In order to determine and study a protein structure, large amounts of it is needed. The easiest way to obtain a protein is to recombinantly overexpress it in the well-studied bacterium Escherichia coli. However, this expression host has one major disadvantage, overexpressed proteins might not be folded or be insoluble. Within the field of structural genomics, protein production has become one of the most challenging problems and the recombinant overexpression of viral proteins has in particular proven to be difficult. The first part of the thesis concerns the recombinant overexpression of troublesome proteins in E. coli. A method has been developed to screen for soluble overexpression in E. coli at the colony level, making it suitable for screening large gene collections. This method was used to successfully screen deletion libraries of difficult mammalian proteins as well as ORFeomes from five herpesviruses. As a result soluble expression of previously insoluble mammalian proteins was obtained as well as crystals of three proteins from two oncogenic human herpesviruses, all linked to DNA synthesis of the viral genome. The second part of the work presented concerns the structural studies of three herpesviral proteins. SOX from Kaposi’s sarcoma associated herpesvirus is involved in processing and maturation of the viral genome. Recently SOX has also been implicated in host shutoff at the mRNA level. With this structure, we propose a substrate binding site and a likely exonucleolytic mechanism. The holoenzyme ribonucleotide reductase is solely responsible for the production of deoxyribonucleotides and regulates the nucleotide pool of the cell. The small subunit, R2, has been solved from both Epstein Barr virus and KSHV. Both structures show disordered secondary structure elements in their apo-and mono metal forms, located close to the iron binding sites in similarity to the p53 induced R2 indicating that these two R2 proteins might play a similar and important role.

No description available.

Membrane proteins are key players in many biological processes. Since most membrane proteins are assembled into oligomeric complexes it is important to understand how they interact with each other. Unfortunately however, the assembly process (i.e. their social life) remains poorly understood. In the work presented in this thesis I have investigated when and how membrane proteins assemble with each other and their cofactors to form functional units. We have shown that that cofactor insertion in the hetero-tetrameric cytochrome bo3 occurs at an early state in the assembly process. We also found that the pentameric CorA magnesium ion channel is stabilised by different interactions depending on the magnesium ion concentration in the cell. These studies indicate that the assembly of a functional unit is a dynamic process, which is a result of many different forces. By studying the assembly of membrane proteins we have obtained a deeper insight into their function, which cannot be explained by static crystal structures. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Nickel insertion into the [NiFe]-hydrogenase requires the accessory protein HypB, which is a GTPase. The GTPase domain of Escherichia coli (E. coli) HypB undergoes dimerization in the presence of GTP. To determine the role of HypB dimerization in hydrogenase biosynthesis, a double mutation L242A/L246A was introduced into full-length E. coli HypB, and the protein was expressed and characterized both in vitro and in vivo. Gel filtration experiments demonstrated that L242A/L246A HypB was monomeric as expected. The inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric L242A/L246A HypB has a similar Ni(II)-binding behavior as that of wild type HypB. Upon the expression of L242A/L246A HypB in vivo the hydrogenase activity is approximately half of the activity of the wild-type control. These experimental results suggest that dimerization of HypB does have a, but not critical, role in hydrogenase biosynthesis.

An air cathode, membrane-less microbial fuel cell (MFC) containing a co-culture of Geobacter sulfurreducens and Escherichia coli was constructed and compared to pure culture MFCs of both organisms. The E. coli containing MFCs were unsparged and relied on E. coli for oxygen removal. The pure G. sulfurreducens MFC had a power output of 128 mW/m2, compared to 63 mW/m2 for the co-culture at an early stage and 56 mW/m2 for the late stage co-culture. The limiting current density is 404 mA/m2 for the pure G. sulfurreducens culture, 184 mA/m2 for the early co-culture, and 282 mA/m2 for the late co-culture, despite an increase in internal resistance between the early and late co-culture cells. Analysis of metabolites has shown that succinate production is likely to have negatively affected current production by G. sulfurreducens, and the removal of succinate is responsible for the increased current density in the late co-culture cell.

Nickel insertion into the [NiFe]-hydrogenase requires the accessory protein HypB, which is a GTPase. The GTPase domain of Escherichia coli (E. coli) HypB undergoes dimerization in the presence of GTP. To determine the role of HypB dimerization in hydrogenase biosynthesis, a double mutation L242A/L246A was introduced into full-length E. coli HypB, and the protein was expressed and characterized both in vitro and in vivo. Gel filtration experiments demonstrated that L242A/L246A HypB was monomeric as expected. The inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric L242A/L246A HypB has a similar Ni(II)-binding behavior as that of wild type HypB. Upon the expression of L242A/L246A HypB in vivo the hydrogenase activity is approximately half of the activity of the wild-type control. These experimental results suggest that dimerization of HypB does have a, but not critical, role in hydrogenase biosynthesis.

An air cathode, membrane-less microbial fuel cell (MFC) containing a co-culture of Geobacter sulfurreducens and Escherichia coli was constructed and compared to pure culture MFCs of both organisms. The E. coli containing MFCs were unsparged and relied on E. coli for oxygen removal. The pure G. sulfurreducens MFC had a power output of 128 mW/m2, compared to 63 mW/m2 for the co-culture at an early stage and 56 mW/m2 for the late stage co-culture. The limiting current density is 404 mA/m2 for the pure G. sulfurreducens culture, 184 mA/m2 for the early co-culture, and 282 mA/m2 for the late co-culture, despite an increase in internal resistance between the early and late co-culture cells. Analysis of metabolites has shown that succinate production is likely to have negatively affected current production by G. sulfurreducens, and the removal of succinate is responsible for the increased current density in the late co-culture cell.

