Caulobacters are gram-negative bacteria that have a biphasic life cycle consisting of a swarmer and a stalked stage. As a result they have elicited interest as a simple developmental model. Less attention has focussed on their role in the environment, although they have been found in almost every aquatic environment as well as in many soils. Caulobacters are often described as oligotrophic bacteria because of their prevalence in pristine waters but have now been isolated from the relatively nutrient-rich wastewater environment. In order to learn more about this population some basic characterization was carried out and an assay system to determine their prevalence in sewage plants was designed. Most of the organisms isolated from sewage treatment facilities had similar gross morphological features, but differed in holdfast composition, total protein profile, antibiotic resistance and restriction fragment length polymorphism, thereby indicating a greater diversity than originally assumed. Most of the organisms hybridized with flagellin and surface array genes that had previously been cloned, and only one of 155 non-Caulobacter sewage isolates hybridized with the flagellin gene probe; consequently these were used in a DNA-based enumeration strategy. DNA was isolated directly from sewage and probed with the flagellin and the surface array gene probes. The signals obtained were compared to standards made up of pooled Caulobacter DNA from the sewage isolates and non-Caulobacter DNA from organisms also present in sewage. Using this assay Caulobacters could only be detected above the 1% level, which was higher than their proportion in the wastewater environment. It appears that this approach will not be useful in monitoring Caulobacters in treatment plants unless a more highly conserved or higher copy number probe is found. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (D.Sc.)--Boston University, Henry M. Goldman School of Dental Medicine, 2004 (Oral Biology). / Includes bibliography (leaves 107-151). / Lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram negative bacteria, induces the secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-a) and interleukin (IL )-1, by monocytes/macrophages. Excessive and uncontrolled secretion of these compounds leads to multiple pathological conditions, such as septic shock. LPS receptors have been shown to be CD14, TLR4 and MD-2. LPS interaction with these receptors mediates many monocyte/macrophage functions. Even though only CD14 was demonstrated to bind to LPS, and TLR4/MD-2 were capable of transducing signals, data only show that LPS and CD 14 were in close proximity to TLR4 and no direct binding was reported. Quite recently, moesin, a member of the ERM family of proteins, has been also found to function as a receptor for LPS. We have shown that anti-moesin antibody inhibited the release of TNFa by LPS stimulated monocytes. Moesin was also found to be necessary for the detection of LPS, where homozygous knockout mice exhibited 3-fold reduction in neutrophil infiltrates in LPS injected sites when compared to their wild type controls. When moesin gene expression was completely suppressed with antisense oligonucleotides, there was a significant reduction of LPS-induced TNF-a secretion. LPS stimulation of mononuclear phagocytes activates several intracellular signaling pathways including the phosphorylation of IKBa, mitogen-activated protein kinase (MAPK) pathways: extracellular signal-regulated kinases (ERK) 1 / 2 (P44/42), p38. These signaling pathways in tum activate a variety of transcription factors including NF-KB, which coordinates the induction of several genes encoding inflammatory mediators. [TRUNCATED]

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Acidithiobacillus ferrooxidans is a gram-negative bacterium that is able to extract energy from oxidation of Fe²⁺ and reduced sulfur compounds and fix carbon dioxide from atmosphere. The facts that A. ferrooxidans thrives in acidic pH (~2), fixes carbon dioxide from the atmosphere and oxidizes Fe²⁺ for energy make it a good candidate in many industrial applications such as electrofuels and biomining. Electrofuels is a new type of bioprocess, which aims to store electrical energy, such as solar power, in the form of chemical bonds in the liquid fuels. Unlike traditional biofuels made from agricultural feedstocks, electrofuels bypass the inefficient photosynthesis process and thus have potentially higher photon-to-fuel efficiency than traditional biofuels. This