Annual Conference 2016
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Annual Conference 2016 POSTER ABSTRACT BOOK 21-24 MARCH 2016 ACC, LIVERPOOL, UK ANNUAL CONFERENCE 2016 SESSION 1 – MEMBRANE TRANSPORTERS S1/P1 the pump in this complex and it is conserved between bacterial species, with an average of 78.5% identity between the DNA Novel tripartite tricarboxylate transporters sequences and approximately 80% similarity between the amino acid sequences amongst Enterobacteriaceae. This pump acts as from Rhodopseudomonas palustris a drug-proton antiporter, four residues have been previously Leonardo Talachia Rosa, John Rafferty, reported as essential for proton translocation in Escherichia coli AcrB: D407, D408, K940 and T978. AcrB of E. coli has an identity David Kelly of 86% and a 94% similarity to that of S. Typhimurium. Based on The University of Sheffield, Sheffield, UK these data, we constructed an AcrB D408A chromosomal mutant in S. Typhimurium SL1344. Western blotting confirmed that the Rhodopseudomonas palustris is a soil non-sulfur purple mutant had the same level of expression of AcrB as the parental bacterium, with ability to degrade lignin-derived compounds and wild type strain. The mutant had no growth deficiencies either in also to generate high yields of hydrogen gas, what raises several LB or MOPS minimal media. However, compared with wild type biotechnological interests in this bacterium. Degradation SL1344, the mutant had decreased efflux activity and was pathways, though, must begin with substrate uptake. In this multi-drug hyper-susceptible. Interestingly, the phenotype of the context, Soluble Binding Proteins (SBP`s) dependant AcrB D408A mutant was almost identical to that of an ΔacrB transporters are responsible for high-affinity and specificity mutant. These data indicate that a single point mutation affecting substrate uptake. The aim of this study is to characterize the proton translocation of AcrB renders the pump inactive in Tripartite Tricarboxylate Transporter (TTT) family network in this Salmonella. organism, which might serve as a new source of diversity for biotechnologically interesting uptake systems. Three different SBP dependent secondary transporters of the TTT family have S1/P3 been characterized so far, named R1, R2 and R3. The strategies to characterize them involve a range of different techniques: Purification and characterisation of Bioinformatics, Protein crystallization, Differential Scanning multidrug resistance PACE family efflux fluorescence, Tryptophan fluorescence and mass spectrometry. R2 was found to bind to C4 dicarboxylic acids, having lower proteins affinity for succinate and fumarate and higher affinity for malate. Irshad Ahmad1, Karl Hassan2, Antonio Q-PCR assays revealed 2.5 to 3 times overexpression of R2 1 2 1 under growth in these carbon sources. For R1 and R3, although Calabrese , Ian Paulsen , Peter Henderson genetic analysis suggested they might be involved in glutaconic 1Astbury Centre for Structural Molecular Biology and School and protocatechuic acid uptake, respectively, binding assays of Biomedical Sciences, University of Leeds, Leeds, UK, performed did not support this hypothesis. R1 was successfully 2Department of Chemistry and Biomolecular Sciences, crystalized and we have solved its structure at less than 2 angstroms resolution. The structure shows a ligand-free open Macquarie University, North Ryde, Australia conformation, in a classical Venus flytrap formation. Comparison Active multi-drug efflux pumps are one of the major with other secondary transporter`s SBP shows R1 lacks mechanisms of bacterial resistance to drugs and are an residues which are key for known substrate coordination and alarming threat to antibiotic therapy. From the widespread may thus bind to novel types of substrates. presence of these pumps in bacterial pathogens, five families of multidrug efflux systems have been identified. These are the MFS (major facilitator superfamily), SMR (small multidrug S1/P2 resistance), ABC (ATP-binding cassette), MATE (multidrug and The D408A substitution in the AcrB toxic efflux), and RND (resistance nodulation division) families [1]. The novel membrane protein AceI (Acinetobacter multi-drug efflux protein of Salmonella chlorhexidine efflux) from the Gram negative bacterium Typhimurium SL1344 confers loss of Acinetobacter baumanii conferred resistance to chlorhexidine [2, 3]. Recently 23-homologues of the AceI protein were grouped in function a new sixth family of bacterial multidrug efflux pumps, Xuan Wang-Kan, Vito Ricci, Jessica M.A. Blair, designated the Proteobacterial Antimicrobial Compound Efflux (PACE) family [4]. We are undertaking the expression screening, Laura J.V. Piddock purification and characterization of PACE family proteins. Institute of Microbiology & Infection, University of Starting with eighteen proteins, small-scale expression screening identified six (STY_3166, Fbal_3166, PFL_4558, Birmingham, Birmingham, UK A1S_1503, Tmarg_opt and PSPTO_3587) that were suitable for Salmonella Typhimurium is one of the most common causes of scaling up to larger cultures, inner membrane preparations and foodborne disease. As with many other pathogens, antibiotic purifications. AceI and four of the homologues were purified and resistance in S. Typhimurium has complicated its treatment, their integrity analysed using CD spectroscopy, which revealed leading to increased morbidity and mortality worldwide. Many high contents of alpha helix. Fluorimetric measurements showed Gram-negative bacteria are inherently resistant to some that the widely used biocide chlorhexidine bound to AceI, antimicrobials due to multi-drug efflux pumps; AcrAB-TolC STY_3166 and Fbal_3166. complex is the main efflux pump in Enterobacteriacea. AcrB is Please note: Abstracts are published as received from the authors and are not subject to editing 2 ANNUAL CONFERENCE 2016 SESSION 1 – MEMBRANE TRANSPORTERS References ability to facilitate the movement of single or many compounds [1] Langton, Henderson, Herbert. 2005. Nat. Prod. Rep. 22:439- (Omote et al., 2006). 451 We have identified two genes encoding 'MATE' proteins in the [2] Hassan, Jackson, Penesyan, Patching, Tetu, Eijkelkamp, model amoeba Dictyostelium discoideum, and are making Brown, Henderson, Paulsen. 2013. Proc. Natl. Acad. Sci. U S A knockout and reporter lines for physiological and imaging 110:20254-20259. studies. Expression of the transporters at different life stages suggests the two have distinct and not redundant functions. The [3] Hassan, Elbourne, Li, Gamage, Liu, Jackson, Sharples, Kolstø , first transporter gene is transcribed in highest levels when cells Henderson, Paulsen. 2015. Front. Microbiol. 6:333. signal to each other and aggregate. The second transporter gene is predominant earlier, when unicellular Dictyostelium cells prey [4] Hassan, Liu, Henderson, Paulsen. 2015. MBio 6(1)e0198. 2-14. on bacteria, and transcription also peaks later when cells have aggregated to form a motile, multicellular slug. S1/P4 We hope to address fundamental questions in the cell biology of this important model organism such as the role of the Improving transport capacity of extracellular matrix and how it is formed; to study localisation of Miscanthus-derived sugars and sugars these proteins in the unicellular and multicellular life cycle; and acids by Escherichia coli also to contribute to work on the mechanisms of a flavonoid therapy for polycystic kidney disease which is under Konstantinos Drousiotis1, Jeff Green2, Gavin H. development by our collaborators (Waheed et al., 2014). In Thomas1 plants, it is hypothesised that so-called 'MATE' transporter proteins may be involved in flavonoid transport: currently there 1University of York, York, UK, 2University of Sheffield, is sparse literature on the transport mechanisms that would Sheffield, UK allow/prevent this family of compounds reaching eukaryotic cellular targets. Constantinos Drousiotis, Jeff Green, Gavin H. Thomas There is increasing demand for biorenewable energy that can be S1/P6 produced using plant biomass as the source of carbon and energy for the bacteria production of valuable chemicals and Pulling on a plug domain: direct mechanical biofuels. One potential biofuel crop of the grass Miscanthus gating of BtuB, an outer-membrane protein which can be processed to release lignocellulosic fractions for bacterial fermentation. The carbon and energy sources in this transporter. feedstock contain both hexose and pentose sugars as well as sugar acids. The efficient utilisation of all of these compounds by Samuel J Hickman, Emanuele Paci, David J the fermenting bacterium is important to ensure maximal Brockwell conversion of biomass to product. Here we have studied the Astbury centre for Structural Molecular Biology, University of ability of bacteria to transport the Miscanthus hemicelluloses components xylopyranose, arabinofuranose and glucuronic acid, Leeds., Leeds, UK using an open-source ethanol-producing strain of Escherichia TonB-dependent transporters (TBDT) are outer-membrane coli. We report data on the construction of E. coli strains to proteins of Gram-negative bacteria responsible for the identify novel transporters for these sugars and our work in the scavenging and import of scarce metallo-organic complexes identification of a novel family of furanose-specific ABC from the environment. The transport process, which occurs transporters called Gaf transporters that are likely important for against a concentration gradient, requires the presence of the efficient uptake of arabinose, which is found exclusively in the