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

RGP035Kinase-Phosphatase Switch of Shewanella oneidensisMR-1 ArcS is mediated by interplay of a sensory PASdomainand a regulatory receiver domainJ. Lassak* 1 , K. Thormann 21 Biocenter, Department Biologie I, Ludwig-Maximilians University Munich,Martinsried, Germany2 Ecophysiology Group, Max Planck Institute for Terrestrial Microbiology,Marburg an der Lahn, GermanyShewanella oneidensis MR-1 is well known for its respiratory versatility. Anenormous amount of alternative electron acceptors are utilized underanaerobic conditions. Among gamma proteobacteria, the anoxic redoxcontrol (arc) system is mediating the response to changes in environmentaloxygen levels. In E. coli, the response regulator ArcA regulates geneexpression upon signal perception from its cognate sensor kinase ArcB.Conversely we have shown by phenotypic mutant characterization,transcriptomic analysis, and protein-protein interaction that in Shewanella,ArcA, HptA, and ArcS constitute an atypical Arc-System. Phylogeneticanalyses suggest that HptA might be a relict of ArcB. In contrast, the sensorkinase ArcS is substantially different with respect to overall sequencehomology and domain organization. Compared to ArcB, the sensory as wellas the catalytic part of ArcS are extended by a PAS domain and a receiverdomain, respectively. In vitro and in vivo studies with ArcS substitutionmutants reveal distinct roles for the two receiver domains. While onereceiver is mediating the phosphorelay to ArcA, the second receiver controlskinase activity presumably through interplay with the sensory PAS domain.Thus, we speculate that ArcS has adopted the role of ArcB after loss of theoriginal sensor kinase as a consequence of regulatory and sensory adaptationto a redox-stratified environment.RGP036YhdA (CsrD) acts on curli fimbriae expression inEscherichia coli via the Rcs phosphorelay and the smallRNA RprAN. Sommerfeldt-Impe*, R. HenggeInstitute für Mikrobiologie, AG Hengge, Free University, Berlin, GermanyThe switch from the motile-planktonic to the adhesive state and therefore tobacterial biofilm formation is stimulated by the second messenger c-di-GMP. Synthesis and degradation of this messenger is controlled bydiguanylate cyclases (DGC, with GGDEF domains) and phosphodiesterases(PDE, with EAL domains), respectively. Escherichia coli possesses 29GGDEF/EAL genes, which most likely encode 12 DGCs, 13 PDEs as wellas four proteins with degenerate GGDEF/EAL motifs and alternativefunctions (Hengge, 2009; Sommerfeldt et al., 2009).YhdA (also known as CsrD) is a degenerate GGDEF/EAL protein known toaffect the turnover of the small RNAs CsrB and CsrC, which, via the CsrAprotein, modulate motility, glycogen formation and other cellular functions(Suzuki et al., 2006). We showed that YhdA also has a positive effect onCsgD/curli formation and motility (Sommerfeldt et al., 2009). Our new dataindicate that this effect is independent of the Csr system. We demonstratethat part of the reducing effect on curli expression of a yhdA::kan insertionis due to increased expression of MreB, an actin homolog encoded by thegene directly downstream of yhdA; nevertheless also a non-polar yhdA::catinsertion, i.e. also the absence of YhdA per se, reduces CsgD/curliexpression. Down-regulation of curli formation as a consequence of a lackof YhdA and/or increased MreB expression (which can also be mimicked byexpressing MreB from a plasmid) is mediated by the Rcs phosphorelaypathway, which triggers increased expression of the small RNA RprA.Knocking out rprA suppresses the effect of yhdA mutations and/or increasedexpression of MreB. This is consistent with recent data showing csgDmRNA to be a direct target of RprA (Mika et al, in preparation).Furthermore, we observed that a few other small RNAs are present at higheror lower levels in a yhdA mutant, suggesting that YhdA may have a morepleiotropic function by affecting additional targets besides the small RNAsCsrB, CsrC and, indirectly, RprA.[1] Hengge, R. (2009): Principles of c-di-GMP signalling in bacteria. Nat. Rev Microbiol. 7(4): 263-73.[2] Sommerfeldt, N. et al (2009): Gene expression patterns and differential input into curli fimbriaeregulation of all GGDEF/EAL domain proteins in Escherichia coli. Microbiology 155(Pt 4):1318-31.