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

RGP006Quorum-sensing control of tropodithietic acidbiosynthesis in Phaeobacter gallaeciensisM. Berger* 1 , A. Neumann 2 , M. Dogs 1 , I. Preuth 1 , S. Schulz 2 , M. Simon 1 ,T. Brinkhoff 11 Institute for Chemistry and Biology of the Marine Environment (ICBM),Carl von Ossietzky University, Oldenburg, Germany2 Institute for Organic Chemistry, Unviersity of Technology, Braunschweig,GermanyProduction of the tropolone antibiotic tropodithietic acid (TDA) is a trait ofsome marine bacteria affiliated to different genera within theRhodobacterales (Alphaproteobacteria) and includes the genome sequencedstrain Phaeobacter gallaeciensis DSM 17395. The synthesis of TDArequires the expression of tdaA-F, as well as six additional genes (cysI,malY, paaIJK, and tdaH). The factors controlling tda gene expression in P.gallaeciensis are not known, but the TDA production correlates with theproduction of acyl-homoserine lactone (AHL) in a growth-phase-dependentmanner. This indicates that TDA production could be controlled by AHLquorum sensing. The genome of P. gallaeciensis codes for a LuxR-LuxItype system with homology to the RaiR-RaiI quorum sensing system ofRhizobium etli. We constructed P. gallaeciensis mutants negative for thecorresponding raiI and raiR homologous genes and determined the chemicalstructure of the signalling molecules to investigate the role of quorumsensing with regard to the TDA biosynthesis. The raiI gene productcatalyzes the production of 3-hydroxy-decanoyl-HSL and is positivelyregulated by RaiR. In contrast to the wild-type P. gallaeciensis, the raiR andthe raiI mutants do not produce TDA when grown in liquid Marine Broth2216 medium. This indicates that the RaiR-RaiI quorum sensing system isrequired for TDA synthesis. Subsequently, we compared the transcriptionlevels of tda genes in the wild-type and the raiR mutant by real-time PCR.The results demonstrated a clear decrease of the expression of allinvestigated tda genes in the raiR mutant, including tdaA, which codes for apotential regulatory protein. Thus, the RaiR-RaiI quorum sensing systemobviously activates the tda gene expression and accordingly the biosynthesisof TDA in P. gallaeciensis in a cell density dependent manner.RGP007Activation of betaine carrier BetP from Corynebacteriumglutamicum in intact cells and in proteoliposomes: aquantitative comparisonS. Maximov*, V. Ott, R. KrämerInstitute of Biochemistry, University of Cologne, Cologne, GermanyIn their natural habitats microorganisms are frequently exposed tohyperosmotic stress. Hyperosmotic stress induces water efflux from a cell,thus increasing the ionic strength of the cytoplasm and reducing the turgor.To counteract dehydration, cells elevate the osmolality of the cytoplasm byimporting inorganic ions or compatible solutes. The best studied transporterfor compatible solutes is the betaine permease (BetP) in the Gram-positivesoil bacterium Corynebacterium glutamicum. BetP is a secondary activetransport protein which imports its specific substrate glycine betaine insymport with two sodium ions from the external medium. Besides transportactivity, BetP comprises the functions of an osmosensor and anosmoregulator. Purified BetP reconstituted in proteoliposomes can detecthyperosmotic stress and regulate its activity in dependence of the externalosmolality. Studies in proteoliposomes and in C. glutamicum demonstratedthat BetP is activated by an increased internal potassium concentration.However, increasing potassium concentration was found not to be the onlystimulus sensed by BetP and not to be sufficient to stimulate the transporterto its maximal activity in C. glutamicum. In vivo studies showed that BetPcan be partially stimulated by high external osmolality also at low internalpotassium concentration. Only a combination of increasing potassiumconcentration and high external osmolality leads to full activation of BetP incells. Consequently, the extent of BetP activity in lipid vesicles might bedifferent than in vivo. To test this hypothesis the molecular activity of BetPin proteoliposmes was compared with the molecular activity (turnover) in C.glutamicum upon the application of different stimuli. To determine themolecular activity of BetP in vivo and in vitro, BetP was quantified in bothC. glutamicum cells and in proteoliposomes. On the basis of thiscomparison, the impact of the different stimuli was analyzed in bothsystems.RGP008Gene conversion in archaeaC. Lange*, C. Hildenbrand, K. Zerulla, T. Stock, S. Breuert, M. Rother,J. SoppaInstitute