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

FGP011Functional genome analysis of Geobacillus sp. HH01, anorganism that secrets a thermostable lipaseS. Wiegand* 1 , U. Köhler 2 ,W.Streit 2 , H. Liesegang 11 Institute for Microbiology and Genetics, Goettingen Genomics Laboratory,Georg-August-University, Goettingen, Germany2 Biocenter Flottbek, University of Hamburg, Hamburg, GermanyThe genus Geobacillus comprises thermophilic bacteria. As members of theBacillaceae Geobacilli are Gram-positive, endospore-forming rods that livefacultative aerob. Geobacillus spp. have been isolated from oilfields as wellas from geothermal volcanic environments or hay compost and diverse otherhabitats. Strains of the genus have been found to utilize a broad range of(polymeric) carbon sources i.e. polysaccharides, proteins and n-alkanes.Some strains of Geobacilli secret proteases and lipases to degrade theirpolymeric substrate extracellularly and are therefore of high interest forindustrial applications.Here we present a functional genome analysis of Geobacillus sp. HH01isolated from soil. The genome size and the GC content are approximately3.5 Mb and 52%, respectively. The initial assembly resulted in 182 contigswith an average coverage of 13. Currently different PCR-based techniquesare employed to close the remaining gaps and to resolve misassembledregions. Gene prediction, annotation and genome comparison are performedas described in Liesegang et al.The focus of the analysis is on putative industrial interesting features. Thegenome will be examined for secretion systems, genetic accessibility,secondary metabolism (PKS/NRPS cluster), and especially on exoenzymessuch as lipases, proteases and amylases.[1] Liesegang, H. et al: Complete genome sequence of Methanothermobacter marburgensis, amethanoarchaeon model organism. J Bacteriol 192: 5850-5851.FGP012Functional genome analysis of the purine-utilizingbacterium Clostridium acidiuriciK. Hartwich*, A. Poehlein, A. Wollherr, G. Gottschalk, R. DanielInstitute for Microbiology and Genetics, Göttingen Genomics Laboratory,Georg-August-University, Göttingen, GermanyClostridium acidiurici is a purine-utilizing organism. It is able to grow withuric acid and xanthine as sole carbon, nitrogen and energy source. The majorfermentation products from these substrates are ammonia, carbon dioxideand acetic acid. C. acidiurici is unable to degrade complex nitrogencontainingsubstrates such as tryptone or yeast extract [1].Raw sequencing of the C. acidiurici genome was done by the GoettingenGenomics Laboratory employing the 454 GS FLX XLR Titaniumpyrosequencing technology. Sequences were assembled into contigs usingthe Newbler assembly tool from Roche. To close remaining gaps and toidentify misassembled regions caused by repetitive sequences, differentPCR-based techniques are currently employed. The estimated genome sizeand the GC content are 3 Mb and 29.74%, respectively.To elucidate the genome features and the unique metabolism of C. acidiuriciannotation and genome comparisons are performed.Automatic annotation indicated the existence of common pathways likeglycolysis/gluconeogenesis and their specific enzymes. However, C.acidiurici did not show any growth on other substrates than purines,including C5- and C6-sugars or amino acids. Further manual annotationsrevealed an incomplete phosphotransferase system, which might be thereason for the organism’s inability to use sugars as substrates.Further growth tests shall reveal the stress response on salts, heavy metalsand antibiotics, which were predicted by automatic and manual annotation.