1,720,993 research outputs found
Expression of pyoverdine genes in Pseudomonas aeruginosa PAO1: intracellular levels of PvdS
No full textIn response to iron-limitation, Pseudomonas aeruginosa PAO1 produces the fluorescent siderophore pyoverdine. Transcription of pyoverdine biosynthetic (pvd) genes depends on the extracytoplasmic function (ECF) sigma factor PvdS, which is negatively regulated by the Fur repressor. An additional level of regulation of pvd genes relies upon the surface signalling mechanism that involves the ferripyoverdine outer membrane receptor FpvA and the pyoverdine molecule itself. In the absence of ferripyoverdine, the activity of PvdS is antagonized by the antisigma factor FpvR. Binding of ferripyoverdine to FpvA initiates a signal transduction cascade that involves FpvR and leads to the transcription of several virulence genes (e.g. pvd, aprA, prpL and toxA) by PvdS-dependent RNA polymerase. To evaluate the competition between PvdS and the major sigma subunit RpoD (s70) for RNA polymerase binding, we have determined the intracellular levels of PvdS and RpoD in iron-starved P. aeruginosa PAO1 cells during the exponential and stationary phase. By means of quantitative immunoblot analysis, we found that PvdS attains 60% of RpoD levels in exponential phase, while it drastically decreases at the stationary phase. PvdS also exhibits reduced stability in the presence of an excess of iron in vivo. Experiments are in progress to investigate the effect surface signalling components on PvdS activity and stability
Transcriptional regulation of pseudobactin synthesis in the plant growth-promoting Pseudomonas B10.
No full textWe have investigated the iron-dependent regulation of the psbA gene, encoding the enzyme L-ornithine N-5-oxygenase in the rhizobacterium Pseudomonas B10. We have cloned and characterized a Pseudomonas B10 gene, designated psbS, required for psbA expression. PsbS is endowed with structural and functional features of extracytoplasmatic function (ECF) sigma factors, and is closely related to the iron starvation sigmas PvdS, PbrA, and PfrI, which mediate the iron-repressible expression of pseudobactin biosynthesis genes in different Pseudomonas species. Expression of psbA was found to be indirectly controlled by Fur, which abrogates psbS transcription in the presence of sufficient iron
Pseudobactin biogenesis in the plant growth-promoting rhizobacterium Pseudomonas strain B10: Identification and functional analysis of the L-Ornithine N-5-oxygenase (psbA) gene
No full textPseudobactin(B10), the fluorescent siderophore produced by the rhizobacterium Pseudomonas strain B10, contains the hydroxamate ligand D-N-5-hydroxyornithine (D-N-5-OH-Orn). We cloned the L-Orn N-5-oxygenase (psbA) gene from a genomic library of Pseudomonas strain B10 and demonstrated that PsbA is involved in the conversion of L-Orn to its N-5-OH derivative. PsbA shows significant similarity to microbial omega-amino acid hydroxylases containing flavin adenine dinucleotide and NADP cofactor-binding sites and the FATGY signature of the putative substrate recognition pocket. The psbA gene is monocistronic, and its transcription is negatively controlled by iron. A site-specific psbA mutant of Pseudomonas strain B10 was biochemically complemented with the precursor L-N-5-OH-Orn, suggesting that L-Orn is hydroxylated before conversion to the D isomer. The L-Orn N-5-hydroxylase-defective mutants of Pseudomonas strain B10 and Pseudomonas aeruginosa PAO1 were much less effective than the parental strains in suppressing the growth of the phytopathogen Erwinia carotovora in iron-poor medium. The extent of in vitro inhibition of E. carotovora was strictly iron dependent and directly correlated with the amount of released siderophores. These data strengthen the role of fluorescent siderophores in biocontrol of deleterious rhizomicroorganisms
Different responses of pyoverdine genes to autoinduction in Pseudomonas aeruginosa and the group Pseudomonas fluorescens-Pseudomonas putida
No full textWe investigated the regulation of the psbA and pvd4 pyoverdine biosynthesis genes, which encode the L-ornithine N-5-oxygenase homologues in Pseudomonas strain 1310 and Pseudomonas aeruginosa PAO1, respectively. We demonstrate that pyoverdine(B10), as the end product of its biosynthetic pathway, is a key participant of the control circuit regulating its own production in Pseudomonas strain B10. In P. aeruginosa PAO1, however, Pyoverdine(PAO1), has no apparent role in the positive regulation of the pvd4 gene
Pyoverdine regulation: a matter of competition between sigma and antisigma factors
No full textTranscription of pyoverdine biosynthetic (pvd) genes in Pseudomonas aeruginosa depends on the ECF sigma factor PvdS. The transmembrane sensor FpvR controls PvdS activity, acting as an antisigma of PvdS in the absence of a signal transmitted from the (ferri)pyoverdine-loaded FpvA receptor. We studied i) the competition between PvdS and RpoD, ii) the effect of FpvR on PvdS stability, and iii) the effect of AlgQ on PvdS-dependent transcription. PvdS attains ca. 60% of RpoD levels in exponentially growing cells, likely reflecting the high PvdS-RNA polymerase (RNAP) demand for recognition of multiple promoters. This explains only in part the changes in transcription pattern, since the major determinant is the ratio between different RNAP holoenzymes. RNAP purification from iron starved cells shows that the PvdS-RNAP complex is poorly represented compared with vegetative holoenzyme; copurification of PvdS-RNAP could only be achieved upon PvdS overexpression. Hence, high levels of PvdS possibly compensate for its weak affinity for RNAP. No significant differences in PvdS levels were observed between wild-type and pyoverdine signalling mutants, suggesting that in the absence of signaling part of the PvdS pool is sequestered by FpvR, but not degraded. Next, we investigated the effect of