Throughout the years lime-induced chlorosis has contributed to untold economic losses. Although this disease has challenged the technical ingenuity of outstanding plant and soil scientists, the exact cause has never been determined; consequently, no preventative measures or permanent cures can, as yet, be recommended. This physiological malady, unchecked, continues its rampage in regions where the disease is prevalent.

Enterohemorrhagic Escherichia coli (EHEC) is a virulent pathotype of E. coli that is associated with major outbreaks of hemorrhagic colitis and the life-threatening kidney disease hemolytic uremic syndrome. For successful host colonization and attachment to the intestinal mucosa, EHEC requires the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (TTSS) responsible for secreting and translocating effector proteins into host colonocytes. Regulation of the LEE is primarily directed through the first operon, LEE1, encoding the locus encoded regulator (Ler), and occurs through the direct and indirect action of several regulators. The 2006 U.S. spinach outbreak of E. coli O157:H7, characterized by unusually severe disease, has been attributed to a strain (TW14359) with enhanced pathogenic potential including elevated virulence gene expression, robust adherence, and the presence of novel virulence factors. Aim 1 of this dissertation proposes a mechanism for the unique virulence expression and adherence phenotype of this strain, and further expands the role for regulator RcsB in control of the E. coli locus of enterocyte effacement (LEE) pathogenicity island. Proteomic analysis of TW14359 revealed a virulence proteome consistent with previous transcriptome studies that included elevated levels of the LEE regulatory protein Ler and type III secretion system (T3SS) proteins, secreted T3SS effectors, and Shiga toxin 2. Basal levels of the LEE activator and Rcs phosphorelay response regulator, RcsB, were increased in strain TW14359 relative to O157:H7 strain Sakai. Deletion of rcsB eliminated inherent differences between these strains in ler expression, and in T3SS-dependent adherence. A reciprocating regulatory pathway involving RcsB and LEE-encoded activator GrlA was identified and predicted to coordinate LEE activation with repression of the flhDC flagellar regulator and motility. Overexpression of grlA was shown to increase RcsB levels, but did not alter expression from promoters driving rcsB transcription. Expression of rcsDB and RcsB was determined to increase in response to physiologic levels of bicarbonate, and bicarbonate-dependent stimulation of the LEE was shown to be dependent on an intact Rcs system and ler activator grvA. The results of this aim significantly broaden the role for RcsB in EHEC virulence regulation. The bicarbonate ion (HCO3-) has been shown to stimulate LEE gene transcription through the LEE1 promoter, and is predicted to serve as a physiologic signal for EHEC colonization. Results from the previous aim demonstrated that bicarbonate induction of the LEE is mediated through the Rcs phosphorelay, and is dependent upon an intact global regulator of virulence grvA gene. However, the direct mechanism through which RcsB-GrvA regulates ler, and the contribution of GrvA to the virulence of EHEC is unknown. In Aim 2, the RcsB-GrvA regulon of EHEC was determined by RNA sequencing, and the contributions of each to virulence and stress fitness was explored. A significant increase in transcription of the gad genes for extreme acid resistance was observed for both EHEC strains TW14359grvA and TW14359rcsBgrvA compared to TW14359, and corresponded with a significant increase in acid survival for TW14359grvA during exponential growth. Therefore, a model by which RcsB-GrvA coordinate LEE expression with acid resistance through GadE was proposed. Finally, the temporal regulation of both rcsDB and grvAB operons in response to bicarbonate was defined using single copy luxE chromosomal reporter fusions. Taken together, these results demonstrate the role of RcsB and GrvA to EHEC virulence, and reveal a novel role for GrvA in of extreme acid resistance and LEE gene expression and in EHEC. Finally, production of the ECP pilus has been demonstrated in enterohemorrhagic Escherichia coli O157:H7 (EHEC), and has been shown to be required for efficient adherence to epithelial cells during colonization. The first gene of the ecpRABCDE operon encodes a transcriptional regulator (EcpR) that positively regulates its own transcription, and promotes transcription and production of the downstream gene, ecpA, encoding the major ECP subunit EcpA. However, the distance between the ecpR and ecpA genes suggests the presence of regulatory elements that control ecpA directly. Therefore, it was hypothesized that an additional promoter was able to direct transcription of ecpA, independent of the promoter upstream of ecpR. To test this, promoter-lacZ transcriptional reporter fusions were created using the regions upstream of ecpR and ecpA to test for promoter activity, coupled with western blot analysis to detect EcpA in both wild-type and ecpR promoter mutant strains. In Aim 3, we showed that an additional promotable element, downstream of the EHEC O157:H7 strain TW14359 ecpR translational start site, is capable of driving transcription of ecpA, and that its activity is independent of an intact ecpR promoter. In addition, site-directed mutagenesis was used to characterize a TW14359 specific single nucleotide polymorphism within the predicted ecpA promoter region. Overproduction of EcpR was observed to increase cytosolic RcsB and Tir, indicating that ecp production is able to stimulate the LEE, and that the ecpA promoter polymorphism may contribute to intrinsically increased rcsB transcription in TW14359. Taken together, the results, and those obtained in Aims 1 and 2, expand the model for regulation of the ecp operon in EHEC O157:H7 strain TW14359, and broaden the model for EcpR and RcsB in the coordinate regulation of E. coli common pilus and type III secretion.

