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

CBP015Profiling of SeqA binding to the Escherichia colichromosome using an improved ChIP-Chip methodT. Waldminghaus*, K. SkarstadDepartment of Cell Biology, Institute for Cancer Research, Oslo, NorwayThe SeqA protein of Escherichia coli is involved in regulation of replicationinitiation and is also proposed to act in organization and segregation ofdaughter chromosomes (Waldminghaus and Skarstad, 2009). SeqA bindsspecifically to hemimethylated GATC sites that are produced during DNAreplication.The DNA remains hemimethylated until the Dammethyltransferase methylates the DNA fully. SeqA dependent regulation ofreplication initiation is based on its binding to recently replicated origins ofreplication. This prevents re-initiation of the new origins and also protectsthe origins from remethylation by Dam. While the role of SeqA in regulationof replication initiation has been investigated in detail its role inchromosome organization and segregation is poorly understood. We applieda published method of chromatin immunoprecipitation combined withmicroarrays (ChIP-Chip) to analyze binding of SeqA to the 20.000 GATCsites found on the E. coli chromosome. Faced with a background signalexceeding the specific signal we reinvestigated the procedure and were ableto reduce the background significantly by modifying the protocol(Waldminghaus and Skarstad, 2010). The new protocol allowed us to profilechromosome wide SeqA binding.[1] Waldminghaus, T. and K. Skarstad (2009): The Escherichia coli SeqA protein. Plasmid, 61, 141-150.[2] Waldminghaus, T. and K. Skarstad (2010): ChIP on Chip: surprising results are often artifacts.BMC Genomics, 11, 414.CBP016Synthetic reconstruction of the chromosome partitioningsystem from Corynebacterium glutamicumB. Sieger*, M. Bramkamp, C. DonovanInstitute for Biochemistry, University of Cologne, Cologne, GermanyCorynebacterium glutamicum is a Gram-positive, MreB-lacking and nonsporulatingmodel organism with high industrial and medical relevance.Compared to other organisms, such as Escherichia coli or Bacillus subtilis,the cell division machinery in C. glutamicum looks much simpler, e.g.proteins for spatial restriction of the divisome such as the Min system andnucleoid occlusion proteins are missing. Further, actin homologues likeMreB are not encoded, implicating, that cell wall synthesis for cellelongation is not governed by an actin-like cytoskeleton. Recent datasuggest that a corynebacterial homologue of the polar determinant DivIVAis responsible for governing of apical growth. We found evidence, thatDivIVA-like proteins in corynebacteria may even be involved inchromosome orientation with the cell via interaction with the ParABpartitioning system, thereby coupling chromosome segregation and cell wallsynthesis.In order to examine the in vitro interaction behaviour of partitioning proteinsof C. glutamicum an expression and purification protocol for DivIVA, ParBand ParA was established. Furthermore, sedimentation experiments formembrane interaction were carried out to establish a synthetic in vitro assayfor reconstruction of the chromosomal segregation machinery. We couldshow that DivIVA likely binds to lipids in a dimeric form. Titration of thepartitioning proteins ParAB to the assay revealed that ParB binds to DivIVAin vitro. Binding of ParA to the complex depends on a pre-existing DivIVA-ParB complex. We will exploid this in vitro setup to unravel the molecularmechanism of chromosome tethering.CBP017Analysis of functional membrane microdomains inbacteriaJ. Bach*, M. BramkampDepartment of Biochemistry, University to Cologne, Cologne, GermanyThe view on plasma membranes has changed dramatically during the lastyears. Initially it was proposed that membranes are a homogeneous mixtureof lipids with embedded proteins. Though, during the last years it could beshown that plasma membranes contain a high degree of lateral organisation.Specialised regions containing different protein and lipid patches wereidentified, termed microdomains or lipid rafts. In eukaryotic cells lipid raftsare characterised by a high content of cholesterol, glycosphingolipids andcharacteristic raft associated proteins such as flotillins and