Kim E. Bettaney
University of Leeds
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Featured researches published by Kim E. Bettaney.
Molecular Microbiology | 2009
Georgios Psakis; Massoud Saidijam; Julia Polaczek; Kim E. Bettaney; Jocelyn M. Baldwin; Stephen A. Baldwin; Ryan J. Hope; Lars-Oliver Essen; Richard C. Essenberg; Peter J. F. Henderson
Helicobacter pylori is a gram-negative pathogenic microaerophile with a particular tropism for the mucosal surface of the gastric epithelium. Despite its obligatory microaerophilic character, it can metabolize D-glucose and/or D-galactose in both oxidative and fermentative pathways via a Na(+)-dependent secondary active transport, a glucokinase and enzymes of the pentose phosphate pathway. We have assigned the Na(+)-dependent transport of glucose to the protein product of the H. pylori 1174 gene. The gene was heterologously expressed in a glucose transport-deficient Escherichia coli strain, where transport activities of radiolabelled D-glucose, D-galactose and 2-deoxy-D-glucose were restored, consistent with the expected specificity of the hexose uptake system in H. pylori. D-mannose was also identified as a substrate. The HP1174 transport protein was purified and reconstituted into proteoliposomes, where sodium dependence of sugar transport activity was demonstrated. Additionally the tryptophan/tyrosine fluorescence of the purified protein showed quenching by 2-deoxy-D-glucose, D-mannose, D-glucose or D-galactose in the presence of sodium ions. This is the first reported purification and characterization of an active glucose transport protein member of the TC 2.1.7 subgroup of the Major Facilitator Superfamily, constituting the route for entry of sugar nutrients into H. pylori. A model is derived of its three-dimensional structure as a paradigm of the family.
Biochemical Society Transactions | 2005
Massoud Saidijam; Kim E. Bettaney; Gerda Szakonyi; Georgios Psakis; K. Shibayama; Shunichi Suzuki; Joanne Clough; Victor Blessie; Atif Abu-bakr; Simon Baumberg; Johan Meuller; C.K. Hoyle; S.L. Palmer; Patrick Butaye; K. Walravens; Simon G. Patching; John O'Reilly; Ng. Rutherford; Roslyn M. Bill; David I. Roper; Mary K. Phillips-Jones; Peter J. F. Henderson
A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides.
PLOS ONE | 2012
Roger Simm; Aniko Vörös; Jaakko Ekman; Marianne Södring; Ingerid Nes; Jasmin K. Kroeger; Massoud Saidijam; Kim E. Bettaney; Peter J. F. Henderson; Mirja Salkinoja-Salonen; Anne-Brit Kolstø
Transcriptional profiling highlighted a subset of genes encoding putative multidrug transporters in the pathogen Bacillus cereus that were up-regulated during stress produced by bile salts. One of these multidrug transporters (BC4707) was selected for investigation. Functional characterization of the BC4707 protein in Escherichia coli revealed a role in the energized efflux of xenobiotics. Phenotypic analyses after inactivation of the gene bc4707 in Bacillus cereus ATCC14579 suggested a more specific, but modest role in the efflux of norfloxacin. In addition to this, transcriptional analyses showed that BC4707 is also expressed during growth of B. cereus under non-stressful conditions where it may have a role in the normal physiology of the bacteria. Altogether, the results indicate that bc4707, which is part of the core genome of the B. cereus group of bacteria, encodes a multidrug resistance efflux protein that is likely involved in maintaining intracellular homeostasis during growth of the bacteria.
