The interplay of StyR and IHF regulates substrate-dependent induction and carbon catabolite repression of styrene catabolism genes in Pseudomonas fluorescens ST

<p>Abstract</p> <p>Background</p> <p>In <it>Pseudomonas fluorescens </it>ST, the promoter of the styrene catabolic operon, P<it>styA</it>, is induced by styrene and is subject to catabolite repression. P<it>styA </it>regulation relies on the StyS/StyR two-component system and on the IHF global regulator. The phosphorylated response regulator StyR (StyR-P) activates P<it>styA </it>in inducing conditions when it binds to the high-affinity site STY2, located about -40 bp from the transcription start point. A <it>cis</it>-acting element upstream of STY2, named URE, contains a low-affinity StyR-P binding site (STY1), overlapping the IHF binding site. Deletion of the URE led to a decrease of promoter activity in inducing conditions and to a partial release of catabolite repression. This study was undertaken to assess the relative role played by IHF and StyR-P on the URE, and to clarify if P<it>styA </it>catabolite repression could rely on the interplay of these regulators.</p> <p>Results</p> <p>StyR-P and IHF compete for binding to the URE region. P<it>styA </it>full activity in inducing conditions is achieved when StyR-P and IHF bind to site STY2 and to the URE, respectively. Under catabolite repression conditions, StyR-P binds the STY1 site, replacing IHF at the URE region. StyR-P bound to both STY1 and STY2 sites oligomerizes, likely promoting the formation of a DNA loop that closes the promoter in a repressed conformation. We found that StyR and IHF protein levels did not change in catabolite repression conditions, implying that P<it>styA </it>repression is achieved through an increase in the StyR-P/StyR ratio.</p> <p>Conclusion</p> <p>We propose a model according to which the activity of the P<it>styA </it>promoter is determined by conformational changes. An open conformation is operative in inducing conditions when StyR-P is bound to STY2 site and IHF to the URE. Under catabolite repression conditions StyR-P cellular levels would increase, displacing IHF from the URE and closing the promoter in a repressed conformation. The balance between the open and the closed promoter conformation would determine a fine modulation of the promoter activity. Since StyR and IHF protein levels do not vary in the different conditions, the key-factor regulating P<it>styA </it>catabolite repression is likely the kinase activity of the StyR-cognate sensor protein StyS.</p

The Quorum-Sensing Negative Regulator RsaL of Pseudomonas aeruginosa Binds to the lasI Promoter

A mutation in the rsaL gene of Pseudomonas aeruginosa produces dramatically higher amounts of N-acyl homoserine lactone with respect to the wild type, highlighting the key role of this negative regulator in controlling quorum sensing (QS) in this opportunistic pathogen. The DNA binding site of the RsaL protein on the rsaL-lasI bidirectional promoter partially overlaps the binding site of the LasR protein, consistent with the hypothesis that RsaL and LasR could be in binding competition on this promoter. This is the first direct demonstration that RsaL acts as a QS negative regulator by binding to the lasI promoter

Dual Role of Response Regulator StyR in Styrene Catabolism Regulation

In Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and repressed by the addition of preferred carbon sources. PstyA is regulated by the StyS/StyR two-component system. The integration host factor (IHF) also plays a positive role in PstyA regulation. Three distinct StyR binding sites, which have different affinities for this response regulator, have been characterized on PstyA. The high-affinity StyR binding site (STY2) is necessary for promoter activity. The DNA region upstream of STY2 contains a lower-affinity StyR binding site, STY1, that partially overlaps the IHF binding site. Deletion of this region, designated URE (upstream regulatory element), has a dual effect on the PstyA promoter, decreasing the styrene-dependent activity and partially relieving the glucose repression. The lowest-affinity StyR binding site (STY3) is located downstream of the transcription start point. Deletion of the URE region and inactivation of the STY3 site completely abolished glucose-mediated repression of PstyA. In the proposed model StyR can act either as an activator or as a repressor, depending on which sites it occupies in the different growth conditions. We suggest that the cellular levels of phosphorylated StyR, as determined by StyS sensor kinase activity, and the interplay of this molecule with IHF modulate the activity of the promoter in different growth conditions

Integration host factor is essential for the optimal expression of the styABCD operon in Pseudomonas fluorescens ST

