Trichloroethene is degraded by ammonia-oxidizing bacteria. In the absence of ammonia, TCE transformation proceeds at moderate rates. Under the experimental conditions tested, the presence of PCE and cis-DCE, competitive inhibitors of TCE, slowed the rate and extent of TCE transformation. The capacity of these microorganisms to degrade TCE in the absence or presence of mixtures is diminished by the inactivation of the cells as a consequence of the transformation of the chlorinated compounds. The potential application of ammonia oxidizers as a stand-alone in situ treatment process for TCE degradation is diminished by the inactivation of the cells and the inhibitory effects that mixtures of TCE and other chlorinated compounds pose to the microorganisms.

In Clostridium acetobutylicum, central-fermentation-pathway enzymes involved in butyryl-CoA synthesis play key roles in acid and solvent production, whereas the acetate production is an important element during acidogenesis and generates ATP. Genes encoding enzymes involved in acetate- and butyryl-CoA-synthesis were cloned, sequenced and expressed. CoA and its derivatives, which are intermediate metabolites for these pathways, were isolated and analyzed to identify regulatory factors for the enzymes involved in such fermentation. Five genes: crt, bcd, etfB, etfA and hbd, which encode the central clostridial pathway enzymes crotonase, butyryl-CoA dehydrogenase (BCD), putative electron-transfer flavoprotein (ETF) $\beta$- and $\alpha$-subunits, and $\beta$-hydroxybutyryl-CoA dehydrogenase, respectively, were cloned and shown to be clustered in the chromosome. These genes are co-transcribed and form an operon, suggesting that BCD in clostridia might interact with ETFs in its redox function. Cloning and sequencing studies reveal that pta and ack, encoding acetate-production-pathway enzymes phosphotransacetylase (PTA) and acetate kinase (AK), respectively, are adjacent in the chromosome. Primer extension analysis suggests an operon arrangement for these tandem genes. Overexpression of ack and pta in C. acetobutylicum shows that the final ratios of acetate to other major products were higher and also results in a greater proportion of two- versus four-carbon-derived products. Formation of a mutant strain by inactivation of the chromosomal pta gene decreased acetate formation. The PTA and AK activities of such a mutant were correspondingly reduced. Intracellular levels of CoA and its derivatives involved in the metabolic pathways were analyzed using reverse-phase high performance liquid chromatography. During the acidogenic-to-solventogenic shift, butyryl-CoA concentration increased rapidly, whereas those for free CoA and acetyl-CoA decreased. These observations were accompanied by a rapid increase of solvent-pathway enzyme activity and a decrease of acid-pathway enzyme activity. Levels of acetoacetyl-CoA, $\beta$-hydroxy-butyryl-CoA and crotonyl-CoA in crude cell extracts were below detectability. The possible roles of CoA and its derivatives in regulating specific enzyme activity were evaluated.

Shutoff of host biosynthesis is one of the earliest and most dramatic events occurring in viral infection and requires the expression of viral early genes. To understand the mechanisms of bacteriophage SPO1 induced host-shutoff, two SPO1 early genes, e3 and e22, were cloned and sequenced, and the roles of e3 in host-shutoff and in phage growth were studied. Both e3 and e22 are novel genes, and are actively expressed during the first few minutes of infection before being promptly shut off. Expression of a plasmid-borne e3 gene, in either B. subtilis or E. coli, caused the inhibition of host DNA, RNA and protein synthesis, and ultimately led to cell death. To identify the primary target of e3-induced shutoff, an e3-resistant E. coli mutant was isolated and analyzed. Plasmid libraries of this mutant's genomic DNA, when screened for genes that could protect wild-type E. coli against e3, yielded the rpoB and dksA genes, which specify the RNA polymerase $\beta$ subunit and a suppressor for DnaK, respectively. The wild-type dksA gene, but not the wild-type rpoB gene, was able to protect against e3, suggesting that the primary e3-resistant mutation was in the rpoB gene and that protection by the dksA gene depended upon overexpression from the plasmid. I suggest that e3 acts by distorting the structure of the host RNA polymerase, thus preventing host transcription, and that this distortion can be prevented or reversed by a chaperonin-like activity specified by dksA. The host-shutoff still occurred normally during infection by an SPO1 mutant which lacked e3 activity, and it occurred much more rapidly than that caused by expression of e3 in uninfected cells. Thus, the e3 product must be only one component of the host-shutoff machinery, which must include elements whose function is redundant to that of e3. At high multiplicities of infection, the mutant SPO1 produced more phage progeny than the wild-type SPO1, suggesting that high concentrations of e3 can be inhibitory to phage growth as well as to host function. Perhaps for that reason, expression of both e3 and e22 is shut off after a brief period of high activity.

