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

contains 6 genome copies in early exponential phase and 10 genome copiesin exponential phase.Methanosarcina acetivorans was found to be polyploid during fast growth(17 copies) and oligoploid during slow growth (3 copies). Methanococcusmaripaludis has the highest ploidy level found for any archaea with 55genome copies in exponential phase and 30 in stationary phase [5].In summary, the results reveal that many polyploid species of archaea andbacteria exist and that monoploidy is exeptional, in contrast to the currentbelief.[1] Webb, C.D. et al (1998): Use of time-lapse microscopy to visualize rapid movement of thereplication origin region of the chromosome during the cell cycle in Bacillus subtilis. Mol Microbiol28(5): 883-892.[2] Bremer, H. and P.P. Dennis (1996): Modulation of chemical composition and other parameters ofthe cell growth rate. In: Neidhardt FC, ed. University of Michigan Medical School. Escherichia coliand Salmonella. ASM Press. Washington.[3] Breuert, S. et al (2006): Regulated polyploidy in halophilic archaea. PLoS ONE 1:e92.[4] Pecararo et al: in press.[5] Hildenbrand, C. et al: Genome copy numbers and gene conversion in methanogenic archaea. JBacteriol: in press.RGP010Regulation of the Escherichia coli sensor histidine kinaseDcuS by direct interaction with the C 4 -dicarboxylatecarriers DctA and DcuBJ. Witan*, G. UndenInstitute for Microbiology and Wine Research, Johannes-Gutenberg-University, Mainz, GermanyEscherichia coli can use various C 4-dicarboxylates as carbon and energysources for aerobic or anaerobic respiration. The two component systemDcuSR activates the transcription of dctA (succinate import), dcuB(fumarate-succinate antiport), fumB (fumarase) and frdABCD (fumaratereductase) in the presence of C 4-dicarboxylates [1]. DcuSR consists of themembrane integral sensor kinase DcuS and the cytoplasmic responseregulator DcuR.Under anaerobic conditions the main transport proteins for C 4-dicarboxylates are DcuA, DcuB and DcuC (1). DctA is the main transportprotein for C 4-dicarboxylates under aerobic conditions. It mediates theuptake of succinate and other C 4-dicarboxylates in symport with protons.DctA and DcuB function as co-sensors of DcuS. Deletion of the carrierscauses constitutive activation of DcuSR [2, 3]. Interaction of the integralmembrane protein DcuS with DctA and DcuB was analysed in vivo with abacterial two-hybrid system based on the Bordetella pertussis adenylatecyclase (BACTH) and by fluorescence resonance energy transfer (FRET).Direct interaction of DctA and DcuB with DcuS was detected. Theinteraction of DcuS with DctA is modulated by fumarate. DctA and DcuBcontain specific sites which are essential for the interaction with DcuS.[1] Zientz et al (1998): J. Bacteriol. 180: 5421-5425.[2] Golby et al (1999): J. Bacteriol 181: 1238-1248.[3] Kleefeld et al (2009): J. Biol. Chem.284:265-275.cryptochromes do not show the photolyase-dependent DNA repair activity.It is known that cryptochromes regulate different processes like theentrainment of the circadian clock in plants and animals. However, abiological function and a complete signalling pathway had not been shownfor a prokaryotic cryptochrome, yet.Earlier we were able to demonstrate that a cryptochrome in R. sphaeroides(CryB) shows an active, light-dependent photocycle, binds FAD as cofactorand is involved in the regulation of photosynthetic apparatus expression [1].We could also identify an RpoH II promoter in front of cryB which brings itsexpression into a singlet oxygen ( 1 O 2) stress-dependent context. We nowpresent a genome wide transcriptional analysis of R. sphaeroides using DNAmicroarrays. For this we compared Rhodobacter wildtype to the cryBdeletion mutant under blue light illumination and under 1 O 2 stressconditions. Furthermore, we were able to identify several putativeinteraction partners to CryB by a Yeast Two Hybrid system. Interestingly,pulldown experiments also revealed an interaction of CryB to AppA whichcould link the cryptochrome in the photosynthesis regulation system. Asindicated by the DNA microarray data, a role of small RNAs in a CryBdependentsignalling pathway is also likely.