Bacteria live in frequently changing environments and have to deal with a multitude of challenges. The chemical challenges to be faced are not only of exogenous origin, but can be the product of the metabolism, as in the case of methylglyoxal (MG), an endogenous electrophile that kills via damage to macromolecules. Escherichia coli (E. coli) has evolved sophisticated protective mechanisms to counteract the toxicity of MG. The glutathione-dependent glyoxalase system, consisting of glyoxalase I and II (GlxI & II), provides the main route for MG detoxification. Protection from MG is highly dependent on the activity of the KefB and KefC protein. KefB and KefC are homologous ligand-gated potassium efflux systems and are maintained inactive by the binding of glutathione, and are activated during MG detoxification by a specific intermediate molecule of the detoxification pathway, S-lactoylglutathione (SLG). The activity of these systems ultimately modulates the cytoplasmic pH. This study assessed the molecular and physiological role of the GlxII-encoding gene (gloB) in E. coli during MG stress. The study emphasises that the degree of KefB and KefC activation is affected by the relative specific activities of GlxI and GlxII via their impact on the SLG molecule. The significance of other genes in protection was poorly understood and this study allowed first insights into the transcriptional response of E. coli to MG stress. ChIPchip studies investigated the genome-wide RNA polymerase distribution of E. coli in response to MG. Furthermore, the contribution of the potassium efflux systems on transcriptional changes was assessed. The data show that E. coli invokes an adaptive transcriptional response that excludes the known key systems for cell survival. The data point to possible novel roles for several other mechanisms known to be involved for example aldehyde detoxification, potassium homeostasis and DNA damage repair.

The main MG detoxification pathway in <i>Escherichia coli </i>consists of two enzymes, the glyoxalases I and II, and is dependent on glutathione (GSH). MG readily conjugates with GSH in a non-enzymatic manner.  Two subsequent enzymatic reactions via the glyoxalases complete a cyclic process that recycles GSH and produces the non-toxic compound, D-lactate.  An intermediate compound in the detoxification pathway, S-lactoylglutathione (SLG), activates potassium efflux systems KefB and KefC.  This triggers a second mechanism of protection mediated by cytoplasmic acidification, enhancing chances of survival.  Therefore, it is important to understand how cells regulate the concentration of this important intermediate compound. A deterministic model was proposed that describes the series of chemical reactions in the MG detoxification pathway, allowing one to predict the flux of all compounds produced during detoxification. Through an iterative process involving model formulation, parameter estimation, data fitting and validation against experimental data, different models were analysed and discriminated in this study.  Mathematical simulations predicted that the glyoxalase pathway is not linear because it involves feedforward mechanisms for the control of SLG, the activator of the potassium efflux systems. The activities of the potassium efflux systems were investigated using deterministic models that describe the interactions between protein and ligand.  From this model, it was possible to quantify the dependence of the possible binding states on the kinetic parameters of the system.  Parameter estimation methods were used for the analysis of experimental data on the gating of the efflux systems, which proved useful for the design of new experimental strategies.

The ligand-gated potassium channels KefC and KefB of <i>Escherichia coli </i>are critical components in protecting cells from toxic electrophiles.  Potassium efflux through these channels is coupled to a decrease in cytoplasmic pH which in turn reduces the damage to DNA by electrophiles.  KefC and KefB are both inhibited by cytoplasmic glutathione and activated by glutathione adducts, such as ESG, formed by conjugation of glutathione with electrophilic compounds. Robust membrane purification protocols were developed to isolate both the wild type full-length KefC and KefB and the mutants required for biophysical analysis.  <i>In vivo </i>K⁺ measurements were performed to ensure that all of the constructs used were fully functional.  Structural and functional analysis used electron paramagnetic resonance (EPR) and stead state emission fluorescence measurements <i>in vivo</i>, on wild type and mutated full-length proteins to elucidate the gating mechanism and test the model generated from crystallographic data.  In particular, EPR spectroscopy combined with site-directed spin labelling revealed a substantial conformational change and thus provided the first insight into coupling between sensing and gating.  Steady state fluorescence spectroscopy was used to precisely measure binding affinities for both activating and inhibitory ligands and characterise nucleotide binding to KefC.  Finally, a variety of chemically diverse glutathione adducts was tested on KefC <i>in vitro </i>to elucidate the mechanism by which these ligands initiate K⁺ flux through the associated transmembrane domain.

HdeA and HdeB are stationary phase periplasmic proteins believed to function as acid-induced chaperones to prevent the aggregation of periplasmic proteins at low pH.  The aims of this project were to examine the importance of HdeA and HdeB for <i>E. coli</i> acid resistance, to determine the fate of periplasmic proteins at acid pH and to establish the extent to which HdeA and HdeB aid protein stability during an acid challenge. This study has demonstrated that HdeA and HdeB are important for acid resistance, but to a lesser extent than is currently described in the literature.  The work presented in this thesis shows that <i>hdeAB </i>mutants retained a degree of acid resistance, albeit at a lower level than the wild type strains.  HdeA and HdeB contribute to, but are not essential for acid resistance.  The temperature at which cells were grown and subsequently acid challenged at was also shown to be an important factor in acid resistance. Cytoplasmic and exponential phase periplasmic proteins were found to be acid sensitive and aggregated after treatment at acid pH.  In contrast, stationary phase periplasmic proteins were resistant to aggregation at acid pH.  The absence of HdeA and HdeB from stationary phase periplasmic extracts did not result in protein aggregation.  This demonstrated that HdeA and HdeB are not required for protein stability at acid pH.  An exception to this was the periplasmic protein, FkpA, which was found to precipitate upon acid treatment in the absence of HdeA and HdeB.  This presented a potential role for HdeA and HdeB in stabilising FkpA upon exposure to acid pH.

