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.

According to the USEPA National Section 303(d) List Fact Sheet, bacterial pathogens are the leading cause of water quality impairments in Texas. The automated Spatially Explicit Load Enrichment Calculation Tool (SELECT) uses spatially variable factors such as land use, soil condition, and distance to streams to characterize pathogen sources across a watershed. The results support development of Total Maximum Daily Loads (TMDLs) where bacterial contamination is of concern. SELECT calculates potential E. coli loads by distributing the contributing source populations across suitable habitats, applying a fecal production rate, and then aggregating the potential load to the subwatersheds. SELECT provides a Graphical User Interface (GUI), developed in Visual Basic for Applications (VBA) within ArcGIS 9.X, where project parameters can be adjusted for various pollutant loading scenarios. A new approach for characterizing E. coli loads resulting from on-site wastewater treatment systems (OWTSs) was incorporated into the SELECT methodology. The pollutant connectivity factor (PCF) module was created to identify areas potentially contributing E. coli loads to waterbodies during runoff events by weighting the influence of potential loading, runoff potential, and travel distance. Simulation results indicate livestock and wildlife are potentially contributing large amounts of E. coli in the Lake Granbury Watershed in areas where these contributing sources are not currently monitored for E. coli. The bacterial water quality violations near Lake Granbury are most likely the result of malfunctioning OWTSs and pet waste in the runoff. The automated SELECT was verified by characterizing the potential E. coli loading in the Plum Creek Watershed and comparing to results from a prior study (Teague, 2007). The E. coli potential load for the watershed was lower than the previous study due to major differences in assumptions. Comparing the average ranked PCF estimated by physical properties of the watershed with the statistical clustering of watershed characteristics provided similar groupings. SELECT supports the need to evaluate each contributing source separately to effectively allocate site specific best management practices (BMPs). This approach can be used as a screening step for determining areas where detailed investigation is merited. SELECT in conjunction with PCF and clustering analysis can assist decision makers develop Watershed Protection Plans (WPPs) and determine TMDLs.

This study evaluated the thermal drying process commonly used by small and very small beef jerky operations in Texas. It was intended to determine the impact of relative humidity on the production of beef jerky and to provide documentation to beef jerky producers to support their Hazard Analysis and Critical Control Point programs. This project was divided into two phases: Phase I provided a low level of relative humidity (15-25%), whereas Phase II provided a high level (100%) for 25% of the cooking cycle. Both phases consisted of three trials, each representing one of the treatments (n=18) applied to the samples. The first treatment served as the control group and included samples that were non-inoculated, while the other two treatments included inoculations of samples with a bovine fecal slurry and rifampicin-resistant Salmonella Typhimurium. Each of the three treatments for both phases was analyzed for reduction of microbial levels in addition to temperature and product composition. Once the two phases had been completed and all data were analyzed, it was concluded that there was not a statistical difference between the level of reduction for Aerobic Plate Counts, coliforms, Escherichia coli and Salmonella provided by Phase I with low humidity and Phase II with high humidity. Both levels of humidity provided similar levels of reduction within each trial, suggesting that the level of humidity does not have a great impact on the level of microbial reduction achieved. However, this study did not provide the adequate level of initial inoculation levels to support the required 6.5 log reduction stated in 9 CFR 318.7. Inoculation levels were lower than 6.5 logs for all three treatments in both phases, resulting in lower levels of overall reduction. Therefore, based upon the information provided by this study, it cannot be concluded that a low level of humidity will achieve a 6.5 log reduction as mandated in 9 CFR 318.17.

