Heterotrophic growth of the cyanobacterium Anabaena (Nostoc) sp. strain PCC7120 and its dependence on a functional cox1 locus encoding cytochrome c oxidase

Cyanobakterien sind Prokaryoten, welche oxygene Photosynthese betreiben. Dennoch können alle Cyanobakterien im Dunkeln atmen. Im Gegensatz zu photosynthetischen Eukaryoten (Algen, höhere Pflanzen), wo Photosynthese und Atmung in Chloroplasten und Mitochondrien räumlich voneinander getrennt stattfinden, laufen diese beiden Prozesse in Cyanobakterien in ein und demselben Kompartiment ab und einige Komponenten werden von beiden Elektronentransportketten verwendet. Die respiratorischen terminalen Oxidasen (RTOs) sind Schlüsselenzyme, da sie nicht direkt an der Photosynthese beteiligt sind, sondern die Elektronen zum terminalen Akzeptor O2 leiten. Respiratorische Oxidasen in Cyanobakterien gehören verschiedenen Klassen an. Wichtige Klassen sind die Häm Kupfer Oxidasen, welche homolog zur Cytochrom c Oxidase in Mitochondrien ist, die Chinon Oxidase, welche homolog zu Cytochrom bd in E. coli ist, sowie die Klasse jener RTOs, die homolog zu Plastid terminalen Oxidasen (Ptox) in Chloroplasten ist. Fast alle Cyanobakterien besitzen zumindest eine Cytochrom c oxidase vom Cytochrom aa3 type. Drei verschiedene Unterklassen von Cytochrom aa3 Typ RTOs sind identifiziert worden: a) echte Cytochrom c Oxidasen, welche die charakteristischen Motive enthalten; b) eine 2. Unterklasse, welche alternative respiratorische terminale Oxidase (ARTO) genannt wird; c) eine Oxidase vom cbb3 Typ (zuerst in Purpurbakterien charakterisiert). Anabaena PCC7120 gehört zu den unverzweigten filamentösen Cyanobakterien, welche zur Zelldifferenzierung befähigt sind. Bei einem Mangel an gebundenem Stickstoff (Nitrat, Nitrit, Ammoniak) können einige vegetative Zellen zu Heterocysten differenzieren (in nicht zufälliger Verteilung innerhalb eines Filaments). PCC7120 hat 5 verschiedene respiratorische terminale Oxidasen: 1 echte Cytochrom c Oxidase (Cox), 2 ARTOs, eine Chinon Oxidase (Qox) und 1 Plastid terminale Oxidase (Ptox). Da die Expression der beiden ARTOs auf die Heterocysten beschränkt zu sein scheint, verbleiben in den vegetativen Zellen 1 Cox, 1 Qox und 1 Ptox. Wenn der cox Locus durch eine Antibiotikumkassette ausgeschaltet wird, verliert der neue Mutantenstamm jegliche Cytochrom c oxidase Aktivität, wie Versuche mit isolierten Membranen aus dem Mutantenstamm und aus Pferdeherz isoliertem Cytochrom c550 bewiesen. Außerdem scheint Cox essentiell für das chemoheterotrophe Wachstum von PCC7120 im Dunkeln zu sein. Trotz bisheriger Untersuchungen, welche diesen Stamm als streng photolithoautotroph klassifizierten, zeigten neue Versuche, dass PCC7120 heterotroph wachsen kann, vorrausgesetzt hohe Mengen an Fruktose wurden dem Medium hinzugesetzt. Offenbar hat PCC7120 eine gewisse Kapazität für heterotrophes Wachstum und Fruktosemetabolismus im Inneren der Zelle. Sehr hohe Konzentrationen im Nährmedium reichen aus, dass Fructose über die Zellwand und die Membranen ins Innere gelangt. Experimente haben gezeigt, dass umso mehr Fruktose eindringt, je höher die Konzentration im äußeren Medium ist. Das Einschleusen des Glukose carrier Gens gtr aus PCC6803 führt zu einem transgenen Stamm, der im Gegensatz zum Wildtyp bereits bei geringeren Fruktosekonzentrationen photoorganoheterotroph wachsen kann. Glukose ist jedoch toxisch für PCC7120gtr+, während der Wildtyp dieser gegenüber tolerant ist. Der Mutantenstamm, in welchem cox ausgeschaltet worden ist, kann nicht mehr chemoheterotroph wachsen. Ähnliche Ergebnisse gibt es auch von Synechocystis PCC6803 und Anabaena variabilis ATCC29413, in welchen Cox ebenfalls notwendig für das chemoheterotrophe Wachstum ist.Cyanobacteria are prokaryotes that perform oxygenic photosynthesis. However, all cyanobacteria respire at dark periods. In eukaryotic phototrophs like algae and higher plants photosynthesis and respiration are separated to the different organells chloroplasts and mitochondria, while in cyanobacteria these two processes occur in the same compartment and many components are shared. The respiratory terminal oxidases (RTOs) are key enzymes because they are not involved directly in photosynthesis but transfer electrons to terminal acceptor O2. Cyanobacterial oxidases belong to different classes. There are the heme copper oxidases homologous to mitochondrial cytochrome c oxidase, the quinol oxidases homologous to cytochrome bd in Escherichia coli and homologues to the plastid terminal oxidases that are insensitive to cyanide. Nearly all cyanobacteria contain at least one heme copper oxidase of the cytochrome aa3 type. There are three different subclasses identified: a) The genuine cytochrome c oxidases, containing the characteristic motives. b) A second class, called alternative respiratory terminal oxidase (ARTO). c) An oxidase of type cbb3 (first characterized in purple bacteria). Anabaena sp. PCC7120 belongs to the unbranched filamentous cyanobacteria that can undergo cell differentiation. If bound nitrogen (nitrate, nitrite, ammonia) is absent some vegetative cells will differentiate into heterocysts with a non random distribution. PCC 7120 has five different respiratory terminal oxidases: one genuine cytochrome c oxidase (Cox), two ARTOs, one quinol oxidase (Qox) and one plastid terminal oxidase (Ptox). As the two ARTOs seem to be restricted to heterocysts, only 1 Cox, 1 Qox and 1 Ptox remain in vegetative cells. When cox is knocked out by an antibiotic resistance cassette the strain loses any cytochrome c oxidase activity as an assay clearly demonstrated with isolated membranes and cytochrome c550 isolated from horse heart. Moreover the Cox seems to be essential for chemoheterotrophic growth in the dark. Despite previous reports, which classify PCC7120 as strictly photolithoautotrophs, experiments revealed that this strain can grow heterotrophically when high amounts of fructose were added to the medium. Evidently PCC7120 has the capacity for heterotrophic growth and fructose metabolism inside its cells, however, heterotrophic growth can only be observed at very high fructose concentrations (at least 50mM) in the medium. Experiments have shown that fructose enters the cells the faster the higher the outside concentration is. Introduction of glucose carrier gene gtr from Synechocystis PCC6803 by conjugation results in a transgenic strain that can grow photoorganoheterotrophically on lower concentrations of fructose than the wild type (WT). However, glucose is toxic for PCC7120gtr+ while the WT is tolerant towards it. The mutant from which cox has been deleted fails to grow chemoheterotrophically. This is analogous to experiments in Synechocystis PCC6803 and Anabaena variabilis ATCC29413, where also the genuine cytochrome c oxidase is essential for chemoheterotrophic growth

