Biofiltration has been shown to be an effective method for the remediation of volatile organic compounds (VOC's), particularly petroleum vapors extracted from the vadose zone. Many bacteria have the enzymatic pathways necessary for aerobic mineralization of VOC's to form cell biomass, carbon dioxide and water. Molecular methods such as nucleic acid hybridizations and the polymerase chain reaction (PCR), are methods that can be applied to environmental samples to characterize bacterial community structure and function. The research presented here reports the use of a field-scale biofilter for the remediation of unleaded gasoline vapors extracted from the vadose zone. An evaluation of contaminant removal efficiency over a five month period showed that the biofilter removed 90% of total petroleum hydrocarbons and greater than 90% of the EPA priority pollutants benzene, toluene, ethylbenzene, and xylene. The bacterial consortium in the biofilter medium readily adapted to increased loading rates, and variations in temperature and moisture. A combination of conventional cultural and molecular methods was used to track the bacterial populations over the course of the experiment. Polymerase chain reaction amplification of the small ribosomal subunit DNA sequence was used for identification of bacterial isolates and the design of DNA hybridization probes. Hybridization of these probes to community DNA samples taken from the biofilter over time revealed changes in specific bacterial populations as bioremediation occurred. Specifically, bacteria that could use gasoline, toluene, ethylbenzene or xylene were prevalent throughout the biofilter. Bacterial populations that could degrade xylene gradually increased over time, while overall total population size was the similar in the background sample and at the end of the study.

Sixty-three of 435 (14.5%) samples collected from broiler chickens were positive for C. jejuni. Twenty-two of 55 samples were from organic chickens (40%) and 41 of 380 samples were from conventional chickens (10.8%). Isolates were subjected to macrorestriction profiling using SmaI and analyzed for their ability to survive in macrophage cells and invade in epithelial cells. Antibiotic testing to cefaclor, ciprofloxacin, tetracycline, erythromycin, gentamicin, trimethroprim/sulfamethoxazole, and ampicillin were performed. Finally, 5 isolates of varying putative in vitro virulence traits were chosen for experimental inoculation of newborn piglets. Five piglets per isolate were tested and examined macroscopically and microscopically upon necropsy. Genotyping of isolates indicated 1 to 3 profiles per flock. Of the 22 organic isolates from chickens, only 3 (13.6%) were able to survive within macrophages. For the conventional isolates, 21 out of 41 (51.2%) were able to survive. However, the majority of isolates (90.5%) from both organic and conventional isolates were not capable of invading epithelial cells. No isolates exhibited resistance to ciprofloxacin or gentamicin. One isolate out of 63 (1.6%) was resistant to erythromycin, 52 (82.5%) to tetracycline, 28 (44.4%) to trimethroprim/sulfamethoxazole, and 6 (9.5%) to cefaclor. In terms of the piglet studies, regardless of the combination of in vitro invasion or survival results or type of flock, most piglets (16/25) in all groups exhibited hyperemia, edema, and hemorrhage in the small intestine or colon upon gross examination. Microscopic examination revealed congested mucosa and erosion of the epithelium in 10 of the 25 piglets from 4 of the 5 groups. In conclusion, this study suggests that C. jejuni isolated from broiler chickens are virulent in piglets and are probably capable of causing disease in humans. Furthermore, the results of the survival and invasion assays did not correlate with the results of the piglet studies and cannot be relied upon to predict degree of virulence. Therefore, another virulence factor is responsible for the pathogenesis, such as a toxin(s). As this is the first study to confirm putative in vitro virulence traits with an animal model, further research is recommended with the piglet model to assess pathogenicity.

