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1.
Environ Microbiol ; 18(10): 3390-3402, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26743546

RESUMO

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner.


Assuntos
Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Oxigênio/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Pseudomonas aeruginosa/metabolismo , Alginatos , Proteínas de Bactérias/genética , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Ácido Glucurônico/biossíntese , Ácidos Hexurônicos , Óperon , Fósforo-Oxigênio Liases/genética , Pseudomonas aeruginosa/genética
2.
Environ Microbiol ; 16(8): 2538-49, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24428244

RESUMO

In Pseudomonas protegens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway controls secondary metabolism and suppression of fungal root pathogens via the expression of regulatory small RNAs (sRNAs). Because of its high cost, this pathway needs to be protected from overexpression and to be turned off in response to environmental stress such as the lack of nutrients. However, little is known about its underlying molecular mechanisms. In this study, we demonstrated that Lon protease, a member of the ATP-dependent protease family, negatively regulated the Gac/Rsm cascade. In a lon mutant, the steady-state levels and the stability of the GacA protein were significantly elevated at the end of exponential growth. As a consequence, the expression of the sRNAs RsmY and RsmZ and that of dependent physiological functions such as antibiotic production were significantly enhanced. Biocontrol of Pythium ultimum on cucumber roots required fewer lon mutant cells than wild-type cells. In starved cells, the loss of Lon function prolonged the half-life of the GacA protein. Thus, Lon protease is an important negative regulator of the Gac/Rsm signal transduction pathway in P. protegens.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Protease La/genética , Pseudomonas/genética , RNA Nuclear Pequeno/genética , Antibacterianos/metabolismo , Antibiose , Proteínas de Bactérias/metabolismo , Cucumis sativus/microbiologia , Mutação , Raízes de Plantas/microbiologia , Protease La/metabolismo , Estabilidade Proteica , Pseudomonas/metabolismo , Pythium/patogenicidade , Pythium/fisiologia , RNA Nuclear Pequeno/metabolismo , Transdução de Sinais
3.
RNA Biol ; 10(6): 1031-41, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23635605

RESUMO

In the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens CHA0, the dimeric RNA-binding proteins RsmA and RsmE, which belong to the vast bacterial RsmA/CsrA family, effectively repress translation of target mRNAs containing a typical recognition sequence near the translation start site. Three small RNAs (RsmX, RsmY, RsmZ) with clustered recognition sequences can sequester RsmA and RsmE and thereby relieve translational repression. According to a previously established structural model, the RsmE protein makes optimal contacts with an RNA sequence 5'- (A)/(U)CANGGANG(U)/(A)-3', in which the central ribonucleotides form a hexaloop. Here, we questioned the relevance of the hexaloop structure in target RNAs. We found that two predicted pentaloop structures, AGGGA (in pltA mRNA encoding a pyoluteorin biosynthetic enzyme) and AAGGA (in mutated pltA mRNA), allowed effective interaction with the RsmE protein in vivo. By contrast, ACGGA and AUGGA were poor targets. Isothermal titration calorimetry measurements confirmed the strong binding of RsmE to the AGGGA pentaloop structure in an RNA oligomer. Modeling studies highlighted the crucial role of the second ribonucleotide in the loop structure. In conclusion, a refined structural model of RsmE-RNA interaction accommodates certain pentaloop RNAs among the preferred hexaloop RNAs.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , RNA Bacteriano/química , RNA Bacteriano/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
4.
Mol Plant Microbe Interact ; 25(11): 1440-9, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23035953

