VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

By the use of their C-terminal domains, DivIVA proteins are thought torecruit a number of binding partners to the septum and the poles that havevarious crucial functions in cell division, peptidoglycan biosynthesis orendospore formation. We decided to analyse the role of DivIVA in celldivision and infectivity of the facultatively intracellular pathogen Listeriamonocytogenes since cellular polarity has been reported to be important forits survival inside eukaryotic host tissues. We found that DivIVA is a crucialtopogenetic factor required for the completion of cross wall formation at thesite of cell division in L. monocytogenes. The severe morphologicalabnormities accompanying the loss of divIVA may explain why these cellsare inable to swarm, severly impaired in biofilm formation at plastic surfacesand clearly attenuated in a cell culture infection assay. We can show that L.monocytogenes cells lacking divIVA are impaired in their ability to enter andto egress eukaryotic cells. Our results suggest that DivIVA proteins mightrepresent a useful target structure for the development of new antibacterialdrugs.CBP012Import and activation of the colicin M protein toxinrequires the periplasmic FkpA prolyl cis-trans isomerase/chaperone in E. coli.V. Braun*, S. Helbig, S. Patzer, C. Römer, K. ZethMax Planck Institute for Developmental Biology, Tübingen, GermanyColicin M (Cma) is a protein toxin that is formed by E. coli strains that carryColBM plasmids. It is imported into the periplasm of sensitive cells via areceptor-dependent energy-coupled process. It kills E. coli cells byinhibition of murein (peptidoglycan) precursor incorporation into theexisting murein in that it cleaves the phosphate ester bond between theprecursor and the lipid carrier that translocates the precursor across thecytoplasmic membrane. The resulting C 55 polyisoprenol no longer enters thereaction cycle, murein synthesis stops and cells lyse. E. coli cells thatsynthesize Cma are protected by an immunity protein, Cmi, which in theperiplasm inactivates Cma.E. coli mutants which are resistant to Cma carry mutations in genes, fhuA,tonB, exbB, exbD, which are involved in Cma import from the outside intothe periplasm. We recently found that an additional type of Cma resistantmutant carries a mutation in fkpA that encodes a periplasmic prolyl cis-transisomerase (PPIase) / chaperone. Spontaneous fkpA deletion and pointmutants in the PPIase domain are completely resistant to high titers (10 5 ) ofCma. The crystal structure of Cma reveals a compact form that must unfoldduring translocation across the outer membrane. It is assumed that thisinvolves a trans-to-cis prolyl isomerisation of Cma that is converted back totrans upon refolding in the periplasm. Cma refolding is catalysed by FkpA.Regardless whether Cma is imported or secreted with a fused signalsequence into the periplasm, it requires FkpA to be active. To identify theresidue that might be cis-trans isomerized, the 15 proline residues wereindividually replaced by alanine. The mutant Cma’s were fully active exceptthree which displayed 1% activity. Two of them are not imported. The onethat remains inactive in the periplasm has a crystal structure identical towild-type Cma which makes it unlikely that the mutation changes thephosphatase active center that is located far from the proline residue. It isproposed that the proline residue of the inactive imported mutant is targetedby FkpA.Sequence and structure of the phosphatase domain of Cma is unique. Theactive center was therefore mapped by random and site-specificmutagenesis. The mutations center in a surface-exposed region. An aspartateresidue was defined as a likely catalytic site since conversion to asparagineor glutamate abolishes Cma activity. The residues implicated in phosphatasecatalysis are highly conserved in Cma-like proteins of other species than E.coli.[1] Hullmann, J. et al (2008): Periplasmic chaperone FkpA is essential for imported colicin Mtoxicity. Mol. Microbiol. 69, 926-937.