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

Based on the recently solved 3D-structure of the periplasmic domain ofCadC and a large scale site-directed mutagenesis approach, a negativelycharged patch was identified that is essential for pH detection. This patch islocated at the dimer interface manifesting the role in proton sensing andsignal transduction.A bioinformatics approach revealed that almost all ToxR-like regulators arevery similar with respect to the cytoplasmic domain that is composed of awinged helix-turn-helix DNA-binding domain and a large unstructured loop.To investigate the role of the loop between the transmembrane domain andthe DNA-binding domain, this part of the protein was gradually truncated orelongated. Our results reveal that the large unstructured loop is important fortransducing the pH signal, but unimportant for lysine signaling.SRV016Signal transduction and gene regulation in response tosurfactant stress in Pseudomonas aeruginosaB. Colley 1 , S. Kjelleberg 1 , J. Klebensberger* 21 Center for Marine Bio-Innovation, University of New South Wales, Schoolof Biotechnology and Biomolecular Sciences, Sydney, Australia2 Institute für Technical Biochemistry, University of Stuttgart, Stuttgart,GermanyBiofilms and cell aggregates are considered to be the predominant form ofmicrobial life in nature. The formation of these multicellular structures oftenproceeds in a sequential manner and is usually a response to the prevailingenvironmental conditions by means of signal transduction pathways. Ourunderstanding of the essential molecular mechanisms underlying thesecomplex regulatory events is currently limited.We previously reported that cell-cell aggregation in response to surfactantstress provides a strategy to increase fitness for Pseudomonas aeruginosaunder unfavourable environmental conditions [1, 2]. Mutagenesisapproaches, overexpression studies and comparative microarray analysisfurther demonstrated, that the second messenger cyclic di-guanosinemonophosphate (c-di-GMP) and a small set of genes, including cupA, psl,cdrAB, PA4623 and the novel signal transduction system siaABCD, areessential for surfactant-induced aggregation [2, 3].In order to decipher the corresponding mechanisms for signal transductionand target gene expression, we performed a systematic mutational analysisof the siaABCD operon. Transcriptional-, biochemical- and physiologicalcharacterisation of these mutants uncovered that the protein encoded by siaBrepresents a repressor of the SiaABCD signalling pathway. Loss of SiaBfunction was found to increase cell aggregation in response to surfactantstress. In contrast, the overexpression of siaB on a multicopy plasmidcompletely abolished cell-cell aggregation during growth in the presence ofsurfactant. Even more interestingly, the non-aggregative phenotype of a∆siaD mutant strain could be complemented by a secondary mutation in thesiaB gene. This suggests that the SiaABCD signal transduction pathway canregulate surfactant-induced aggregation by a bifunctional mechanism. One,which is dependent on SiaD, a putative di-guanylate cyclase involved in thesynthesis of c-di-GMP, and one which is independent of SiaD but mostlikely requires a functional siaA gene, encoding a putative PP2C-likephosphatase. A model for the regulatory mechanism of signal transduction,target gene expression, and the interconnection of the SiaABCD pathwaywith other global regulatory systems will be discussed.[1] Klebensberger et al (2006): Arch Microbiol 185: 417-427.[2] Klebensberger et al (2007): Environ Microbiol 9: 2247-2259.[3] Klebensberger et al (2009): Environ Microbiol 11: 3073-3086.SRP001Glycogen deficiency affects the response to nitrogenstarvation in the cyanobacterium Synechocystis sp. PCC6803Y. Zilliges*, M. Gründel, R. Scheunemann, W. LockauDepartment of Biology,Humboldt-University, Berlin, GermanyGlycogen is a branched polymer of glucose that is present as a carbon andenergy reserve compound in many organisms. Cyanobacteria usuallysynthesize this storage carbohydrate during the day and catabolize it duringthe night. The polymer accumulates massively under conditions ofunbalanced growth, e.g. when cells are starved for nitrogen. Furthermore,the most abundant cyanobacterial protein complexes, the light-harvestingphycobilisomes, are degraded in order to supply amino acids for synthesis ofproteins that may be essential under