thesis covers the development of a novel bioprocess involving A. ferrooxidans to make electrofuels, i.e. isobutyric acid and heptadecane. There are four major steps: characterization of wild-type cells, engineering of medium for improved electrochemical performance, genetic modification of A. ferrooxidans and optimization of operating conditions to enhance biofuel production. Each is addressed in one of the chapters in this thesis. In addition, applications of A. ferrooxidans in biomining processes will be briefly discussed. An economic analysis of various applications including electrofuels and biomining is also presented. Wild-type A. ferrooxidans were first characterized in both batch and continuous cultures. A modified 9-K medium suggested by American Type Culture Collection (ATCC) was used as a starting point which has 72 mM Fe²⁺ at pH 1.8. The Fe²⁺ concentration and pH were varied in the experiments to assess their impacts on growth rate, cell yield (g cells/g Fe²⁺) and maintenance (energy used to keep cell viability). Citrate was added to the growth medium to dissolve precipitates which can be problematic in a continuous operation. It was found out that cells exhibited higher cell yield (g cells/g Fe²⁺) and lower maintenance with higher pH and addition of citrate. This indicates that cells grow in a more energy-efficient manner at such conditions since cells spend less energy in maintenance and more energy in biomass formation. Next the growth medium containing 72 mM Fe³⁺ and 70 mM citrate at pH 2.2 was explored during the electrochemical reduction of Fe³⁺. It turned out that electrochemical reduction of Fe³+ could not be carried out effectively due to a low electrolyte conductivity and low energy density of the medium. Citrate was also found to negative affect electrochemical performance due to a strong complexation with Fe³⁺. The conductivity was improved by adding 500 mM Mg²⁺ to the medium. Vanadium was used as an alternative redox mediator that has a much better solubility than Fe³⁺ to improve the energy density. Genetic modification was achieved by introducing genes from two foreign pathways i.e. valine synthesis and fatty acid synthesis into A. ferrooxidans to enable cells to produce either isobutyric acid (IBA) or heptadecane. Transformed cells were characterized based on the findings in wild-type cells. Isobutyric acid production was found to increase with increasing pH and Fe²⁺ concentration and addition of citrate. Further optimization of the growth medium was done by increasing Fe²⁺ to 288 mM, holding pH at 2.2 and using gluconate as the iron chelator instead of citrate. An economic analysis was performed on the electrofuel process and applications of genetically modified A. ferrooxidans in copper biomining processes. At electricity prices of $0.05/kWh, further improvement in biological efficiency needs to be achieved before the electrofuel process may become economically viable. The use of genetically modified cells in copper biomining process could open new opportunities to co-produce valuable chemicals and copper from the reduced material associated with the copper ores. The chemicals co-produced during copper processing could be sold for additional revenue or used on-site.

by Chin Miu Ling, Nathalie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 130-143). / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.iii / LIST OF TABLES --- p.vii / LIST OF FIGURES --- p.x / INTRODUCTION --- p.1 / Chapter A --- Aminoglycosides --- p.1 / Chapter 1 --- Structure --- p.1 / Chapter 2 --- Classification --- p.1 / Chapter 3 --- Mode of action --- p.2 / Chapter 4 --- Types --- p.9 / Chapter B --- Mechanisms of aminoglycoside-resistance --- p.13 / Chapter C --- Aminoglycoside-modifying enzymes --- p.14 / Chapter 1 --- Classification --- p.19 / Chapter i --- Phosphotransferases --- p.19 / Chapter ii --- Adenylytransferases --- p.21 / Chapter iii --- Acetyltransferases --- p.22 / Chapter 2 --- Genes encoding AMEs --- p.24 / Chapter 3 --- Applications --- p.27 / Chapter D --- Prevalence of aminoglycoside-resistance --- p.28 / Chapter E --- Methods for the determination of aminoglycoside-modifying enzymes --- p.34 / Chapter 1 --- Examination of resistance phenotype --- p.35 / Chapter 2 --- Phosphocellulose paper binding assay --- p.38 / Chapter 3 --- Hybridization with specific gene probes --- p.39 / Chapter 4 --- Antibiotic inactivation --- p.44 / Chapter 5 --- High performance liquid chromatography (HPLC) --- p.44 / Chapter F --- Prevalence of