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

PSP006Investigation of PEP-PTS homologous proteins inRalstonia eutropha H16C. Kaddor*, A. SteinbüchelInstitute for Molecular Microbiology and Biotechnology (IMMB),Westphalian Wilhelms-University, Münster, GermanyRalstonia eutropha H16 is a facultative chemolithoautotrophic, H 2-oxidizingβ-Proteobacterium. The genome consists of two chromosomes and themegaplasmid pHG1 and its nucleotide sequence was published in 2006 [1].The genome sequence was investigated to identify by in silico analysiscomponents of the phosphoenolpyruvate-carbohydrate phosphotransferasesystem (PEP-PTS), an important method of sugar uptake in many bacteria.Seven gene loci were found to encode for putative PEP-PTS proteins.Besides the N-acetylglucosamine-specific PEP-PTS (nagFE), a completePEP-dependent phosphoryl transfer chain is lacking in strain H16. Based onthese findings, we generated single and multiple deletion mutants defectivein the PEP-PTS genes and gene regions known to be responsible for fructosetransport (frcACB) to investigate their influence on carbon source utilization,growth behavior and PHB accumulation. In many cases no effect oncarbohydrate uptake was observed. As supposed, the H16 ∆frcACB and H16∆nagFEC mutants exhibited no growth when cultivated on fructose and N-acetylglucosamine, respectively. In addition to the altered utilization ofcarbon sources, different phenotypes and modified PHB contents wereobserved in many mutants. The fruA, ptsH and ptsI single, double and triplemutants stored much less PHB than the wild type and caused reduced PHBsynthesis in mutants lacking the H16_A2203, H16_A0384, frcACB ornagFEC genes. Mutant strain H16 ∆H16_A0384 accumulated 11.5%(wt/wt) more PHB in the cells than the wild type when grown on gluconateand suppressed partially the negative effect of the fruAptsHI mutant on PHBsynthesis. In contrast, deletion of gene H16_A2203 resulted in no significantdifference to the wild type regarding growth and storage behavior. Based onour experimental data we confirmed that the PEP-PTS homologous proteinspresent in R. eutropha H16 are not exclusively involved in the complexsugar transport system but also in cellular regulatory functions.[1] Pohlmann et al (2006): Nat Biotechnol 24:1257-1262.PSP007Two stators contribute to the motility of Shewanellaputrefaciens CN-32S. Held*, A. Paulick, S. Bubendorfer, K. ThormannEcophysiology Group, Max Planck Institute for Terrestrial Microbiology,Marburg, GermanyMany bacteria are motile by rotating flagella, which generally consist ofthree major parts: filament, hook, and basal body. The latter includes statorand rotor elements which create torque to drive the flagellum. The rotation isenergized by gradients of either protons or sodium-ions across themembrane. The preference of the stators for the driving ions specifies thetwo major subtypes of flagellar motors. Genome analysis revealed that inseveral bacteria the number of encoded stator complexes exceeds thenumber of motor systems. In contrast, Shewanella putrefaciens CN-32harbors two complete flagella gene clusters encoding a putative polarflagella system and a putative lateral flagella system along with two sets ofstator elements: the putative sodium-driven PomAB and the putative protondependentMotAB complex. By tagging the stator components MotB andPomB with GFP we demonstrated that PomB predominantly localizes at thepole of the cell whereas MotB-GFP has a lateral and polar localizationpattern. The deletion of the respective stator genes revealed that each statorelement is sufficient to maintain motility. Uncoupling of either the sodiumgradientby the addition of phenamil or collapsing the proton motive forcewith the protonophor CCCP resulted in a reduced but not abolished motilityof the wild type cells. A study using a fusion of GFP to the promoter of thestators indicate a substrate-dependent regulation of the stator elements.Since most S. putrefaciens CN-32 cells possess only a single polar flagellumunder