The purpose of this study was (1) to determine whether a correlation exists among the protein profiles, extracted from cell membranes of mutants belonging to five pigment cluster groups, (2) to locate the protein moiety and cartenoprotein complex in the membranes of wild type and colorless mutant (designated W-19) of C. poinsettae and to show whether there are any structural differences between cell membranes of the wild type and a colorless mutant, (3) to determine the effect of six antibiotics on cartenoid gene expression.

The aerobic transformation of TCE and cis-DCE by a tetrabutoxysilane-grown microorganism (Vancheeswaran et al., 1999) led to the investigation of novel substrates, including benzyl alcohol, for promoting cometabolism. The culture grew on carboxylic compounds and alcohols, but did not grow on formate, methanol, methane, propane, butane, ethylene, benzene, toluene, or p-xylene. Cis-DCE transformation was observed when the culture grew on butyrate, glucose, 1-propanol, 1-butanol, ethanol, benzyl alcohol, and phenol, and effectively transformed TCE, cis-DCE, and vinyl chloride when grown on phenol or benzyl alcohol. Several cycles of growth on benzyl alcohol led to increases in TCE transformation rates and transformation capacities. Products of benzyl alcohol degradation shifted from benzaldehyde to 2-hydroxy benzyl alcohol (2HBA) during the several cycles of growth. In resting cells studies, 2HBA production rates were highly correlated with TCE transformation rates. TCE transformation and 2HBA production rates doubled when the culture was grown on phenol and rates of TCE transformation were correlated with 2HBA production rates. Benzyl alcohol- and phenol-grown cells oxidized toluene to o-cresol, which indicated the similarity between benzyl alcohol ortho-monooxygenase, phenol hydroxylase, and toluene ortho-monooxygenase. 2-Butyne and 1-hexyne (but not acetylene) inhibited benzyl alcohol- and phenol-grown cells similarly, indicating the same ortho-monooxygenase was responsible for TCE cometabolism. Resting cell kinetic studies were performed with cells grown on phenol or benzyl alcohol. Benzyl alcohol degradation followed a Monod kinetics while phenol degradation followed a Haldane kinetics. The maximum transformation rates (k[subscript max]) of TCE, cis-DCE, and VC achieved by phenol-grown cells were about a factor of two higher than achieved with benzyl alcohol-grown cells, while the half-saturation constants (K[subscript s]) were in a similar range. Transformation capacities (Tc) for TCE, cis-DCE, and VC were about a factor of two to four higher with phenol-grown cells. The modeling of TCE, cis-DCE, and VC transformation using independently measured k[subscript max] and K[subscript s] values matched well with observed data from batch tests. Benzyl alcohol was shown to be an effective novel substrate for the aerobic cometabolism of TCE, cis-DCE, and vinyl chloride. Being a non-regulated compound, it might have applications for in-situ bioremediation. / Graduation date: 2004

Thesis (M.S.)--Virginia Polytechnic Institute, 1953. / Vita. Includes bibliographical references (leaves ix-xii). Also available via the Internet.

Thesis (Ph. D.)--Michigan State University. Dept. of Chemistry, 2008. / Title from PDF t.p. (viewed on Aug. 20, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 148-159).

