30S PIC presents the mRNA for targeted recognition and degradation. Ribonuclease activity on PIC-bound ompD is critically dependent on the recruitment of RNase E into the multi-enzyme RNA degradosome, and our data suggest a process of substrate capture and handover to catalytic sites within the degradosome, in which sequential steps of seed matching and duplex remodelling contribute to cleavage efficiency. Our findings support a putative mechanism of surveillance at translation that potentially terminates gene expression efficiently and rapidly in response to signals provided by regulatory RNA.
Assuntos
Endorribonucleases , Complexos Multienzimáticos , Polirribonucleotídeo Nucleotidiltransferase , Porinas , RNA Helicases , Estabilidade de RNA , RNA Mensageiro , Endorribonucleases/metabolismo , Endorribonucleases/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Estabilidade de RNA/genética , Porinas/metabolismo , Porinas/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Pequeno RNA não Traduzido/metabolismo , Pequeno RNA não Traduzido/genética , Regulação Bacteriana da Expressão GênicaRESUMO
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.
Assuntos
Caulobacter crescentus , Pequeno RNA não Traduzido , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Pequeno RNA não Traduzido/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , Proteínas de Membrana Transportadoras/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
In the past decades, Caulobacter crescentus has been extensively studied, mostly regarding its dimorphic, asymmetric life cycle. Its distinct mode of reproduction and the need to optimally adapt to ever-changing environmental conditions require tight coordination of gene regulation. Post-transcriptional regulation through non-coding RNAs and RNA-binding proteins constitutes an important layer of expression control in bacteria, but its principles and mechanisms in Caulobacter have only recently been explored. RNA-binding proteins including the RNA chaperone Hfq and ribonuclease RNase E contribute to the activity of regulatory RNAs. Riboswitches and RNA thermometers govern expression of downstream open reading frames, while the small regulatory RNAs CrfA, ChvR and GsrN instead control targets encoded in trans by direct base-pairing interactions.
Assuntos
Caulobacter crescentus , Pequeno RNA não Traduzido , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , RNA Mensageiro , Pequeno RNA não Traduzido/genéticaRESUMO
Vibrio cholerae, the cause of cholera disease, exhibits a characteristic curved rod morphology, which promotes infectivity and motility in dense hydrogels. Periplasmic protein CrvA determines cell curvature in V. cholerae, yet the regulatory factors controlling CrvA are unknown. Here, we discover the VadR small RNA (sRNA) as a post-transcriptional inhibitor of the crvA mRNA. Mutation of vadR increases cell curvature, whereas overexpression has the inverse effect. We show that vadR transcription is activated by the VxrAB two-component system and triggered by cell-wall-targeting antibiotics. V. cholerae cells failing to repress crvA by VadR display decreased survival upon challenge with penicillin G indicating that cell shape maintenance by the sRNA is critical for antibiotic resistance. VadR also blocks the expression of various key biofilm genes and thereby inhibits biofilm formation in V. cholerae. Thus, VadR is an important regulator for synchronizing peptidoglycan integrity, cell shape, and biofilm formation in V. cholerae.
Assuntos
Resistência Microbiana a Medicamentos/genética , RNA Bacteriano/genética , Vibrio cholerae/citologia , Vibrio cholerae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Vibrio cholerae/fisiologiaRESUMO
Regulation at the post-transcriptional level is an important mode of gene expression control in bacteria. Small RNA regulators (sRNAs) that act via intramolecular base-pairing with target mRNAs are key players in this process and most often sequester the target's ribosome binding site (RBS) to down-regulate translation initiation. Over the past few years, several exceptions from this mechanism have been reported, revealing that sRNAs are able to influence translation initiation from a distance. In this issue of Molecular Microbiology, Azam and Vanderpool show that repression of the manY mRNA by the sRNA SgrS relies on an unconventional mechanism involving a translational enhancer element and ribosomal protein S1. Binding of S1 to an AU-rich sequence within the 5' untranslated region of the manY transcript promotes translation of the mRNA, and base-pairing of SgrS to the same site can interfere with this process. Therefore, instead of blocking translation initiation by occluding the manY RBS, SgrS reduces ManY synthesis by inhibiting S1-dependent translation activation. These findings increase the base-pairing window for sRNA-mediated gene expression control in bacteria and highlight the role of ribosomal protein S1 for translation initiation.