[2] Zeth, K.et al (2008): Crystal structure of colicin M, a novel phosphatase specifically imported byEscherichia coli. J. Biol. Chem. 283, 25324-25331.CBP013Does RAS-1 regulate adelylate cyclase activity?S. Gutiérrez*, P. Rangel, W. HansbergInstitute of Cellular Physiology, Department of Cell and DevelopmentalBiology, National Autonomous University, Mexicomorphogenetic transitions take place: hyphae adhesion, aerial hyphaegrowth and conidia development [1]. Each transition is started by anunstable hyperoxidant state and results in growth arrest, autophagy,antioxidant response and a dioxygen insulation process. These responsesstabilize the system and, once stable, growth can start again [2,3].In a solid medium the band mutant (bd) exhibits a conidiation band every 22h [4] resulting from a Thr79Ile substitution in ras-1 [5]. The same behavioris observed in a Δsod-1 mutant strain. In both strains, N-acetyl-cysteinesuppresses the conidiation rhythm and paraquat shortens its period.Compared to Wt, ras-1 bd has increased ROS formation during conidiationresulting in increased aerial mycelium growth and increased submergedconidiation.Our hypothesis is that RAS-1 acts as a switch between growth andconidiation in N. crassa. Only three proteins have a predicted RASassociation domain: NRC-1, STE50p orthologue and adenylate cyclase(AC). A Δcr-1 mutant strain decreases grow of vegetative and aerial hyphaeand increases conidia formation. Upon exposure to air, cAMP levels in amycelial mat follow a similar pattern to protein oxidation, loss ofNAD(P)(H)-reducing power and glutathione oxidation [6]. cAMP levelsdecrease during the hyperoxidant state, both at the start of hyphal adhesionand of aerial hyphae formation, and recover thereafter. AC and the lowaffinity phosphodiesterase (NCU00237) activity regulation explained cAMPdecrease. However, during conidia formation, cAMP decrease was due toregulation of AC and the high affinity phosphodiesterase (NCU00478).[1] Toledo, I. et al (1986): Aerial growth in Neurospora crassa: caracterization of an experimentalmodel system. Exp Mycol. 10: 114-125. [2] Hansberg, W. and J. Aguirre (1990): Hyperoxidant statescause microbial cell differentiation by cell isolation from dioxygen. J Theoret Biol 142: 201-221.[3] Aguirre, J. et al (2005): Reactive oxygen species and development in microbial eukaryotes. TIM13: 111-118.[4] Loros, JJ and JC Dunlap (2001): Genetic and molecular analysis of circadian rhythms inNeurospora, Annu Rev Physiol 63: 757-794.[5] Belden, WJ et al (2007): The band mutation in Neurospora crassa is a dominant allele of ras-1implicating RAS signaling in circadian output. Genes Dev 21: 1494-1505.[6] Hansberg, W. et al (2008): Cell differentiation as a response to oxidative stress. In: Stress inYeasts & Filamentous Fungi (Ed. Avery; Stratford; van West) Elsevier IBSN 978-0-12-374184-4.CBP014The complex assembly of the Actinobacterial RieskeproteinR. Keller*, T. PalmerCollege of Life Sciences, University of Dundee, Dundee, United KingdomProtein export and assembly is essential for the bacterial cell and is generallyrealized by two distinct operating translocases, called the Sec and Tatsystems. Proteins are transported via the Sec pathway in an unfoldedconformation. In contrast, proteins are transported through the Tat (twinarginine translocation) pathway in a folded state and are targeted to the Tatpathway by N-terminal signal peptides harboring consecutive, invariantarginine residues. One of the most important Tat-dependent membraneproteins is the Rieske protein, a fundamental component of the essentialenergy transduction cytochrome bc 1 complex in the respiratory chain ofmany bacteria. Usually the Rieske protein is composed of a singletransmembrane helix at its N-terminus which is preceded by the Tat motifand followed by an iron-sulphur domain. However, in actinomycetes andother pathogenic relatives such as mycobacteria the Rieske protein has threetransmembrane domains (TMD) prior to the iron-sulphur cluster.Interestingly and very unusually sequence alignment revealed an internal Tatmotif preceding the third TMD, which suggests that the Tat system isrequired for the transport of the folded iron-sulphur domain across themembrane but probably not for the membrane insertion of the first twotransmembrane helices. To investigate the assembly of the TMD of theRieske protein into the cytoplasmic membrane, a reporter system has beenused, whereby the iron-sulphur domain of the Rieske protein ofStreptomyces coelicolor is replaced with maltose binding protein of E. coli.Thus, using different molecular biology and biochemical approaches wedemonstrated that the assembly of this chimeric protein is dependent on theTat pathway. But our data also implies that an additional protein insertionpathway co-operates with the Tat pathway in the assembly of the RieskeTMD.In Neuropspora crassa, conidiation is started when an aerated liquid cultureis filtered and the resulting mycelial mat is exposed to air. Threespektrum | Tagungsband 2011