It is generally known that media containing simple sugars (sucrose, glucose) and organic nitrogen sources (ammonium) when buffered to acidic pH stimulates aflatoxin production in Aspergillus flavus & A. parasiticus while lactose, nitrate and an alkaline pH inhibit aflatoxin biosynthesis. It has been shown that pH of the growth medium is the most important regulatory factor for aflatoxin biosynthesis since media containing stimulatory carbon and/or nitrogen sources (sucrose and ammonia) do not enhance aflatoxin (or sterigmatocystin) production at alkaline pH. RNA interference (in A. flavus) of the pH regulatory transcription factor, PacC, resulted in aflatoxin production under acidic and alkaline pH conditions whilst wildtype Aspergillus flavus produced aflatoxins only under acidic conditions. This conclusively proved that PacC negatively regulates aflatoxin production at alkaline pH in A. flavus. However the exact mechanism involved in PacC repression of aflatoxin biosynthesis at alkaline pH still remains unknown. The AflR protein is essential for expression of several genes in the aflatoxin biosynthetic cluster. In the current study, sequence analysis of the aflR promoter indicated the presence of two putative PacC binding sites within the aflR promoter of A. flavus 3357WT located at positions -162 and -487 bp from the start codon. The presence of the PacC binding sites in the aflR promoter indicated a possible link between aflR expression and PacC regulation under alkaline conditions. Thus, in this study, it was hypothesized that at alkaline pH, PacC inhibits aflR expression by binding to one or both of the PacC binding sites within the aflR promoter. This in turn, would result in inhibition of aflatoxin biosynthesis since expression of several aflatoxin biosynthetic pathway genes is dependent on activation by AflR. The aim and objective of this study was to test the validity of this hypothesis i.e. that at alkaline pH PacC binds to one or both of its recognition sites within the aflR promoter thereby inhibiting aflR expression which subsequently would result in inhibition of aflatoxin biosynthesis. This was done by first mutating each individual and then both PacC binding sites in the A. flavus 3357 aflR promoter via Single-Joint PCR (SJ-PCR) and fusing the wildtype and each mutated aflR promoter to the Green Fluorescent Protein (gfp) gene and the trpC terminator to yield a functional expression vector. These constructs were then transformed into A. flavus 3357.5. Positive transformants were confirmed to express GFP by fluorescence microscopy and spectrofluorometry. Quantification of GFP protein levels of the various transformants in this study indicated that PacC negatively regulated aflR promoter activity at alkaline pH. RT-qPCR was performed on positive transformants after growth on SLS medium at acidic and alkaline pH to determine if PacC negatively regulated aflR promoter activity at alkaline pH and to determine whether PacC binds preferentially to one or both recognition sites within the aflR promoter. RT-qPCR analysis suggest that PacC binds non-preferentially to both recognition sites within the aflR promoter on sucrose and lactose media at alkaline pH, although mutation of PacC binding site 2 results in a slightly higher expression compared to mutation of PacC binding site 1. Increasing the concentration of an aflatoxin conducive nitrogen source stimulated aflR promoter activity but this was not sufficient to overcome negative regulation by PacC. It is generally known that repression of aflR expression results in repression of aflatoxin biosynthesis irrespective of pH. The results of this study strongly suggest that PacC negatively regulates aflR promoter activity at alkaline pH by binding to one or both PacC recognition sites within the aflR promoter. Since aflR promoter activity is repressed by PacC at alkaline pH, this substantiates the hypothesis that PacC represses aflatoxin biosynthesis by inhibiting expression of aflR. Furthermore, the results of this study indicated that there may be some PacC protein present in the active form at acidic pH irrespective of the carbon source and nitrogen source used in the growth medium. RT-qPCR analysis indicated that any active PacC present at acidic pH may cause repression of the aflR promoter based on the position of the PacC binding site relative to the aflR start codon, although it appears that PacC may have a higher affinity for PacC binding site 2 (which is closer to the aflR start codon).

Activation of otherwise unreactive substrates is a common strategy in chemistry and in nature. Adenylate-forming enzymes use adenosine monophosphate to activate the hydroxyl of their carboxylic substrate, creating a better leaving group. In a second step this reactive group is replaced in a nucleophilic elimination reaction to form esters, amides or thioesters. Recent studies have revealed that NRPS- independent siderophore (NIS) synthetases are also adenylate-forming enzymes, but are not included in the current superfamily description. NIS enzymes are involved in biosynthesis of high-affinity iron chelators which are used for iron acquisition by many pathogenic microorganisms. This is an important area of study, not only for potential therapeutic intervention, but also to illuminate new enzyme chemistries. Here the structural and biochemical studies of AcsD from Pectobacterium chrysanthemi are reported. AcsD is a NIS synthetase involved in achromobactin biosynthesis. The co-complex structures of ATP and citrate provide a mechanism for the stereospecific formation of an enzyme-bound citryl-adenylate. This intermediate reacts with L-serine to form a likely achromobactin precursor. A detailed characterization of AcsD nucleophile profile showed that it can not only catalyze ester formation, but also amide and possibly thioester formation, creating new stereospecific citric acid derivatives. The structure of a N-citryl-ethylenediamine product co-complex identifies the residues that are important for both recognition of L-serine and for catalyzing ester formation. The structural studies on the processive enzyme AlcC, which is involved in the final step of alcaligin biosynthesis of Bordetella pertussis, show that it has a similar topology to AcsD. It also shows that ATP is coordinated in a manner similar to that seen in AcsD. Biochemical studies of a substrate analogue establish that AlcC is not only capable of synthesizing substrate dimers and trimers, but also able to assemble the respective dimer and trimer macrocycles. A series of docked binding models have been developed to illustrate the likely substrate coordination and the steps along dimerization and macrocyclization formation. Structural and mechanistic comparison of NIS enzymes with other adenylate-forming enzymes highlights the diversity of the fold, active site architecture, and metal coordination that has evolved. Hence, a new classification scheme for adenylate forming enzymes is proposed.