Numerical models of flow and transport are commonly applied for the sustainable management of water resources and for the selection of appropriate remediation techniques. However, these numerical models are not always accurate due to uncertain parameters and the disparity of scales across which observations are made, hydrological processes occur, and modeling is conducted. The modeling framework becomes further complex because hydrologic processes are coupled with chemical and biological processes. This dissertation focuses on the most widespread contaminants of surface and ground water, which are E. coli and nitrate, respectively. Therefore, this research investigates the linkages between bio-chemical and hydrologic processes for E. coli transport, explores the spatio-temporal variability of nitrate, quantifies uncertainty, and develops models for both E. coli and nitrate transport that better characterize these biogeochemical linkages. A probabilistic framework in the form of Bayesian Neural Networks (BNN) was used to estimate E. coli loads in surface streams and was compared with a conventional model LOADEST. This probabilistic framework is crucial when water quality data are scarce, and most models require a large number of mechanistic parameters to estimate E. coli concentrations. Results indicate that BNN provides better characterization of E. coli at higher loadings. Results also provide the physical, chemical, and biological factors that are critical in the estimation of E. coli concentrations in Plum Creek, Texas. To explore model parameters that control the transport of E. coli in the groundwater (GW) and surface water systems, research was conducted in Lake Granbury, Texas. Results highlight the importance of flow regimes and seasonal variability on E. coli transport. To explore the spatio-temporal variability of nitrate across the Trinity and Ogallala aquifers in Texas, an entropy-based method and a numerical study were employed. Results indicate that the overall mean nitrate-N has declined from 1940 to 2008 in the Trinity Aquifer as opposed to an increase in the Ogallala Aquifer. The numerical study results demonstrate the effect of different factors like GW pumping, flow parameters, hydrogeology of the site at multiple spatial scales. To quantify the uncertainty of nitrate transport in GW, an ensemble Kalman filter was used in combination with the MODFLOW-MT3DMS models. Results indicate that the EnKF notably improves the estimation of nitrate-N concentrations in GW. A conceptual modeling framework with deterministic physical processes and stochastic bio-chemical processes was devised to independently model E. coli and nitrate transport in the subsurface. Results indicate that model structural uncertainty provides useful insights to modeling E. coli and nitrate transport.

The ultimate goal in the field of metabolic engineering is improving cellular processes in a rational manner using engineering design principles and molecular biology techniques. The syntheses of several industrially useful compounds are cofactor-dependent. The reducing equivalent NADPH is required in several enzymatic reactions leading up to the synthesis of high-value compounds like polymers, chiral alcohols, and antibiotics. However, it’s a highly costly compound with limited intracellular availability. This study focuses on the genetic manipulation of a whole-cell system using the two transhydrogenase isoforms pntAB and udhA. Two model systems are used: 1) the production of (S)-2-chloropropionate and 2) the production of poly(3-hydroxybutyrate). Results suggest that the presence of udhA increases product yield and NADPH availability while the presence of pntAB has the opposite effect. A maximum product yield of 1.4 mole-product/mole-glucose was achieved aerobically in a pntAB-deletion strain with udhA overexpression, a 150% improvement over the wild-type control strain.

Background: Access to improved water and sanitation are key measures of the World Health Organizations. However, while a community can be classified as having access to improved water and sanitation, the possibility of microbiological contaminations exists. Globally, there is a need to assess the quality of drinking water to better classify levels of microbiological quality in attempts to reduce diarrheal disease burden. Utilizing the test for hydrogen sulfide (H2S) producing bacteria test is a cost effective and easy to use method that may be comparable to the traditional yet more costly method (IDEXX Colilert Quantitray). Due to a paucity of data on the test for H2S producing bacteria, this study was performed to examine how well the test for hydrogen sulfide (H2S) producing bacteria compared to traditional measure of fecal indicator bacteria total coliforms and E. coli in drinking water. Furthermore, an analysis of the ability of the test for H2S producing bacteria to predict diarrheal disease was also examined. Methods: The following conditions for the H2S were examined in the study: 2 volumes (10mL or 90mL), 2 incubation times (24 and 48 hours) and the use of a semi-quantitative scoring system that measured the intensity of the black precipitate formed (H2S). To examine how well these conditions compared to E. coli and total coliform results, the following analyses were performed: 1) analysis of sensitivity and specificity to examine presence/absence of bacteria in both samples, 2) linear regression to examine how well a semi-quantitative H2S scoring system predicted bacterial concentrations and 3) logistic regression to examine how well the H2S test predicted risk of diarrheal disease. Results: Within the dataset, there were 816 observations among the 7 communities involved in the study. The H2S test condition that had the highest sensitivity and specificity (94.23% and 36.07% respectively) for total coliforms was 90mL volume at 48 hours. This test condition also produced the highest sensitivity and specificity for E. coli (97.82% and 78.67%, respectively). An analysis using linear regression demonstrated that a semi-quantitative H2S scoring system was able to predict both total coliform and E. coli concentrations in the same samples. In a logistic regression analysis of diarrheal disease, the test of H2S producing bacteria suggested an increase in diarrheal disease risk for higher levels of H2S (OR of 1.18 (p=0.03; 1.02 – 1.35)). Discussion: The initial results here suggest that the use of the test for H2S producing bacteria has potential with high sensitivity (>90%) for E. coli and total coliforms. The application of the semi-quantitative scoring system may also have applications in predicting concentration of E. coli and total coliforms and well as possibly predicting diarrheal disease. However, more work needs to be completed to standardize the semi-quantitative approach to reduce subjectivity of scoring as well as examine the role of the test in additional epidemiologic studies. INDEX WORDS: waterborne disease, E. coli, Dominican Republic, microbial testing