A detailed mechanistic model, with a large number of metabolites and parameters, describing growth of a single cell of the bacterium Escherichia coli is simulated and the resulting predictions are compared with available reported experimental observations. The effects of small time-invariant perturbations in each of the parameters on final model predictions of metabolite concentrations and doubling times are systematically investigated using the theory of sensitivity analysis. This is quantified by defining (linear) sensitivity coefficients $\underline{\Lambda},$ and are subsequently used to identify sensitive parameters in the model description. Growth rate effects due to parametric perturbations are studied by comparing the total phase shift obtained with that for a nominal system description. The prevailing behavior of kinetic and polymerization rate constants compared to that of saturation constants is established. Results obtained are successfully compared with available experimental data. Implications to Metabolic Control Analysis are also discussed.

A literature search and laboratory treatability study were conducted to determine the potential of commercial surfactants to enhance the bioremediation of PAH-contaminated sediments. Phenanthrene was used as a model PAH and an inoculum of PAH-degrading microorganisms enriched from an aquatic sediment was used in sediment-water slurry microcosm biodegradation experiments. The abilities of nonionic surfactants to reduce surface tension and enhance phenanthrene solubility, the effect of sediment on the physical behavior of surfactants, and the effects of surfactants on phenanthrene biodegradation were examined. Of the surfactants tested, all except Triton X-100 severely inhibited phenanthrene mineralization. Adding Triton X-100 to microcosms with or without sediment significantly enhanced the extent of phenanthrene mineralization after 1 week. These experimental results suggest that this surfactant may be useful in the bioremediation of PAH-contaminated sediments and that further studies using pilot-scale systems or a field demonstration are warranted.

Adenoviral vectors have great potential for use in gene therapy and genetic immunization. The targeting of Ad vectors to the relevant tissue and cell types in vivo could greatly improve their safety and performance by lowering the effective dosage required for therapeutic levels of gene expression. Redirection of Ad vector tropism will require physical modifications of the adenoviral capsid but direct genetic modification of the Ad capsid has so far been limited to small peptides. A novel system for the attachment of targeting ligands to the Ad capsid, based on the extremely strong avidin-biotin interaction, is described herein. The genetic insertion of a biotin acceptor peptide (BAP) into the fiber, protein IX, or hexon components of the Ad capsid has resulted in vectors that are metabolically biotinylated upon production in host cells. Avidin-dependent redirection of transduction through a variety of biotinylated ligands is greatly dependent on the nature of the biotinylated capsid protein. While targeted transduction via the fiber was efficient through a broad array of ligand-receptor interactions, redirection of binding and uptake through the more abundant protein IX and hexon resulted in poor transduction. Although the basis of these differences has not been determined, it most likely reflects functional differences between the capsomeres during the process of vector uptake and trafficking. This study represents the first direct comparison of transduction through the various capsomeres and strongly suggests that future targeting efforts should be focused on fiber modification. In addition to the functional studies on Ad-IX-BAP, structural analysis by cryoelectron microscopy and particle reconstruction is presented. The C-terminal BAP fusion was used as a structural tag to visualize the position of IX within the capsid. Results contradict all previous reports on the location of IX and suggest the surface accessible density currently assigned as IIIa is actually attributable to protein IX. These studies highlight the need for a more thorough analysis of adenoviral structure and the complex interactions between its components.

The outer membranes of Gram-negative bacteria are highly asymmetric, with the inner leaflet composed of phospholipids and the outer leaflet mostly of lipopolysaccharides (LPS). The outer membrane is thought to act as a protective and permeability barrier, but is somehow permeable to various antimicrobial peptides. To study this problem we prepared oriented multilamellae of bilayers composed of pure LPS. X-ray diffraction showed that the samples produced good spectrum. The liquid crystalline to gel transition was observed by the appearance of a sharp peak corresponding to 4.23A and coexistence of two lamellar diffraction series. Phase diagrams of mutant LPS's were constructed as functions of temperature and the level of hydration. Electron density profiles were constructed, and compared with the program calculated model electron profiles, the peak position in the electron density profile was determined as the position of the phosphate atom in LPS molecule. Inorganic ions like Sodium(Na +), Potassium(K+), and Barium(Ba++) were added into the sample. Results showed that with the presence of the ions, the phase transition temperature of LPS mutant decreased, and the ions bound to the site where the phosphate atom located. Also, beta-sheet peptide protegrin-1 was added in the LPS sample. Results implied the peptide inserted in the headgroup of the LPS bilayer.