RESUMO

In Pseudomonas fluorescens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is instrumental for secondary metabolism and biocontrol of root pathogens via the expression of regulatory small RNAs (sRNAs). Furthermore, in strain CHA0, an imbalance in the Krebs cycle can affect the strain's ability to produce extracellular secondary metabolites, including biocontrol factors. Here, we report the metabolome of wild-type CHA0, a gacA-negative mutant, which has lost Gac/Rsm activities, and a retS-negative mutant, which shows strongly enhanced Gac/Rsm-dependent activities. Capillary electrophoresis-based metabolomic profiling revealed that the gacA and retS mutations had opposite effects on the intracellular levels of a number of central metabolites, suggesting that the Gac/Rsm pathway regulates not only secondary metabolism but also primary metabolism in strain CHA0. Among the regulated metabolites identified, the alarmone guanosine tetraphosphate (ppGpp) was characterized in detail by the construction of relA (for ppGpp synthase) and spoT (for ppGpp synthase/hydrolase) deletion mutants. In a relA spoT double mutant, ppGpp synthesis was completely abolished, the expression of Rsm sRNAs was attenuated, and physiological functions such as antibiotic production, root colonization, and plant protection were markedly diminished. Thus, ppGpp appears to be essential for sustaining epiphytic fitness and biocontrol activity of strain CHA0.


Assuntos
Pseudomonas fluorescens/metabolismo , Pseudomonas fluorescens/patogenicidade , Cucumis sativus/microbiologia , Eletroforese Capilar , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/metabolismo , Pseudomonas fluorescens/genética
5.
Mol Microbiol ; 80(4): 868-85, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21375594

RESUMO

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.


Assuntos
Pseudomonas aeruginosa/metabolismo , Quinolonas/metabolismo , Percepção de Quorum , RNA Bacteriano/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Northern Blotting , Regulação Bacteriana da Expressão Gênica , Análise em Microsséries , Fases de Leitura Aberta/genética , Pseudomonas aeruginosa/genética , Análise de Sequência de RNA , Transdução de Sinais , Transativadores/metabolismo
6.
Appl Environ Microbiol ; 78(14): 5016-20, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22562990

RESUMO

In Pseudomonas aeruginosa, the catabolite repression control (Crc) protein repressed the formation of the blue pigment pyocyanin in response to a preferred carbon source (succinate) by interacting with phzM mRNA, which encodes a key enzyme in pyocyanin biosynthesis. Crc bound to an extended imperfect recognition sequence that was interrupted by the AUG translation initiation codon.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Metiltransferases/metabolismo , Pseudomonas aeruginosa/metabolismo , Piocianina/biossíntese , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Repressão Catabólica , Códon de Iniciação , Metiltransferases/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Piocianina/genética , Piocianina/metabolismo , Proteínas Repressoras/genética , Ácido Succínico/metabolismo
7.
Appl Environ Microbiol ; 78(6): 1658-65, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22247157

RESUMO

The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as noncoding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small RNA (sRNA) genes rsmX, rsmY, and rsmZ. Naturally occurring mutations in the gacS-gacA system accounted for phenotypic switching, which was characterized by downregulation of antifungal secondary metabolites (2,4-diacetylphloroglucinol and cyanide), indoleacetate, exoenzymes (lipase and protease), and three different N-acyl-homoserine lactone molecules. Moreover, in addition to abrogating these biocontrol traits, gacS and gacA mutations resulted in reduced expression of the type VI secretion machinery, alginate biosynthesis, and biofilm formation. In a gacA mutant, the expression of rsmX was completely abolished, unlike that of rsmY and rsmZ. Overexpression of any of the three sRNAs in the gacA mutant overruled the pleiotropic changes and restored the wild-type phenotypes, suggesting functional redundancy of these sRNAs. In conclusion, our data show that phenotypic switching in P. brassicacearum results from mutations in the gacS-gacA system.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas/fisiologia , RNA Bacteriano/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/metabolismo , Antifúngicos/metabolismo , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/genética , Análise em Microsséries , Fenótipo , Pseudomonas/genética
8.
Nat Struct Mol Biol ; 14(9): 807-13, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17704818

RESUMO

Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5'-A/UCANGGANGU/A-3' sequence in the mRNA. When tightly gripped by RsmE, the ANGGAN core folds into a loop, favoring the formation of a 3-base-pair stem by flanking nucleotides. We validated these findings by in vivo and in vitro mutational analyses. The structure of the complex explains well how, by sequestering the Shine-Dalgarno sequence, the RsmA/CsrA proteins repress translation.


