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

SRP016Effect of the sRNA repeat RSs0680a-d on global generegulation in Rhodobacter sphaeroidesF. Billenkamp*, B.A. Berghoff, G. KlugInstitute for Micro- and Molecular Biology, Justus-Liebig-University,Giessen, GermanyIn bacteria small RNAs (sRNAs) play an important role in response to stresssituations by means of posttranscriptional gene regulation. Although thereare few sRNAs that bind proteins, common sRNAs bind to target messengerRNAs (mRNAs) and modulate the stability and/or translation of the mRNA[1]. There are two classes of sRNAs interacting with mRNAs, the so calledcis-encoded antisense RNAs that show perfect base pairing with their targetmRNAs and the trans-encoded sRNAs which show limited complementarityto their target mRNAs. To facilitate the interaction, the hexameric proteinHfq is needed in case of trans-encoded sRNAs to overcome the limited basepairing [1]. In Rhodobacter sphaeroides the expression of several suchsRNAs is related to oxidative stress [2]. One sRNA that shows increasedexpression levels under oxidative stress is RSs0680a, which is cotranscribedwith 3 homologous sRNAs (RSs0680b-d) and one hypothetical protein(RSP_6037). To realize stress dependent induction, theRSP_6037/RSs0680a-d operon is controlled by an RpoH I/RpoH II-dependentpromoter [3]. We could show that constitutive overexpression of RSs0680adin R. sphaeroides leads to enhanced resistance to oxidative stress.Transcriptome and proteome analyses revealed serveral mRNAs andproteins with a changed abundance in the R. sphaeroides RSs0680a-doverexpression strain. Combination of those experiments with bioinformaticapproaches revealed putative target mRNAs. Most of them show a putativerelation to sugar transport or to aerobic respiration, which is a major sourcefor oxidative stress. Especially a putative operon of four genes (RSP_2876 -RSP_2879) shows lower levels of both the expressed mRNAs as well as therespective proteins. The genes in this operon are subunits of a putativeaerobic carbon monoxide dehydrogenase and one hypothetical protein. Apossible function of the genes in this operon is related to the oxidation ofcytochrome b561, which is part of the membrane bound electron transportchain. Interestingly possible binding sites for RSs0680a can be detected 2-6bp upstream of the AUGs of the four genes. Presently in vitro sRNA:mRNAinteraction studies including Hfq are performed, in order to prove theputative binding of RSs0680a to the mRNA of the putative operon.[1] Waters, L.S. and G. Storz (2009): Regulatory RNAs in Bacteria. Cell 136: 615-628.[2] Berghoff, B.A. et al (2009): Photooxidative stress induced and abundant small RNAs inRhodobacter sphaeroides. Mol Microbiol 74: 1497-512.[3] Nuss, A.M. et al (2010): Overlapping Alternative Sigma Factor Regulons in the Response toSinglet Oxygen in Rhodobacter sphaeroides. J Bacteriol 192: 2613-2623.SRP017Transcriptional analyses of steady-state cells ofClostridium acetobutylicum in a chemostat cultureH. Janssen* 1 , C. Voigt 1 , C. Grimmler 2 , A. Ehrenreich 2,3 , H. Bahl 1 , R.-J. Fischer 11 Institute of Biological Sciences, Division of Microbiology, University ofRostock, Rostock, Germany2 Institute of Microbiology and Genetics, Department Genomic and AppliedMicrobiology, Georg-August-University, Göttingen, Germany3 Department of Microbiology, Technical University Munich, Freising,GermanyClostridium acetobutylicum is well known for its acetone-butanol (AB)fermentation. Using phosphate limited chemostat cultures at pH 5.7, C.acetobutylicum was kept at a steady state in the acidogenesis, whereas at pH4.5, the cells showed stable solventogenesis without sporulation. Weinvestigated steady-state transcriptomes of pH 5.7 and pH 4.5 using DNAmicro array analyses to provide new insights into the metabolic changes (1).Stable growth rates and constant exogenous parameters during the chemostatfermentation process enabled homogeneity of bacterial cells and the pH assingle parameter was changed to switch from acidogenesis tosolventogenesis.All in all, 53 genes were significantly repressed, while 95 genes showed asignificant upregulation in the steady-state of the solventogenesis at pH 4.5.The respective genes are suitable candidates for a basic mathematic modelof the solventogenic shift, which will be developed within the COSMIC2project (www.sysmo.net).Furthermore, a transcriptional analysis of butanol stressed steady-state cellsof the acidogenesis will be presented using DNA micro array analysesenabling the differentiation of genes, which transcription pattern wasinfluenced by lowering the pH value or by butanol stress.