This thesis focuses on the heterologous expression of alcohol acetyltransferase (AATase) genes in Escherichia coli and Clostridium acetobutylicum for the production of the esters isoamyl acetate, butyl acetate and butyl butyrate. Isoamyl acetate, butyl acetate and butyl butyrate are esters that confer a fruity aroma and taste to the materials in which they are found. AATases are a class of enzymes that have been found to enzymatically catalyze the reaction between alcohols and acyl-CoAs to produce the corresponding ester. Butanol, acetyl-CoA, and butyryl-CoA, the substrates necessary for butyl acetate and butyl butyrate production, are produced in high concentrations by C. acetobutylicum, making it an ideal host for the expression of genes for AATases. Previous studies have characterized AATases in yeast, and expressed them in E. coli, but this is the first report of ester production by AATase activity in C. acetobutylicum . The genes ATF1 and ATF2, encoding AATase I and AATase II from the yeast Saccharomyces cerevisiae, and SAAT encoding a strawberry alcohol acetyltransferase, Saatp, have previously been sequenced. Biochemical studies have shown them to possess AATase activity. For this thesis, ATF1, ATF2, and SAAT were subcloned and expressed in E. coli. Ester production was determined by head-space gas analysis on a gas chromatagraph. AATase I in E. coli cultures and cell-free extracts produced more ester than AATase II did with each alcohol substrate that was investigated. Saatp produced less ester than AATase I, but more than AATase II. The ester butyl butyrate was detected in cell-free extracts of E. coli expressing SAAT, but not in cell-free extracts expressing ATF1 or ATF2 . ATF2 was also expressed in C. acetobutylicum for the production of isoamyl acetate and butyl acetate. Ester concentrations in C. acetobutylicum strains expressing ATF2 were lower than ester concentrations in E. coli strains expressing ATF2. The expression of ATF2 in C. acetobutylicum demonstrates the viability of using this organism for natural ester production via microbial fermentation.

Acetoin is a product of fermentation by C. acetobutylicum, and a component of several natural and artificial flavors, including butter flavor. Acetolactate synthase (ALS) catalyzes the production of acetolactate from pyruvate. Acetolactate is converted to acetoin enzymatically or chemically. A putative catabolic acetolactate synthase was cloned from C. acetobutylicum . The gene is 1692 nucleotides long. His-tagged ALS protein was purified from E. coli. In vivo experiments in E. coli and C. acetobutylicum and in vitro experiments failed to detect activity for the acetolactate synthase, though it probably is involved in Clostridial acetoin production. Introduction of a B. subtilis ALS under control of its own promoter into C. acetobutylicum cells was also found to have no effect on solvent production. Acetoin reductase (AR) catalyzes the production of 2,3-butanediol from acetoin. 2,3-butanediol is a compound of commercial interest for use in making polyurethane foams, moistening agents, carriers for pharmaceuticals, and liquid fuel, among other products. The K. pneumoniae CG21 acetoin reductase gene was cloned into a Clostridial/E. coli shuffle vector and expressed in both hosts. The nucleotide sequence of the gene is 768 bp long. SDS-PAGE analysis of expressed protein indicates a molecular weight of 31,000 Da. Activity of the K. pneumoniae acetoin reductase gene in C. acetobutylicum, did not result in 2,3-butanediol synthesis during fermentation, as determined by analysis of culture supernatants. However, addition of racemic acetoin to Clostridial cells expressing the ar , yielded small amounts of 2,3-butanediol. The stereoisomer of acetoin produced by C. acetobutylicum may be incompatible with the acetoin reductase or enzyme activity in cells may be too low. A B. subtilis oxidoreductase gene, ywrO, was cloned and analyzed as a putative acetoin reductase. In vitro and in vivo experiments conducted in E. coli suggest it is not an acetoin reductase. E. coli expressing the ar of K. pneumoniae grown in the presence of racemic acetoin make 2,3-butanediol. Expression of the K. pneumoniae als and ar from a Clostridial promoter was examined, but did not promote 2,3-butanediol production in E. coli and C. acetobutylicum.

We have studied spontaneous mutations in a chromosomally-located, single copy HSV-1 thymidine kinase (TK) gene in a human AK 143 TK$ sp-$ cell line. We have used three anti HSV-1 thymidine kinase nucleotide analogues, namely Trifluorothymidine (TFT), Acyclovir (ACV) and DHPG (9-(1,3-dihydroxy-2-propoxy-methyl)guanine), to select for $TK sp-$ mutants. The spontaneous mutation rate for the TK gene was very high in this system. The mutation frequency was 5 $ times$ 10$ sp{-3}$ per cell per generation when Acyclovir (ACV) was used as a selective agent and 5 $ times$ 10$ sp{-4}$ per cell per generation when Trifluorothymidine (TFT) was used. The spontaneous mutation frequency dropped to 1 $ times$ 10$ sp{-5}$ per cell per generation when a combination of TFT plus ACV was used and to 1 $ times$ 10$ sp{-6}$ per cell per generation when a combination of TFT plus DHPG (9-(1,3-dihydroxy-2-propoxy-methyl)guanine) was used as selective agents.