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

PSP011Growth rate-dependent physiology of Aromatoleumaromaticum EbN1 in anaerobic, benzoate-limitedchemostatsK. Trautwein* 1,2 , K. Mangelsdorf 3 , A. Steinbüchel 4 , R. Reinhardt 5 ,R. Rabus 1,21 Institute for Chemistry and Biology of the Marine Environment (ICBM),General and Molecular Microbiology, Oldenburg, Germany2 Department of Microbiology, Max Planck Institute for MarineMicrobiology, Bremen, Germany3 German Research Centre for Geosciences (GFZ), Section 4.3 OrganicGeochemistry, Potsdam, Germany4 Institute for Molecular Microbiology and Biotechnology, WestphalianWilhelms-University, Münster, Germany5 Genome Center Cologne at Max Planck Institute for Plant BreedingResearch, Cologne, GermanyThe growth rate-dependent physiological and proteomic response ofAromatoleum aromaticum EbN1 was analyzed in anaerobic chemostatsunder benzoate-limited conditions. Constrained by a defined and constantsupply of the limiting nutrient at a specific rate (dilution rate), the bacterialpopulation in the chemostat approached a steady state characterized by aspecific growth rate and stable growth parameters (optical density, cellnumber, nitrate and nitrite concentration). Stabilization of growth parameterswas observed after 3 to 5 residence times (calculated as the inverse of thedilution rate).To analyze global changes in response to different growth rates, cells wereharvested from continuous cultures during steady state (12 +/- 0.9 residencetimes) at low (0.036 h -1 ), medium (0.108 h -1 ) or high (0.180 h -1 ) growthrates, and from batch cultures during growth at the maximum specificgrowth rate (0.20 h -1 ). For each growth condition four biological replicateswere comparatively analyzed by two-dimensional difference gelelectrophoresis (2D DIGE). This revealed dynamic, growth rate-dependentchanges in the protein abundance of more than 160 proteins including alsoproteins involved in benzoate catabolism. Compared to benzoate-limitedgrowth at a high growth rate, the most dramatic changes were observed at alow growth rate, e.g. up-regulation of several periplasmic binding proteinsinvolved in nutrient uptake and in proteins related to other aromaticcatabolic pathways. In addition, growth rate-dependent changes in themembrane phospholipid composition and polyhydroxybutyrate (PHB)content were also observed for each of the four growth conditions.PSP012Analysis of antibiotic tolerance in Staphylococcus aureus -towards the characterization of S. aureus persister cellsS. Lechner* 1 , M. Vulic 2 , K. Lewis 2 , R. Bertram 11 Institute of Microbiology and Infection Medicine (IMIT), Eberhard-Karls-University, Tübingen, Germany2 Antimicrobial Discovery Center, Biology, Northeastern University, Boston,USABacterial cultures contain subpopulations of dormant cells, so calledpersisters, able to survive antibiotic treatment without acquiring heritableresistance. Persisters are not mutants, but reversible phenotypic variants ofnormally growing cells. We aimed to study mechanisms governing persisterformation and their physiologic, cellular, and genetic properties in S. aureus.Different planktonically grown S. aureus strains were treated in log orstationary growth phase with various antibiotics. Strains included SA113,the small colony variants (SCVs) hemB and menD, as well as HG001,HG002, and HG003. Antibiotics applied were daptomycin, tobramycin,ciprofloxacin, rifampin, and penicillin in a range of 1-100-fold MIC.Time-dependent CFU analyses revealed widely minimal killing ofstationary-phase cells, almost irrespective of the strain or the kind andconcentration of antibiotic. Hence, the persister state may be thepredominant S. aureus phenotype in stationary-phase. Two strikingexceptions were observed: I) Treatment of SA113 with 100-fold MIC ofdaptomycin eradicated about 99.98 % of cells within 1 h, whereas theremaining population appeared less vulnerable over time. The biphasictemporal killing kinetics are highly indicative of persister cells. II) Upontreatment with 100-fold MIC tobramycin, the menD culture displayed asimilar, albeit less pronounced effect.In exponentially growing cultures daptomycin killed SA113 cellscompletely within 4 h at 10-fold MIC or 1 h at 100-fold MIC, while SCVkilling was retarded. HG001-003 strains were efficiently killed after 1 h at100-fold MIC of daptomycin. Intriguingly, tobramycin