Assuntos
Proteínas de Bactérias/fisiologia , Pseudomonas aeruginosa/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo
9.
Proc Natl Acad Sci U S A ; 106(51): 21866-71, 2009 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-20080802

RESUMO

In the metabolically versatile bacterium Pseudomonas aeruginosa, the RNA-binding protein Crc is involved in catabolite repression of a range of degradative genes, such as amiE (encoding aliphatic amidase). We found that a CA-rich sequence (termed CA motif) in the amiE translation initiation region was important for Crc binding. The small RNA CrcZ (407 nt) containing 5 CA motifs was able to bind the Crc protein with high affinity and to remove it from amiE mRNA in vitro. Overexpression of crcZ relieved catabolite repression in vivo, whereas a crcZ mutation pleiotropically prevented the utilization of several carbon sources. The sigma factor RpoN and the CbrA/CbrB two-component system, which is known to maintain a healthy carbon-nitrogen balance, were necessary for crcZ expression. During growth on succinate, a preferred carbon source, CrcZ expression was low, resulting in catabolite repression of amiE and other genes under Crc control. By contrast, during growth on mannitol, a poor carbon source, elevated CrcZ levels correlated with relief of catabolite repression. During growth on glucose, an intermediate carbon source, CrcZ levels and amiE expression were intermediate between those observed in succinate and mannitol media. Thus, the CbrA-CbrB-CrcZ-Crc system allows the bacterium to adapt differentially to various carbon sources. This cascade also regulated the expression of the xylS (benR) gene, which encodes a transcriptional regulator involved in benzoate degradation, in an analogous way, confirming this cascade's global role.


Assuntos
Carbono/metabolismo , Pseudomonas aeruginosa/metabolismo , RNA Bacteriano/metabolismo , Sequência de Bases , Genes Bacterianos , Dados de Sequência Molecular , Pseudomonas aeruginosa/genética , Processamento Pós-Transcricional do RNA , Transdução de Sinais
10.
J Bacteriol ; 193(11): 2784-92, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21478360

RESUMO

In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein. Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and σ(54) RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sítios de Ligação , Análise Mutacional de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Fatores Hospedeiros de Integração/metabolismo , Lipase/metabolismo , Ligação Proteica , RNA Polimerase Sigma 54/metabolismo
11.
Antimicrob Agents Chemother ; 55(7): 3399-405, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21537014

RESUMO

Azithromycin at clinically relevant doses does not inhibit planktonic growth of the opportunistic pathogen Pseudomonas aeruginosa but causes markedly reduced formation of biofilms and quorum-sensing-regulated extracellular virulence factors. In the Gac/Rsm signal transduction pathway, which acts upstream of the quorum-sensing machinery in P. aeruginosa, the GacA-dependent untranslated small RNAs RsmY and RsmZ are key regulatory elements. As azithromycin treatment and mutational inactivation of gacA have strikingly similar phenotypic consequences, the effect of azithromycin on rsmY and rsmZ expression was investigated. In planktonically growing cells, the antibiotic strongly inhibited the expression of both small RNA genes but did not affect the expression of the housekeeping gene proC. The azithromycin treatment resulted in reduced expression of gacA and rsmA, which are known positive regulators of rsmY and rsmZ, and of the PA0588-PA0584 gene cluster, which was discovered as a novel positive regulatory element involved in rsmY and rsmZ expression. Deletion of this cluster resulted in diminished ability of P. aeruginosa to produce pyocyanin and to swarm. The results of this study indicate that azithromycin inhibits rsmY and rsmZ transcription indirectly by lowering the expression of positive regulators of these small RNA genes.


Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Northern Blotting , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Pseudomonas aeruginosa/genética
12.
Appl Microbiol Biotechnol ; 91(1): 63-79, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21607656