aminoglycoside-modifying enzymes --- p.45 / Chapter G --- Objectives --- p.52 / MATERIALS AND METHODS --- p.53 / Materials --- p.53 / Chapter A --- Bacterial strains --- p.53 / Chapter 1 --- Standard strains --- p.53 / Chapter 2 --- Clinical isolates --- p.53 / Chapter B --- "Antibiotic, media, chemicals and instruments" --- p.55 / Methods --- p.55 / Chapter A --- Orevalence of aminoglycoside-resistance --- p.55 / Chapter B --- Susceptibility testing --- p.55 / Chapter C --- Characterization of aminoglycoside-modifying enzymes (AMEs) --- p.61 / Chapter 1 --- Extraction of enzymes --- p.61 / Chapter 2 --- Substrate profile analysis by the phosphocellulose paper binding assay --- p.62 / Chapter D --- Localization of resistance genes --- p.64 / Chapter 1 --- Genetic study --- p.64 / Chapter 2 --- Molecular studies --- p.67 / Chapter i --- Preparation of crude plasmid extracts --- p.68 / Chapter ii --- Agarose gel electrophoresis --- p.68 / Chapter E --- Plasmid profile analysis --- p.69 / Chapter F --- Plasmid fingerprinting --- p.69 / Chapter 1 --- Preparation of purified plasmid --- p.69 / Chapter 2 --- Restriction endonuclease digestion of plasmid DNA --- p.70 / Plan to achieve objectives --- p.71 / results --- p.73 / Chapter A --- Prevalence of aminoglycoside-resistant Gram-negative bacteria isolated in the Prince of Wales Hospital from 1989 to1992 --- p.73 / Chapter B --- "Susceptibility to 12 aminoglycosides of aminoglycoside-resistant E. coli, K pneumoniae and Ps. aeruginosa" --- p.78 / Chapter C --- "Aminoglycoside-modifying enzymes (AMEs) produced by E. coli, K pneumoniae and Ps. aeruginosa" --- p.88 / Chapter D --- Plasmid profile analysis --- p.93 / Chapter E --- Localization of aminoglycoside-resistance genes --- p.102 / discussion --- p.114 / Chapter A --- Aminoglycoside-resistance --- p.114 / Chapter B --- Mechanisms of aminoglycoside-resistance --- p.118 / Chapter C --- Genetic location of aminoglycoside-resistance and plasmid profiles --- p.122 / Chapter D --- Characterization of AMEs --- p.126 / Chapter E --- Areas for future research --- p.128 / references --- p.130 / appendix --- p.144

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Phillip E. Klebba / Siderophore uptake systems are one the most prominent methods of Fe³+-iron acquisition in Gram negative bacteria. The catecholate siderophore enterobactin is synthesized and utilized by many members of Enterobacteriaceae as well as several of the ESKAPE pathogens. The outer membrane (OM) transporter of ferric enterobactin (FeEnt), FepA is a ligand-gated porin (LGP) that requires interaction with the inner membrane (IM) protein TonB in order to accomplish active transport. TonB is thought to transduce the electrochemical energy created by the proton gradient across the IM to LGPs like FepA in the OM, to promote siderophore transport through their occluded channels. However, we do not yet have a clear picture of either how TonB transfers energy to FepA, or what kind of conformational changes occur in the occluding domain of FepA to allow ligand passage. The experiments described herein investigate these two questions, building on previously outlined models and observations. Using fluorescence labeling of strategically substituted cysteines in the surface loops of FepA, we unraveled a hierarchy of loop motion during binding of FeEnt to FepA. Additionally, by rendering parts of the FepA protein immobile as a result of engineered disulfide bonds, I identified residues or regions within its occluding domain that may normally unfold to open a size-specific channel for FeEnt. I also elucidated the role of the peptidoglycan polymer beneath the OM a framework for protein-protein interactions between IM and OM proteins. This includes the proposed interaction between a rotating TonB and FepA, or other LGPs, that may transfer kinetic energy to the OM transporter. The role of iron in microbial survival and pathogenesis makes iron-uptake pathways an attractive target for therapeutic intervention. Using the FeEnt-FepA uptake system as a model, we used a fluorescence based high-throughput screening method to identify novel small molecule inhibitors of TonB action in E. coli. The approach used can be potentially adopted to screen bigger chemical libraries as well as used to find inhibitors of ESKAPE pathogens that use FeEnt such as, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Finally, we discoverd a TonB-dependent OM transporter of heme/hemoglobin called HutA in the oligotrophic bacterium Caulobacter crescentus.

