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Autumn Meeting 6–9 September 2010 University of Nottingham – www.sgmnottingham2010.org.uk POSTER ABSTRACTS ↑CONTENTS NT01 CONT. NT01/37 Copper enhances organic hydroperoxide and hydrogen peroxide killing in Xanthomonas campestris pv. campestris SKORN MONGKOLSUK 1,3,4 , Nisa Patikarnmonthon 1 , Sirikan Nawapan 2 , Sarinya Buranajitpakorn 1 & Paiboon Vattanaviboon 1,2 1 Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok 10210, Thailand; 2 Program in Applied Biological Sciences: Environmental Health, Chulabhorn Graduate Institute, Bangkok, Thailand; 3 Dept of Biotechnology, 4 Center of Emerging Bacterial Infections, Faculty of Science, Mahidol University, Bangkok, Thailand Copper (Cu)-based biocides are important chemical controls for both fungal and bacterial diseases in crop fields. During plant microbe interaction, bacteria are exposed to reactive oxygen species produced by plant as a part of host defence responses. Hence, exposure to metals could modulate the oxidative stress response. Cu ions at a sub inhibitory concentration for growth (100 μM) enhances t-butyl hydroperoxide (tBOOH) and hydrogen peroxide (H 2 O 2 ) killing of phytopathogen Xanthomonas campestris pv. campestris (Xcc). Addition of an anti-lipid peroxidation agent (α-tocopherol) and hydroxyl radical scavengers (glycerol and dimethylsulfoxide) partially protected the bacteria from the Cu-enhanced tBOOH and H 2 O 2 killing, respectively. Furthermore, alkyl hydroperoxide reductase mutant is more sensitie to kiliing concentrations of CuSO 4 . The phenotype could be partially suppressed by an H 2 O 2 scavenger (pyruvate) and α-tocopherol. This suggests that Cu ions promote the formation of hydroxyl radicals, and lipid peroxidation which could be responsible for the Cu ion-enhanced H 2 O 2 and organic hydroperoxide killing effects. NT01/38 Roles of RirA and Irr in controlling iron uptake and the oxidative stress response of Agrobacterium tumefaciens Nantaporn Ruangkiattikul 1 , Sakkarin Bhubhanil 1 , ROJANA SUKCHAWALIT 2,3,4 , & Skorn Mongkolsuk 4,5 1 Environmental Toxicology, 2 Applied Biological Sciences, Chulabhorn Graduate Institute; 3 Laboratory of Biotechnology, Chulabhorn Research Institute; 4 Center of Excellence on Environmental Health, Toxicology & Management of Chemicals (ETM); 5 Dept of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand (Email rojana@cri.or.th; Tel. +66 02 5740622) Agrobacterium tumefaciens causes crown gall tumor disease in dicotyledonous plants. In order to survive and cause disease, phytopathogens have to successfully combat both iron deprivation and oxidative bursts, which are environmental stresses that are normally encountered during interaction with host plants. A. tumefaciens RirA (rhizobial iron regulator) functions in concert with Irr (iron response regulator) in controlling iron uptake. RirA repressed iron uptake under high-iron conditions thereby preventing iron overloadinduced toxicity, whereas Irr acted as a positive regulator under low-iron conditions. Therefore, during iron starvation the expression of iron uptake genes were induced via derepression of RirA and activation through Irr that together help to increase efficiency of iron acquisition. Inactivation of the rirA led to deregulation of iron uptake genes. Consequently, the rirA – strain had increased sensitivity to H 2 O 2 -killing via the Fenton reaction. In contrast, an irr inactivation-mutant strain showed increased resistance to H 2 O 2 -killing, at least in part, due to the overproduction of a membrane bound ferritin (MbfA). The data indicated that RirA and Irr are important for adaptation to iron and oxidative stress in A. tumefaciens. NT01/39 The 2-Cys peroxiredoxin, alkyl hydroperoxide reductase C binds haem and participates in its intracellular availability in Streptococcus agalactiae DELPHINE LECHARDEUR 1 , Annabelle Fernandez 1 , Bruno Robert 2 , Philippe Gaudu 1 , Patrick Trieu-Cuot 3 , Gilles Lamberet 1 & Alexandra Gruss 1 1 Institut de la Recherche Agronomique, Institut Micalis, UMR 1319, Jouy-en-Josas, France; 2 Commissariat à l’Energie Atomique, Institut