for Molecular Bio Science, Goethe-University, Frankfurt, GermanyThe halophilic archaeon Haloferax volcanii and the methanogenic archaeonMethanococcus maripaludis were recently shown to be polyploid [1, 2]. H.volcanii contains 18 genome copies during exponential growth and 10genome copies in the stationary phase, M. maripaludis 30 to 55 chromosomecopies with a maximum in the transition phase. This raises the question ifthe alleles on the multiple genome copies are homozygous and if yes, howthe sequences are harmonized.To address this question, a heterozygous M. maripaludis strain containing awild type copy of the essential selD gene as well as an allele interrupted by apuromycin resistance cassette (selD::PM R ) was used [3]. Cultivation in thepresence of different puromycin concentrations revealed that the number ofalleles encoding the resistance depends on the puromycin concentration. Inthe absence of antibiotic, the initial ratio of about 25 selD::PM R alleles to 2selD wild type alleles reversed within 14 generations [2].To study gene conversion in more detail, a H. volcanii model straincontaining two different selectable alleles was constructed on the basis of atrpA deletion mutant [4]. Part of the leuB alleles were interrupted by afunctional trpA copy (leuB::trpA) preserving some wild type leuB copies inthe same cell. This strain is only prototroph for tryptophan as well as leucineas long as it keeps the two different genome copies. Southern blots showedthat cultivation in the presence of only one amino acid leads to a loss of the„unnecessary” allele. The presence of both amino acids caused a geneconversion to the leuB allele which can be explained by the differentamounts of DNA to be synthesized, 53 bp to convert leuB::trpA to leuB and958 bp vice versa. Real time PCR quantification of genome copies [1] atdifferent time points of the experiment revealed that also the velocity ofgene conversion depends on DNA synthesis. The conversion fromleuB::trpA to leuB took 4 days in the presence of tryptophan and 9 days inthe presence of tryptophan and leucine. The conversion from leuB toleuB::trpA was not totally finished after 37 days, 1 of 25 initial leuB copieswas left.The experiments clearly demonstrate that the genome copies of polyploidarchaeal species are permanentlyharmonized by gene conversion, favouringthe evolutionary advantageous variant.[1] Breuert, S. et al (2006): PLoS ONE 1: e92.[2] Hildenbrand, C. et al (2010): JBac doi10.1128/JB.01016-10[3] Stock, T. et al (2010): Mol Microbiol 75:149-16.[4] Allers, T. et al (2004): Appl Env Microbiol 70:943-953.RGP009Polyploidy in ProkaryotesK. Zerulla*, V. Pecoraro, C. Hildenbrand, M. Griese, C. Lange, J. SoppaInstitute for Molecular Bio Science, Goethe-University, Frankfurt, GermanyIn addition to diploid species many polyploid species of eukaryotes exist. Incontrast, prokaryotes are believed to be generally monoploid and to containone copy of a circular chromosome. This assumption is mainly based ongeneralization of the results obtained with the model species Bacillussubtilis and Escherichia coli [1;2]. A literature survey revealed that theploidy level has been determined for a very limited number of species. Morethan half of them have several copies of the chromosome, indicating thatpolyploidy might be more widespread in prokaryotes than anticipated.To get a better overview of the distribution of ploidy levels, genome copynumbers were quantified in 11 bacterial and archaeal species of variousgroups. A recently developed PCR approach [3], originally applied tohaloarchaea, was optimized for the characterization of bacteria. It wasvalidated using slow-growing (t D 103 min) and fast-growing (t D 25 min) E.coli cultures. The copy numbers of the origin and terminus region werequantified and the results were in excellent agreement with published data[2].The approach was applied to determine the ploidy levels of Caulobactercrescentus (α-proteobacterium) and Wolinella succinogenes (εproteobacterium),both of which are monoploid. In contrast, Pseudomonasputida (γ-proteobacterium) contains 20 genome copies and is thus polyploid.A survey of proteobacteria with experimentally-determined genome copynumbers revealed that monoploidy is not typical for proteobacteria [4].The cyanobacteria Synechococcus elongatus and Synechococcus sp. werefound to be polyploid with 4 genome copies, while Synechocystis sp. ishighly polyploid with 58 genome copies. Of two gram-positive speciesCorynebacterium glutamicum is monoploid, while Staphylococcus carnosusspektrum | Tagungsband 2011