Biosynthesis and regulation of cyclic lipopeptides in Pseudomonas fluorescens

Cyclic lipopeptides (CLPs) are surfactant and antibiotic metabolites produced by a variety of bacterial genera. For the genus Pseudomonas, many structurally different CLPs have been identified. CLPs play an important role in surface motility of Pseudomonas strains, but also in virulence and attachment/detachment to and from surfaces. In this Ph.D. thesis project, two new CLP biosynthesis clusters were identified in Pseudomonas fluorescens and fully sequenced. In P. fluorescens strain SBW25, the viscosin biosynthesis cluster was identified by bioinformatic analyses of the genome followed by genetic and chemical analyses. For P. fluorescens strain SS101, the genes for massetolide biosynthesis were identified via random mutagenesis followed by cloning, sequencing and chemical analyses. Biosynthesis of viscosin and massetolide is governed by three nonribosomal peptide synthetase (NRPS) genes, designated viscABC and massABC, respectively. The viscosin and massetolide biosynthesis gene clusters are very similar, but different from CLP gene clusters described for other Pseudomonas as the viscA and massA genes are physically disconnected from viscBC and massBC, respectively. Viscosin differs from massetolide A only at position number four in the peptide moiety, which is a valine in viscosin and an isoleucine in massetolide A. Because of the modular structure of the NRPSs and the co-linearity of the assembly process, transfer of the mass genes of strain SS101 into strain SBW25 resulted in the production of both massetolide A and viscosin, demonstrating that the assembly line for CLP biosynthesis in Pseudomonas can be altered leading to the production of non-native products. Compared to the understanding of CLP biosynthesis, not so much is known about the regulation. This thesis shows that the GacA/GacS two-component system regulates massetolide and viscosin biosynthesis in strains SS101 and SBW25, respectively. No indications were found that massetolide or viscosin biosynthesis is regulated by quorum sensing via N-acylhomoserine lactones. Site-directed mutagenesis of the LuxR-type regulator genes luxR-vA and luxR-vBC flanking the viscosin biosynthesis cluster resulted in a loss of viscosin production, indicating that both LuxR-type transcriptional regulators are important for viscosin biosynthesis in strain SBW25. Phylogenetic analyses further suggested that these LuxR-type transcriptional regulators do not contain the autoinducerbinding domain found for the quorum sensing-associated LuxR regulator in Vibrio fischeri. Instead, the LuxR-type regulator genes flanking the massetolide and viscosin biosynthesis genes are closely related to the LuxR-type regulators identified for syringomycin/ syringopeptin biosynthesis and appear to belong to a separate LuxR-type regulator subfamily, different from the autonomous effector domain protein GerE. Via random mutagenesis and subsequent screening for massetolide-deficient mutants, also other regulator genes were identified including clpP. ClpP is a serine protease that plays a crucial role in intracellular refolding and degradation of proteins, which is an essential process for the viability of cells. ClpP was shown to affect transcription of luxR-mA, thereby regulating transcription of the massetolide biosynthesis genes. Results further suggested that, at the transcriptional level, ClpPmediated regulation of massetolide biosynthesis operates independently from regulation by the GacA/GacS two-component system. In conclusion, the results of this thesis led to the identification of several genes and previously unknown pathways involved in regulation of CLP biosynthesis and highlighted the complexity of the signaling cascades underlying CLP biosynthesis in Pseudomonas. CLPs have diverse functions for the producing bacterial strains, including a role in motility, biofilm formation, antimicrobial activity and virulence. Also in establishment and persistence in plant environments, CLPs were shown to confer a competitive advantage. A new function of CLPs, identified in a collaboration with Mark Mazzola (USDA) and presented in this thesis, is their protective effects against predation by protozoa. In vitro assays showed that both massetolide and viscosin can lyse the trophozoites of Naeglaria americana and that wild type strains SS101 and SBW25 were substantially less sensitive to protozoan grazing than their CLP-deficient mutants. Also in soil containing N. americana, population densities of wild type strains SS101 and SBW25 were significantly higher compared to the massetolide and viscosin-deficient mutants, showing that CLP production confers a competitive advantage in survival in complex environments. Moreover, transcription of the CLP-biosynthesis genes increased significantly upon protozoan grazing, indicating that the Pseudomonas strains sense the protozoa and react by producing CLPs as defense compounds. Which signal triggers the induction of the CLP biosynthesis genes is not known yet and currently under investigation. Based on these results, we postulate that CLPs are an important component of the preingestional defense mechanisms of bacteria against protozoan predation, not only due to their lytic effects on protozoa, but also because CLPs contribute to evasion of protozoan grazing via altered cell surface properties, swimming and swarming, and microcolony and biofilm formation. <br/

Random matrices with external source and KP τ\tau functions

In this paper we prove that the partition function in the random matrix model with external source is a KP $\tau$ function.Comment: 12 pages, title change