RESUMO

Small RNAs (sRNAs) exert important functions in pseudomonads. Classical sRNAs comprise the 4.5S, 6S, 10Sa and 10Sb RNAs, which are known in enteric bacteria as part of the signal recognition particle, a regulatory component of RNA polymerase, transfer-messenger RNA (tmRNA) and the RNA component of RNase P, respectively. Their homologues in pseudomonads are presumed to have analogous functions. Other sRNAs of pseudomonads generally have little or no sequence similarity with sRNAs of enteric bacteria. Numerous sRNAs repress or activate the translation of target mRNAs by a base-pairing mechanism. Examples of this group in Pseudomonas aeruginosa are the iron-repressible PrrF1 and PrrF2 sRNAs, which repress the translation of genes encoding iron-containing proteins, and PhrS, an anaerobically inducible sRNA, which activates the expression of PqsR, a regulator of the Pseudomonas quinolone signal. Other sRNAs sequester RNA-binding proteins that act as translational repressors. Examples of this group in P. aeruginosa include RsmY and RsmZ, which are central regulatory elements in the GacS/GacA signal transduction pathway, and CrcZ, which is a key regulator in the CbrA/CbrB signal transduction pathway. These pathways largely control the extracellular activities (including virulence traits) and the selection of the energetically most favourable carbon sources, respectively, in pseudomonads.


Assuntos
Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Pseudomonas/metabolismo , RNA Bacteriano/metabolismo , RNA não Traduzido/metabolismo , Pseudomonas/genética , RNA Bacteriano/genética , RNA não Traduzido/genética
13.
J Bacteriol ; 192(16): 4251-5, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20543073

RESUMO

L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.


Assuntos
Aminobutiratos/metabolismo , Antibacterianos/biossíntese , Antimetabólitos/metabolismo , Vias Biossintéticas/genética , Família Multigênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Ordem dos Genes , Genes Bacterianos , Mutagênese Sítio-Dirigida
14.
J Biol Chem ; 284(50): 34976-85, 2009 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-19840935

RESUMO

Pseudomonas fluorescens CHA0, an antagonist of phytopathogenic fungi in the rhizosphere of crop plants, elaborates and excretes several secondary metabolites with antibiotic properties. Their synthesis depends on three small RNAs (RsmX, RsmY, and RsmZ), whose expression is positively controlled by the GacS-GacA two-component system at high cell population densities. To find regulatory links between primary and secondary metabolism in P. fluorescens and in the related species Pseudomonas aeruginosa, we searched for null mutations that affected central carbon metabolism as well as the expression of rsmY-gfp and rsmZ-gfp reporter constructs but without slowing down the growth rate in rich media. Mutation in the pycAB genes (for pyruvate carboxylase) led to down-regulation of rsmXYZ and secondary metabolism, whereas mutation in fumA (for a fumarase isoenzyme) resulted in up-regulation of the three small RNAs and secondary metabolism in the absence of detectable nutrient limitation. These effects required the GacS sensor kinase but not the accessory sensors RetS and LadS. An analysis of intracellular metabolites in P. fluorescens revealed a strong positive correlation between small RNA expression and the pools of 2-oxoglutarate, succinate, and fumarate. We conclude that Krebs cycle intermediates (already known to control GacA-dependent virulence factors in P. aeruginosa) exert a critical trigger function in secondary metabolism via the expression of GacA-dependent small RNAs.


Assuntos
Ciclo do Ácido Cítrico/fisiologia , Metabolismo Energético , Regulação Bacteriana da Expressão Gênica , Pseudomonas fluorescens/metabolismo , RNA/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Teste de Complementação Genética , Humanos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Mutagênese , Pseudomonas fluorescens/genética , RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
15.
Appl Environ Microbiol ; 76(5): 1497-506, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20048056

RESUMO

The Gac/Rsm signal transduction pathway positively regulates secondary metabolism, production of extracellular enzymes, and biocontrol properties of Pseudomonas fluorescens CHA0 via the expression of three noncoding small RNAs, termed RsmX, RsmY, and RsmZ. The architecture and function of the rsmY and rsmZ promoters were studied in vivo. A conserved palindromic upstream activating sequence (UAS) was found to be necessary but not sufficient for rsmY and rsmZ expression and for activation by the response regulator GacA. A poorly conserved linker region located between the UAS and the -10 promoter sequence was also essential for GacA-dependent rsmY and rsmZ expression, suggesting a need for auxiliary transcription factors. One such factor involved in the activation of the rsmZ promoter was identified as the PsrA protein, previously recognized as an activator of the rpoS gene and a repressor of fatty acid degradation. Furthermore, the integration host factor (IHF) protein was found to bind with high affinity to the rsmZ promoter region in vitro, suggesting that DNA bending contributes to the regulated expression of rsmZ. In an rsmXYZ triple mutant, the expression of rsmY and rsmZ was elevated above that found in the wild type. This negative feedback loop appears to involve the translational regulators RsmA and RsmE, whose activity is antagonized by RsmXYZ, and several hypothetical DNA-binding proteins. This highly complex network controls the expression of the three small RNAs in response to cell physiology and cell population densities.