Gbx2 and Otx2 Interact with the WD40 Domain of Groucho/Tle Corepressors

One of the earliest organizational decisions in the development of the vertebrate brain is the division of the neural plate into Otx2-positive anterior and Gbx2-positive posterior territories. At the junction of these two expression domains, a local signaling center is formed, known as the midbrain-hindbrain boundary (MHB). This tissue coordinates or “organizes” the development of neighboring brain structures, such as the midbrain and cerebellum. Correct positioning of the MHB is thought to depend on mutual repression involving these two homeobox genes. Using a cell culture colocalization assay and coimmunoprecipitation experiments, we show that engrailed homology region 1 (eh1)-like motifs of both transcription factors physically interact with the WD40 domain of Groucho/Tle corepressor proteins. In addition, heat shock-induced expression of wild-type and mutant Otx2 and Gbx2 in medaka embryos demonstrates that Groucho is required for the repression of Otx2 by Gbx2. On the other hand, the repressive functions of Otx2 on Gbx2 do not appear to be dependent on corepressor interaction. Interestingly, the association of Groucho with Otx2 is also required for the repression of Fgf8 in the MHB. Therefore Groucho/Tle family members appear to regulate key aspects in the MHB development of the vertebrate brain

Photoheterotrophic growth of unicellular cyanobacterium Synechocystis sp. PCC 6803 gtr− dependent on fructose

Although cyanobacteria have specialized for a photolithoautotrophic mode of life during evolution many cyanobacterial strains have been identified as being capable of photoheterotrophy or even chemoheterotrophy. The mutant strain of Synechocystis sp. PCC 6803, which lacks the gtr gene coding for the strain’s glucose/fructose permease, has been believed to be a strict photolithoautotroph in the past as it has lost the wild type’s facility to use external glucose for both photoheterotrophy and light-induced chemoheterotrophy. However, recent experiments revealed the strain’s capacity to use fructose for mixotrophic and photoheterotrophic growth, a sugar which is toxic for the wild type. Both the growth rate and the amount of fructose incorporated into the cells increased along with the fructose concentrations in the surrounding medium. Furthermore an increase of the total carbon mass of the cells within a liquid culture over a period of photoheterotrophic growth could be demonstrated. Contrary to the wild type, glucose could not be used for photoheterotrophic growth, and chemoheterotrophic growth failed with fructose as well as with glucose.© The Author(s) 201