VAAM-Jahrestagung 2012 18.–21. März in Tübingen

127therefore resistance development. Other gyrase inhibitors, namely theaminocoumarines bind the GyrB subunit which leads to competitiveinhibition of the ATPase activity of gyrase but not to double strand brakes.Here we observe partially antagonistic effects of quinolones(ciprofloxacin) and aminocoumarines (novobiocin) with regard to RecAinduction, SOS response, mutation rate and phage induction in the humanpathogen Staphylococcus aureus.Site-specific mutants (recA, lexA) as well as an inducible recA mutantwere constructed and the combined action of gyrase inhibitors analysed bytranscriptional analysis and Western blots. In addition effects on phageinduction and mutation frequencies were assessed.We could show that ciprofloxacin results in a RecA dependentderepression of LexA target genes such as the error prone polymeraseSACOL1400. In contrast the aminocoumarine novobiocin leads to adecrease in RecA expression on protein as well as transcript level.Interestingly, the combination of ciprofloxacin and novobiocin results alsoin decrease of RecA. However, by combination of both antibioticsalthough RecA expression is significantly repressed the SOS response isstill induced as shown by the induction of the LexA target gene coding forthe error-prone polymerase SACOL1400. Also phage induction was notaltered by RecA repression. An artificially dose-dependent recAexpression system showed us, that induction of the lexA genes as well asphage induction is clearly correlated to the RecA expression level.In summary, the result indicate that there are additional RecA independentmechanisms involved in lexA autocleavage induced by a mix ofciprofloxacin and novobiocin.To identify this second activator or pathwayis very important, since it is involved in generating resistant bacteria andneeds to be considered during antibacterial therapy.MPP077Comparative proteome analysis of Staphylococcus aureusstrains co-internalized into S9 cellsH. Pförtner* 1 , M. Burian 1 , P. Hildebrandt 2 , J. Liese 3 , C. Wolz 3 , F. Schmidt 2 ,U. Völker 11 University Greifswald, Department of Functional Genomics, Greifswald,Germany2 University Greifswald, Junior Research Group Applied Proteomics of the ZIK-FunGene , Greifswald, Germany3 University Tübingen, Interfaculty Institute for Microbiology and InfectionMedicine, Tübingen, GermanyStaphylococcus aureus, the cause of a wide spectrum of severecommunity-acquired and nosocomial infections, is acknowledged as anintracellular pathogen, as it can be internalized and persist in nonprofessionalphagocytic cells in cell culture experiments [1] . During theinternalization process, S. aureus has to adapt to the intracellularenvironment to survive or even persist within the host, but still little isknown about these adaptive changes on proteome level. S. aureusvirulence factors, which are important to establish an infection are tightlycontrolled by global regulators. The accessory gene regulator (agr) is oneof the major global regulators of S. aureus virulence. RNAIII, the effectormolecule of the agr system, positively controls the production ofexoproteins and negatively controls cell surface bound proteins during thepost exponential growth phase [2] . There is evidence that this regulatorysystem plays a role in the establishment of an infection and host cellkilling. For instance, expression of agr is initially increased in the acutephase of infection in non-professional phagocytic cells [3] .Furthermore,agr mutants are attenuated in their virulence in several mouse models [4,5,6,7] .The aim of this study is to comparatively investigate the adaptive andcompetitive response of S. aureus HG001 wild type and its isogenic agrmutant upon co-internalization by human bronchial epithelial cells (S9).The striking advantage of such a co-infection assay is that both strains areinternalized simultaneously and adapt to the host under exactly the sameconditions.Proteome analysis of the co-internalized S. aureus are performed with thewell established workflow, which combines a classical infection assaywith high capacity cell sorting and gel-free proteomics [8] .To make the internalized Staphylococci accessible, they have to beseparated from debris of lysed S9 cells and distinguished between wildtype and mutant by FACS. After validation, the fluorescent markergpCerulean of the agr mutant showed a clear distinction to the GFPexpression of the wild type. Accordingly, we are now able to sort cointernalizedS. aureus parallel in distinct wells of a 96-well plate.In conclusion, with this setting we are able to monitor co-internalizedHG001 wild type and agr mutant and make them with FACS-sorting andon membrane digest accessible for proteome analysis.[1] Garzoni C. et al. 2009. Trends Microbiol, 17, 59-65.[2] Dunman PM. et al. 2001. Journal of Bacteriology, 24, 7341-7353.[3] Tuchscherr L. et al. 2011. EMBO Mol. Med., 3, 129-141.[4] Abdelnour A. et al. 1993. Infection and Immunity, 9, 3879-3885.[5] Cheung AL. et al. 1994. Journal of Clinical Investigation, 94, 1815-1822.[6] Gillaspy AF. et al. 1995. Infection and Immunity, 63 (9), 3373-3380.[7] Wright JS. 3 rd et al. 2005. PNAS, 102 (5), 1691-1696.