Assuntos
Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Pseudomonas fluorescens/fisiologia , RNA Bacteriano/biossíntese , RNA não Traduzido/biossíntese , Ativação Transcricional , Sequência de Bases , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica
16.
J Bacteriol ; 190(20): 6706-17, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18708503

RESUMO

Restricted bioavailability of copper in certain environments can interfere with cellular respiration because copper is an essential cofactor of most terminal oxidases. The global response of the metabolically versatile bacterium and opportunistic pathogen Pseudomonas aeruginosa to copper limitation was assessed under aerobic conditions. Expression of cioAB (encoding an alternative, copper-independent, cyanide-resistant ubiquinol oxidase) was upregulated, whereas numerous iron uptake functions (including the siderophores pyoverdine and pyochelin) were expressed at reduced levels, presumably reflecting a lower demand for iron by respiratory enzymes. Wild-type P. aeruginosa was able to grow aerobically in a defined glucose medium depleted of copper, whereas a cioAB mutant did not grow. Thus, P. aeruginosa relies on the CioAB enzyme to cope with severe copper deprivation. A quadruple cyo cco1 cco2 cox mutant, which was deleted for all known heme-copper terminal oxidases of P. aeruginosa, grew aerobically, albeit more slowly than did the wild type, indicating that the CioAB enzyme is capable of energy conservation. However, the expression of a cioA'-'lacZ fusion was less dependent on the copper status in the quadruple mutant than in the wild type, suggesting that copper availability might affect cioAB expression indirectly, via the function of the heme-copper oxidases.


Assuntos
Proteínas de Bactérias/biossíntese , Cobre/metabolismo , Perfilação da Expressão Gênica , Pseudomonas aeruginosa/fisiologia , Aerobiose , Fusão Gênica Artificial , Proteínas de Bactérias/genética , Meios de Cultura/química , Regulação para Baixo , Deleção de Genes , Genes Reporter , Glucose/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Regulação para Cima , beta-Galactosidase/biossíntese , beta-Galactosidase/genética
17.
BMC Genomics ; 9: 167, 2008 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-18405392

RESUMO

BACKGROUND: Small RNAs (sRNAs) are widespread among bacteria and have diverse regulatory roles. Most of these sRNAs have been discovered by a combination of computational and experimental methods. In Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium and opportunistic human pathogen, the GacS/GacA two-component system positively controls the transcription of two sRNAs (RsmY, RsmZ), which are crucial for the expression of genes involved in virulence. In the biocontrol bacterium Pseudomonas fluorescens CHA0, three GacA-controlled sRNAs (RsmX, RsmY, RsmZ) regulate the response to oxidative stress and the expression of extracellular products including biocontrol factors. RsmX, RsmY and RsmZ contain multiple unpaired GGA motifs and control the expression of target mRNAs at the translational level, by sequestration of translational repressor proteins of the RsmA family. RESULTS: A combined computational and experimental approach enabled us to identify 14 intergenic regions encoding sRNAs in P. aeruginosa. Eight of these regions encode newly identified sRNAs. The intergenic region 1698 was found to specify a novel GacA-controlled sRNA termed RgsA. GacA regulation appeared to be indirect. In P. fluorescens CHA0, an RgsA homolog was also expressed under positive GacA control. This 120-nt sRNA contained a single GGA motif and, unlike RsmX, RsmY and RsmZ, was unable to derepress translation of the hcnA gene (involved in the biosynthesis of the biocontrol factor hydrogen cyanide), but contributed to the bacterium's resistance to hydrogen peroxide. In both P. aeruginosa and P. fluorescens the stress sigma factor RpoS was essential for RgsA expression. CONCLUSION: The discovery of an additional sRNA expressed under GacA control in two Pseudomonas species highlights the complexity of this global regulatory system and suggests that the mode of action of GacA control may be more elaborate than previously suspected. Our results also confirm that several GGA motifs are required in an sRNA for sequestration of the RsmA protein.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Pseudomonas/genética , Pseudomonas/metabolismo , RNA Bacteriano/genética , RNA não Traduzido/genética , Sequência de Bases , DNA Intergênico/genética , Genômica , Dados de Sequência Molecular , Estresse Oxidativo/genética , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , RNA Bacteriano/metabolismo , RNA não Traduzido/metabolismo , Fator sigma/metabolismo
18.
Appl Environ Microbiol ; 74(6): 1902-8, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18203852