Assuntos
Bactérias/genética , Iniciação Traducional da Cadeia Peptídica , RNA Bacteriano/fisiologia , Pequeno RNA não Traduzido/genética , Proteínas Ribossômicas/genética , Regiões 5' não Traduzidas , Pareamento de Bases/genética , Sítios de Ligação , Elementos Facilitadores Genéticos , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/fisiologiaRESUMO
Hfq (host factor for phage Q beta) is key for posttranscriptional gene regulation in many bacteria. Hfq's function is to stabilize sRNAs and to facilitate base-pairing with trans-encoded target mRNAs. Loss of Hfq typically results in pleiotropic phenotypes, and, in the major human pathogen Vibrio cholerae, Hfq inactivation has been linked to reduced virulence, failure to produce biofilms, and impaired intercellular communication. However, the RNA ligands of Hfq in V. cholerae are currently unknown. Here, we used RIP-seq (RNA immunoprecipitation followed by high-throughput sequencing) analysis to identify Hfq-bound RNAs in V. cholerae Our work revealed 603 coding and 85 noncoding transcripts associated with Hfq, including 44 sRNAs originating from the 3' end of mRNAs. Detailed investigation of one of these latter transcripts, named FarS (fatty acid regulated sRNA), showed that this sRNA is produced by RNase E-mediated maturation of the fabB 3'UTR, and, together with Hfq, inhibits the expression of two paralogous fadE mRNAs. The fabB and fadE genes are antagonistically regulated by the major fatty acid transcription factor, FadR, and we show that, together, FadR, FarS, and FadE constitute a mixed feed-forward loop regulating the transition between fatty acid biosynthesis and degradation in V. cholerae Our results provide the molecular basis for studies on Hfq in V. cholerae and highlight the importance of a previously unrecognized sRNA for fatty acid metabolism in this major human pathogen.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Ácidos Graxos/metabolismo , Regulação Bacteriana da Expressão Gênica , Pequeno RNA não Traduzido/metabolismo , Vibrio cholerae/genética , Regiões 3' não Traduzidas/genética , Acil-CoA Desidrogenase/metabolismo , Proteínas de Bactérias/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Interferência de RNA , RNA Bacteriano/isolamento & purificação , Proteínas Repressoras/metabolismo , Vibrio cholerae/metabolismoRESUMO
In the pathogen Pseudomonas aeruginosa, LasR is a quorum sensing (QS) master regulator that senses the concentration of secreted autoinducers as a proxy for bacterial cell density. Counterintuitively, previous studies showed that saturating amounts of the LasR ligand, 3OC12-HSL, fail to induce the full LasR regulon in low-density liquid cultures. Here we demonstrate that surface association, which is necessary for many of the same group behaviors as QS, promotes stronger QS responses. We show that lasR is upregulated upon surface association, and that surface-associated bacteria induce LasR targets more strongly in response to autoinducer than planktonic cultures. This increased sensitivity may be due to surface-dependent lasR induction initiating a positive feedback loop through the small RNA, Lrs1. The increased sensitivity of surface-associated cells to QS is affected by the type IV pilus (TFP) retraction motors and the minor pilins. The coupling of physical surface responses and chemical QS responses could enable these bacteria to trigger community behaviors more robustly when they are more beneficial.
