Characterization of the citrate/acetate antiporter CitW of Klebsiella pneumoniae

The genome of Klebsiella pneumoniae contains at least three different genes encoding citrate transporters. Recently, a third and hitherto unknown gene encoding a citrate transport system ( citW) was identified. Escherichia coli transformed with a plasmid expressing citW was able to grow on citrate as sole carbon and energy source, identifying CitW as a citrate carrier. In this report, we provide evidence that further specifies CitW as a Na(+)-independent citrate/citrate and citrate/acetate exchanger. Kinetic analysis of citrate uptake at different pH values identified Hcitrate(2-) as the transported citrate species, with a K(m) of 25 microM. Since citW is expressed under anoxic conditions and acetate is the main end-product of citrate fermentation in K. pneumoniae, citrate/acetate exchange might be its in vivo function. Sequence similarity searches identified CitW (454 amino acids, 48.15 kDa) as a member of the 2-hydroxycarboxylate transporter family (TC 2.A.24). The substrate specificity seems to partially contradict this phylogenetic classification, but appears logical with respect to the putative functional role of CitW in the citrate fermentation pathway of K. pneumoniae.     

The Na+-dependent citrate carrier of Klebsiella pneumoniae: high-level expression and site-directed mutagenesis of asparagine-185 and glutamate-194

The Na+-dependent citrate carrier of Klebsiella pneumoniae (CitS) is a member of the 2-hydroxycarboxylate transporter family. Within the highly conserved helix Vb region, Asn-185 of CitS was mutated to Val and Glu-194 was mutated to Gln. The wild-type and mutant proteins were synthesised in Escherichia coli DH5alpha or C43(DE3) as biotinylated or His-tagged CitS-fusions, respectively. The synthesis and purification procedure yielded 6.5 mg pure CitS per litre culture. The fusion proteins were characterised with E. coli cell suspensions or after reconstitution of the purified enzymes into proteoliposomes. The E194Q mutation had almost no effect on the kinetics of Na+ or citrate transport. In contrast, aberrant citrate transport kinetics were found for the N185V mutant. The apparent K(m) value for the citrate species H-citrate(2-) was increased about nine-fold, whereas the apparent Vmax value and the effect of Na+ on the transport kinetics were comparable to the wild-type. Asn-185 of CitS appears therefore to participate in the binding of H-citrate(2-).     

A Quantitative and Dynamic Model of the Arabidopsis Flowering Time Gene Regulatory Network

Various environmental signals integrate into a network of floral regulatory genes leading to the final decision on when to flower. Although a wealth of qualitative knowledge is available on how flowering time genes regulate each other, only a few studies incorporated this knowledge into predictive models. Such models are invaluable as they enable to investigate how various types of inputs are combined to give a quantitative readout. To investigate the effect of gene expression disturbances on flowering time, we developed a dynamic model for the regulation of flowering time in Arabidopsis thaliana. Model parameters were estimated based on expression time-courses for relevant genes, and a consistent set of flowering times for plants of various genetic backgrounds. Validation was performed by predicting changes in expression level in mutant backgrounds and comparing these predictions with independent expression data, and by comparison of predicted and experimental flowering times for several double mutants. Remarkably, the model predicts that a disturbance in a particular gene has not necessarily the largest impact on directly connected genes. For example, the model predicts that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) mutation has a larger impact on APETALA1 (AP1), which is not directly regulated by SOC1, compared to its effect on LEAFY (LFY) which is under direct control of SOC1. This was confirmed by expression data. Another model prediction involves the importance of cooperativity in the regulation of APETALA1 (AP1) by LFY, a prediction supported by experimental evidence. Concluding, our model for flowering time gene regulation enables to address how different quantitative inputs are combined into one quantitative output, flowering time.     