Wetlands containing aquatic plants have been found to economically provide a mechanism for improving the microbial and other quality of wastewater. The purpose of this study was to elucidate the effect of vegetation and sedimentation on the survival of enteric microorganisms in constructed wetlands. The first part of this study was designed to determine the effect of vegetation on the survival of Escherichia coli, Salmonella typhimurium , bacteriophage MS-2 and poliovirus in wetlands. The organisms were added to the water from six wetland systems, containing different aquatic plants. The wetland systems received either fresh water or secondary sewage. The presence of aquatic plants significantly increased the die-off of E. coli, S. typhimurium, bacteriophage MS-2, and poliovirus in fresh water and secondary sewage. Additional work on the survival of E. coli in non-sterile, filter sterilized, and autoclaved wetland water indicated that one of the plausible mechanisms of bacterial die-off in constructed wetlands is through increased microbial competition or predation. The next phase of this study investigated the survival of indicator microorganisms in wetlands similar to field conditions. E. coli, bacteriophage MS-2, and PRD-1 were added to tanks which were unvegetated or contained different aquatic plants. E. coli die-off in unvegetated tanks was greater than the vegetated tanks. Temperature was found to be significantly correlated with the die-off of E. coli. Inactivation of bacteriophage MS-2 in unvegetated tanks was also higher than the vegetated tanks. In contrast, PRD-1 inactivation was greater in the vegetated tanks compared to the unvegetated tanks. Thus, a combination of unvegetated and vegetated wetland would probably result in the greatest reduction of microorganisms by the wetlands. The last phase of this study was to examine the relative occurrence and survival of indicator microorganisms and pathogens in the water column and sediments of two constructed surface flow wetlands. On a volume/wet weight basis the concentration of fecal coliforms and coliphage in the water column and sediment was similar. Giardia and Cryptosporidium concentration in the sediment were one to three logs higher in the sediment compared to the water column. The die-off rates of all the organisms were greater in the water, except for Giardia muris. Giardia die-off in the sediment was greater than in the water column. These results suggest that sedimentation may be the primary removal mechanism for the larger organisms in surface flow wetlands. Overall, these studies suggest that vegetation, microbial competition or predation, temperature/sunlight, and sedimentation play important roles in microbial reduction in constructed wetlands. The effect of vegetation on microbial survival appeared to be indirect, through increasing microbial competition. However, vegetation in constructed wetlands may offset the effect of temperature and sunlight, resulting in a longer survival of microorganisms. Thus a combination of non-vegetated and vegetated wetland would probably result in the greatest reduction of microorganisms from wetlands. Future experiments are needed to examine such combined wetlands.

The control of gene expression is greatly influenced by regulatory events at post-transcriptional steps. In my studies of these post-transcriptional control steps, I have utilized the tools provided by the model organism Saccharomyces cerevisiae, including genetic and molecular tools as well as the complete genome sequence. Based on genetic evidence, I hypothesized that the products of the SKI2, SKI3, and SKI8 genes were involved in a pathway of mRNA degradation that acts in the 3' to 5' direction. I demonstrated that mutations in any of the three genes lead to a stabilization of an mRNA species that degrades 3' to 5'. I further demonstrated that components of a protein complex, the exosome, were also required for 3' to 5 ' mRNA degradation. I went on to demonstrate that mutations that disrupt 3' to 5' mRNA degradation are synthetically lethal with mutations that disrupt another pathway that operates in the 5' to 3' direction. This last observation leads to two conclusions. First, these two mechanisms are likely to be the only major methods of mRNA degradation in Saccharomyces cerevisiae. Second, mRNA degradation is an essential process in this organism. I developed a computational method that uses statistical analysis of oligonucleotide frequencies to identify potential cis-acting elements. Application of my method to a group of genes allows the identification of sequences that may be involved in directing co-regulation. Unlike similar methods, my method accounts for oligonucleotide usage in the genes that are not observed to be co-regulated, ensuring that elements common to all genes will not be erroneously detected. After demonstrating that the method detected several known splicing elements in a group of genes containing introns, I went on to characterize the performance of the method under several conditions designed to simulate 'real world' experiments. Finally, I utilized the method to identify an element in a group of nuclear genes that encode mitochondrial proteins.