planktonic conditions, we propose that both stators might besimultaneously incorporated and function in a single motor system.PSP008Multiple β-ketothiolases of Ralstonia eutropha H16N. Lindenkamp* 1 , E. Volodina* 1 , K. Peplinski 1 , A. Ehrenreich 2 ,A. Steinbüchel 11 Institute for Molecular Microbiology and Biotechnology (IMMB),Westphalian Wilhelms-University, Münster, Germany2 Insitute of Microbiology, Technical University Munich, Freising, Germanyβ-Ketothiolases catalyze the first step of poly(3HB) synthesis in bacteria bycondensing two molecules of acetyl-CoA to acetoacetyl-CoA. Analyses ofthe genome sequence of Ralstonia eutropha H16 revealed 15 isoenzymes ofPhaA in this bacterium. In this study, we generated knockout mutants ofvarious phaA homologues to investigate their role and contributions topoly(3HB) metabolism and to suppress biosynthesis of 3HB-CoA forobtaining enhanced molar 3-mercaptopriopionate (3MP) contents inpoly(3HB-co-3MP) copolymers. Additionally, to examine the role of singlehomologue, each gene was cloned for heterologous expression in E.coli,protein purification and enzyme characterization. In silico sequence analysisof PhaA homologues and transcriptome data recommended the homologuesphaA, bktB, H16_A1713/H16_B1771, H16_A1528, H16_B1369,H16_B0381 and H16_A0170 for further analysis. Single and multipledeletion mutants were generated to investigate the influence of these β-ketothiolases on growth and polymer accumulation. The deletion of singlegenes resulted in no significant differences to the wild type duringcultivation on gluconate or gluconate plus 3MP. Deletion of phaA plus bktB(= H16∆2 mutant) resulted in approximately 30% less polymeraccumulation than in the wild type. Deletion of H16_A1713/H16_B1771,H16_A1528, H16_B0381 and H16_B1369 in addition to phaA and bktBgave no differences in comparison to the H16∆2 mutant. In contrast,deletion of H16_A0170 additionally to phaA and bktB yielded a mutantwhich accumulated about 30% poly(3HB) (wt/wt, of CDW). We coulddemonstrate that PhaA, BktB and H16_A0170 are majorly involved inpoly(3HB) synthesis in R. eutropha H16. We were not able to suppresspoly(3HB) biosynthesis completely, but the copolymer compositions couldbe altered significantly to a lower percentage of 3HB (from 85 to 52 mol%)and a higher percentage of 3MP (from 15 to 48 mol%), respectively.PSP009Penicillin Binding Protein 4b of Escherichia coli is not aD,D-carboxypeptidase but rather an N-acetylmuramyl-Lalanineamidase involved in cell wall recyclingA. Schneider*, R. Peichert, C. MayerDepartment of Molecular Microbiology, University of Konstanz, Konstanz,GermanyPenicillin-binding proteins (PBPs) are characterized by their affinity forpenicillin and constitute a group of enzymes required for the biosynthesisand modification of the bacterial cell wall. They either catalyze the crosslinkingof peptidoglycan (D,D-amino acid transpeptidation) or have D,Dpeptidaseactivity. PBP4b of E. coli has been reported to bind penicillin butpossesses only very low D,D-carboxypeptidase activity (Vega & Ayala,2006, Arch. Microbiol. 185: 23-27). Here we report that this protein is ratheran N-acetylmuramyl-L-alanine amidase (D,L-peptidase). It cleaves the D-lactyl-L-alanine bond of N-acetylmuramic acid (MurNAc)-peptidesincluding muramyl dipeptide (MDP). The PBP4b-encoding gene yfeW islocated in a putative operon together with the genes encoding the MurNAcspecifictransporter MurP and the MurNAc etherase MurQ. Therefore a rolein MurNAc-peptide recovery is proposed for PBP4b. Interestingly, theenzyme does not accept anhydro-MurNAc-peptides or muropeptides (Nacetylglucosamine(GlcNAc)-MurNAc-peptides),indicating a critical role ofthe MurNAc residue for substrate specificity. Reinvestigation of substraterequirement and a biochemical characterization of PBP4b was enabled bythe development of novel highly sensitive coupled assay that bases on theradioactive phosphorylation of MurNAc or anhydro-MurNAc.PSP010Will not be presented!spektrum | Tagungsband 2011