GPI anchoredproteins. In contrast lipid rafts in bacterial cell membranes are hardlyunderstood.Previously, we have described a bacterial flotillin, YuaG that is involved inthe signalling pathway which leads to Spo0A phosphorylation. YuaGlocalises in discrete foci in the membrane and these foci are highly dynamic.Purification of detergent resistant membranes (DRM) revealed that YuaG isfirmly associated with negatively charged phospholipids. Here we have useda YuaG-SNAP construct to isolate proteins that are associated with YuaG inbacterial membrane microdomains. One of the identified proteins is YqfA,which also has a flotillin like structure and might be part of a heterooligomericcomplex of flotillins that are scaffolding the lipid microdomains.CBP018SMC shows high condensin-like DNA binding dynamicsin Bacillus subtilis cellsL. Simon*, H. Hummel, P.L. GraumannFaculty for Biology, Department of Microbiology, Albert-Ludwigs-University, Freiburg, GermanyChromosomes must be compacted to fit into the bacterial cell, and thetopology of DNA must be regulated to allow efficient transcription andreplication to go on. The compaction of the DNA is regulated by a numberof proteins including histones, histone-like proteins, topoisomerases and theSMC (structural maintenance of chromosomes) complex.The Bacillus subtilis SMC complex consists of an SMC dimer, an ATPasewith ABC-transporter related head domains, and a subcomplex of the twoaccessory proteins ScpA and ScpB. This complex is essential forchromosome segregation and condensation. Deletion causes severe defectsin chromosome organization, whereas overexpression of SMC in Bacillussubtilis leads to an excessive overcondensation of the nucleoid.The SMC complex localizes cell cycle dependent in a bipolar manner todiscrete centers on the nucleoid. The mode of formation and function ofthese Centers is unclear. We observed in Fluorescence Recovery afterPhotobleaching (FRAP) experiments of SMC-GFP, ScpB-GFP and ScpA-YFP (performed in exponentially growing cells) rapid recovery of the fociwithin few minutes. This shows that there is ongoing exchange of the SMCcomplexes between bound and unbound molecules, and in between thecenters. Thus the SMC complex binds transiently and highly dynamically toDNA. We provide evidence that these dynamics depend, to a considerabledegree, on de novo protein synthesis. These findings have importantimplications on the mode of DNA compaction through the SMC complex.We further created a dominant negative point mutant in the head domain ofSMC, which shows severe effects in chromosome segregation, anddisordered formation of the discrete Centers. This mutant will allow us torevealing if dynamic binding is important for the function of the SMCcomplex.CBP019On the role of a new member of the CDK9 kinase familyin Aspergillus nidulansC. Kempf*, F. Bathe, R. FischerDepartment of Microbiology, Karlsruhe Institute of Technology (KIT),Karlruhe, GermanyCyclin dependent kinases (CDKs) are a large group of protein kinases whichare regulated by association with cyclins. Members of the Cdk9 family havebeen described from yeast to human and are known to be part of the basaltranscription elongation machinery. Their regulatory subunits are differentcyclins (cyclin T1, T2a, T2b and K), which do not oscillate during the cellcycle.In A. nidulans the cyclin PclA has been characterized as a cyclin involved indevelopment. PclA interacts with the main regulator of the cell cycle, NimXand may help to adjust the cell cycle during asexual sporulation [1]. In atargeted approach it was found that PclA also interacts with another kinase,a Cdk9 family member (PtkA) [2]. Deletion of the ptkA gene causes a lethaldefect and the mutant arrests in a short germling state. PtkA localizes tonuclei during interphase. PtkA does also interact with a cyclin T (PchA) as itdoes in other organsims, suggesting a conserved role in transcriptionregulation.Performing Y2H screens with PtkA, we identified two more interactionpartners, one protein kinase and surprisingly, another Pcl cyclin. Theseinteractions occurred most interestingly only in metulae and phialides andare thus restricted to asexual development. These results point to thespektrum | Tagungsband 2011