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

of aconitases regulates a number of cellular processes, especially in responseto iron limitation and oxidative stress. The plant pathogen Xanthomonascampestris pv. vesicatoria (Xcv) is an obligately aerobic γ- proteobacteriumthat causes bacterial spot disease on pepper and tomato plants. The genomeof Xcv encodes three aconitases (2 AcnA and 1 AcnB enzyme). The acnBgene and one of the acnA genes are divergently organized in the genome ofXcv. While this acnA gene is monocistronic, the acnB gene forms an operonwith two small genes xcv1925 (orfX) and xcv1926 (orfY). This operonorganistaion of orfX-orfY-acnB is highly conserved in the related genera ofplant pathogenic bacteria Xanthomonas and Xylella. The orfX gene producthas similarity to AbrB-type transition-state regulators while OrfY sharesamino acid similarity with virulence-associated proteins (Vap) found in anumber of pathogenic bacteria [2, 3]. Although the function of neitherprotein in Xcv is currently known, both are predicted to bind nucleic acids.Mutants lacking acnB or orfX-orfY-acnB both show normal growth whencultured under laboratory conditions but exhibit reduced growth in planta.They also show a reduced disease progression on susceptible pepper plantsand a decreased hypersensitive reaction (HR) on resistant plants. Platesensitivity assays showed that the mutants exhibit reduced growth in thepresence of the superoxide generator menadione, suggesting that AcnBmight have a role in sensing superoxide stress, which might possibly be aplant response to pathogen infection. An orfX-orfY mutant, in contrast, wasunaffected for growth on menadione but synthesized higher levels of AcnBthan the wildtype, perhaps suggesting a regulatory role for the OrfX and/orOrfY proteins in controlling the levels of AcnB.[1] Williams et al (2002): Nat Struct Biol 9(6):447-52.[2] Hamon et al (2004): Mol Microbiol 52(3):847-60.[3] Miallau et al (2008): J Biol Chem 284(1) :276-83.MPP053In vitro tests of the essential YycFG (WalRK/VicRK) twocomponentregulatory system of Staphylococcus aureusM. Türck*, C. Seifert, P. Sass, G. BierbaumInstitute of Medical Microbiology, Immunology & Parasitology (IMMIP),Friedrich-Westphalian Wilhelms-University, Bonn, GermanyEspecially Gram-positive bacteria are characterized by a massive cell wall,which is consisting of multiple layers of peptidoglycan. Since the structuralintegrity of the cell wall directly affects the viability of the bacterium itself,biosynthesis of peptidoglycan is beyond doubt one of the tightest controlledmechanisms in bacterial growth. This is one of the reasons, why cell walltargeting antibiotics have such an importance in clinical treatment ofbacterial infections. While our knowledge of cell wall composition,biosynthesis and mode of action of targeting antibiotics is comprehensive,much less is known in detail about how bacteria control the tricky interplaybetween degradation and new synthesis of peptidoglycan to coordinate celldivision and cell growth.In recent years it has been shown for S. aureus that the essential YycFG(WalRK/VicRK) two-component regulatory system (TCS) plays a majorrole in the homeostasis of cell wall by controlling the expression ofpeptidoglycan degrading enzymes (autolysins) [1]. Inside the group ofGram-positive bacteria with low G+C-content the orthologues of YycFG(WalRK/VicRK) are almost ubiquitously distributed [1, 2]. Despite this fact,the specific signal(s) by which this TCS is activated is/are still unknown andin vitro tests of the YycG (WalK/VicK) Kinase have been limited to the useof truncated proteins, which comprised only the cytoplasmic regions of theprotein.For this reason, we established an experimental approach that utilizes thepurified full-length kinase in in vitro phosphorylation assays. This systemhas been used to determine the phosphorylation properties of the wild typeYycG (WalK/VicK) kinase and two mutated versions of this protein,harboring an amino acid exchange in the cytoplasmic PAS and HATPase_cdomains, respectively.[1] Dubrac et al (2008): Mol. Microbiol., 70(6), 1307 - 1322.