Bacteria in the gastrointestinal tract play an important role in intestinal motility, inflammation, homeostasis, and behavior. Bacteria, through the natural synthesis of neuroactive compounds and secondary metabolites, can modulate the host immune system and communicate with the host along the signaling pathway along the gut-brain axis. Here, we functionally design, develop, test, and characterize a platform for the study of microbial-host interactions using advancements in the field of synthetic biology. First, we describe the engineering of Escherichia coli Nissle to biosynthesize serotonin within the mammalian gut using a native-plasmid optimized approach. Serotonin is crucial for neurotransmission throughout the body and may be playing a role in microbial gut-brain communication. In the gastrointestinal tract, serotonin regulates intestinal motility, cell turnover, intestinal inflammation, and gastrointestinal homeostasis. Upon serial daily oral gavages, our engineered bacterium populates a murine colon to produce serotonin locally in the mucosa layers along the epithelial lining. Changes in host physiology were observed including decreased gastrointestinal motility, increased colonic Muc2 expression, induction of host TPH2, responsible for serotonin biosynthesis in enteric neurons, and upregulation of serotonin receptors HTR3, HTR4, and HTR7 in the colon. Behavioral tests revealed a statistically significant decrease in anxiety and depression in stress-induced environments in mice treated with the engineered bacterium. This work suggests that gut bacteria engineered to modulate host gut-brain axis may have both scientific and clinical uses to study microbial-host interactions and treat gastrointestinal and behavioral mood disorders in humans. Second, we engineered bacteria to produce exogenous butyrate and other SCFAs in the murine gut. Short chain fatty acids (SCFAs) play an important role in intestinal homeostasis, fluid dynamics, inflammation, oxidative stress, and intestinal hypersensitivity and motility. With this development, we characterized the effects of our butyrate-producing bacteria on a high-fat diet and DSS-induced colitis model within the colon. Although energetically burdensome to produce, our strains produced butyrate in the colon at higher density in an actively inflamed colitis model. After 14 days of oral administration, our engineered strain (EcN:B) increased the colon length of normal wild-type mice, in high fat fed mice, and in mice with recovering and actively inflamed DSS-induced colitis. EcN:B increased mucosal barrier thickness, upregulated gene expression of the barrier integrity markers Cldn1, Ocln, Zo1, and altered crypt and villus height during inflammation recovery. Furthermore, as butyrate is known to induce Foxp3+ Regulatory T cells, we saw a 13.01% percent increase in Foxp3+ cells in the colon of mice fed our engineered bacteria. This work suggests that synthetic gut bacteria engineered to produce short chain fatty acids may have future clinical uses to treat patients with inflammatory bowel disease including Crohn’s and Colitis with future potential to serve as a therapeutic for irritable bowel syndrome, idiopathic constipation, obesity, and colorectal cancer. This platform, with the use of synthetic biology to natively engineer Escherichia coli Nissle to produce bioactive compounds in the distal gastrointestinal tract, creates a framework for future characterization of bacterial-host communication and future microbial-based therapeutics.

The increasing frequency of enterococci as a major cause of nosocomial infections and the transmission of these organisms amongst hospital patients demands a greater awareness of the Enterococcus. Therapy of enterococcal infections is complicated by the pathogens continually changing resistance patterns to many broad-spectrum antibiotics. In addition, the ability of enterococci to cause serious invasive infections including endocarditis and septicaemia with associated high mortality rates; prompted this study which was aimed at identifying the biological properties of enterococci isolated from blood cultures of patients admitted at King Edward VIII hospital, Durban. Enterococci were identified to species level by the API 20 Strep system which identified 68% and a conventional biochemical system of Facklam and Collins which identified 100% of the isolates.The emergence of beta-Iactamase producing enterococci in other countries encouraged the testing of all isolates for this enzyme. All were beta-Iactamase negative. The reported false susceptibility for aminoglycosides and cephalosporins with blood enriched media encouraged the testing of these antibiotics with and without the supplementation of 5% lysed blood. The results showed that an average false susceptibility of 55 % occurred for gentamicin and 35% for tobramycin and netilmicin. The cephalosporins affected, cefotaxime and cefuroxime showed a false susceptibility of 28% and 17% respectively. The choice of treatment for serious enterococcal infections is a syllergistic combination of a beta-Iactam antibiotic plus an aminoglycoside for enterococci with intrinsic low-level resistance. The development of high-level aminoglycoside resistance, MIC 22000,ug/ml results in loss of synergism. This study showed that 26.4 % of enterococcal isolates displayed high level aminoglycoside resistance i.e. to gentamicin and streptomycin. Time-kill study showed reduced killing rate for these organisms for the beta-Iactams and glycopeptides with low-level gentamicin resistance. The results confirmed that a cell-wall active agent combined with gentamicin can be successfully used for enterococcal therapy if the organism has intrinsic low-level resistance to this amino glycoside. Pulsed-field gel electrophoresis (PFGE) carried out on a selected number of Enterococcus faecalis and Enterococcus faecium with high-level aminoglycoside resistance showed a variability in the restriction endonucelase digestion patterns. This suggests independent development of high-level gentamicin resistance and not clonal expression. The ease and reliability with which enterococcal isolates may be typed using this technique to compare different strains represent a significant advance. / Thesis (M.Med.Sc.)-University of Natal, 1999.

