RESUMEN
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 (σS) 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.
RESUMEN
Bioinformatic approaches employed to analyse intergenic regions of Pseudomonas aeruginosa O1 (PAO1) for small RNAs (sRNAs) revealed a putative RNA gene encoded upstream of the nitrate assimilation operon nirBD-PA1779-cobA. Here, we show that this RNA, termed nitrogen assimilation leader A (NalA), represents the leader RNA of the nirBD-PA1779-cobA operon, and that nalA transcription is σ(54)- and NtrC-dependent. A PAO1 nalA deletion strain and a strain bearing a deletion in ORF PA1785 failed to grow on nitrate. PA1785 was identified as a homologue of the Azotobacter vinelandii nasT gene, the product of which is required for transcription of the A. vinelandii nitrite/nitrate reductase operon. Collectively, these studies reveal that transcriptional antitermination of the leader RNA NalA is required for expression of the PAO1 nitrate assimilation operon, and that this process is governed by conserved functions in PAO1 and A. vinelandii.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Nitrato-Reductasa/genética , Nitratos/metabolismo , Operón , Pseudomonas aeruginosa/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Nitrato-Reductasa/metabolismo , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Alineación de Secuencia , Transactivadores/genéticaRESUMEN
Quorum sensing, a cell-to-cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans-encoded RNAs has become a focal issue in studies of virulence gene expression of bacterial pathogens. In this study, we have identified the small RNA PhrS as an activator of PqsR synthesis, one of the key quorum-sensing regulators in P. aeruginosa. Genetic studies revealed a novel mode of regulation by a sRNA, whereby PhrS uses a base-pairing mechanism to activate a short upstream open reading frame to which the pqsR gene is translationally coupled. Expression of phrS requires the oxygen-responsive regulator ANR. Thus, PhrS is the first bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing, which may impact on oxygen-limited growth in P. aeruginosa biofilms.
Asunto(s)
Pseudomonas aeruginosa/metabolismo , Quinolonas/metabolismo , Percepción de Quorum , ARN Bacteriano/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Northern Blotting , Regulación Bacteriana de la Expresión Génica , Análisis por Micromatrices , Sistemas de Lectura Abierta/genética , Pseudomonas aeruginosa/genética , Análisis de Secuencia de ARN , Transducción de Señal , Transactivadores/metabolismoRESUMEN
Inactivation of the Pseudomonas aeruginosa (PAO1) hfq gene, encoding the Sm-like Hfq protein, resulted in pleiotropic effects that included an attenuated virulence. As regulation by Hfq often involves the action of small regulatory RNAs (sRNAs), we have used a shotgun cloning approach (RNomics) and bioinformatic tools to identify sRNAs in strain PAO1. For cDNA library construction, total RNA was extracted from PAO1 cultures either grown to stationary phase or exposed to human serum. The cDNA libraries were generated from small-sized RNAs of PAO1 after co-immunoprecipitation with Hfq. Of 400 sequenced cDNA clones, 11 mapped to intergenic regions. Band-shift assays and Northern blot analyses performed with two selected sRNAs confirmed that Hfq binds to and affects the steady-state levels of these RNAs. A proteome study performed upon overproduction of one sRNA, PhrS, implicated it in riboregulation. PhrS contains an ORF, and evidence for its translation is presented. In addition, based on surveys with structure-based bioinformatic tools, we provide an electronic compilation of putative sRNA and non-coding RNA genes of PAO1 based on their evolutionarily conserved structure.
Asunto(s)
Proteína de Factor 1 del Huésped/genética , Pseudomonas aeruginosa/genética , ARN Bacteriano/genética , ARN no Traducido/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Northern Blotting , Biología Computacional/métodos , Ensayo de Cambio de Movilidad Electroforética , Biblioteca de Genes , Genes Bacterianos , Genoma Bacteriano , Genómica/métodos , Humanos , Datos de Secuencia Molecular , Plásmidos , Unión Proteica , Proteoma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
In Pseudomonas aeruginosa the Rsm system is involved in regulation of quorum-sensing and virulence gene expression. Our recent studies revealed that the stability and abundance of the non-coding RNA RsmY, which antagonizes the translational regulator RsmA, is dependent on Hfq. Here, we show that Hfq and RsmA bind concurrently to RsmY. Enzymatic probing of RsmY RNA in the presence of RsmA and Hfq verified the proposed -GGA- motifs as RsmA binding sites and located Hfq binding sites in single-stranded regions adjacent to stem-loop structures, respectively. We conclude that distinct binding of Hfq and RsmA on RsmY RNA permits RsmY-mediated RsmA titration upon binding to and stabilization of RsmY RNA by Hfq. In addition, we provide evidence that Hfq sequesters RNase E cleavage sites on RsmY, which explains the previously observed dependence of RsmY RNA stability on Hfq.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli/genética , Proteína de Factor 1 del Huésped/genética , Pseudomonas aeruginosa/genética , ARN no Traducido/genética , Proteínas de Unión al ARN/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Represoras/genética , Transcripción Genética/genética , Sitios de UniónRESUMEN
The Pseudomonas aeruginosa quorum-sensing (QS) systems, Las and Rhl, control the production of several virulence factors and other proteins, which are important to sustain adverse conditions. A comparative transcriptome analysis of a rpoS (-) and a rpoS(-)hfq( -) strain indicated that the Sm-like RNA-binding protein Hfq affects approximately 5% of the P. aeruginosa O1 transcripts. Among these transcripts 72 were identified to be QS regulated. Expression studies revealed that Hfq does not control the master regulators of the Las system, LasR and LasI. Upon entry into stationary phase, Hfq exerted a moderate stimulatory effect on translation of the rhlR gene and on the qscR gene, encoding a LasR/RhlR homologue. However, Hfq considerably stimulated translation of the rhlI gene, encoding the synthetase of the autoinducer N-Butyryl-homoserine lactone (C4-HSL). Correspondingly, the C4-HSL levels were reduced in a hfq(-) strain. To elucidate the stimulatory effect of Hfq on rhlI expression we asked whether Hfq affects the stability of the regulatory RNAs RsmY and RsmZ, which have been implicated in sequestration of the translational repressor RsmA, which in turn is known to negatively regulate RhlI synthesis. We demonstrate that Hfq binds to and stabilizes the regulatory RNA RsmY, which is further shown to bind to the regulatory protein RsmA. A model for the Hfq regulatory network is presented, wherein an alleviation of the negative effect of RsmA accounts for the observed stimulation of rhlI expression by Hfq. The model is corroborated by the observation that a rsmY(-) mutant mimics the hfq(-) phenotype with regard to rhlI expression.