[1] Janssen et al (2010): A Proteomic and Transcriptional View of Acidogenic and SolventogenicSteady-State Cells of Clostridium acetobutylicum in a Chemostat Culture. Appl. Microbiol.Biotechnol. 87:2209-2226.SRP018Peptide antibiotic sensing and detoxification modules inFirmicutes bacteria: Co-evolution of ABC-transportersand two component systemsS. Dintner*, A. Staroń, T. Petri, T. Mascher, S. GebhardBiology Department I, Ludwig-Maximillians-Unviversity Munich, Planegg-Martinsried, GermanyThe genome of Bacillus subtilis contains three loci (bceRSAB, psdRSAB,yxdJKLM), which are very similar in gene organization and in sequence, andinvolved in resistance to various peptide antibiotics. The encoded systemsare comprised of a two-component regulatory system (TCS) and an ATPbinding-cassette(ABC) transporter. Both the permease and sensor kinasecomponents of these modules show unusual domain architecture: thepermeases contain ten transmembrane helices with a large extracellular loopbetween helices 7 and 8, while the sensor kinases lack any obvious inputdomain. Strikingly, in the Bce and Psd modules the ABC-transporter andTCS have an absolute and mutual requirement for each other in both sensingof and resistance to their respective antimicrobial compounds. A search ofseveral non-redundant protein databases revealed the existence of 265 ABCtransporterswith homology to BceB and PsdB, 80% of which wereassociated with a TCS homologous to BceRS and PsdRS. All but four ofthese were found in Firmicutes bacteria. Parallel phylogenetic analysis of thepermease and sensor kinase components revealed a tight evolutionarycorrelation, displayed as a congruence of the two phylogenetic trees. Ourfindings suggest direct protein-protein interactions between the ABCtransportersand TCSs in mediating resistance. Based on this correlation, wecould identify putative corresponding two-component systems fortransporters lacking a regulatory system in their immediate neighborhood.Taken together, our results show that these types of ABC-transporters andTCSs have co-evolved to form self-sufficient detoxification modules againstantimicrobial peptides, restricted to and conserved among Firmicutesbacteria.SRP019Will not be presented!SRP020Guanidino-ectoine: a new member of the incompatiblesolute familyK. Sell*, E.A. GalinskiInstitute for Microbiology und Biotechnology, Friedrich-WestphalianWilhelms-University, Bonn, GermanyThe guanidinium function has been identified as a protein-backboneinteracting group which displays the opposite effect to that of compatiblesolutes [1, 2]. This also seems to apply for guanidinium groups in aminoacids like arginine [3]. In previous studies, inhibitory effects of theguanidinium compound creatine on bacterial growth have beendemonstrated. Under elevated salinities, creatine is „mistaken” for thestructurally related compatible solute betaine and taken up with the help ofbetaine transport systems. Despite its negative effect on cellular metabolism,creatine is accumulated to high cytoplasmic concentrations. Such a solutehas since been named an incompatible solute.To investigate whether other compounds which serve as compatible solutesalso lose their protecting feature when a guanidinium group is introduced, anew derivate of the compatible solute ectoine, 2-amino-3,4,5,6-tetrahydro-4-pyrimidinecarboxylic acid (abbreviated guanidino-ectoine), was synthesizedand growth experiments were performed in media supplemented with thenew substance. The effect of guanidino-ectoine on bacterial growth, itsimpact on the intracellular solute-pool and the uptake systems of E. coliwere characterized.Contrary to ectoine, guanidino-ectoine did not support cell growth atelevated salinity. Instead, increasing concentrations led to enhancedinhibitory effects. In the presence of both solutes, the compatible andspektrum | Tagungsband 2011