Quelques remarques sur l'holisme dans la pensée grecque

Summary The conception of the world with Greek thinkers generally differs from contemporary thought in the idea of the limitation of the world whose essence manifests itself to speculative thinking. The cosmology which culminates inPlato's Timaios attributes to the whole of being the properties of finiteness, uniqueness, life and perfection.Plato's holism subsumes inanimate and living nature under the idea of cosmic life, the parts of which are constituted by life-phenomena given in experience. His world-conception, constructed from within and intuitively, which ranges phenomena in descending order, is opposed to the atomism ofDemocritos which reduces the higher structures to the one equal material of all things. This opposition manifests itself in all essential points: toDemocritos there are worlds in infinite number, only accumulations of atoms are real, uniqueness and perfection are lacking as well as the idea of Life as a central reality.Modern research foregoes the attempt to determine the whole speculatively, but holistic and atomistic ways of thinking, after having been dogmatically applied by the Greeks, continue to exist as opposed types of mental attitude. Contemporary holism may be empirical and limited in application, but it is directed by the same intuition of life articulating itself organically as wasPlato's. It would be incorrect, however, to characterize holism as synthetical, since all synthetics is based upon analysis. The bounds of synthesis are never fixed ones, nor does it ever determine essence. Holism's way of subsuming single life-phenomena under more comprehensive wholes is an intuitional one, the connections which it creates between phenomena are not external ones, but apprehended intuitively in analogy to inner experience.—In distinction to atomism, which only admits as real atoms and their mutual relations, holism poses groups of unities, classified in grades, the higher of which dominate the lower, without debasing them to elements. As metaphysics is concerned to discover a connection between Nature and human life, it must be highly interested in the empirical demonstration, such as holism offers, of connections of life, surpassing individual being.

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Zusammenfassung Die griechische Weltauffassung unterscheidet sich van der modernen durch den Gedanken der Begrenztheit des Weltalls, das sich dem spekulativen Blick seinem Wesen nach erschliesst. Die inPlaton's Timaios gipfelnde Kosmologie bestimmt das Weltall nach den Wesensgesichtspunkten der Endlichkeit, der Einzigkeit, der Lebendigkeit und der Vollkommenheit.Platon's Holismus bezieht die leblose und die lebende Natur unter die Idee des Lebens des Kosmos, dessen Teile die in der Erfahrung gegebenen Lebenserscheinungen sind. Sein von innen her und intuitiv aufgebautes Weltbild, das die Erscheinungen von oben herab hierarchisch ordnet, tritt dem AtomismusDemokrits gegenüber, der in umgekehrtem Verfahren die höheren Strukturen auf den einen Baustoff aller Dinge reduziert. Der Gegensatz zum Holismus offenbart sich in allen Bestimmungen: fürDemokrit sind der Kosmoi unbegrenzt viele, es gibt nur Ansammlungen von Atomen, es fehlen Einzigkeit und Vollkommenheit, sowie ein Begriff des Lebens als des zentralen Geschehens.Die neuzeitliche Forschung verzichtet auf die spekulativen Bestimmungen des Ganzen, aber der bei den Griechen dogmatische Gegensatz holistischer und atomistischer Betrachtungsweise lebt fort als Gegensatz geistiger Einstellung. Der neuzeitliche Holismus ist ein empirischer, beschränkter, aber bei ihm liegt dieselbe Intuition des sich organisch gestaltenden Gesamtlebens zu Grunde wei beiPlaton. Nur wäre es, unrichtig, diesen Holismus als synthetisch zu bezeichnen. Synthetisches Denken erhebt sich immer auf analytischem Boden. Jede Synthese hat eine offene Grenze. Die Art aber, wie der Holismus die vereinzelten Lebenserscheinungen auf umfassendere Ganzheiten bezieht, ist eine intuitive. Das Band der Erscheinungen wird nicht durch äusseres Beziehen gestiftet, sondern geschaut nach Analogie einer inneren Erfahrung.Im Gegensatz zum Atomismus, der als real nur die Atome und deren Verhältnisse gelten lässt, erkennt der Holismus stufenartig aufgebaute Einheitsarten an, in denen die höheren die niedrigeren beherrschen, ohne sie zu Elementen herabzudrücken. Die Metaphysik, auf der Suche nach dem Zusammenhang zwischen dem Menschenleben und der Natur ist an der empirischen Aufweisung das Einzelne übersteigender Lebensverbände wie sie der Holismus zu Tage fordert aufs Höchste interessiert.

     

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris Wg2.

An endopeptidase has been purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that includes diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography over an anion-exchange column and a hydrophobic-interaction column. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular mass of the purified enzyme of 70,000 Da. The endopeptidase can degrade several oligopeptides into various tetra-, tri-, and dipeptides. The endopeptidase has no aminopeptidase, carboxypeptidase, dipeptidase, or tripeptidase activity. It is optimally active at pH 6.0 to 6.5 and in the temperature range of 30 to 38 degrees C. The enzyme is inactivated by the chemical agents 1,10-phenanthroline, ethylenedinitrilotetraacetate, beta-mercaptoethanol, and phenylmethylsulfonyl fluoride and is inhibited by Cu2+ and Zn2+. The ethylenedinitrilotetraacetate- or 1,10-phenanthroline-treated enzyme can be reactivated by Co2+. Immunoblotting with specific antibodies raised against the purified endopeptidase indicated that the enzyme is also present in other Lactococcus spp., as well as in Lactobacillus spp. and Streptococcus salivarius subsp. thermophilus.