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

The histidine-containing protein (HPr) is a central phosphotransfer component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) that transports carbohydrates across the cell membrane of bacteria. There are two HPr phosphorylation events investigated in this thesis. Firstly, BPr from Gram-positive species may undergo a regulatory phosphorylation of an absolutely conserved Ser46 residue. There are numerous metabolic consequences to this phosphorylation, including inducer exclusion and expulsion, inhibition of PTS sugar uptake and catabolite repression. While HPr from Gram-negative sources cannot undergo phosphorylation of Ser46 'in vivo' or ' in vitro' it is possible to mimic the phosphorylation through the Ser46Asp mutation. To determine the structural consequences of the mutation the crystallographic structure of the 'E. coli'. Ser46Asp HPr was determined at 1.5 Å resolution. The structure revealed that no significant structural rearrangements are induced by the mutation and the inability to accept phosphotransfer from Enzyme I is due to electrostatic disruption of the interaction of these proteins. Phosphorylation of an absolutely conserved His15 for the purpose of phosphotransfer represents the second phosphorylation event to be investigated. The absolute requirement for histidine at the 15 position was investigated through mutagenesis. The mutation of His15Asp of 'E. coli' HPr was able to accept a phosphoryl group from Enzyme I and further transfer the phosphoryl group to Enzyme IIAglc. None of the other mutations of the fifteen position were able to be phosphorylated. The His15Asp mutant had a Vmax of 0.1% and a ten-fold increase in Kin with respect to wild type HPr. As a consequence of the phosphorylation of His15Asp HPr a third protein species of higher pI than the original protein was identified. This high pI species seemed to share numerous similarities to succinimides which are known to be involved in deamidation. The inability to detect the known degradation products of succinimides suggested that the high pI species may involve isoimide formation. Isoimides have been proposed, but never experimentally demonstrated in proteins. A mechanism through which the phosphoacyl intermediate may catalyze isoimide formation is proposed. In addition the potential involvement of isoimide formation as a mechanism in physiological regulatory signaling is discussed.

Antimicrobial interventions implemented in slaughter establishments for the reduction of enteric pathogens on beef carcasses must be validated to demonstrate efficacy under commercial operation conditions. Validation studies can be conducted using surrogates which are nonpathogenic organisms that respond to a particular treatment in a manner equivalent to a target pathogen. The purpose of this study was to identify surrogates for enteric pathogens to validate antimicrobial interventions on beef carcasses. The growth, attachment, resistance properties as well as the response to interventions on beef carcasses of nonpathogenic fluorescent protein-marked E. coli strains were evaluated and compared to E. coli O157:H7 and Salmonella strains. Growth curves were performed in tryptic soy broth at 37°C and it was demonstrated that in general, growth parameters were not different among surrogates and target pathogens. Thermal resistance was compared in phosphate buffered saline (PBS) at 55, 60 and 65°C; D-values of surrogates were not different or were higher than those of target pathogens. The acid resistance of surrogates was not different to that of E. coli O157:H7 in PBS acidified with lactic acid at pH 2.5, 3.0 and 3.5. Some Salmonella serotypes were found to be less acid resistant than the surrogates. Survival of surrogates after storage at low temperatures (4°C and -18°C) was not different or was longer than survival of E. coli O157:H7 and Salmonella. Additionally, the cell surface hydrophobicity and attachment to beef carcasses surfaces was not different among surrogates and pathogens. Antimicrobial interventions were applied on carcass surfaces under laboratory controlled conditions. After application of hot water washes, D-values were not different among surrogates and pathogens, while no differences were observed in log reductions (CFU/cm2) among surrogates and pathogens when 2% L-lactic acid sprays at 25 and 55°C were applied, regardless of the temperature and volume of the acid solution. The response of surrogates to water washes and lactic acid sprays on beef carcasses was also evaluated in commercial slaughter facilities. Reductions of surrogates were not different to those of aerobic plate count, coliforms and E. coli. However, the surrogates showed less variation and provided more consistent results than traditional indicators.

Autoinducer 2 (AI-2) molecules produced by bacterial cells are thought to be involved in controlling a variety of bacterial cellular processes by coordinated gene and protein expression. Previous work in our laboratory has shown that ground beef contains compounds that can interfere with AI-2-mediated bioluminescence expression in Vibrio. harveyi. The underlying hypothesis of this work was that AI-2 molecules affect the protein expression in Escherichia coli O157:H7 and AI-2 inhibitory molecules negate the influence of AI-2 molecules. The main objectives of this study were to identify, characterize, and isolate the factors responsible for inhibition of AI-2 molecules from ground beef extracts, elucidate the role of LuxS/AI-2 cell signaling system in E. coli O157:H7 protein expression, and determine if inhibitory factors present in ground beef extract can negate the influence of AI-2 molecules on the protein expression. Using a solvent extraction procedure and gas chromatography analysis, AI-2 inhibitory factors present in ground beef extracts were identified as both medium and long chain fatty acids. When identified fatty acids were tested at different concentrations for AI-2 inhibition, AI-2 inhibition ranging from 25% to 90% was observed. Both ground beef extracts and mixture of selected fatty acids also resulted in 2- to 4-fold reduced AI-2 influenced biofilm formation by E. coli K12 cells. Identification of LuxS/AI-2-mediated protein expression in E. coli O157:H7 was conducted using two dimensional gel electrophoresis. Protein expression analysis showed that the LuxS/AI-2 system modulates the expression of proteins involved in different cellular processes such as carbohydrate and amino acid metabolism, stress response, and formation of flagella and motility. When AI-2 inhibitory factors were added along with AI-2 molecules, the expression patterns of three AI-2-influenced proteins (GlmS, SpeE, and NikA) were changed suggesting that AI-2 inhibitors can negate the influence of AI-2 molecules on protein expression of selected proteins. Collectively, these results highlight that proteins associated with different cellular processes in E. coli O157:H7 can be modulated depending on whether cells are in contact with AI-2 molecules in the presence or absence of AI-2 inhibitory factors.