Abstract not available

High bicarbonate (HCO3-) content and associated high pH of irrigation water is detrimental to plant growth. Sustain ableagricultural/horticultural production will increasingly have to rely on economically feasible and environmentally sound solutions to the problems associated with high levels of HCO3- in irrigation water. The ability of a mixed Glomus Tulasne & Tulasne species inoculum of arbuscular mycorrhizal fungi (AMF), Glomus ZAC-19 (containing Glomus albidum Walker & Rhodes, Glomus claroideum Schenck & Smith, and Glomus diaphanum Morton & Walker), to enhance plant tolerance to HCO3- was tested on the growth and nutrient uptake of Rosa multiflora Thunb. ex J. Murr. cv. Burr (rose). Arbuscular mycorrhizal colonized and non-inoculated (non-AMF) R. multiflora cv. Burr were treated with 0, 2.5, 5, and 10 mM HCO3-. Increasing HCO3- concentration and associated high pH reduced R. multiflora cv. Burr growth, nutrient uptake, and acid phosphatase activity (ACP), while increasing alkaline phosphatase activity (ALP). Inoculation with AMF enhanced plant tolerance to HCO3- as indicated by greater growth, nutrient uptake, leaf chlorophyll content, higher mycorrhizal inoculation effect (MIE), lower root iron reductase activity, and generally lower soluble and wall-bound ALP activity. While AMF colonization (arbuscules, vesicles, and hyphae formation) was reduced by increasing HCO3- concentration, colonization still occurred at high HCO3- concentration. At 2.5 mM HCO3-, AMF plant growth was comparable to plants at 0 mM HCO3-, further indicating the beneficial effect of AMF for alleviation of HCO3- plant stress.

The concentration of carbon dioxide in natural seawater is sufficient for microalgal growth, but insufficient for high algal-density culture due to limitation of photosynthesis in the artificial medium. This study was adjusted the initial pH to 5.5, 6.5 and 7.5 by adding CO2 or HCl in 1L flask cultured stagnantly with continuous illumination. The best growth of Isochrysis galbana was found in the culture (CO2-6.5i) with initial pH 6.5 using CO2 adjustment and maintaining the same during day 2 to day 5, while the worst was in CO2-5.5i. Furthermost, we adjusted the pH of cultures daily to the set values; the best growth was also found in CO2-6.5e having the cell number 221% of the blank. Initial addition of NaHCO3 with doses of 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/ L and 1g/ L in the culture, showed the lowest cell number after 5 days culture is in the group of 1.0 g/L and no significant difference among the rest groups. Addition of 0.5 g/L NaHCO3 and adjusted the pH to 6.5 by HCl in the beginning promoted algal growth and resulted in the culture having 212 % cell number of the blank. Using the feedback control system, 100 L algal cultures with aeration and providing CO2 gas or HCl liquid to maintain the pH as 5.5, 6.5 and 7.5 or 7.5 individually were conducted to test the effect of pH control on the mass culture of I. galbana. Better growth was found in the culture with CO2 feedback control than HCl-control in duplicate experiments. It also showed significant difference among the groups adjusted pH between 6.5-7.5. The cell concentration could reach 1100-1400 x 104 cells/ml and was about double the amount of the blank without pH control cultured in 7 days. Meanwhile, the NO3-N concentration was nearly exhausted while the PO4-P still replete. This study reveals the high concentration and fast growth of I. galbana can be maintained under the suitable physical condition providing the carbon source in an optimal pH.