Assuntos
Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias , Propriedades de Superfície , Regulação para CimaRESUMO
Altering membrane protein and lipid composition is an important strategy for maintaining membrane integrity during environmental stress. Many bacterial small RNAs (sRNAs) control membrane protein production, but sRNA-mediated regulation of membrane fatty acid composition is less well understood. The sRNA RydC was previously shown to stabilize cfa (cyclopropane fatty acid synthase) mRNA, resulting in higher levels of cyclopropane fatty acids in the cell membrane. Here, we report that additional sRNAs, ArrS and CpxQ, also directly regulate cfa posttranscriptionally. RydC and ArrS act through masking an RNase E cleavage site in the cfa mRNA 5' untranslated region (UTR), and both sRNAs posttranscriptionally activate cfa In contrast, CpxQ binds to a different site in the cfa mRNA 5' UTR and represses cfa expression. Alteration of membrane lipid composition is a key mechanism for bacteria to survive low-pH environments, and we show that cfa translation increases in an sRNA-dependent manner when cells are subjected to mild acid stress. This work suggests an important role for sRNAs in the acid stress response through regulation of cfa mRNA.IMPORTANCE Small RNAs (sRNAs) in bacteria are abundant and play important roles in posttranscriptional regulation of gene expression, particularly under stress conditions. Some mRNAs are targets for regulation by multiple sRNAs, each responding to different environmental signals. Uncovering the regulatory mechanisms governing sRNA-mRNA interactions and the relevant conditions for these interactions is an ongoing challenge. In this study, we discovered that multiple sRNAs control membrane lipid composition by regulating stability of a single mRNA target. The sRNA-dependent regulation occurred in response to changing pH and was important for cell viability under acid stress conditions. This work reveals yet another aspect of bacterial physiology controlled at the posttranscriptional level by sRNA regulators.
Assuntos
Bactérias/genética , Metiltransferases/genética , Pequeno RNA não Traduzido/genética , Regiões 5' não Traduzidas , Bactérias/enzimologia , Ciclopropanos/metabolismo , Ácidos Graxos/metabolismo , Regulação Bacteriana da Expressão Gênica , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Mensageiro/genéticaRESUMO
We have solved the X-ray crystal structure of the RNA chaperone protein Hfq from the alpha-proteobacterium Caulobacter crescentus to 2.15-Å resolution, resolving the conserved core of the protein and the entire C-terminal domain (CTD). The structure reveals that the CTD of neighboring hexamers pack in crystal contacts, and that the acidic residues at the C-terminal tip of the protein interact with positive residues on the rim of Hfq, as has been recently proposed for a mechanism of modulating RNA binding. De novo computational models predict a similar docking of the acidic tip residues against the core of Hfq. We also show that C. crescentus Hfq has sRNA binding and RNA annealing activities and is capable of facilitating the annealing of certain Escherichia coli sRNA:mRNA pairs in vivo. Finally, we describe how the Hfq CTD and its acidic tip residues provide a mechanism to modulate annealing activity and substrate specificity in various bacteria.
Assuntos
Proteínas de Bactérias , Caulobacter crescentus , Fator Proteico 1 do Hospedeiro , RNA Bacteriano , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/química , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Cristalografia por Raios X , Fator Proteico 1 do Hospedeiro/química , Fator Proteico 1 do Hospedeiro/metabolismo , Modelos Moleculares , Chaperonas Moleculares , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismoRESUMO
Bacteria use quorum sensing to monitor cell density and coordinate group behaviours. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, quorum sensing is connected to virulence gene expression via the two autoinducer molecules, AI-2 and CAI-1. Both autoinducers share one signal transduction pathway to control the production of AphA, a key transcriptional activator of biofilm formation and virulence genes. In this study, we demonstrate that the recently identified autoinducer, DPO, also controls AphA production in V. cholerae. DPO, functioning through the transcription factor VqmA and the VqmR small RNA, reduces AphA levels at the post-transcriptional level and consequently inhibits virulence gene expression. VqmR-mediated repression of AphA provides an important link between the AI-2/CAI-1 and DPO-dependent quorum sensing pathways in V. cholerae. Transcriptome analyses comparing the effect of single autoinducers versus autoinducer combinations show that quorum sensing controls the expression of â¼400 genes in V. cholerae and that all three autoinducers are required for a full quorum sensing response. Together, our data provide a global view on autoinducer interplay in V. cholerae and highlight the importance of RNA-based gene control for collective functions in this major human pathogen.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Homosserina/análogos & derivados , Cetonas , Vibrio cholerae/genética , Virulência/genética , Biofilmes/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Homosserina/genética , Lactonas , Regiões Promotoras Genéticas , Percepção de Quorum/genética , Transdução de Sinais/genética , Vibrio cholerae/patogenicidadeRESUMO
Bacterial small RNAs (sRNAs) are a heterogeneous group of post-transcriptional regulators that often act at the heart of large networks. Hundreds of sRNAs have been discovered by genome-wide screens and most of these sRNAs exert their functions by base-pairing with target mRNAs. However, studies addressing the molecular roles of sRNAs have been largely confined to gamma-proteobacteria, such as Escherichia coli. Here we identify and characterize a novel sRNA, ChvR, from the alpha-proteobacterium Caulobacter crescentus. Transcription of chvR is controlled by the conserved two-component system ChvI-ChvG and it is expressed in response to DNA damage, low pH, and growth in minimal medium. Transient over-expression of ChvR in combination with genome-wide transcriptome profiling identified the mRNA of the TonB-dependent receptor ChvT as the sole target of ChvR. Genetic and biochemical analyses showed that ChvR represses ChvT at the post-transcriptional level through direct base-pairing. Fine-mapping of the ChvR-chvT interaction revealed the requirement of two distinct base-pairing sites for full target regulation. Finally, we show that ChvR-controlled repression of chvT is independent of the ubiquitous RNA-chaperone Hfq, and therefore distinct from previously reported mechanisms employed by prototypical bacterial sRNAs. These findings have implications for the mechanism and evolution of sRNA function across bacterial species.
Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Caulobacter crescentus/metabolismo , Dano ao DNA , Perfilação da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismoRESUMO
The ability of bacteria to thrive in diverse habitats and to adapt to ever-changing environmental conditions relies on the rapid and stringent modulation of gene expression. It has become evident in the past decade that small regulatory RNAs (sRNAs) are central components of networks controlling the bacterial responses to stress. Functioning at the posttranscriptional level, sRNAs base-pair with cognate mRNAs to alter translation, stability, or both to either repress or activate the targeted transcripts; the RNA chaperone Hfq participates in stabilizing sRNAs and in promoting pairing between target and sRNA. In particular, sRNAs act at the heart of crucial stress responses, including those dedicated to overcoming membrane damage and oxidative stress, discussed here. The bacterial cell envelope is the outermost protective barrier against the environment and thus is constantly monitored and remodeled. Here, we review the integration of sRNAs into the complex networks of several major envelope stress responses of Gram-negative bacteria, including the RpoE (σE), Cpx, and Rcs regulons. Oxidative stress, caused by bacterial respiratory activity or induced by toxic molecules, can lead to significant damage of cellular components. In Escherichia coli and related bacteria, sRNAs also contribute significantly to the function of the RpoS (σS)-dependent general stress response as well as the specific OxyR- and SoxR/S-mediated responses to oxidative damage. Their activities in gene regulation and crosstalk to other stress-induced regulons are highlighted.
Assuntos
Enterobacteriaceae/metabolismo , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo/fisiologia , RNA Bacteriano/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Regulon , Proteínas Repressoras/metabolismo , Fator sigma/metabolismo , Estresse Fisiológico , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Model enteric bacteria such as Escherichia coli and Salmonella enterica express hundreds of small non-coding RNAs (sRNAs), targets for most of which are yet unknown. Some sRNAs are remarkably well conserved, indicating that they serve cellular functions that go beyond the necessities of a single species. One of these 'core sRNAs' of largely unknown function is the abundant â¼100-nucleotide SdsR sRNA which is transcribed by the general stress σ-factor, σS and accumulates in stationary phase. In Salmonella, SdsR was known to inhibit the synthesis of the species-specific porin, OmpD. However, sdsR genes are present in almost all enterobacterial genomes, suggesting that additional, conserved targets of this sRNA must exist. Here, we have combined SdsR pulse-expression with whole genome transcriptomics to discover 20 previously unknown candidate targets of SdsR which include mRNAs coding for physiologically important regulators such as the carbon utilization regulator, CRP, the nucleoid-associated chaperone, StpA and the antibiotic resistance transporter, TolC. Processing of SdsR by RNase E results in two cellular SdsR variants with distinct target spectra. While the overall physiological role of this orphan core sRNA remains to be fully understood, the new SdsR targets present valuable leads to determine sRNA functions in resting bacteria.
