VAAM-Jahrestagung 2012 18.–21. März in Tübingen

226labelled hydrocarbons or potential intermediates of the methanogenicdegradation pathway, combined with molecular and biochemical analyses,we are attempting to reveal the carbon flow as well as the active microbialcommunity in the enrichment cultures.SSV001Metabolic pathway fluxes of the marine model bacteriumDinoroseobacter shibae under changing environmental conditionsA. Bartsch*, A. Klingner, J. Becker, C. WittmannInstitut für Bioverfahrenstechnik, TU Braunschweig, Braunschweig, GermanyThe Roseobacter clade is one of the most prevalent bacteria in marinehabitats and is living in various ecological niches [1]. As indicated fromrecently sequenced genomes, they comprise a rich repertoire of metabolicpathways [2, 3]. Dinoroseobacter shibae as prominent member of theRoseobacterclade is additionally known for its ability to grow in asymbiotic relationship with algae, to produce acetylated homoserinelactones (AHL), and to perform aerobic anoxygenic photosynthesis. Firststudies of the central carbon metabolism showed that glucose ismetabolized exclusively via the Entner-Doudoroff pathway [3]. Thisunusual flux distribution differs from most terrestrial microorganisms [3,4]. For a more detailed view into the carbon core metabolism of D. shibae,state of art 13 C metabolic flux analysis was applied [5]. This comprised thecreation of a metabolic network model from available pathway information(databases from Kyoto Encyclopedia of Genes and Genomes and JointGenome Institute). The model was then integrated into the modellingsoftware platform OpenFLUX [6]. For the first time, this allowed toinvestigate the physiological response of D. shibae on the flux level tochanges in environmental conditions such as nutrient status, temperature orsalt level, providing a first systems level insight into this important marinemodel organism. In conclusion fluxes remained quite unaffected byenvironmental perturbation, which indicates a distinct homeostasis as wellas a high robustness of D.shibae. This might partly explain the enormoussuccess of this bacteria and its related species in the marine realm.Acknowledgements: The work is funded by the German ResearchFoundation within the subproject C4 in the SFB TRR51 “Ecology,Physiology and Molecular Biology of the Roseobacter clade: Towards aSystems Biology Understanding of a Globally Important Clade of MarineBacteria”.[1] Buchan et al. (2005): Overview of the marine Roseobacter lineage. Appl Environ Microbiol, 71(10):5665-5677[2] Wagner-Döbler et al. (2010): The complete genome sequence of the algal symbiont Dinoroseobactershibae: a hitchhiker's guide to life in the sea. ISME J, 4: 61-77[3] Fürch et al. (2009): Metabolic fluxes in the central carbon metabolism of Dinoroseobacter shibae andPhaeobacter gallaeciensis, two members of the marine Roseobacter clade. BMC Microbiology, 9: 209[4] Tang et al. (2009): Carbohydrate Metabolism and Carbon Fixation in Roseobacter denitrificans OCh114.PLoS ONE, 4:12[5] Kohlstedt et al. (2010): Metabolic fluxes and beyond-systems biology understanding and engineering ofmicrobial metabolism. Appl Microbiol Biotech, 88:1065-1075.[6] Quek et al. (2009): OpenFLUX: efficient modeling software for 13 C-based metabolic flux analysis.Microbial Cell Factories, 8:25.SSV002Glucosyl-glycerate is a nitrogen stress-dependent carboncapacitatorin Mycobacterium smegmatisV. Behrends* 1 , K.J. Williams 2 , V.A. Jenkins 2 , B.D. Robertson 2 , J.G. Bundy 21 Imperial College, Biomolecular Medicine, London, United Kingdom2 Imperial College, London, United KingdomQuestion: Nutrient depletion often requires an organism to drastically alterits physiology and metabolism. We investigated the response to nutrientdepletion in the form of nitrogen starvation of the bacteriumMycobacterium smegmatis, an important model for the study of the humanpathogen M. tuberculosis.Methods: We profiled the metabolic response of M. smegmatis to nitrogenstarvation, by quantifying the changes in exo- and endometabolome overtime using NMR spectroscopy as well as mass spectrometry. Additionally,we replenished nitrogen and quantified the metabolic consequences of thisnitrogen up-shift.Results: Interestingly, cells of M. smegmatis continued to divide and growafter the extracellular nitrogen source is depleted (albeit at a slower rate)hinting at the presence of an intracellular storage molecule. Concomitantwith extracellular nitrogen run-out, levels of glycerone showed a transientincrease. Inside the cells, low nitrogen triggers the accumulation ofglycogen and other carbon storage molecules including the disaccharidetrehalose and the hexose-conjugate glycosyl-glycerate (GGA), whichaccumulates to high (approx. 