these conditions. This process iscommonly designated chlorosis.The particular role of glycogen in the interconnected carbon and nitrogenmetabolism in cyanobacteria is not fully understood yet. A detailed analysisof glycogen-deficiency via the analysis of knockout mutants provided newinsights into the cyanobacterial carbon metabolism. Mutants of the modelorganism Synechocystis sp. PCC 6803, defective in genes of ADP glucosepyrophosphorylase and glycogen synthases, respectively, were impaired inphycobilisome degradation under nitrogen starvation (non-bleachingphenotype). Moreover, glycogen-deficient mutants massively excretedpyruvate and 2-oxoglutarate. The latter organic acid is the key metabolitesensor of the cyanobacterial nitrogen response. Glycogen deficiency likeheterotrophic growth on glucose might originate a metabolic switch inSynechocystis sp. PCC 6803. The properties of the glycogen-deficientmutants suggest that an as yet unknown metabolic signal is involved in thecyanobacterial nitrogen response. The impact of this putative metabolicsignal on transcription and expression of key proteins involvedphycobilisome degradation was further examined with respect to the actionof sRNA´s and transcription factors.SRP002The two sides of the medal: impact of carbon dioxide onpH homeostasis and anaplerotic reactions inCorynebacterium glutamicumK.M. Kirsch*, M. Follmann, S. Faust, R. Krämer, K. MarinDepartment of Biochemistry, University of Cologne, Cologne, GermanyDuring industrial fermentations e.g. glutamate and lysine production usingC. glutamicum, increased CO 2 concentrations occur [1]. This phenomenon iscaused by high hydrostatic pressure resulting in a higher solubility of CO 2and by insufficient mixing at the bottom region of large bioreactors. It iswell known that this causes acidification of the medium, however, theimpact of CO 2 on the internal pH of bacterial cells is scarcely understood. Atneutral and alkaline pH, C. glutamicum tolerates up to 20% CO 2 [2]. Underacidic conditions the spontaneous reaction of CO 2 with H 2O leading toHCO 3 - and H + should cause an additional decrease of the internal pH. Weestablished a method to monitor changes in pHi by measuring thefluorescence of GFP variants and applied the technique at different externalCO 2 concentrations. We show that under acidic conditions, pH homeostasisfails in a CO 2 dependent manner. Subsequently, we address the role of thecarbonic anhydrase, responsible for the conversion of CO 2. A deletionmutant of C. glutamicum lacking the ß-type carbonic anhydrase cg2954 didnot show improved pH homeostasis at low pH and high CO 2 concentrationsbut, is unable to grow unless the CO 2 concentration is raised to 10%. This isin agreement with earlier findings at neutral pH [3]. In conclusion, twoaspects have to be considered. On the one hand CO 2 is required in particularfor anaplerotic reactions but, on the other hand high CO 2 concentrationstrigger the collapse of pH homeostasis. We will discuss whether theenzymatic formation of HCO 3 - from CO 2 is essential for growth, especiallyat low pH and whether the lack of carbonate is a bottleneck for C.glutamicum under acidic stress conditions.[1] Mostafa and Gu (2003): Biotechnol. Prog.[2] Bäumchen et al. (2007): J. Biotechnol.[3] Mitsuhashi et al. (2004): Appl. Microbiol. Technol.SRP003Stress responses in the soil bacteria Bradyrhizobiumjaponicum relating to temperature, pH and saltK. Lang*, M. GöttfertInstitute of Genetics, University of Technology, Dresden, GermanyBradyrhizobium japonicum is able to establish a symbiotic interaction withsoybean and is used for inoculation of this crop. During symbiosis, bacteriareduce atmospheric nitrogen to ammonia, which is used by the plant asnitrogen source. The natural habitat of B. japonicum is the soil, a complexand dynamic ecological system with changing parameters like pH, saltconcentration, nutrition availability and a temperature gradient between dayand night. Because these parameters may influence symbiosis, a wholegenome microarray (AffymetrixGeneChip ® ) was used for studying thetranscriptome of B. japonicum in response to heat shock, heat and salt stress,pH 4.0 and pH 8.0. This revealed global as well as specific stress responses.The pH of the growth medium strongly influenced the expression pattern.After incubation for four hours at pH 8.0, more than 1600 genes werespektrum | Tagungsband 2011