Application of Chelex Standard Beads in Integrated Morphometrical and X-Ray Microanalysis

Chelex ion exchange beads loaded with a known amount (18.3% weight percentage (w/w)) of platinum, have been co-embedded with a mouse peritoneal cell population. To establish the influence of the various deconvolution methods applied, upon the platinum concentration in cytoplasmic granules and erythrocytes these cross-sectioned beads are used as a standard. It is concluded that irrespective of the deconvolution method chosen 1) the Pt concentration inside the particles is identical when the particles and the co-embedded Chelex Pt standard, are analysed strictly under the same instrumental conditions 2) the Pt concentration outside the particle is zero, or virtually zero when that element is absent there (erythrocyte surrounded by Epon) 3) the Pt concentration outside the particle in the surrounding cytoplasm was identical (when the element Pt was present there). The information about the elemental concentration obtained by point analysis in the STEM-mode in homogeneous objects was compared with the mean value obtained by the reduced raster method. The ratio between these values were constant. As an example of a heteromorphic, heterogeneous cell organelle population, the application of the method of integrated morphometrical and chemical (X-ray) analysis is demonstrated on lysosomes within a single human liver parenchymal cell, containing iron and cerium. It was shown that the cerium concentration (from the cytochemical reaction to detect acid phosphatase activity in lysosomes) was rather homogeneously distributed over the small population and in the individual lysosomes. The iron distribution was very inhomogeneous, both in its distribution over the lysosomal area, and among the lysosomes in the population

De Zorgrelatie ontrafeld; Conceptueel onderzoek naar de zorgrelatie tussen jeugdige en professional in de jeugdsector in Nederland vanuit zorgethisch perspectief.

Deze thesis is een literatuuronderzoek, gericht op de persoonlijke zorgrelatie in de jeugd-sector, vanuit zorgethisch perspectief. Het doel van dit onderzoek is de uitwerking van concepten beschrijven, die het begrip zorgrelatie en hoe deze tot stand kan komen, kunnen verhelderen. Deze uitwerking vindt plaats aan de hand van zorgethische literatuur en empirisch kwalitatief onderzoek. Kennis van deze concepten en verkregen inzichten kunnen in de toekomst mogelijk een bijdrage leveren aan ontwikkelingen in de jeugdzorgsector en daarmee indirect aan goede zorg. De concepten ‘aansluiten’, ‘erkenning’ en ‘vertrouwen’ blijken essentieel te zijn in het aangaan van een goede zorgrelatie met jeugdigen. De uitwerking van de concepten op basis van zorgethische literatuur laat een verdieping, explicitering en onderlinge verwevenheid van deze processen zien. De concepten krijgen hun betekenis in een juist begrip en toepassing ervan in de praktijk. Concepten worden op diverse manieren ingevuld met als risico vervlakking, ongewenste reductie en communicatiestoornissen in de zorgrelatie. Hoewel wederkerigheid als centraal begrip door alle concepten heen zichtbaar wordt, is de actieve rol van de jeugdige niet nadrukkelijk uitgewerkt in de bestudeerde literatuur. De zorgrelatie vindt bovendien plaats in parallelle processen en ketens van relaties. Zowel individuele kenmerken, interactie tussen alle betrokkenen als de bredere context, zijn van invloed op de zorgrelatie. Specifieke kenmerken van jeugdigen in de jeugdsector compliceren de zorgrelatie nog verder. De beschrijving van de concepten in hoofdstuk drie geeft echter inspiratie en handvatten om die uitdaging aan te gaan en invloed te kunnen hebben op de situatie. Dit onderzoek maakt duidelijk dat naast vaktechnische competenties ook kennis en toepassing van de uitwerking van deze concepten, ethisch bewustzijn en reflectie kunnen helpen een goede zorgrelatie tot stand te brengen

Правове регулювання повторного шлюбу у римському та ранньовізантійському праві

В статті досліджується традиційна проблема інституту повторного шлюбу в контексті його еволюції на засадах античних та християнських цінностей.В статье исследуется традиционная проблема института повторного брака в контексте его эволюции на принципах античных и христианских ценностей.In the article the traditional problem of institute of the deuterogamy is probed in the context of its evolution on principles of ancient and christian values

What is a crystal?

Almost 25 years have passed since Shechtman discovered quasicrystals, and 15 years since the Commission on Aperiodic Crystals of the International Union of Crystallography put forth a provisional definition of the term crystal to mean ``any solid having an essentially discrete diffraction diagram.'' Have we learned enough about crystallinity in the last 25 years, or do we need more time to explore additional physical systems? There is much confusion and contradiction in the literature in using the term crystal. Are we ready now to propose a permanent definition for crystal to be used by all? I argue that time has come to put a sense of order in all the confusion.Comment: Submitted to Zeitschrift fuer Kristallographi