Chan, Kwok Ho. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 124-129). / Abstracts in English and Chinese. / Statement --- p.I / Acknowledgements --- p.II / Abstract --- p.III / 摘要 --- p.IV / Table of Contents --- p.V / Abbreviations --- p.XIII / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- GTPase in general --- p.1 / Chapter 1.2 --- G proteins and GTP switch --- p.2 / Chapter 1.3 --- Structural similarities in GTPase --- p.3 / Chapter 1.4 --- G proteins in bacteria --- p.3 / Chapter 1.5 --- Background information of the protein family EngB --- p.4 / Chapter 1.6 --- Basic information of EngB in Thermotoga maritima --- p.5 / Chapter 1.7 --- Objectives of this work --- p.6 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Chemical reagents --- p.8 / Chapter 2.1.2 --- Buffers / Chapter 2.1.2.1 --- Preparation of buffers --- p.10 / Chapter 2.1.2.2 --- Buffers for common use --- p.11 / Chapter 2.1.3 --- Expression strains and plasmids --- p.14 / Chapter 2.1.4 --- Primer list --- p.14 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Preparation of competent cells --- p.15 / Chapter 2.2.2 --- Cloning / Chapter 2.2.2.1 --- Cloning of target genes by PCR --- p.15 / Chapter 2.2.2.2 --- Agrose gel electrophoresis --- p.17 / Chapter 2.2.2.3 --- Extraction and purification of DNA from agarose gel --- p.17 / Chapter 2.2.2.4 --- Restriction digestion of DNA --- p.18 / Chapter 2.2.2.5 --- Ligation of digested insert and expression vector --- p.18 / Chapter 2.2.2.6 --- Transformation and plating out transformants for miniprep --- p.19 / Chapter 2.2.2.7 --- Verification of insert by PCR --- p.20 / Chapter 2.2.2.8 --- Mini-preparation of plasmid DNA --- p.21 / Chapter 2.2.2.9 --- Confirmation of miniprep product by restriction enzyme digestion..… --- p.22 / Chapter 2.2.2.10 --- Sequencing of the plasmid DNA --- p.23 / Chapter 2.2.3 --- Expression of the recombinant MBP-TM EngB protein and SBP-CBP EC EngB / Chapter 2.2.3.1 --- Transformation for protein expression --- p.23 / Chapter 2.2.3.2 --- Preparation of starter culture --- p.24 / Chapter 2.2.3.3 --- Expression of recombinant protein --- p.24 / Chapter 2.2.3.4 --- Cell harvesting --- p.24 / Chapter 2.2.3.5 --- Releasing the cell content --- p.25 / Chapter 2.2.3.6 --- Check for protein expression by SDS-PAGE --- p.25 / Chapter 2.2.4 --- Purification of TM EngB / Chapter 2.2.4.1 --- SP ion-exchange chromatography --- p.27 / Chapter 2.2.4.2 --- Thrombin digestion to remove MBP tag --- p.28 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.29 / Chapter 2.2.4.4 --- Gel filtration chromatography --- p.29 / Chapter 2.2.5 --- Purification of SBP-CBP EC EngB / Chapter 2.2.5.1 --- SP ion-exchange chromatography --- p.30 / Chapter 2.2.5.2 --- Gel filtration chromatography --- p.31 / Chapter 2.2.6 --- Protein concentration quantitation --- p.32 / Chapter 2.2.7 --- Crystallography of TM EngB / Chapter 2.2.7.1 --- Crystallization preparation --- p.32 / Chapter 2.2.7.2 --- Crystallization screening by sitting drop method --- p.32 / Chapter 2.2.7.3 --- Optimization of crystallization conditions --- p.33 / Chapter 2.2.7.4 --- X-ray diffraction --- p.33 / Chapter 2.2.8 --- Thermodynamics studies of proteins / Chapter 2.2.8.1 --- Preparation of protein sample --- p.34 / Chapter 2.2.8.2 --- Guanidine-induced denaturation experiment --- p.34 / Chapter 2.2.8.3 --- Thermal-induced denaturation experiment --- p.35 / Chapter 2.2.9 --- Binding assay to study affinity for ligands --- p.36 / Chapter 2.2.9.1 --- Using GDP analogue mant-GDP to detect formation of enzyme-ligand complex (TM EngB-mant-GDP) --- p.36 / Chapter 2.2.9.2 --- Basic information of Fluorescence spectroscopy --- p.36 / Chapter 2.2.9.3 --- Determination of λem and λex --- p.37 / Chapter 2.2.9.4 --- Studying ligand affinity by titration with ligand analogue --- p.37 / Chapter 2.2.10 --- Pull down experiment to study interacting partner of E. coli EngB --- p.38 / Chapter 2.2.10.1 --- Preparing protein extracts from E. coli --- p.38 / Chapter 2.2.10.2 --- Preparing streptavidin resin --- p.39 / Chapter 2.2.10.3 --- Binding of dual-tagged E. coli EngB to streptavidin resin --- p.39 / Chapter 2.2.10.4 --- Purifying protein using the prepared streptavidin resin --- p.40 / Chapter 2.2.10.5 --- Preparing calmodulin resin --- p.41 / Chapter 2.2.10.6 --- Binding of dual-tagged E.coli EngB to calmodulin resin --- p.41 / Chapter 2.2.10.7 --- Analysis of dual-tag affinity purified protein --- p.42 / Chapter 2.2.11 --- Silver staining of acrylamide gel / Chapter 2.2.11.1 --- Staining reagents --- p.42 / Chapter 2.2.11.2 --- Staining procedures --- p.43 / Chapter Chapter 3 --- Structure determination of T. maritima EngB by X-ray crystallography / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Generation of TM EngB expression construct --- p.45 / Chapter 3.3 --- Expression and purification of TM EngB --- p.46 / Chapter 3.4 --- TM EngB was crystallized with freshly purified TM EngB --- p.47 / Chapter 3.5 --- Data processing of diffraction data and structure refinement of TM EngB …… --- p.48 / Chapter 3.6 --- Apo-form TM EngB was obtained by unfolding and refolding --- p.49 / Chapter 3.7 --- Crystallization of apo-form TM EngB --- p.50 / Chapter 3.8 --- Data processing of diffraction data and structure refinement of apo-form TM EngB --- p.51 / Chapter 3.9 --- Producing EngB-GDP complex crystal from apo-from EngB --- p.52 / Chapter 3.10 --- TM EngB is a monomer in solution --- p.54 / Chapter 3.11 --- Summary of chapter three --- p.55 / Tables and figures of chapter three --- p.57 / Chapter Chapter 4 --- Structural details of TM EngB / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Overall fold of TM EngB --- p.67 / Chapter 4.3 --- Mode of nucleotide binding of TM EngB --- p.68 / Chapter 4.4 --- Structural differences in switch I region between chain A and chain B in crystal structure of TM EngB/GDP complex --- p.70 / Chapter 4.5 --- Structural difference between TM EngB/GDP complex and apo TM EngB --- p.73 / Chapter 4.6 --- Summary of chapter four --- p.73 / Tables and figures of chapter four --- p.76 / Chapter Chapter 5 --- Purified TM EngB is Active for binding guanine nucleotide but inactive for GTPase hydrolysis activity / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Studying ligand affinity by competitive binding experiment --- p.88 / Chapter 5.3 --- GDP binds to TMEngB with higher affinity than GTPyS --- p.91 / Chapter 5.4 --- TM EngB showed very low intrinsic GTPase activity --- p.92 / Chapter 5.5 --- Discussion --- p.93 / Tables and figures of chapter five --- p.95 / Chapter Chapter 6 --- Thermostability of EngB of T. maritima / Chapter 6.1 --- Introduction --- p.98 / Chapter 6.2 --- Guanidine hydrochloride - induced unfolding --- p.98 / Chapter 6.3 --- Thermal-induced unfolding --- p.99 / Chapter 6.4 --- Structural comparison of thermophilic and mesophilic EngB --- p.100 / Chapter 6.5 --- Discussion --- p.102 / Tables and figures of chapter six --- p.105 / Chapter Chapter 7 --- Construction of a dual-tag affinity pull-down system for finding interacting partner of EngB / Chapter 7.1 --- Introduction --- p.112 / Chapter 7.2 --- Preparation of dual-tagged E.coli EngB / Chapter 7.2.1 --- Cloning of SBP-CBP-EC EngB expression construct --- p.113 / Chapter 7.2.2 --- Expression and purification of SBP-CBP-EC EngB --- p.114 / Chapter 7.3 --- Pull down using dual tagged E.coli EngB as bait to isolate potential interacting partners of EngB --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Tables and figures of chapter seven --- p.117 / Chapter Chapter 8 --- Conclusion --- p.122 / References --- p.124

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 170-185).