AlgQ, the Escherichia coli Rsd othologue, on pyoverdine regulation. AlgQ is a global regulatory protein which activates alginate, ppGpp, and polyP synthesis through a cascade involving nucleoside diphosphate kinase (Ndk). AlgQ was also shown to interact with region 4 of RpoD. We demonstrate that AlgQ modulates the transcription of pvd genes by an Ndk- and PvdS-independent mechanisms. Increased intracellular concentration of RpoD mimicked the DalgQ phenotype, whereas PvdS overexpression suppressed the algQ mutation. E. coli rsd could partly substitute for algQ in transcriptional modulation of pvd genes. Thus, AlgQ acts as an antisigma for RpoD, eliciting RNAP recruitment by PvdS and transcription initiation at pvd promoters. Collectively, these data indicate that PvdS activity is finely controlled by two antisigma factors capable of directly antagonizing or indirectly promoting PvdS binding to RNA
Sub-cellular and molecular topology of L-ornithine Nd- oxygenase (PvdA) in Pseudomonas aeruginosa PAO1
No full textPyoverdines (PVDs), the fluorescent siderophores of rRNA group I Pseudomonas spp., are composed of an invariant dihydroxyquinoline chromophore linked to a variable peptide chain. Both the chromophore and the peptide backbone are assembled by non-ribosomal peptide synthetases (NRPSs). The peptide chain of Pseudomonas aeruginosa PAO1 PVD results from the condensation and partial cyclization of eight amino acids. In the complex scenario of PVDPAO1 biogenesis, a primary precursor-generating reaction is driven by L-ornithine Nd-oxygenase (PvdA), an w–aminoacid monooxygenase (EC 1.14.13.-) encoded by the pvdA gene, catalysing the Nd-hydroxylation of L-ornithine. We investigated the molecular and sub-cellular topology of PvdA. Membrane topogenic determinants of PvdA were identified by computational analysis and exploited to construct a series of translational fusions with alkaline phosphatase (PhoA). The inferred topological model suggests a single N-terminal transmembrane domain anchored to the inner membrane, and a long hydrophilic loop exposed to the cytosol. The model was corroborated by mapping PvdA in the membrane compartment with anti-PvdA monoclonal antibodies (MAbs) in immunoelectron microscopy studies. Western blot analysis of membrane fractions confirmed PvdA location within the inner membrane. Binding to the membrane was confirmed by an in vitro transcription/translation assay and characterised as a FAD/NAD(P)H-assisted process. We speculate that the membrane-associated state of PvdA would facilitate oxygen and NAD(P)H recruitment for the oxygenase reaction while providing an anchoring site for the pyoverdine biosynthetic multienzyme complex, likely facilitating an export-coupled biosynthetic proces
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
SEARCHING FOR NEW GUT FRIENDS: IN VITRO CHARACTERIZATION OF THE PROBIOTIC PROPERTIES OF HUMAN COLONIC ISOLATED ESCHERICHIA COLI.
No full textIntroduction. Inflammatory bowel diseases (IBDs) are chronic inflammatory condition of the gastrointestinal tract (GI), and include ulcerative colitis (UC) and Crohn’s disease (CD) both characterized by an altered mucosa structure, systemic biochemical disorders due to intestinal altered permeability and extended modification of the gut microbial composition. Patients present reduced microbial diversity often in combination with a lower abundance of obligate anaerobic bacteria and an expansion of facultative anaerobic bacteria, particularly from the Proteobacteria phylum. Indeed, stable colonization by Escherichia coli pathotypes (e.g. Adherent Invasive E. coli (AIEC) or E. coli belonging to the B2 phylogroup) is frequently observed in patients suffering from IBDs. Then, the replacement of E. coli pathotypes with commensal strains is considered a valuable approach for the treatment of IBDs. Specific formulations of probiotics were shown to be effective if administrated in conjunction with standard therapies in patients suffering from IBDs and one of the most used probiotic is the E. coli strain Nissle 1917 (EcN). Although EcN possesses all the characteristics to be the ideal probiotic, the results in terms of contributing to/maintaining the remission of inflammatory diseases as well as preventing the relapse of the diseases are extremely variable affecting its efficacious applications. Hence, this project aimed at selecting several heterogeneous E. coli isolates collected from the colonic mucosa of healthy subjects to develop a new probiotic product enriched in commensal strains beneficial for IBD patients. Materials and Methods. From our previously described E. coli collection, genotypically different isolates sharing common phenotypic features were assayed for their probiotic properties including fitness-associated factors, such as (i) metabolic requirements (competitive capability to metabolize simple sugars and dietary proteins in comparison to different E. coli pathotypes), (ii) production of microcins and colicines and (iii) evaluation of competitive planktonic and sessile growth against selected E. coli pathotypes. EcN was used as control. Results. Commensal E. coli isolates can be classified as faster or slower growing depending on the sugar metabolized in comparison to EcN. As EcN, 4 isolates were able to produce and release thermolabile molecules showing inhibitory activity against different E. coli pathotypes. Competitive planktonic and sessile growth indicated the capability of commensal E. coli isolates to restrict E. coli pathotypes growth. Conclusions. Our in vitro characterization demonstrates that intra-species competition represents an efficient strategy to reduce the burden of E. coli pathotypes. This feature could help the development of new E. coli-based probiotic formulations whose efficacy should be tested to improve the quality of life of patients suffering from IBDs
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
- …