This study measured and compared different temperatures and dwell times of hot water treatment on the reduction of Escherichia coli O157:H7 and Salmonella Typhimurium on beef carcass surfaces. Two different types of beef surfaces, lean and fat, were inoculated with a fecal slurry containing E. coli O157:H7 and S. Typhimurium at ca. 7-log CFU/g, washed to remove gross fecal matter, and rinsed with hot water between 66 and 82 degrees C (150 to 180 degrees F water) for either 5, 10, or 15 s. There were no differences (P > 0.05) in the log reductions of S. Typhimurium and E. coli O157:H7 on the lean surfaces for all three temperature treatments (66, 74, and 82 degrees C). Although the 15 s treatment resulted in a numerically higher log reduction than the other treatments, each of the times resulted in at least a 1 log reduction of both S. Typhimurium and E. coli O157:H7 for lean surfaces. For the fat surfaces, all time treatments for the 82 degrees C and the 10 and 15 s treatments for the 74 degrees C resulted in the highest log reduction for S. Typhimurium. The 5 and 10 s dwell times for treatments at 66 degrees C and the 5 s dwell time at 74 degrees C resulted in the lowest log reduction of S. Typhimurium and E. coli O157:H7. For E. coli O157:H7 all temperature and time treatments resulted in at least a 1 log reduction for the fat surfaces of the outside round. Therefore, hot water treatment is a proven method for reducing both coliforms and pathogenic bacteria.

Due to the seasonal nature of E. coli O157:H7 shedding and of hormone production by the thyroid gland, two studies were initiated to determine whether chemical inhibition of the thyroid gland influences fecal shedding of Escherichia coli O157:H7. Twenty-four crossbred sheep (68.6 kg BW) were randomly assigned to pen and either 0.0 mg/kg BW PTU or 20 mg/kg BW PTU for 5, 11, or 14 days. Sheep were experimentally infected (d 0) with E. coli O157:H7 11 days prior to PTU treatment. Fecal and serum samples were collected for bacterial enumeration and for analysis of T3 and T4, respectively. Sheep were humanely euthanized and tissue and content samples were collected from the rumen, ileum, colon and rectum. Detection of E. coli O157:H7 increased toward the terminal end of the GI tract. In the treatment group, serum T3 levels decreased to an overall lower level than the control group. A correlation was seen between T3 levels and daily O157:H7 bacterial shedding (P=0.003; r=0.37). In experiment 2, 12 growing lambs (41.04 kg BW) were exposed to either 0.0 mg/kg BW PTU or 40 mg/kg BW PTU for 21 days. Fecal samples were collected for analysis of generic E. coli and body weights were recorded on days 0, 7, 14, and 21. Feed intake was recorded throughout the experiment. Animals were experimentally infected with E. coli O157:H7 on day 15. Sheep were humanely euthanized on day 21 and GI tract tissue and content was collected from the rumen, ilium, colon and rectum. A date by treatment interaction was observed for T4 (P=0.0016) and hormone levels decreased in treated animals. Thyroxine and E. coli O157:H7 display a multivariate treatment (P=0.0005) and date effect (P=0.0174) but no significant interaction. Triiodothyronine and E. coli O157:H7 shedding have a slight date trend (P=0.065) but no significant treatment or treatment by date interaction. Generally, the treatment group shed genreric E. coli at higher levels throughout the study period with slightly more than a log count difference between groups at the last collection point (control = 3.8 CFU/gram of feces (log10); treatment = 4.9 CFU/gram of feces (log10)). Results from these experiments suggest that correlations exist between both E. coli O157:H7 and generic E. coli shedding in sheep.

Microbiological detection of foodborne pathogens is ineffective for monitoring critical control points (CCP) within a slaughter/processing Hazard Analysis and Critical Control Point (HACCP) system. Pathogens are usually absent from carcass surfaces and their uneven distribution makes it difficult to obtain a representative sample. However, microbiological testing can be applied within a HACCP plan to validate and verify the effectiveness of decontamination procedures designed to control hazards. With proper data collection, the reduction of an indicator group at a point in processing can indicate that a specific pathogen is being effectively controlled, especially when pathogen levels are too low to allow confirmation of process control, as they typically are in beef slaughter processing. Since E. coli O157:H7 has been shown to have some acid resistance, the ability of typical indicator organisms to accurately predict the reduction of this pathogen by carcass decontamination procedures has been a concern. Obtaining potential indicator bacteria from the same environmental reservoir as E. coli O157:H7 may provide non-pathogenic indicators with similar heat- and acid-resistance characteristics suitable for use in processing plant environments for validation and verification of carcass decontamination treatments within HACCP plans. Potential indicator bacteria were isolated from hides of cattle at slaughter facilities in Arizona, Georgia, and Texas and compared with isolates of E. coli O157:H7 from the same locations to determine similarity in acid- and heat-resistance characteristics. After evaluation at 2 heating temperatures (55 and 65??C) and 3 pH levels (3.0, 4.0, and 5.0), it was determined that several potential indicator bacteria were slightly more resistant than E. coli O157:H7 to heating and acid treatment. The greatest reduction in numbers for E. coli O157:H7 and indicator bacteria occurred at pH 3.0 and temperature of 65??C. Counts of bacteria grown at pH 4.0 and 5.0 were not significantly different. Testing indicated that several of the isolates from cattle hides would make good process control indicators since the indicator bacteria were reduced by heating or acid conditions at similar or greater rates when compared to E. coli O157:H7, providing an increased level of security that pathogens have been reduced in processing.