Protein-protein interactions participate in the assembly, regulation, and processivity of all molecular "machines." In this dissertation, a series of genetic and biochemical approaches have been used to analyze the protein-protein interactions which participate in the formation of the Herpes Simplex Virus Type 1 replisome. The emphasis of these studies lies on the herpes helicase-primase complex, its assembly, regulation, and processivity. The herpes helicase-primase is a heterotrimeric complex encoded by the viral UL5, UL52, and UL8 genes. The yeast two-hybrid system was used to generate a protein linkage map of the herpes replisome and to characterize the interactions among the three subunits of the helicase-primase (UL5, UL52, and UL8). Deletion analysis and co-immunoprecipitation, were used to show that a 548 amino acid carboxy-terminal fragment of UL52 interacts with UL8, while a 350 amino acid N-terminal fragment is required for interaction with UL5 and may also be involved in the regulation of the strength of interaction with UL8. Comparative sequence analysis suggested that the functional interactions among the subunits of the helicase-primase encoded by alpha-herpesviruses may differ from those of the helicase-primases encoded by the beta- and gamma-herpesviruses. Expression and purification of the UL5 subunit of the helicase-primase in the absence of UL52 resulted in an inactivating but reversible catalytic deficiency in UL5. UL52 corrected this deficiency when added subsequently. Based on these results, a proposal is suggested here that UL52 regulates the activity of UL5 by inducing conversion of UL5 from an inactive to an active conformation, and/or by contributing amino acid residues to the catalytic site. Finally, evidence from a combination of biochemical approaches suggested that the HSV-1 helicaseprimase is a monomeric ATPase with a non-globular shape and that it belongs to the group of helicases which use an inchworming mechanism for unwinding DNA.

The Atacama Desert of northern Chile is one of the most arid regions on Earth. The central plateau, between the coastal escarpment and the Andes, is devoid of vegetation and receives only millimeters of rain every few years. Though plants are absent in the soils of this desert, perhaps bacteria can survive, and even thrive, in these hyperarid conditions. This dissertation represents the first comprehensive study of bacterial diversity in the driest central latitudes (approximately 24°S) of the Atacama Desert. Study 1 covers the development of a soil DNA extraction method for the study of soil bacterial populations. This method was field tested in an ecology study in the Santa Catalina Mountains of southern Arizona. In Study 2, Atacama soils were sampled in two transects at approximately 24°S and 25°S. The first transect runs across the absolute (plantless) desert and through several narrow bands of sparse vegetation at high altitudes in the Andes. The second transect is within the well-developed fog zone near Paposo on the Pacific coastal escarpment, where an endemic plant community called lomas is established. Analysis of DGGE profiles of bacterial !6S rRNA genes extracted from these soils with Kruskal's Isotonic Multidimensional Scaling indicates that the bacterial populations cluster into several groups, including the low diversity populations of the core absolute desert, and the higher diversity high elevation Andean populations influenced by the vegetation of Andean biomes. Only one group clustered in the lomas; the rest of the profiles were unique, demonstrating the high diversity of bacterial populations within this diverse vegetation community. Soil 3107, which is within the absolute desert, clustered with the Andean bacterial populations. This soil lies within the transition zone between the low precipitation of the absolute desert (approximately 2.4 mm per year) and the higher precipitation of the high elevation Andes (approximately 47.1 mm per year). This Andean bacterial population may extend further into the absolute desert than the Andean vascular plants due to superior aridity tolerance. Alternatively, this bacterial population may be a relic from when the Andean vegetation advanced through this elevation in a wet period 3000 years ago.

