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

202attracted the attention of molecular microbiologists and there have beenseveral approaches to uncover their signaling landscape.Bacillus subtilis encodes three putative diadenylate cyclases. One of them,DisA, checks DNA integrity to control cell division and sporulation. Incontrast, the function of the other proteins, CdaA and CdaS, is stillunknown. Moreover, B. subtilis encodes two diguanylate cyclases, CdgAand CdgB. In many bacteria, c-di-GMP governs the lifestyle switchbetween biofilm formation and motility. However, nothing is known aboutthe function of c-di-GMP in B. subtilis.We have studied the role of these enzymes in the metabolism of cyclicdinucleotides in B. subtilis. Both signaling molecules have a tremendouseffect on the production of flagellin (Hag), thus they are involved in thecontrol of motility. In contrast, cyclic dinucleotides have no effect onbiofilm formation. This similarity of the phenotypes of the mutants hasnever been observed before and suggests a link between the signalingpathways of the two cyclic signaling molecules.Another unprecedented result of our studies is the essential function of B.subtilis for the formation of c-di-AMP. The genes encoding the twovegetative diadenylate cyclases, DisA and CdaA, could be deleted onlywhen the sporulation-specific enzyme CdaS was artificially expressed inthe logarithmic phase. Thus, our results indicate a key role for cyclicdinucleotide signaling in B. subtilis.RSP035Role of alternative sigma factor PP4553 in stress response andbiofilm formation ofPseudomonas putidaKT2440B. Bugert*, J. OverhageKarlsruhe Institute of Technology - KIT, Institute of Functional Interfaces,Eggenstein-Leopoldshafen, GermanyPseudomonas putidais a Gram-negative, aerobic, flagellated and nonpathogenicsoil bacterium, which is well known for its extremelymetabolic versatility. Because of this, P. putidaoffers a considerablepotential for biotechnological applications. The remarkable versatility ofthis bacterium is at least in parts driven by sophisticated and coordinatedregulation of gene expression mediated by a repertoire of transcriptionalregulators, in particular the so called sigma factors. Sigma factors areessential for prokaryotic transcription initiation and enable specific bindingof the RNA polymerase to the respective promoter recognition sites.Bacteria generally contain one housekeeping sigma factor and a pool ofalternative sigma factors which are activated in response to different andoften stressful conditions. The genome of P. putida exhibits with 24 astriking number of putative sigma factors, one of which is open readingframe PP4553. To analyze this putative sigma factor in more detail, weconstructed a gene knock-out deletion mutant of PP4553 inP. putida KT2440. Further characterization of this PP4553-mutant revealeda twofold increase in attachment as well as biofilm formation on abioticsurfaces in comparison to the wild type strain. Moreover, growth analysesof wild type and PP4553-mutant strain under different stressful conditionssuggested that PP4553 is also involved in stress response ofP. putidaKT2440. To gain a deeper insight into the regulatory circuit of the putativesigma factor PP4553, we performed transcriptome analysis using Illuminasequencing.RSP036Cloning and heterologous expression of naphthoate-CoA ligasefrom the sulphate-reducing culture N47P. Tarouco* 1 , H. Mouttaki 1 , J. Kölschbach 1 , A. Geerlof 2 , R. Meckenstock 11 Helmholtz Zentrum München, Institute of Groundwater Ecology, Munich,Germany2 Helmholtz Zentrum München, Institute of Structural Biology, Munich,GermanyThe anaerobic metabolism of naphthalene by the sulphate-reducing cultureN47 is initiated by carboxylation to 2- naphtoic acid. N47 is an enrichmentculture composed mainly of one member of the deltaProteobacteria. In cellextracts of this culture we have been able to measure a specific 2-naphthoate-CoA-ligase activity which is responsible for the activation ofthe carboxyl group with HS-CoA. Blasting the amino acid sequence ofbenzoate-CoA ligase of Rhodopseudomonas palustris[1] against the N47genome were identified 9 putative 2-naphthoate-CoA ligase candidates.Here, we aim at the purification and characterization of the N47 2-naphthoate-CoA-ligase. The putative genes will be cloned and expressedinE. coli. Primers were designed to remove the native stop codon and toplace the gene of interest in frame with an N-terminal His-tag of theexpression vector. The gene will be amplified and transferred into anexpression system. Functional naphthoate-CoA ligase shall be purified viathe His-tag and characterized.1. Gibson J, Dispensa M, Fogg GC, Evans DT & Harwood CS. 4-Hydroxybenzoatecoenzyme Aligase fromRhodopseudomonas palustris: purification, gene sequence, and role in anaerobicdegradation.J Bacteriol.176(1994) 634-641.RSP037The sensor kinase