RESUMO

Pseudomonas aeruginosa undergoes spontaneous mutation that impairs secretion of several extracellular enzymes during extended cultivation in vitro in rich media, as well as during long-term colonization of the cystic fibrosis lung. A frequent type of strong secretion deficiency is caused by inactivation of the quorum-sensing regulatory gene lasR. Here we analyzed a spontaneously emerging subline of strain PAO1 that exhibited moderate secretion deficiency and partial loss of quorum-sensing control. Using generalized transduction, we mapped the secretion defect to the vfr gene, which is known to control positively the expression of the lasR gene and type II secretion of several proteases. We confirmed this secretion defect by sequencing and complementation of the vfr mutation. In a reconstruction experiment conducted with a 1:1 mixture of wild-type strain PAO1 and a vfr mutant of PAO1, we observed that the vfr mutant had a selective advantage over the wild type after growth in static culture for 4 days. Under these conditions, spontaneous vfr emerged in a strain PAO1 population after four growth cycles, and these mutants accounted for more than 40% of the population after seven cycles. These results suggest that partial or complete loss of quorum sensing and secretion can be beneficial to P. aeruginosa under certain environmental conditions.


Assuntos
Proteínas de Bactérias/genética , Proteína Receptora de AMP Cíclico/genética , Mutação , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes Reguladores , Teste de Complementação Genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/genética , Transdução Genética
19.
Nat Rev Microbiol ; 3(4): 307-19, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15759041

RESUMO

Particular bacterial strains in certain natural environments prevent infectious diseases of plant roots. How these bacteria achieve this protection from pathogenic fungi has been analysed in detail in biocontrol strains of fluorescent pseudomonads. During root colonization, these bacteria produce antifungal antibiotics, elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors. Before engaging in these activities, biocontrol bacteria go through several regulatory processes at the transcriptional and post-transcriptional levels.


Assuntos
Antibiose , Fungos/crescimento & desenvolvimento , Controle Biológico de Vetores/métodos , Doenças das Plantas/microbiologia , Pseudomonas/fisiologia , Microbiologia do Solo , Antibacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Raízes de Plantas/microbiologia , Pseudomonas/genética , Transcrição Gênica
20.
Adv Exp Med Biol ; 631: 54-79, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18792682

RESUMO

Two-component systems (TCSs) allow bacteria to monitor diverse environmental cues and to adjust gene expression accordingly at the transcriptional level. It has been recently recognized that prokaryotes also regulate many genes and operons at a posttranscriptional level with the participation of small, noncoding RNAs which serve to control translation initiation and stability of target mRNAs, either directly by establishing antisense interactions or indirectly by antagonizing RNA-binding proteins. Interestingly, the expression of a subset of these small RNAs is regulated by TCSs and in this way, the small RNAs expand the scope of genetic control exerted by TCSs. Here we review the regulatory mechanisms and biological relevance ofa number of small RNAs under TCS control in Gram-negative and -positive bacteria. These regulatory systems govern, for instance, porin-dependent permeability of the outer membrane, quorum-sensing control of pathogenicity, or biocontrol activity. Most likely, this emerging and rapidly expanding field of molecular microbiology will provide more and more examples in the near future.


Assuntos
Bactérias/genética , Bactérias/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Carbono/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Enterobacteriaceae/patogenicidade , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Biológicos , Iniciação Traducional da Cadeia Peptídica , Pseudomonas/genética , Pseudomonas/metabolismo , Pseudomonas/patogenicidade , Percepção de Quorum , Estabilidade de RNA , RNA Antissenso/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Virulência
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