This manuscript presents a thorough investigation and description of metabolic control dynamics in vivo and in silico using as a model de novo pyrimidine biosynthesis. Metabolic networks have been studied intensely for decades, helping develop a detailed understanding of the way cells carry out their biosynthetic and catabolic functions. Biochemical reactions have been defined, pathway structures have been proposed, networks of genetic control have been examined, and mechanisms of enzymatic activity and regulation have been elucidated. In parallel with these types of traditional biochemical analysis, there has been increasing interest in engineering cellular metabolism for commercial and medical applications. Several different mathematical approaches have been developed to model biochemical pathways by combining stoichiometric and/or kinetic information with probabilistic analysis, or deciphering the comparative logic of metabolic networks using genomic-derived data. However, most of the research performed to date has relied on theoretical analyses and non-dynamic physiological states. The studies described in this dissertation provide a unique effort toward combining mathematical analysis with dynamic transition experimental data. Most importantly these studies emphasize the significance of providing a quantitative framework for understanding metabolic control. The pathway of de novo biosynthesis of pyrimidines in Escherichia coli provides an ideal model for the study of metabolic control, as there is extensive documentation available on each gene and enzyme involved as well as on their corresponding mechanisms of regulation. Biochemical flux through the pathway was analyzed under dynamic conditions using middle-exponential growth and steady state cultures. The fluctuations of the biochemical pathway intermediates and end products transitions were quantified in response to physiological perturbation. Different growth rates allowed the comparison of rapid versus long-term equilibrium shifts in metabolic adaptation. Finally, monitoring enzymatic activity levels during metabolic transitions provided insight into the interaction of genetic and biochemical mechanisms of regulation. Thus, it was possible to construct a robust mathematical model that faithfully represented, with a remarkable predictability, the nature of the metabolic response to specific environmental perturbations. These studies constitute a significant contribution to the fields of quantitative biochemistry and metabolic control, which can be extended to other cellular processes as well as different organisms.

HDGF (hepatoma-derived growth factor) is a novel growth factor,identified from conditioned medium of hepatoma cell line. HDGF has growth stimulating activity for fibroblast and some hepatoma cells. HDGF, a novel defined growth factor with mitogenic effect, has homology protein sequence as HMG (high mobility group) protein and their three dimension structures appeared to be similar to each other. Recently, elevated HDGF expression was found in developing kidneys but less was found in adult kidney. In addition, HDGF expression was found to be correlated with angiogenic status of tissues. Thus, it is speculated that HDGF plays a role during embryonic development and angiogenesis. HDGF also plays a role in cell-cell interaction and cell migration. HDGF is a growth factor that is involved in stimulating vascular smooth muscle cells (SMCs)proliferation during development and in disease. HDGF contains a true bipartite nuclear localization sequence necessary for nuclear targeting. HDGF is sciential factor in stimulating DNA replication and cell proliferation of vascular smooth muscle cell.In this study,we used E. coli strain BL21 (DE3) to express the recombinant protein hepatoma derived growth factor(HDGF). To find out the optimal production conditions,we studied on the different temperature and fermentor to calculate all cost .