Transkriptionskontrolle des nuoA-N Operons (NADH-Dehydrogenase I) von Escherichia coli

Das Elektronentransportsystem von E. coli enthält zwei verschiedene NADH-Dehydrogenasen. Die NADH-DehydrogenaseI (nuoA-N) koppelt im Gegensatz zur NADH-DehydrogenaseII die Oxidation von NADH an eine Protonentranslokation und trägt zur Energiekonservierung bei. Die NADH-DehydrogenaseI wird über die Promotoren P1 und P2 exprimiert und besitzt mehrere Bindestellen für verschiedene Regulatoren.Die separate Klonierung der Promotoren, lacZ-Fusionen, Inaktivierung von Transkriptionsfaktoren, sowie die Nutzung mutierter Regulatorbindestellen in vivo zeigen, dass P1 im wesentlichen die Expressionshöhe bestimmt und ist unter aeroben und anaeroben Bedingungen aktiv. P2 trägt in wesentlich geringerem Maße als P1 zur Expression des Enzyms bei. Er ist stark abhängig von ArcA und IHF. Beide Promotoren wirken nicht additiv.Unter anaeroben Bedingungen wird die Transkription von nuo durch das Zweikomponenten-System ArcB/A reprimiert. ArcA bindet unabhängig und mit unterschiedlicher Affinität an die beiden Bindestellen arc1 und arc2. Von den 8 ArcA-Konsensussequenzen führen nur Mutationen der Konsensussequenzen arc1ab in vitro zu verminderter Bindungsaffinität von ArcA an die Bindestelle arc1. Dieselben führen in vivo unter anaeroben Bedingungen zur Derepression des Promotors P1 bzw. P1+P2. Unter aeroben Bedingungen zeigen nur Mutationen in arc2 eine Derepression, die nicht durch ArcA vermittelt wird. Der veröffentliche ArcA-Konsensus scheint deshalb hier in dieser einfachen Form nicht gültig zu sein.

Anaerobic pathways in the Porifera: Strombine dehydrogenase, an opine dehydrogenase, from the sponge Suberites domuncula

The study presented here encompasses identification, analysis and characterization of the strombine dehydrogenase (StDH) from the sponge S. domuncula, on the gene and protein level. StDH is an opine dehydrogenase which is involved in opine production pathways found mainly in marine invertebrates. These anaerobic pathways are regarded as analogues to the classical anaerobic glycolytic pathway (lactate production pathway), which is predominant in vertebrates. The StDH was previously annotated as a tauropine dehydrogenase (TaDH) on the basis of its 68% identity with the TaDH protein from Halichondria japonica. Subsequent enzymatic assays showed that S. domuncula opine dehydrogenase is in fact strombine dehydrogenase which possesses specific characteristics not found in other proteins of the same family. It is described here for the first time the StDH gene in Eukaryotes. Two allelic variants have been identified which are present in the different specimens either as a homozygotic or a heterozygotic. Phylogenetic analyses supported with enzymatic assays indicate that S. domuncula StDH is only distantly related to the opine dehydrogenases from marine invertebrates. StDH showed that the protein is highly specific to glycine and inhibited by the substrate pyruvate. Furthermore, S. domunucla StDH has a dimeric structure (~75 kDa) which is not observed in so far described OpDHs that are monomeric proteins. This enzyme showed similarities to the OCD/mu-cristallyin protein family. Results showed that a sponge StDH is unusual enzyme that belongs to the independent enzyme class. In addition, expression studies revealed that the StDH is down-regulated with aeration. Immunohistology analyses showed high expression of the protein in almost all sponge cells. A strong accumulation of the enzyme was seen around the bacteria indicating that under aerobic conditions the bacteria might metabolize strombine (end product of the reaction). In conclusion, the data documented here shed new light on the anaerobic pathways in marine invertebrates. Potential mutual influences between bacteria and sponge are discussed as well. Hopefully, these results could have a small but important contribution to the better understanding of the evolution in the animal kingdom.