[1] Hendrischk, A.K. et al (2009): A cryptochrome-like protein is involved in the regulation ofphotosynthesis genes in Rhodobacter sphaeroides, Mol. Microbiol. 74 (4), 990-1003.RGP012Role of the small RNA RSs2430 in the regulation ofphotosynthesis genes in Rhodobacter sphaeroidesN. Mank*, B. Berghoff, G. KlugInstitute for Micro- and Molecular Biology, Justus-Liebig-University,Gießen, GermanySmall RNAs (sRNAs) play a regulatory role in the adaptation of variousbacteria to changing environmental conditions. The identification of sRNAs,using RNA-seq based on 454 pyrosequencing, in the phototrophic bacteriumRhodobacter sphaeroides (1) was of major interest because of its highmetabolic versatility. In particular, synthesis of the photosynthetic apparatusis regulated in an oxygen- and light-dependent manner. In a physiologicalscreen the sRNA RSs2430 was also found to be influenced by the oxygentension. Induction of RSs2430 depends on the PrrB/PrrA system, which is amajor regulatory system for redox control of photosynthesis genes. Here wepresent how overexpression of RSs2430 influences the expression ofphotosynthesis genes in Rhodobacter sphaeroides. Northern blots showedthat RSs2430 is processed, whereby different 3’ends are generated. Thedifferent 3’ends were identified by 3’RACE. Interestingly, only theprocessed RSs2430-fragments, not the primary transcript, were enriched inthe overexpression strain. By using real time RT-PCR and microarrayanalyses we showed that overexpression of RSs2430 results in a decreasedexpression of photosynthesis genes.[1] Berghoff, B.A. et al (2009): Photooxidative stress-induced and abundant small RNAs inRhodobacter sphaeroides. Mol. Microbiol., 74(6), 1497-512.RGP011Identification of cryptochrome-dependent signallingpathways in Rhodobacter sphaeroides - Genome wideanalysis under blue light and singlet oxygen stressconditionsS. Frühwirth*, S. Metz, G. KlugMolecular Microbiology, AG Klug, Justus-Liebig-University, Gießen,GermanyRhodobacter sphaeroides belongs to the alpha subdivision of proteobacteria.The bacterium is known for its high metabolic versatility, as it can, besidesrespiration, also perform anoxygenic photosynthesis. To prevent theformation of reactive oxygen species (ROS), the formation of thephotosynthetic apparatus has to be tightly controlled. ROS are generatedwhen light, oxygen and a photosensitizer (e.g. chlorophyll) are presentsimultaneously.The blue light photoreceptor AppA belongs to the BLUF domain proteinsand plays a major role in the regulation of photosynthetic apparatusformation. This protein shows dual sensing abilities, sensing both, light andoxygen.Besides AppA other blue light photoreceptors were identified in R.sphaeroides, recently. Cryptochromes belong to a superfamily together withphotolyases. Although both exhibit a high sequence homology,RGP013Examination of a timing mechanism in RhodobactersphaeroidesY. Hermanns*, A. Wilde, G. KlugInstitute for Micro- and Molecular Biology, Justus-Liebig-University,Gießen, GermanyTiming mechanisms are known for over 250 years in eukaryotes. Until nowamongst prokaryotes only cyanobacteria could be shown to possess a systemto measure time. In Synechococcus elongatus a circadian clock builds uponan oscillator of three proteins, KaiA, KaiB and KaiC. A phosphorylation ofKaiC in a circadian manner could be shown in vitro [1]. All three proteinsare essential for clock function. Accordingly, most cyanobacteria possess atleast one copy of each gene. An exception is the marine cyanobacteriumProchlorococcus marinus, which has suffered a stepwise deletion of thekaiA gene [2] but retains a 24 hour rhythm in DNA replication, which isstrongly synchronized by alternation of day and night cycles. Surprisingly,the facultative phototrophic proteobacterium Rhodobacter sphaeroidespossesses a cluster of kaiBC genes similar to Prochlorococcus. Therefore ithas been hypothesized that R. sphaeroides may exhibit a rhythmic behaviorin gene expression. Such a rhythm has been reported earlier via a luciferasereporter gene system [3]. We were able to show a rhythmic expression ofphotosynthesis genes for over 4 days in a continuously growing R.sphaeroides culture which had been entrained by a 12 hour light and darkspektrum | Tagungsband 2011