treatment appearedto eradicate SA113 wt less efficiently as SCV strains at both 10- and 100-fold MIC. Killing curves indicated a large fraction of SA113 persisters 1 hafter tobramycin treatment, lasting for 5-7 h. Tobramycin treatment at 10-fold MIC of HG001-003 resulted in SCV-like-cells upon cultivation on solidmedia.Thus, growth phase, strain background, and genotype appear to be importantfactors in the formation of S. aureus persister cells. We suggest that S.aureus tolerance to antibiotics in stationary-phase is strongly associated withelevated levels of persisters.PSP013The membrane protein MusI is indispensable for maltoseuptake in Corynebacterium glutamicum by the ABCtransportsystem MusEFGK 2A. Henrich, N. Kuhlmann, R. Krämer, G.M. Seibold*Institute of Biochemistry, University of Cologne, Cologne, GermanyThe disaccharide maltose is efficiently used by the Gram-positiveCorynebacterium glutamicum as substrate for growth and amino acidproduction. Furthermore maltose can be used as an additive in L-valinefermentations to increase the overall productivity of C. glutamicum strains[1]. Maltose is metabolized in C. glutamicum by a pathway requiringmaltodextrin and glucose formation by the 4-α-glucanotransferase MalQwith maltose as substrate, glucose phosphorylation by the glucose kinasesGlk and PPgk and maltodextrin degradation via the reactions ofmaltodextrin phosphorylase and α-phosphoglucomutase [2, 3]. Maltoseuptake is accomplished by an ABC transport system encoded by musK(cg2708), musE (cg2705), musF (cg2704), and musG (cg2703).We here analysed the transcriptional organisation of the mus genes usingNorthern Blots and RT-PCRs: Whereas musK and musE are transcribedmonocistronically in C. glutamicum, musF and musG are part of an operon,which also includes the orf cg2701 (musI). The gene musI encodes aputative membrane protein, which shares no homologies to so farcharacterised proteins. Characterisation of growth and of 14 C-maltose uptakein the musI-mutant strain C. glutamicum IMcg2701 showed that maltoseutilisation and uptake were abolished. Plasmid encoded expression of musIand of musI-strep (encodes a N-terminal Streptavidin tagged version ofMusI) fully complemented C. glutamicum IMcg2701. In Western blotexperiments the tagged MusI protein was detected exclusively in themembrane fraction of C. glutamicum.From these results we conclude, that the musI encoded protein encodes anovel essential component of the maltose ABC-transporter of C.glutamicum, which should be therefore designated MusEFGK 2I.[1] Krause, F. S. et al (2010): Increased glucose utilization in Corynebacterium glutamicum by use ofmaltose, and its application for the improvement of L-valine productivity. Appl Environ Microbiol76:370-374.[2] Lindner, S. N. et al (2010): Cg2091 encodes a polyphosphate/ATP-dependent glucokinase ofCorynebacterium glutamicum. Appl Microbiol Biotechnol 87:703-13.[3] Seibold, G. M.et al (2009): Roles of maltodextrin and glycogen phosphorylases in maltoseutilization and glycogen metabolism in Corynebacterium glutamicum. Microbiology 155:347-358.PSP014Characterization of a novel subtilisin-like serine proteaseof Pseudomonas aeruginosaA. Pelzer* 1 , F. Rosenau 2 , K.-E. Jaeger 1 , S. Wilhelm 11 Institute for Molecular Enzyme Technology, Heinrich-Heine-University,Jülich, Germany2 Institute of Pharmaceutical Biotechnology, University of Ulm, Ulm,GermanyP. aeruginosa is ubiquitously distributed, living in wet or humidsurroundings ranging from soil to human and produces a huge variety ofextracellular proteins including several proteases. Some of these proteaseslike Elastase and Protease IV are well characterized but others exist of whichnothing is known so far. Proteases in general are highly relevant fortechnical enzyme applications. Subtilases for example are typical detergentproteases and are defined as serine proteases belonging to the peptidase_S8family. These subtilases are encoded as preproenzymes carrying a signalpeptide which drives their translocation through the cytoplasmic membraneand a propeptide acting as a folding mediator required to give the proteaseits final native conformation.By homology we have identified the open reading frame PA1242 in thegenome sequence of P. aeruginosa PAO1 encoding a so far hypotheticalprotein as a putative member of the subtilisin-like serine protease family S8.spektrum | Tagungsband 2011