500 mM) concentrations inside the cytosol.Following nitrogen up-shift, the metabolism of the cells was drasticallyaltered, leading to a sharp increase in glutamate and trans-aconitate. Thiscoincided with a decrease in GGA. Interestingly, a mutant unable tosynthesise GGA is not viable in low nitrogen concentrations despite themolecule itself not containing any nitrogen.Conclusion: Our study shows that the mycobacterial responses to nitrogenstarvation are not yet fully understood, and potentially involve novelmetabolic regulation. We found that extracellular nitrogen availabilitycontrols intracellular carbon turnover, but surprisingly is not an absoluteprerequisite for growth. Instead, the ability to synthesise a carbon storagemolecule that accumulates during nitrogen shortage is essential for growthin low nitrogen concentrations.SSV003Flavohemoprotein Hmp of Corynebacterium glutamicum isinvolved in nitrosative stress resistanceL. Platzen*, A. Michel, B. Weil, M. Brocker, M. BottInstitut für Bio- und Geowissenschaften, Forschungszentrum JülichGmbH, IBG-1: Biotechnologie, Jülich, GermanyCorynebacterium glutamicum is a Gram-positive soil bacterium, which isused in industrial biotechnology for the production of amino acids [1].Only recently it was discovered that it can also grow under anaerobicconditions by means of nitrate respiration [2,3]. In this process nitrite isformed, which cannot be reduced further by C. glutamicum and thereforeaccumulates in the medium. Nitrate respiration and the presence of nitritecan trigger the formation of reactive nitrogen species, which are toxic forthe cell. Hence, nitrosative stress tolerance has become of interest in orderto improve anaerobic growth of C. glutamicum. We could show that nitriteinhibited aerobic growth of C. glutamicum in a concentration-dependentmanner. The NO-donating agent sodium nitroprusside (SNP) alsodecelerated aerobic growth. Studies on the impact of nitrite on global geneexpression under aerobic conditions revealed that the gene cg3141 (hmp)was 10-fold upregulated. In other organisms, e.g. E. coli, flavohemoproteinHmp has been shown to mediate resistance towards nitric oxide [4].Deletion of hmp in C. glutamicum ATCC13032 resulted in a strain (hmp)which is more sensitive towards nitrite and SNP than the wild type. Thisphenotype was complemented successfully by plasmid-based expression ofhmp. Anaerobic growth with nitrate of the hmp mutant was also retardedin comparison to the wild type. These results demonstrate that theflavohemoprotein Hmp of C. glutamicum is important for nitrosative stresstolerance under aerobic and anaerobic conditions.1. Eggeling, L. and M. Bott, Handbook of Corynebacterium glutamicum 2005: CRC Press, Taylor & FrancisGroup, Boca Raton, Florida, USA.2. Nishimura, T., et al., Anaerobic growth of Corynebacterium glutamicum using nitrate as a terminalelectron acceptor. Appl Microbiol Biotechnol, 2007.75(4): p. 889-97.3. Takeno, S., et al., Anaerobic growth and potential for amino acid production by nitrate respiration inCorynebacterium glutamicum. Appl Microbiol Biotechnol, 2007.75(5): p. 1173-82.4. Gardner, P.R., et al., Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc Natl AcadSci U S A, 1998.95(18): p. 10378-83.SSV004Drug efflux as a surviving strategy in response to theanaerobic stress in E. coliA. YanThe University of Hong Kong, School of Biological Sciences, Hong Kong,Hong KongMultidrug efflux pumps are well known for their ability of removingintracellular antibiotics from bacteria and causing antibiotic and multidrugresistance during the infectious diseases treatment. Bioinformatics andgenome-wide studies have revealed that efflux genes indeed are widelydistributed in all living organisms and constitute from 6% to 18% of alltransporters in bacterial genomes, suggesting a more general role of thisclass of gene products in bacterial physiology beyond just causingantibiotic resistance. In pursue of these physiological functions especiallyduring the process of bacterial stress response, we examined the expressionof all 20 efflux systems encoded in E. coli genome under the anaerobicstress conditions. This led to the identification of a dramatic up-regulationof an efflux pump, MdtEF, under this condition, which is independent ofantibiotic exposure. Expression of MdtEF is found to be up-regulated morethan 20 fold by the global regulator ArcA under anaerobic conditions,resulting in increased efflux activity and enhanced drug tolerance inE. coliunder this condition. To explore physiological functions of MdtEF, weconstructed mdtEF strain and found that E. coli K-12 cells lacking theMdtEF efflux pump display a significantly decreased survival rate whencells reduce nitrate via anaerobic respiration. Replacing nitrate withfumarate as the terminal electron acceptor, or deletion of the genes tnaABwhich are responsible for the biosynthesis of indole, restores the viabilityof the mdtEF strain under anaerobic respiratory conditions. Furtherinvestigation revealed that cells lacking the MdtEF efflux pump aresusceptible to indole nitrosated compounds, a class of toxic by-productswhich are formed and accumulated during the nitrate respiration in E. coliowing to the generation of reactive nitrogen species (RNS) under thiscondition. Taken together, we propose that E. coli activates the multidrugefflux pump MdtEF to remove the toxic nitrosated indole derivativesduring its anaerobic respiration of nitrate, thus providing a survivingstrategy against nitrosative damages during its lifestyle in the anaerobicecological niches.BIOspektrum | Tagungsband 2012