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

about 600 S. aureus proteins from 3x10 6 to 6x10 6 bacteria. However,secreted proteins which are of particular interest in the interplay between S.aureus and its host were not covered by this approach. In order to study therole of secreted bacterial proteins after internalization by human epithelialcells, we adapted our established workflow to allow detection of secretedstaphylococcal proteins. Since S. aureus was shown to be located inphagosomes [5], we isolated these phagosomes by density gradientcentrifugation and analyzed the proteome of the internalized bacterialpathogen at three time points after internalization. Beside the identificationand quantitation of more than 500 intracellular proteins with this approach,about 25 secreted virulence factors were monitored which could not becaptured by earlier workflows.Moreover, selected proteins were also quantified with the aid of syntheticheavy-isotope labeled peptides or proteins, which were added as externalstandards to each sample.Thus, high precision MS approaches combined with phagosome enrichmenttechniques provide new insights into the virulence factors repertoire ofinternalized S. aureus and thus its interaction with its host.[1] Lowy, F.D. (1998): Staphylococcus aureus infections. N. Engl. J. Med; 339: 520-532.[2] Garzoni, C. and W.L. Kelley (2009): Staphylococcus aureus: new evidence for intracellularpersistence. Trends Microbiol.; 17(2), 59-65.[3] Ong, S.E. et al (2002): Stable isotope labelling by amino acids in cell culture, SILAC, as a simpleand accurate approach to expression proteomics. Mol Cell Proteomics, 2002; 1(5): 376-386.[4] Schmidt, F. et al (2010): Time resolved quantitative proteome profiling of host−pathogeninteractions: The response of S. aureus RN1HG to internalisation by human airway epithelial cells.Proteomics; 10(15): 2801-11.[5] Sinha, B. and M. Fraunholz (2010): Staphylococcus aureus host cell invasion and post-invasionevents. International Journal of Medical Microbiology; 300(2-3):170-5.FGP004Biosynthesis of the siderophore rhodochelin requires thecoordinated expression of three independent geneclusters in Rhodococcus jostii RHA1M. Bosello*, L. Robbel, U. Linne, X. Xie, M.A. MarahielDepartment of Chemistry, Philipps-University, Marburg, GermanyThe biosynthesis and the secretion of siderophores is one of the the mainiron-mobilizing strategies used by microorganisms to cope with ironlimitingconditions [1]. Here, we report the isolation, the structuralcharacterization and the genetic analysis of the biosynthetic origin ofrhodochelin, a unique mixed-type catecholate-hydroxamate siderophoreisolated from Rhodococcus jostii RHA1, which is assembled through anNRPS-dependent pathway [2]. Rhodochelin structural elucidation wasaccomplished via MS n - and NMR-analysis and revealed the tetrapeptide tocontain an unusual ester bond between an L-δ-N-formyl-δ-Nhydroxyornithinemoiety and the sidechain of a threonine residue. Genedeletions within three putative biosynthetic gene clusters abolishrhodochelin production, proving that the ORFs responsible for rhodochelinbiosynthesis are located in different chromosomal loci. These results givedetailed insights into natural product biosynthesis and represent the firstexample of NRPS crosstalk involving three separate genomic regions.[1] Miethke, M. and M.A. Marahiel (2007): Siderophore-based iron acquisition and pathogen control.Microbiol Mol Biol Rev. 71(3): p. 413-51.[2] McLeod, M.P. et al (2006): The complete genome of Rhodococcus sp. RHA1 provides insightsinto a catabolic powerhouse. Proc Natl Acad Sci USA. 103(42): p. 15582-7.FGP005In vivo mobilization of fosmid (meta)genomic librariesB. Leis*, A. Angelov, W. LieblDepartment of Microbiology, Technical University Munich, Freising,GermanyCurrently, genomic libraries are most often produced in E. coli and they lackappropriate mobilizing and selection elements which would allow theirtransfer to other host organisms. The Cre/loxP system has been reported tobe very effective for site-specific insertion of controlling elements intolarge-insert genomic libraries from artificial chromosomes (BACs/PACs) foreukaryotic cells [1, 2]. To our knowledge, there are no Cre/loxP -basedfosmid modification systems established for bacterial hosts other than E.coli. Here, we report the Cre/loxP mediated modification of (meta)genomicfosmid libraries by in vivo recombination and their mobilization into theextremely thermophilic bacterium