PA4398 of Pseudomonas aeruginosa PA14regulates swarming motility and biofilm formationJ. Strehmel*, J. OverhageKIT, Institute for Functional Interfaces, Eggenstein-Leopoldshafen,GermanyMulticellular behavior is an important process central to the pathogenesisof P. aeruginosa. In addition to biofilm formation, swarming motilityrepresents a second surface-associated community behavior of this humanpathogen. Recently, we have shown that swarming can be considered as adistinct physiological state with a tailored metabolic lifestyle or a complexadaptation of P. aeruginosa in response to a viscous environment(arguably similar to the mucus rich CF lung) leading to increased antibioticresistance and virulence gene expression. During an extensive mutantlibrary screening for swarming deficient mutants, we identified a twocomponentsensor kinase transposon mutant (PA4398) in P. aeruginosaPA14 with defects in the ability to swarm on semisolid surfaces (1). Toanalyze the function of this sensor kinase in more detail, we constructed aknock-out deletion mutant of PA4398 in P. aeruginosa PA14 andphenotypically characterized this sensor kinase mutant. In addition to itsswarming defect, this mutant also exhibited a decreased production ofbiofilm mass in comparison to wildtype cells after 24 hours of growth. Incontrast, no differences regarding growth rate, twitching and swimmingmotility were observed. First preliminary microarray analysis revealed theinvolvement of this sensor kinase in the regulation of nitrogen and ironmetabolism.(1) Yeung AT et al., 2009. J. Bacteriol. 191:5592-602.RSP038Will not be presented!RSP039Exploring subtilin/nisin hybrid-peptidesT. Spieß* 1 , B. Sattler 1 , K.-D. Entian 1,21 Goethe Universität Frankfurt, Institute of Molekular Biosiences, Frankfurt a.M., Germany2 Goethe Universität Frankfurt, Cluster of Excellence:MacromolecularComplexes, Frankfurt a. M., GermanyLantibiotics [1], such as subtilin and nisin are gene-encoded antimicrobialactive peptides [2]. These peptides are ribosomally synthesized by Bacillussubtilis and Lactococcus lactis, as inactive prepeptides [1]. The inactiveprepeptides undergo an extensive posttranslational modification togenerate active peptides [3, 4]. The posttranslational modifications resultin the formation of non-proteinogenic amino acids lanthionine and 3-methyllanthionin, as well as didehydroamino acids. Nisin and subtilin havea similar lanthionin ring structure and they differ in 14 amino acids.Despite their structural similarity, they are highly specific for theirrespective autoinduction system. In the presence of extracellular subtilin ornisin, a two component system consistent of a histidine kinase and aresponse regulator interacts with the respective lantibiotic. The histidinekinase is autoinduced in a quorum sensing manner and phosphorylates theresponse regulator, which in turn induces the expression of the lantibioticstructural gene, the genes of the lantibiotic biosynthesis machinery and theself immunity genes[5].So far the interaction between subtilin or nisin with their correspondinghistidine kinases has not been characterized. To identify the specificbinding motif of subtilin and nisin, a -galactosidase based reporter systemfor lantibiotic autoinduction was constructed. In addition, a plasmid basedexpression system was created, which enables the heterologous expressionof subtilin/nisin hybrid peptides. These hybrid molecules will be used toanalyse and optimize the lantibiotic properties with respect to the activity,stability and solubility. Additionally, these molecules will also identify thespecific binding motif between the lantibiotic and its histidine kinase byvirtue of their specific autoinduction.1. Schnell, N., et al.,Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic withfour sulphide-rings.Nature, 1988.333(6170): p. 276-8.2. Chatterjee, C., et al.,Biosynthesis and mode of action of lantibiotics.Chem Rev, 2005.105(2): p.633-84.3. Siegers, K., S. Heinzmann, and K.D. Entian,Biosynthesis of lantibiotic nisin. Posttranslationalmodification of its prepeptide occurs at a multimeric membrane-associated lanthionine synthetasecomplex.J Biol Chem, 1996.271(21): p. 12294-301.4. Kiesau, P., et al.,Evidence for a multimeric subtilin synthetase complex.J Bacteriol, 1997.179(5):p. 1475-81.5. Kleerebezem, M.,Quorum sensing control of lantibiotic production; nisin and subtilinautoregulate their own biosynthesis.Peptides, 2004.25(9): p. 1405-14.RSP040The phytochrome regulon of Pseudomonas aeruginosaS. Heine* 1 , K. Barkovits 1 , M. Sheer 2 , N. Frankenberg-Dinkel 11 Ruhr-University Bochum, Physiology of Microorganisms, Bochum, Germany2 Technical University , Institue of Microbiology, Braunschweig, GermanyPhotoreceptors are able to sense light with specific wavelengths. One ofthe most familiar biliproteins are red/far-red light sensing phytochromes.BIOspektrum | Tagungsband 2012