During fermentation, the yeast Saccharomyces cerevisiae produces a broad range of aroma-active esters that are important for the desirable complex flavour of beer. The sensory threshold levels of these esters in beer are low, ranging from 0.2 ppm for isoamyl acetate to 15-20 ppm for ethyl acetate. Although esters are only present in trace amounts in beer, they are extremely important as minor changes in their concentration may have dramatic effects on beer flavour. Therefore, optimization of the concentrations of these aroma-active esters in beer is of interest in beer brewing. The number and concentration of esters in beer may be influenced by the fermentation parameters, nutritional composition of fermentation medium and yeast strain type. Therefore, this study investigated the influence of fermentation temperature, pH, and wort nutritional supplements (amino acids and zinc) on the production of yeast-derived ester compounds. In addition, the overall fermentation performance was evaluated based on the reducing sugar and Free Amino Nitrogen (FAN) utilization, ethanol production and yeast cell density. These parameters were analysed using the Dinitrosalicyclic acid method, Ninhydrin assay, Gas Chromatography and standard spread plate technique. The concentration and stability of ethyl acetate, isoamyl acetate, phenyl ethyl acetate, ethyl hexanoate, ethyl decanoate and ethyl octanoate was monitored during storage at 4 °C and room temperature (RT), in the final beer by Chromatography. The expression levels of the ester synthetase genes under conditions that resulted in the highest increase in ester production were quantified by Real-Time PCR. For the lager beer, the best fermentation performance was achieved at RT (±22.5°C), resulting in the utilization of the highest amount of nutrients and production of 4.86% (v/v) ethanol. This was accompanied by the highest production of acetate and ethyl esters, which were 40.86% and 87.21%, respectively, higher than that of the control. Spent yeast density ranged from 2.492 to 3.358 mg/ml for all parameters tested, with the highest yield produced when wort was supplemented with 0.120 g/l zinc sulphate. Fermentations at 14 °C yielded the highest foam head stability and spent yeast viability with a foam head rating of 2.67 and a spent yeast viability of 3.85 × 107 cfu/ml. Ester compounds were relatively stable at 4 °C than at room temperature decreasing by only 7.93% after three months. Of all the volatile esters produced, ethyl decanoate was the least stable, with a 36.77% decrease in concentration at room temperature. For the ale beer, the best fermentation performance which resulted in the highest nutrient utilization was achieved when wort was supplemented with 0.75 g/l L-leucine resulting in the utilization of the highest amount of nutrients (51.25% FAN and 69.11% reducing sugar utilization) and production of 5.12% (v/v) ethanol. At the optimum fermentation pH of 5, 38.27% reducing sugars and 35.28% FAN were utilized, resulting in 4.32% ethanol (v/v) production. Wort supplemented with 0.12 g/l zinc sulphate resulted in 5.01% ethanol (v/v) production and 54.32% reducing sugar utilization. Spent yeast density ranged from 1.985 to 2.848 mg/ml for all parameters tested with the highest yield produced when wort was supplemented with 0.120 g/l zinc sulphate. This was also accompanied by the highest yeast viability of 2.12 × 107 cfu/ml achieved on day 3 of fermentation. Supplementation with 0.75 g/l L-leucine yielded the highest foam head stability with a rating of 2.67. Overall, ester compounds were relatively more stable at 4 °C than at RT decreasing by only 6.93% after three months, compared to a decrease of up to 16.90% observed at RT at the same time. Of all the volatile esters produced, ethyl octanoate was the least stable, with a 32.47% decrease in concentration at RT, phenyl ethyl acetate was the most stable ester at RT, decreasing by 9.82% after three months. Wort supplemented with 0.75 g/l L-leucine resulted in an increase in isoamyl acetate and phenyl ethyl acetate production by 38.69% and 30.40%, respectively, with a corresponding high expression of alcohol acetyltransferases, ATF2 (133.49-fold higher expression than the control). Elevation of fermentation temperature to RT resulted in the upregulation of ATF2 (27.11-fold), and producing a higher concentration of isoamyl acetate. These findings indicate that ester synthesis during fermentation is linked to both substrate availability and the regulation of gene expression. Therefore, it would be possible to manipulate the expression of certain ester synthestase genes to create new yeast strains with desirable ester production characteristics. Results from this study also suggest that supplementing wort with essential nutrients required for yeast growth and optimizing the fermentation conditions could be effective in controlling aroma-active ester concentrations to a desired level in beer. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.