High bicarbonate (HCO3-) content and associated high pH of irrigation water is detrimental to plant growth. Sustain ableagricultural/horticultural production will increasingly have to rely on economically feasible and environmentally sound solutions to the problems associated with high levels of HCO3- in irrigation water. The ability of a mixed Glomus Tulasne & Tulasne species inoculum of arbuscular mycorrhizal fungi (AMF), Glomus ZAC-19 (containing Glomus albidum Walker & Rhodes, Glomus claroideum Schenck & Smith, and Glomus diaphanum Morton & Walker), to enhance plant tolerance to HCO3- was tested on the growth and nutrient uptake of Rosa multiflora Thunb. ex J. Murr. cv. Burr (rose). Arbuscular mycorrhizal colonized and non-inoculated (non-AMF) R. multiflora cv. Burr were treated with 0, 2.5, 5, and 10 mM HCO3-. Increasing HCO3- concentration and associated high pH reduced R. multiflora cv. Burr growth, nutrient uptake, and acid phosphatase activity (ACP), while increasing alkaline phosphatase activity (ALP). Inoculation with AMF enhanced plant tolerance to HCO3- as indicated by greater growth, nutrient uptake, leaf chlorophyll content, higher mycorrhizal inoculation effect (MIE), lower root iron reductase activity, and generally lower soluble and wall-bound ALP activity. While AMF colonization (arbuscules, vesicles, and hyphae formation) was reduced by increasing HCO3- concentration, colonization still occurred at high HCO3- concentration. At 2.5 mM HCO3-, AMF plant growth was comparable to plants at 0 mM HCO3-, further indicating the beneficial effect of AMF for alleviation of HCO3- plant stress.

The effects of different types and amounts of sodium bicarbonate (NBC) were evaluated during the processing of flour tortillas. Fat encapsulated NBC, BS199, BS195, BS193, BS180, BS184, HM50 and HM70, and different particle-sized, non-encapsulated NBC, grade 1, grade 2, grade 3 and Tortilla Blend TM, were tested at different levels and combinations. Longer shelf stable tortillas with lower opacity were obtained when the level of NBC decreased. Tortillas with higher opacity, diameter and volume were obtained using the correct types of NBC. Increased tortilla opacity, thickness and shelf stability were obtained using 3 g encapsulated NBC/kg compared to non-encapsulated NBC. Encapsulation of NBC enables a temperature-triggered leavening reaction during baking of tortillas. Combinations of different levels and ratios of fast- and slow-release NBC did not yield significant improvements in tortilla properties. Tetrasodium pyrophosphate (TSPP, 0.15%) was added to modify protein functionality in tortilla dough containing less NBC. Tortillas with improved opacity, thickness and shelf stability resulted using TSPP with 3 g Grade 1 NBC/kg but similar improvements were not seen with encapsulated NBC. The effects of TSPP with low levels of NBC and slow reacting leavening acids were tested. Slight improvements in opacity were observed with sodium aluminum phosphate (SALP) compared to sodium aluminum sulfate (SAS) using encapsulated and non-encapsulated NBC. Tortillas produced with these formulations have longer shelf stabilities with similar properties. This benefits the consumers. The manufacturing costs for ingredients are 1.2% less using 0.15% TSPP with 3 g non-encapsulated NBC/kg flour. The manufacturer gains not only by the reduced cost of ingredients but also by the increased ease of attaining target diameter of tortillas. This could also be accomplished by using less dough to form the tortilla.

No description available.

Amantadine is a cationic aliphatic primary amine eliminated by the kidneys, excreted predominantly unchanged into the urine, and undergoes limited metabolism. Renal tubule secretion has an important role in its elimination. We studied two aspects of amantadine disposition, firstly acetylation, by developing a model to induce the enzyme spermidine/spermine N1-acetyltransferase (SSAT1) with N1, N11-diethylnorspermine (DENSPM) and alcohol (Alc) as representative agents reported to induce its activity, and secondly renal secretion, by studying the effect of intravenous bicarbonate infusion on its renal elimination. We drew two conclusions, firstly, longer exposure to Alc combined with DENSPM administration provided the greatest potentiation of SSAT1 enzyme activity than each agent alone, which indicates a high likelihood of synergy between Alc and DENSPM; and secondly, bicarbonate load administered to healthy male volunteers impairs amantadine renal secretion in the absence of a clinically important change in blood pH, serum creatinine concentration or urinary creatinine clearance.

Amantadine is a cationic aliphatic primary amine eliminated by the kidneys, excreted predominantly unchanged into the urine, and undergoes limited metabolism. Renal tubule secretion has an important role in its elimination. We studied two aspects of amantadine disposition, firstly acetylation, by developing a model to induce the enzyme spermidine/spermine N1-acetyltransferase (SSAT1) with N1, N11-diethylnorspermine (DENSPM) and alcohol (Alc) as representative agents reported to induce its activity, and secondly renal secretion, by studying the effect of intravenous bicarbonate infusion on its renal elimination. We drew two conclusions, firstly, longer exposure to Alc combined with DENSPM administration provided the greatest potentiation of SSAT1 enzyme activity than each agent alone, which indicates a high likelihood of synergy between Alc and DENSPM; and secondly, bicarbonate load administered to healthy male volunteers impairs amantadine renal secretion in the absence of a clinically important change in blood pH, serum creatinine concentration or urinary creatinine clearance.