Assuntos
Regulação Bacteriana da Expressão Gênica , Interferência de RNA , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Salmonella/genética , Sequência de Bases , Sítios de Ligação , Biologia Computacional/métodos , Expressão Gênica , Perfilação da Expressão Gênica , Genes Reporter , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Mensageiro/química , RNA Mensageiro/genética , Pequeno RNA não Traduzido/química , Reprodutibilidade dos TestesRESUMO
Horizontal transfer of genetic information is a major driving force of evolution. In bacteria, genome plasticity is intimately linked to the ability of the bacterium to integrate novel material into existing gene expression circuits. Small RNAs (sRNAs) are a versatile class of regulatory molecules, and have recently been discovered to perform important tasks in the interplay between core genomic elements and horizontally-acquired DNA. Together with auxiliary proteins such as the RNA-chaperone Hfq and cellular ribonucleases, sRNAs typically act post-transcriptionally to either promote or restrict the expression of multiple target genes. Bacterial sRNAs have been identified in core and peripheral (acquired) genome sequences, and their target suites may likewise include genes from both locations. In this review, we discuss how sRNAs influence the expression of foreign genetic material in enterobacterial pathogens, and outline the processes that foster the integration of horizontally-acquired RNAs into existing regulatory networks. We also consider potential benefits and risks of horizontal gene transfer for RNA-based gene regulation.
Assuntos
Enterobacteriaceae/genética , Sequências Repetitivas Dispersas , Sequências Reguladoras de Ácido Ribonucleico , Regulação Bacteriana da Expressão Gênica , Transferência Genética Horizontal , RNA Bacteriano/metabolismo , RNA Citoplasmático Pequeno/genéticaRESUMO
Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a 'seed' region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at a 3.48 Å resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein-RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target.
Assuntos
Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/metabolismo , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/química , Salmonella/genética , Salmonella/metabolismoRESUMO
A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, σ(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by σ(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Porinas/biossíntese , Pequeno RNA não Traduzido/metabolismo , Salmonella/genética , Fator sigma/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Endorribonucleases/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Dados de Sequência Molecular , Porinas/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/genética , Salmonella/metabolismo , Alinhamento de Sequência , Estresse Fisiológico/genética , Transcrição GênicaRESUMO
P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. P-bodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.
Assuntos
ADP Ribose Transferases/metabolismo , Estruturas Celulares/metabolismo , Estruturas Celulares/microbiologia , Salmonella/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Inativação Gênica , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Camundongos , MicroRNAs/biossíntese , Salmonella/citologia , Infecções por Salmonella/metabolismo , Vesículas Secretórias/metabolismo , Shigella/metabolismo , Fatores de VirulênciaRESUMO
Small regulatory RNAs (sRNAs) commonly act to downregulate gene expression. In bacteria, however, sRNAs have also been shown to activate genes by a variety of direct or indirect mechanisms. Several sRNAs (DsrA, GlmZ, RNAIII, RprA, RyhB, and Qrr) act as direct translational activators by an 'anti-antisense mechanism' in the 5' mRNA region to liberate a sequestered ribosome binding site, while pairing of GadY sRNA to the 3'-end alters processing and increases stability of its target mRNA. Indirect activation includes cases of RNA mimicry in which degradation of the activating GlmZ sRNA is suppressed by the highly similar GlmY sRNA, or where a pseudo-target mRNA traps MicM sRNA to derepress porin synthesis.
Assuntos
Fenômenos Fisiológicos Bacterianos , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ativação Transcricional , Modelos Biológicos , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
The human malaria parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within the host erythrocyte, including the erythrocyte cytoplasm, plasma membrane and membrane profiles of parasite origin termed Maurer's clefts. Most of the exported proteins contain a conserved pentameric motif termed plasmodial export element (PEXEL)/vacuolar transfer signal (VTS) that functions as a cleavable sorting signal permitting export to the host erythrocyte. However, there are some exported proteins, such as the skeleton-binding protein 1 (PfSBP1) that lack the PEXEL/VTS motif and that are not N-terminally processed, suggesting the presence of alternative sorting signals and/or mechanisms. In this study, we have investigated trafficking of PfSBP1 to the Maurer's clefts. Our data show that the transmembrane domain of PfSBP1 functions as an internal signal sequence for entry into the parasite's secretory pathway and for transport to the parasite plasma membrane. Trafficking beyond the parasite's plasma membrane required additional N-terminal domains, which are characterized by a high negative net charge. Biochemical data indicate that these domains affect the solubility and extraction profile, the orientation of the protein within the membrane and the subcellular localization. Our findings suggest new principles of protein export in P. falciparum-infected erythrocytes.