[3] Suzuki, K. et al (2006): Identification of a novel regulatory protein (CsrD) that targets the globalregulatory RNAs CsrB and CsrC for degradation by RNase E. Genes Dev. 20(18):2605-17.[4] Mika, F. et al:A key role for the small regulatory RNA RprA in the σS/CsgD/Rcs biofilm controlnetwork of Escherichia coli (in preparation).RGP037NADPH oxidases (Nox) as source of endogenous reactiveoxygen species (ROS) - a proteomic analysisK. Tuppatsch* 1 , P. Hortschansky 1 , A.A. Brakhage 1,21 Department of Molecular and Applied Microbiology, Hans-Knöll-Institute(HKI), Jena, Germany2 Department of Microbiology, Friedrich-Schiller-University, Jena,GermanyOxidative stress and redox regulation play a key role in development andhyphal growth in Aspergillus nidulans. The ROS-signalling networkcontrols apical growth and development of A. nidulans. Within this networkNADPH oxidases are important members due to their function as source forendogenous ROS which have signalling function.Here we describe recent results concerning mutants of NADPH oxidase ofA. nidulans designated Nox. It is known that deletion of noxA/ noxR causesdistinct phenotypes in growth, sexual and asexual development [1].Therefore, proteome analyses of wild-type A. nidulans and noxA/ noxRdeletion strains were performed to identify key proteins associated withthese mutations and therefore with an altered ROS-level in the cell. Wecompared wild-type A. nidulans and noxA/ noxR deletion strains with thehelp of 2-D gel proteome analysis to identify proteins with higher or lowerabundance in the cellular extracts. Protein spots were identified by MALDI-TOF-MS/MS and classified by their cellular function. This gave us anoverview about the global effect of endogenous ROS on the A. nidulansproteome. Furthermore, we established the first 2-D reference map for wildtypeA. nidulans which possesses 435 spots representing 364 proteins.[1] Semighini CP and Harris SD (2008): Genetics 179:1919-1932.RGP038ncRNA Syr1 is a possible regulator of Alb3 in thecyanobacterium Synechocystis sp. PCC6803E. Kuchmina* 1 , D. Dienst 2 , N. Schürgers 1 , A. Wilde 11 Institute for Micro- and Molecular Biology, Justus-Liebig-University,Giessen, Germany2 Institute for Genetics, Humboldt-University, Berlin, GermanyNon-coding RNAs (ncRNA) are known as novel regulators of geneexpression in different bacteria, including cyanobacteria. Using a tilingmicroarray about 60 ncRNAs and 73 asRNAs were identified in the modelcyanobacterium Synechocystis sp. PCC 6803 [1], 28 of which were verifiedby further methods.One of these ncRNAs Syr1 is a 135 nt long ncRNA located in the 206 ntlong intergenic spacer between the fabF and hoxH genes. It is not cotranscribedwith fabF as judged by Northern blot.Our aim is to investigate the function of Syr1 in the regulatory network ofSynechocystis sp.PCC 6803. Bioinformatic analyses revealed a possibleinteraction with the 5’ region of alb3 gene (slr1471). The putative bindingsite of Syr1 ncRNA overlaps the ribosome binding site of alb3 mRNA,possibly destabilizing mRNA or preventing its translation. Alb3 is ahomologue of the YidC/OxaI/Alb3 protein family, which play an essentialrole in the insertion of a wide range of membrane proteins in bacteria andmitochondria, respectively. In thylakoids chloroplasts the homologue of thisprotein family Albino3 (Alb3) facilitates the insertion of a specialized subsetof proteins, involved in photosynthesis [2].We over-expressed the ncRNA Syr1 under control of inducible by thecopper depletion promoter P petJ on the self-replicating pVZ321 vector inSynechocystis. Over-expression led to a bleaching phenotype and retardedgrowth of the mutant culture. Using antibody against the Alb3 protein, wedemonstrated the reduced amount of Alb3 in an Syr1 over-expressing strain.The content of phycobilisomes was also strongly reduced as shown by SDS-PAGE and pigment measurements. This phenotype affirms the previousbioinformatic prediction.We concluded that the ncRNA Syr1 plays a role in regulation of thephotosynthetic apparatus in Synechocystis through expression control of theAlb3 protein.[1] Georg, J. et al (2009): Evidence for a major role of antisense RNAs in cyanobacterial generegulation.Mol Syst Biol. 5:30[2] Spence, E. et al (2004): A homolog of Albino3/OxaI is essential for thylakoid biogenesis in thecyanobacterium Synechocystis sp. PCC6803. Biol Chem. 279:55792-800.spektrum | Tagungsband 2011