Thermus thermophilus HB27.In summary, E.coli EPI300 clones carrying pCC1FOS (meta)genomicfosmid libraries from Spirochaeta thermophila were successfully modifiedin vivo by co-transforming a Cre recombinase expression vector and thecorresponding suicide plasmid. The integration into the fosmid was specificto the single loxP site, hence false recombination, deletion or undesiredmodifications were not observed. The pCC1FOS vectors were transferredinto T. thermophilus HB27 via natural competence, resulting inchromosomal integrants via homologous recombination that were stablymaintained with antibiotic selection at 60°C. Furthermore, we currentlyevaluate the use of the Cre/loxP system for the mobilization of genomiclibraries in Bacillus as a gram positive model host.We speculate that large-insert DNA fragments with an existing loxP site canbe mobilized in a variety of bacterial host organisms. With this specific andefficient in vivo recombination system, additional cloning procedures can beomitted and no further modifications of the DNA libraries are needed. Infuture, we apply the Cre/loxP system in order to screen and identify novelgenes from (meta)genomes in Thermus thermophilus as an alternativeexpression host.[1] Mejía J.E. and Larin Z. (2000): The Assembly of Large BACs by in Vivo Recombination;Genomics (70): 165-170.[2] Magin-Lachmann, C. (2003): Retrofitting BACs with G418 resistance, luciferase, and oriP andEBNA-I - new vectors for in vitro and in vivo delivery; BMC Biotechnology (3): 2-13.FGP006Functional analysis of the Synechocystis sp. PCC 6803ycf34 gene product, an ortholog of a conservedchloroplast open reading frameT. Wallner* 1 , J. Kopečná 2 , A. Wilde 11 Institute for Micro- and Molecular Biology, Justus-Liebig-University,Giessen, Germany2 Institute of Microbiology, Department of Autotrophic Microorganisms,Trebon, Czech RepublicCyanobacteria are the ancestors of the chloroplasts due to an endosymbioticevent that occurred 2 billions years ago. For nearly all proteins that areencoded by chloroplast genomes orthologs exist in cyanobacteria. Plant andalgal chloroplast genomes still mainly contain genes involved inphotosynthesis and housekeeping of the organelle. The remaining genesinclude open reading frames of unknown function and have been designatedycf for hypothetical chloroplast open reading frame. No final conclusion canbe illustrated about these ycfs without functional analysis of the resultinggene products. Ycf34 (locus ssr1425) is a hypothetical open reading framethat is conserved in all cyanobacterial lines and in the chloroplast genomesof Cyanophora paradoxa, red algae and some brown algae harboring redalgal derived plastids . No orthologs of this gene are found in the nucleargenome of higher plants. We report here on the phenotypical and functionalanalysis of the cyanobacterial ycf34 gene product using a mutant ofSynechocystis sp. PCC 6803 lacking the gene product. We show that Ycf34is a new small protein tightly bound to the thylakoid membrane. It ispossibly involved in the adaptation of the cyanobacterial light harvestingantenna, the phycobilisomes, to different light conditions. The mutant has asignificantly reduced level of phycocyanin as revealed by 77K fluorescencespectroscopy under light conditions, which require changes in thecomposition of the phycobilisomes. The wild-type phenotype was restoredby expression of an epitope-tagged Ycf34 fusion protein. Geneticallyengineered strains of Synechocystis expressing the FLAG-tagged Ycf34fusion protein were used for the localisation of Ycf34. A GST fusion ofYcf34 was used for heterologous expression in E.coli and the purified Ycf34protein was used for different activity assays which will be shown.FGP007Directed and undirected mutagenesis in BacilluslicheniformisM. Rachinger* 1 , M. Bauch 1 , S. Evers 2 , J. Bongaerts 2 , R. Daniel 3 ,H. Liesegang 3 , W. Liebl 1 , A. Ehrenreich 11 Department of Microbiology, Technical University Munich, Freising,Germany2 Henkel AG & Co. KGaA, Düsseldorf, Germany3 Insitute of Microbiology and Genetics, Georg-August-University,Göttingen, GermanyBacillus licheniformis is an organism of great scientific and biotechnologicalpotential. For further improvement of this strain we established anddeveloped methods for markerless deletions and insertions in B.licheniformis. Especially for the introduction of markerless insertions in thegenome, DNA transfer is a central problem due to the larger vector size.spektrum | Tagungsband 2011