Objetivos: Determinar la prevalencia de Escherichia coli BLEE en urocultivos del Hospital Central FAP en el periodo Enero-Junio 2016. Material y métodos: Se realizó un estudio observacional, descriptivo, analítico, retrospectivo y transversal. Se revisaron 1772 aislamientos bacterianos en orina (urocultivos) generados desde el laboratorio de microbiología del Hospital en el periodo comprendido entre Enero a Junio del 2016. Resultados: Encontramos 1175 urocultivos positivos de los cuales el 26.5% resultaron ser urocultivos E coli BLEE (+).El 80 % de los urocultivos E coli BLEE (+) fueron de género femenino y el 20% masculino. La media de la edad de los pacientes con E coli BLEE (+) fue de 64+/-22,2 años, siendo la mínima de 1 año y la máxima de 102 años. En la mujer, la prevalencia de Escherichia coli BLEE (+), fue más frecuente en el grupo etáreo de 35 a 64 años (34,7%), y en el hombre fue más frecuente en el grupo etáreo mayor de 80 años (42,6%). Observamos que la media de edad de los BLEE (+) fue mayor en relación a los BLEE negativos (64 vs 60.9 años). Encontramos una mayor frecuencia de sensibilidad antimicrobiana de los aislados de Escherichia coli BLEE (+) con amikacina (91,7%), e imipenem (91,5%); y la frecuencia de mayor resistencia fue encontrada con el ácido Nalidíxico (94%), y Cefalotina (89,8%). En los BLEE negativos hubo mayor sensibilidad con la nitrofurantoína (95,5%), Cefalotina (85,1%) y gentamicina (80%), asimismo en el mismo grupo se evidenció mayor resistencia al ácido nalidíxico (64.3%). Conclusiones: La prevalencia de Escherichia coli BLEE fue del 26.5%, afectando principalmente al sexo femenino y el grupo etáreo de 35 a 64 años. En los urocultivos E. coli BLEE (+), la media de edad fue de 64 años, y en los urocultivos E. coli BLEE (-) fue 60 años. Hubo una mayor frecuencia de sensibilidad antimicrobiana de los aislados de Escherichia coli BLEE positivos con amikacina, e imipenem; y de mayor resistencia con el ácido Nalidíxico, y Cefalotina; y de los BLEE negativos hubo mayor sensibilidad con la gentamicina, Cefalotina, nitrofurantoína y mayor resistencia a ácido nalidíxico.

No description available.

Integral membrane proteins perform a wide range of biological processes, including respiration, signal transduction and molecular transport. Structural information is necessary for a full understanding of the mechanisms by which integral membrane proteins work. Ferrous iron transporter protein B (FeoB) is an integral membrane protein of Escherichia coli which is considered to transport ferrous iron in to bacteria. But there are no definite proofs or clear indications of the precise mechanism of ferrous transport. By expressing and crystallizing the G-protein domain (FeoGP) and FeoB, it will be helpful to know about the iron transport system. In order to express FeoB and FeoGP, expression vector pFeoB (FeoB in pGEX-4T-1) and pFeoGP (FeoB in pGEX-4T-1) were made. FeoB and FeoGP proteins were expressed and purified. Using vapour diffusion method crystallization trials of FeoB and FeoGP were done. Crystals of FeoGP are observed and no crystal formation for FeoB. Native crystals of FeoGP diffracted to 2.2 ?? resolution, and mant-GMPPNP crystals to 2.6 ??. Preliminary data processing indicate space group P212121 for native crystals, with cell dimensions 46 x 119 x 146 ??. The data set is 100% complete, Rmerge 0.08, and I/ ?? 3.2.

1 v. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1992

<p>Stafylococcus aureus, Stafylococcus epidermidis, Escherichia coli, Streptococcus</p><p>pyogenes och Bacillus sp. är bakterier som förekommer i vår normalflora eller runt om i</p><p>vår omgivning. De är i de allra flesta fall harmlösa men kan orsaka sjukdom ifall de</p><p>hamnar i t.ex. sår eller kontaminerar mat. Vissa av dessa bakterier har även en tendens att</p><p>utveckla resistens mot antibiotika, infektioner orsakade av resistenta bakterier kan bli</p><p>allvarliga och väldigt svåra att behandla. Detta är det främsta skälet till varför vi har valt</p><p>att undersöka ifall de ovan nämnda bakterierna förkommer på avdelningen SET på</p><p>Halmstad högskola. För att kunna identifiera de bakterier vi fann använde vi oss av en rad</p><p>olika biokemiska tester och resistensbestämningen utfördes bl.a. med hjälp av en PCR</p><p>körning. Vi fann alla de bakterier vi sökte efter förutom S. pyogenes och ingen av de</p><p>funna visade på någon resistens mot antibiotika.</p>

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.