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Rosch T.C.,LOEWE Zentrum fur Synthetische Mikrobiologie SYNMIKRO | Graumann P.L.,LOEWE Zentrum fur Synthetische Mikrobiologie SYNMIKRO | Graumann P.L.,University of Marburg
Journal of Biological Chemistry | Year: 2015

Conjugation of plasmid pLS20 from Bacillus subtilis is limited to a time window between early and late exponential growth. Genetic evidence has suggested that pLS20-encoded protein RcoLS20 represses expression of a large conjugation operon, whereas Rap protein RapLS20 relieves repression. We show that RapLS20 is a true antirepressor protein that forms dimers in vivo and in vitro and that it directly binds to the repressor protein RcoLS20 in a 1:1 stoichiometry. We provide evidence that RapLS20 binds to the helix-turn-helix-containing domain of RcoLS20 in vivo, probably obstructing DNA binding of RcoLS20, as seen in competitive DNA binding experiments. The activity of RapLS20 in turn is counteracted by the addition of the cognate PhrLS20 peptide, which directly binds to the Rap protein and presumably induces a conformational change of the antirepressor. Thus, a Rap protein acts directly as an antirepressor protein during regulation of plasmid conjugation, turning on conjugation, and is counteracted by the PhrLS20 peptide, which, by analogy to known Rap/Phr systems, is secreted and taken back up into the cells, mediating cell density-driven regulation. Finally, we show that this switchlike process establishes a population heterogeneity, where up to 30% of the cells induce transcription of the conjugation operon. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Ritter C.,University of Marburg | Nett N.,University of Marburg | Acevedo-Rocha C.G.,University of Marburg | Acevedo-Rocha C.G.,Max-Planck-Institut fur Kohlenforschung | And 11 more authors.
Angewandte Chemie - International Edition | Year: 2015

Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations. This proof-of-principle study points towards the utility of bioorthogonal enzyme/protecting group pairs for applications in the life sciences. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Ritter C.,University of Marburg | Nett N.,University of Marburg | Acevedo-Rocha C.G.,Max Planck Institute for Chemistry | Lonsdale R.,Max-Planck-Institut fur Kohlenforschung | And 6 more authors.
Angewandte Chemie - International Edition | Year: 2015

Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations. This proof-of-principle study points towards the utility of bioorthogonal enzyme/protecting group pairs for applications in the life sciences. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Gonzalez N.H.,University of Marburg | Felsner G.,University of Marburg | Schramm F.D.,University of Marburg | Klingl A.,LOEWE Zentrum fur Synthetische Mikrobiologie SYNMIKRO | And 2 more authors.
PLoS ONE | Year: 2011

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1. © 2011 Gonzalez et al. Source


Lill R.,University of Marburg | Srinivasan V.,LOEWE Zentrum fur Synthetische Mikrobiologie SYNMIKRO | Muhlenhoff U.,University of Marburg
Current Opinion in Microbiology | Year: 2014

Mitochondria are indispensable in eukaryotes because of their function in the maturation of cytosolic and nuclear iron-sulfur proteins that are essential for DNA synthesis and repair, tRNA modification, and protein translation. The mitochondrial Fe/S cluster assembly machinery not only generates the organelle's iron-sulfur proteins, but also extra-mitochondrial ones. Biogenesis of the latter proteins requires the mitochondrial ABC transporter Atm1 that exports a sulfur-containing compound in a glutathione-dependent fashion. The process is further assisted by the cytosolic iron-sulfur protein assembly machinery. Here, we discuss the knowns and unknowns of the mitochondrial export process that is also crucial for signaling the cellular iron status to the regulatory systems involved in the maintenance of cellular iron homeostasis. © 2014 Elsevier Ltd. Source

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