de Biologie et de Technologie de Saclay, CNRS, URA2096 CNRS, Gif sur Yvette, France; 3 Institut Pasteur, Unité de Biologie des Bactéries Pathogènes à Gram-Positif, CNRS, URA 2172, Paris, France (Email delphine.lechardeur@jouy.inra.fr; Tel. +33 1 34 65 20 91) Haem is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular haem partners were identified to date and are not well-conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (Group B Streptococcus; GBS) is a haem-auxotroph, which acquires exogenous haem to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a haem binding protein. AhpC binds hemin with a K d of 0.5 µM and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with haem in vitro and in vivo. A GBS ahpC mutant displayed attenuation of two haem-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in haem intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a haem-binding protein. Please note: Abstracts are published as received from the authors and are not subject to editing. society for general microbiology 83 sgmconferences
Autumn Meeting 6–9 September 2010 ↑CONTENTS University of Nottingham – www.sgmnottingham2010.org.uk NT03 POSTER ABSTRACTS Streptococci NT03 ↑CONTENTS NT03/01 SzeF, SzeN and SzeP: three novel Streptococcus zooepidemicus superantigens ROMAIN PAILLOT 1 , Alistair Darby 2 , Emma Anderson 1,3 , Nicola Wright 1 , Karen Steward 1 , Katy Webb 1 , Ken Smith 3 , Simon Priestnall 3 , Sandra Schöeniger 3 , Kerstin Erles 3 , Androulla Efstratiou 4 , Carl Robinson 1 & Andrew Waller 1 1 Dept of Infectious Diseases, Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU; 2 School of Biological Sciences, University of Liverpool, Liverpool; 3 Dept of Pathology & Infectious Diseases, Royal Veterinary College, Hatfield; 4 Health Protection Agency, London The horizontal transfer of mobile genetic elements and their associated cargo genes between streptococcal species can significantly alter the virulence properties of recipient strains. Gain of prophage elements by Streptococcus pyogenes that contain superantigen-encoding genes has been linked with increased morbidity and mortality in humans. Similarly, the acquisition of φSeq3 (containing seeL, seeM) and φSeq4 (containing seeH, seeI) by Streptococcus equi have been suggested to represent key steps in the evolution of this host-restricted equine pathogen from an ancestral Streptococcus zooepidemicus strain. Streptococcus equi subspecies zooepidemicus is the most frequently isolated opportunistic pathogen of the horse. Streptococcus zooepidemicus also infects a wide range of other animal species including cattle, sheep, pigs, monkeys and humans. Culture supernatants from several strains of Streptococcus zooepidemicus possess superantigen activity, but these strains lack known superantigen-encoding genes. Superantigens are bacterial toxins that trigger unspecific T-cell proliferation and overzealous inflammatory cytokine production in the host. We sequenced the genome of one of these putative superantigen-producing isolates, S. zooepidemicus strain BHS5 (SzBHS5). The strain SzBHS5 has been recently associated with several cases of fatal haemorrhagic pneumonia in dogs. SzBHS5 contains three novel superantigen-encoding genes, szeF, szeN and szeP, the products of which share 59%, 34% and 49% amino acid sequence identity with SpeH, SpeL and SpeM, respectively. To determine the prevalence of Streptococcus zooepidemicus sAgs, we screened by quantitative PCR (qPCR) a panel of Streptococcus equi and Streptococcus zooepidemicus strains that are representative of the wider population as defined by MLST. This included 26 isolates of Streptococcus equi (representing 2 STs) and 165 isolates of S. zooepidemicus (representing 111 STs). Surprisingly, 35% (57 of 165) of Streptococcus zooepidemicus isolates examined contained szeF, 19% (31 of 165) contained szeN and 21% (35 of 165) contained szeP, with szeN and szeP almost always occurring together. The presence of superantigen-encoding genes was significantly associated with mitogenic activity in the Streptococcus zooepidemicus population (P
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