[8] Schmidt F. et al. 2010. Proteomics, 10, 2801-2811.MPP078Comparative dRNA-seq analysis of multiple Campylobacterjejuni strainsG. Dugar* 1 , A. Herbig 2 , K. Förstner 1 , N. Heidrich 1 , R. Reinhardt 3 , K. Nieselt 2 ,C. Sharma 11 University of Würzburg, Research Centre of Infectious Diseases, Würzburg,Germany2 University of Tübingen, Integrative Transcriptomics, ZBIT (Center forBioinformatics Tübingen), Tübingen, Germany3 Max Planck Institute for Plant Breeding Research, Cologne, GermanyCampylobacter jejuni, a Gram-negative spiral-shapedEpsilonproteobacterium, is one of the most common causes of bacterialgastroenteritis in humans [1]. While it is a commensal of chicken, it hasalso been associated with the development of autoimmune disorders likeGuillain-Barré and Miller-Fisher syndromes in humans. Themicroaerophilic, foodborne pathogen is able to survive under various stressconditions imposed by the environment and the host. The small genome ofC. jejuni (1.65 Mb) carries only a few transcriptional regulators and almostnothing is known about the role of non-coding RNAs in this pathogen.Like the related human pathogen Helicobacter pylori, C. jejuni also lacksthe RNA chaperone, Hfq, which plays a pivotal role in sRNA-mediatedregulation in many bacteria.Massively parallel cDNA sequencing (RNA-seq) has been revolutionizingtranscriptome analysis in both eukaryotes and prokaryotes and hasrevealed a wealth of novel information about microbial transcriptomes [2].Recently, we have developed a novel differential approach (dRNA-seq)selective for the 5’ end of primary transcripts, which revealed anunexpectedly complex transcriptional output and massive antisensetranscription from the small and compact genome of the relatedEpsilonproteobacterium H. pylori [3]. This method allowed us to define agenome-wide map of transcriptional start sites (TSS) and operons, andrevealed more than 60 sRNAs including potential regulators of cis- andtrans- encoded mRNAs in H. pylori.Here we present a comparative dRNA-seq approach to analyze thetranscriptome structure and TSS conservation of four different C. jejunistrains. This comparative study reveals that the majority of TSS isconserved among all strains but that there are also several strain-specificTSS indicating divergent transcription patterns among different strains.Moreover, Northern blot analysis confirmed similar and differentialexpression patterns of several conserved and strain specific sRNAcandidates in C. jejuni. This is the first comparative analysis of the primarytranscriptomes and sRNA repertoire of multiple C. jejuni strains and willprovide new insights into riboregulation in this bacterial pathogen.1. Young, K. T., L. M. Davis & V. J. Dirita, (2007)Campylobacter jejuni: molecular biology andpathogenesis.Nat Rev Microbiol 5: 665-679.2. Croucher, N. J. & N. R. Thomson, (2010)Studying bacterial transcriptomes using RNA-seq.Curr OpinMicrobiol 13: 619-624.3. Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiß S, Sittka A, Chabas S, Reiche K,Hackermüller J, Reinhardt R, Stadler PF, Vogel J (2010) The primary transcriptome of the major humanpathogen Helicobacter pylori. Nature, 464(7286):250-5MPP079How a thioredoxin-like protein influences the susceptibility toß-lactam antibiotics in Staphylococcus aureus.N. Göhring* 1 , I. Fedtke 1 , G. Xia 1 , A.M. Jorge 2 , M.G. Pinho 2 , U. Bertsche 3 ,A. Peschel 11 Interfaculty Institute of Microbiology and Infection Medicine, University ofTübingen, Cellular and Molecular Microbiology, Tübingen, Germany2 Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa,Laboratory of Bacterial Cell Biology, Oeiras, Portugal3 Interfaculty Institute of Microbiology and Infection Medicine, University ofTübingen, Microbial Genetics, Tübingen, GermanyAs a human pathogen, Staphylococcus aureus is capable of colonizing thehostile ecological niche of the anterior nares in humans and has thereforedeveloped different strategies in order to survive during variousenvironmental stresses. During the process of infection, S. aureus isexposed to multiple antimicrobial compounds such as oxidative burstproducts and antibiotics. The underlying regulatory pathways governingsusceptibility or resistance are complex and still remain only superficiallyunderstood. Within this tightly balanced resistance network a thioredoxinlikeprotein YjbH has been shown to control disulfide stress response inBacillus subtilis by monitoring the controlled degradation of thetranscriptional stress regulator Spx via the proteasome-like ClpXPprotease. Similar functions could be attributed to the S. aureus YjbHhomolog using the disulfide stress-inducing agent diamide as in B. subtilis.Further experiments revealed the indispensable role of conserved cysteineresidues within the YjbH protein for this activity. In addition, inactivationof YjbH led to moderate resistance to oxacillin and other -lactamantibiotics, which was associated with an increase in peptidoglycan crosslinkingand higher penicillin-binding protein 4 levels. Of note, the impactof YjbH on -lactam susceptibility was still observed when the conservedcysteines of YjbH were mutated indicating that the roles of YjbH indisulfide stress and -lactam resistance rely on different types ofBIOspektrum | Tagungsband 2012