[2] Winkler and Hoch (2009): J. Bacteriol., 190(8), 2645 - 2648.MPP054Colonisation of the host plant root by MagnaportheoryzaeR. Kist* 1 , N. Requena 11 Plant-Microbe Interactions, Karlsruhe Institute of Technology (KIT),Karlsruhe, GermanyThe well known leaf pathogen Magnaporthe oryzae has also been shown tobe able to invade the plant via the roots. However the fungus undergoes adifferent hyphal differentiation in this interaction then it does in the leafinfection (Dufresne et.al, 2001, Sesma & Osbourn, 2004). This hyphaldifferentiation is a crucial step for all root colonising fungi andconsequentially it was shown that some of these components are shared withother fungi (Heupel et al., 2010). In our work we are interested in thecharacterisation of underlying differences in hyphal morphogeneticdevelopment which are leading to root colonisation. For this we did ascreening for root infection defective insertional mutants and, in a secondapproach, microarray analysis, comparing root infection with leaf infection.In this transcriptomic approach we found several genes that are apparentlyspecific for the colonisation of plant roots. Many of them are secretedproteins and so are good candidates to be fungal effector proteins. For themost interesting ones further analysis, like creation of knock out strains andin planta characterisation are on the way.MPP055Phosphorylation activities of the VraSR two-componentsystem of Staphylococcus aureus - employing the fulllengthVraS histidine kinase -L. Mildenberger, M. Türck*, A. Berscheid, G. BierbaumInstitute of Medical Microbiology, Immunology & Parasitology (IMMIP),Friedrich-Westphalian Wilhelms-University, Bonn, GermanyS. aureus is a major cause of nosocomial infections and clinical therapybecomes more and more difficult because of the increase in antibioticresistance displayed by this pathogen. Two-component regulatory systems(TCSs), consisting of a sensor histidine kinase (HK) and a correspondingresponse regulator protein (RR), play an important role in bacterial growth,enabling them to respond and to adapt to environmental stress conditions.In S. aureus the VraSR (vancomycin-resistance associated sensor/regulator)TCS is involved in resistance to vancomycin and β-lactam antibiotics [1, 2,3]. In this context, it has been shown that VraSR is involved in amechanism, which senses inhibition of bacterial cell wall (peptidoglycan)biosynthesis [4].Signal transduction via phosphoryl group transfer follows a general schemein TC systems. Upon activation by a specific signal the HK (VraS)undergoes autophosphorylation at a conserved His residue and in a secondstep the phosphoric group is transferred to a conserved Asp residue of theRR (VraR), thereby changing its activity in controlling DNA transcription.Since VraS is a membrane protein, that possesses two TM domains at the N-terminus, purification of the entire protein is difficult. For that reason,previous experiments [4] have been carried out with truncated versions ofVraS. Here, we want to present the recombinant expression of the full-lengthVraS kinase and VraR response regulator protein of S. aureus NCTC8325and their activity tests in in vitro phosphorylation assays. For this purpose adetergent-micelle system was used, which has been previously establishedfor initial studies of the phosphorylation behavior of the essential YycFG(WalRK/VicRK) TCS of S. aureus.[1] Kuroda et al (2000): Biochem. Biophys. Res. Commun. 269, 485 - 490.[2] Kuroda et al (2003): Mol. Microbiol. 49, 807 - 821.[3] Boyle-Vavra et al (2006): FEMS Microbiol. Lett. 262, 163 - 171.[4] Belcheva and Golemi-Kotra (2007): J. Biol. Chem., 283, 12354 - 12364.MPP056Staphylococcus saprophyticus: localisation of fibronectinbinding in the A domain of SdrIS. Neumann*, M. Korte, L. Marlinghaus, F. Szabados, M. Kaase,S. GatermannInstitute for Hygiene and Microbiology, Department of MedicalMicrobiology, Ruhr-University, Bochum, GermanyStaphylococcus saprophyticus is a gram-positive and coagulase-negativepathogenic staphylococcus causing urinary tract infections in young women.It is hydrophobic, able to bind fibronectin, laminin and collagen andspektrum | Tagungsband 2011