The StyS/StyR two-component regulatory system of Pseudomonas fluorescens ST controls the expression of the styABCD operon coding for the styrene degradation upper pathway. In a previous work we showed that the promoter of the catabolic operon (PstYA) is induced by styrene and repressed to differing extents by organic acids or carbohydrates. In order to study the mechanisms controlling the expression of this operon, we performed a functional analysis on 5' deletions of PstyA by the use of a promoter-probe system. These studies demonstrated that a palindromic region (sty box), located from nucleotides -52 to -37 with respect to the transcriptional start point is essential for PstyA activity. Moreover, additional regulatory regions involved in the modulation of PstyA activity were found along the promoter sequence. In particular, deletion of a putative StyR binding site, homologous to the 3' half of the sty box and located upstream of this box, resulted in 65% reduction of the induction level of the reporter gene. Additionally, we performed bandshift assays with a DNA probe corresponding to PstyA and protein crude extracts from P. fluorescens ST, using specific DNA fragments as competitors. In these experiments we demonstrated that IHF binds an AT-rich region located upstream of the sty box. On the basis of this finding, coupled with the results obtained with PstYA functional analysis, we suggest that the role of the IHF-mediated DNA bend is to bring closer, in an overlapping position, the upstream StyR putative binding site and the downstream sty box, and that the formed complex enhances transcription. (C) 2002 Editions scientifiques et medicales Elsevier SAS. All rights reserved

Occurrence and properties of glutathione S-transferases in phenol-degrading Pseudomonas strains

Pseudomonas sp. strains, able to degrade aromatic compounds such as phenol, were chosen to investigate the occurrence and characteristics of glutathione S-transferases (GSTs). Affinity chromatography purification showed the presence of at least one GST in each studied strain. The purified proteins exhibited a great variety in the N-terminal sequences and different enzyme activities with the standard GST substrates tested. Two Pseudomonas strains, M1 and CF600, were chosen to investigate the GST activities under different growth conditions. Therefore, cells were grown either on phenol or on different nonaromatic carbon sources in the presence and absence of increasing phenol concentrations. In strain M1 a strong correlation between the activities of the catechol 1,2-dioxygenase and GST was observed in all the tested conditions. Moreover, growth on different organic acids also affected GST activity levels, with a negative correlation with the specific growth rate determined by each substrate. These results suggest a possible function of GST as a response to specific metabolic conditions determined by phenol toxicity and/or catabolism and the metabolic status of the cells. The same experiments performed with the CF600 strain did not show induction of GST activity in any of the tested conditions, indicating that GST_CF600 probably has a different role in cell metabolism. Native gel electrophoresis gave indications that GST dimerization could be an important process in the modulation of GST activity. 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

Enfisema polmonare progressivo successivo ad inalazione di arachide senza rilievo di persistenza del corpo estraneo. Progressive pulmonary emphysema after inhalation of peanuts without persistence of the foreign body

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Biosensor for N-3-oxo-dodecanoyl-homoserin lactone detection

La presente invenzione concerne la costruzione di un biosensore per la rilevazione di 3OC12-HSL, il principale segnale di quorum sensing (QS) prodotto da Pseudomonas aeruginosa, in campioni di laboratorio ed in campioni clinici, e per lo screening di inibitori del sistema 3OC12-HSL-dipendente di Pseudomonas aeruginosa

Styrene-catabolism regulation in Pseudomonas fluorescens: phosphorylation of StyR induces dimerization and cooperative DNA-binding

Styrene is an important chemical extensively used in the petrochemical and polymer industries. In Pseudomonas fluorescens ST, styrene metabolism is controlled by a two-component regulatory system, very uncommon in the degradation of aromatic com- pounds. The two-component regulatory proteins StyS and StyR regulate the expression of the styABCD operon, which codes for styrene degradation. StyS corresponds to the sensor kinase and StyR to the response regulator, which is essential for the activation of PstyA, the promoter of the catabolic operon. In two-component systems, the response regulator is phosphorylated by the cognate sensor kinase. Phosphorylation activates the response regulator, inducing DNA-binding. The mechanism underlying this activation hasbeenreportedonlyforaveryfewresponseregulators.Here,theeffectofphosphorylationontheoligomericstateandonthe DNA-binding properties of StyR has been investigated. Phosphorylation induces dimerization of StyR, the affinity of dimeric StyR for the target DNA is higher than that of the monomer, moreover dimeric StyR binding to the DNA target is cooperative. Fur- thermore, StyR oligomerization may be driven by the DNA target. This is the first direct demonstration that StyR response reg- ulator binds to the PstyA promoter