Molecular Membrane Biology | 2013
Kim E. Bettaney; Preethi Sukumar; Rohanah Hussain; Giuliano Siligardi; Peter J. F. Henderson; Simon G. Patching
Abstract A systematic approach was used for the cloning and amplified expression in Escherichia coli of the genes for each of three inositol transport proteins (IolF, IolT, YfiG) from Bacillus subtilis that are evolutionarily-related to human transporters. Inducible amplified expression of each was achieved to levels of ∼ 10–15% of total protein in E. coli inner membrane preparations. The functional integrity of each heterologously-expressed protein was demonstrated by measuring the kinetics of 3H-myo-inositol transport into energized whole cells; this confirmed that IolT is the major inositol transporter, IolF is an inefficient transporter of this substrate and demonstrated that YfiG is an inositol transport protein for the first time. Competition for 3H-myo-inositol transport by 17 unlabelled compounds revealed all three proteins to be highly specific in recognizing inositols over sugars. IolT was confirmed to be highly specific for both myo- and D-chiro-inositol and IolF was confirmed to prefer D-chiro-inositol over myo-inositol. YfiG selectively recognized myo-inositol, D-chiro-inositol and, uniquely, L-chiro-inositol. All three proteins were successfully solubilized and purified in milligram quantities from inner membrane preparations and their suitability for inclusion in crystallization trials was assessed by analysis of structural integrity and thermal stability using circular dichroism spectroscopy followed by examination for monodispersity using gel filtration chromatography.
Frontiers in Microbiology | 2015
Jasmin K. Kroeger; Karl A. Hassan; Aniko Vörös; Roger Simm; Massoud Saidijam; Kim E. Bettaney; Andreas Bechthold; Ian T. Paulsen; Peter J. F. Henderson; Anne-Brit Kolstø
Phylogenetic classification divides the major facilitator superfamily (MFS) into 82 families, including 25 families that are comprised of transporters with no characterized functions. This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a member of the “unknown major facilitator family-2” (UMF-2). BC3310 was shown to be a multidrug efflux pump conferring resistance to ethidium bromide, SDS and silver nitrate when heterologously expressed in Escherichia coli DH5α ΔacrAB. A conserved aspartate residue (D105) in putative transmembrane helix 4 was identified, which was essential for the energy dependent ethidium bromide efflux by BC3310. Transport proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF-2 proteins revealed that they carry a variant of the MFS motif A, which may be used as a marker to distinguish easily between this family and other MFS proteins. Genes orthologous to bc3310 are highly conserved within the B. cereus group of organisms and thus belong to the core genome, suggesting an important conserved functional role in the normal physiology of these bacteria.
Methods | 2018
Marvin V. Dilworth; Mathilde S. Piel; Kim E. Bettaney; Pikyee Ma; Ji Luo; David Sharples; David R. Poyner; Stephane R. Gross; Karine Moncoq; Peter J. F. Henderson; Bruno Miroux; Roslyn M. Bill
Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture.
Current Drug Targets | 2006
Massoud Saidijam; Giulia Benedetti; Qinghu Ren; Zhiqiang Xu; Christopher J. Hoyle; S.L. Palmer; Alison Ward; Kim E. Bettaney; Gerda Szakonyi; Johan Meuller; Scott Morrison; Martin K. Pos; Patrick Butaye; Karl Walravens; Kate P. Langton; Richard B. Herbert; Ronald A. Skurray; Ian T. Paulsen; John O'Reilly; Nicolas G. Rutherford; Melissa H. Brown; Roslyn M. Bill; Peter J. F. Henderson
Journal of Antimicrobial Chemotherapy | 2007
Gerda Szakonyi; Dong Leng; Pikyee Ma; Kim E. Bettaney; Massoud Saidijam; Alison Ward; Saeid Zibaei; Alastair T. Gardiner; Richard J. Cogdell; Patrick Butaye; Anne-Brit Kolstø; John O'Reilly; Ryan J. Hope; Nicholas G. Rutherford; Christopher J. Hoyle; Peter J. F. Henderson
Advances in Enzymology and Related Areas of Molecular Biology | 2011
Massoud Saidijam; Kim E. Bettaney; Dong Leng; Pikyee Ma; Zhiqiang Xu; Jeffrey N. Keen; Nicholas G. Rutherford; Alison Ward; Peter J. F. Henderson; Gerda Szakonyi; Qinghu Ren; Ian T. Paulsen; Ingerid Nes; Jasmin K. Kroeger; Anne-Brit Kolstø
Archive | 2006
Joanne Clough; Massoud Saidijam; Kim E. Bettaney; Gerda Szakonyi; Simon G. Patching; Johan Meuller; Shun’ichi Suzuki; Mark Bacon; Emma Barksby; Marie Groves; Richard B. Herbert; Mary K. Phillips-Jones; Alison Ward; Frank Gunn-Moore; John O’Reilly; Nick Rutherford; Roslyn M. Bill; Peter J. F. Henderson