[1] Vogels, G. D. and C. van der Drift (1976): Degradation of Purines and Pyrimidines byMicroorganisms. Bacteriol. Rev. 40(2): 403-468.FGP013Proteomic and transcriptomic elucidation of mutantRalstonia eutropha G+1 with regard to glucose utilizationM. Raberg* 1 , K. Peplinski 1 , S. Heiss 1 , A. Ehrenreich 2 , B. Voigt 3 , C. Döring 4 ,M. Bömeke 4 , M. Hecker 3 , A. Steinbüchel 11 Institute for Molecular Microbiology and Biotechnology (IMMB),Westphalian Wilhelms-University, Münster, Germany2 Department of Microbiology,Technical University, München, Germany3 Department of Microbiology, Ernst-Moritz-Arndt-University, Greifswald,Germany4 Institute for Microbiology und Genetics, Georg-August-University,Göttingen, GermanyTaking advantage of the available genome sequence of R. eutropha H16,glucose uptake in the UV generated glucose-utilizing mutant R. eutrophaG+1 was investigated by transcriptomic and proteomic analyses. Datarevealed clear evidence that glucose is unspecifically transported by a notstrictly specific N-acetyl glucosamine phosphotransferase system (PTS)-typetransport system, which is overexpressed probably due to a derepression ofthe encoding nag operon by an identified insertion mutation in geneH16_A0310 (nagR) in this mutant. Phosphorylation of glucose issubsequently mediated by NagF (cytosolic PTS component EIIA-HPr-EI) orGlK (glucokinase), respectively. The inability of the defined deletion mutantR. eutropha G+1 ∆nagFEC to utilize glucose strongly confirms this finding.In addition, secondary effects of glucose, which is now intracellularyavailable as carbon source, on the metabolism of the mutant cells in thestationary growth phase occurred: Intracellular glucose degradation isstimulated by stronger expression of enzymes involved in the 2-keto-3-deoxygluconate 6-phosphate (KDPG) pathway and subsequent reactionsyielding pyruvate. The intermediate phosphoenolpyruvate (PEP) in turnsupports further glucose uptake by the Nag-PTS. Pyruvate is thendecarboxylated by the pyruvate dehydrogenase multienzyme complex toacetyl CoA, which is directed to poly(3-hydroxybutyrate), PHB, which issynthesized in greater extent as indicated by the upregulation of variousenzymes of PHB metabolism. The larger amounts of NADPH required forPHB synthesis are delivered by significantly increased quantities of protontranslocatingNAD(P) transhydrogenases. This current study successfullycombined transcriptomic and proteomic investigations to unravel thephenotype of this hitherto undefined glucose-utilizing mutant.FGP014Genome-analysis of ClostridiumsaccharoperbutylacetonicumA. Poehlein* 1 , A. Grimaldo 2 , A. Thürmer 1 , K. Hartwich 1 , S. Offschanka 1 ,G. Gottschalk 1 , H. Liesegang 1 , P. Dürre 3 , R. Daniel 11 Institute for Microbiology and Genetics, Göttingen Genomics Laboratory,Georg-August-University, Göttingen, Germany2 Biologic Sciences Faculty, Autonomous University of Nuevo LeónMonterrey, Mexico3 Institute for Microbiology und Biotechnology, University of Ulm, Ulm,GermanyClostridium saccharoperbutylacetonicum strain N1-4, is known as abutanol-hyperproducing bacterium. Various organic compounds arefermented, such as glucose, fructose, saccharose, xylose and cellobiose, butalso sorbitol, dulcitol and inositol. The industrial strains of C.saccharoperbutylacetonicum are used in the fermentation processes for theproduction of the solvents acetone, butanol, and ethanol from a variety ofsugar- and starch-based substrates.The economics of butanol production is primarily affected by raw materialsused, yields and concentrations of solvents as well as productivity. One ofthe most important economic factors in solvent fermentation is the cost ofsubstrate. Thus, the availability of an inexpensive raw material is essential ifsolvent fermentation is to become economically viable.C. saccharoperbutylacetonicum N1-4 is a hyperamylolytic strain andcapable of producing solvents efficiently from cassava starch and cassavachips which represents an alternative cheap carbon source for fermentationprocesses.To extend our knowledge on the biochemistry and physiology of thisinteresting organism, we completely sequenced the genome of C.saccharoperbutylacetonicum N1-4. The strain has two replicons, achromosome with the size of 6.5 Mb and a megaplasmid of 135 kb; the G+Ccontent of the DNA is 29.53 mol%. Some features of this organism apparentfrom the genome sequence will be reported.spektrum | Tagungsband 2011