Current thinking is that co-contaminated sites (i.e., sites with both organic and metallic pollutants) are difficult to bioremediate because the metal toxicity is such that organic degradation is inhibited. The objective of this research was to evaluate the potential of bioaugmentation with metal-detoxifying microbial populations as a viable remediative approach for such sites. Divided into three sections, this research found that metal-detoxifying microorganisms could facilitate the remediation of co-contaminated systems. The objective of the first study was to compare the microbial community response to cadmium exposure between metal-contaminated and uncontaminated soils. This study found that while cadmium adversely affected the numbers of culturable microorganisms in all soils, cadmium-resistant isolates were found in each soil, regardless of prior metal exposure. However, the metal-contaminated soil microbial communities were more resistant than the uncontaminated soil community. In one metal-stressed soil, resistance increased with increasing cadmium stress. A cadmium-resistant Pseudomonas spp. was found to increase in numbers with increasing cadmium, suggesting a different mechanism of cadmium resistance at high cadmium concentrations. The second study evaluated the diversity of cadmium-resistance/detoxification mechanisms in six cadmium-resistant isolates found in the first study. Genetic and microscopic analyses found several different approaches to cadmium resistance. Two mechanisms known to confer resistance were observed, including exopolymer and biosurfactant production. Two other isolates demonstrated intracellular cadmium accumulation via as yet unknown mechanisms. The mechanism of resistance for one isolate could not be identified. Four out of the six isolates detoxified cadmium as part of their resistance. Since metal detoxification is necessary to allow for organic degradation, these four isolates were included in 2,4-D degradation studies under co-contaminated conditions. The last study examined the use of cadmium-detoxifying microorganisms to enhance organic degradation under co-contaminated conditions. In pure culture and laboratory soil microcosms with cadmium and 2,4-dichlorophenoxyacetic acid (2,4-D) as model contaminants, four cadmium-detoxifying isolates supported the degradation of 2,4-D by the cadmium-sensitive 2,4-D degrader Alcaligenes eutrophus JMP134 in the presence of toxic levels of cadmium. Ina pilot field study, a cadmium-detoxifying Pseudomonas isolate enhanced 2,4-D degradation by A. eutrophus JMP 13 4 in the presence of cadmium.

In order to test hypotheses of the selective advantage of sex, I have investigated three subjects: the timing of a sexual process, natural genetic transformation, in the bacterium Bacillus subtilis, the relationships between sex, dispersal and dormancy in a variety of organisms, and the evolutionary history of natural genetic transformation, in all living things. My investigation of B. subtilis concerns the relationship between the spore state and competence, the ability to undergo genetic transformation. I show that competence and spore-formation are alternative processes. This is unusual, since in most microorganisms, sex and sporulation are associated. The tradeoff between sex and the spore state found in B. subtilis contradicts ecological hypotheses for sex. These hypotheses predict that, when sex is facultative, it should be associated with the spore state, because that state is more dormant and more dispersible. I discovered that other microorganisms also violate these predictions: sex is either unassociated with dormancy, or unassociated with dispersal, or both. However, in most facultatively sexual organisms, sex, dormancy and dispersal are still associated. Two popular hypotheses for the selective advantage of sex make the wrong prediction for the usual dormancy-dispersal-sex relationship. Here I deduce that the red queen hypothesis and the sib-competition hypothesis incorrectly predict that sex should usually not be associated with dormancy and dispersal. Other hypotheses that I analyze make the correct prediction. Some of these hypotheses of sex make predictions about the history of (bacterial) sex, such as that competence should be ubiquitous. To test these predictions, I have reconstructed the evolutionary history of natural competence. My results show that competence is taxonomically widespread phylogenetically primitive, easy-to-lose, evolutionarily variable, and negatively correlated with certain habitats. These results confirm that competence is ubiquitous. However, the most notable result is that competence is primitive: the most parsimonious evolutionary hypothesis is that the universal ancestor of life was competent.

The use of DNA typing and fingerprinting methods to identify and discriminate strains of bacteria, including Salmonella, has increased dramatically in recent years. Traditional typing methods, including serotyping and phage typing, have often not adequately discriminated strains, nor have they always identified virulent or antibiotic resistant strains. In a literature review, DNA-based methods, including plasmid analysis, restriction fragment length polymorphism (RFLP) analysis, and polymerase chain reaction (PCR) fingerprinting methods were evaluated. Plasmid analysis, including plasmid profiles and plasmid fingerprints have been shown to be useful primarily in short-term investigations of disease outbreak. However, plasmid profiles or possession of individual plasmids have generally not been good indicators of cell phenotypes overall. RFLP fingerprinting of Salmonella utilizing probes from ribosomal DNA, insertion sequence IS200, or random sequences has been reported. Ribotypes detected with ribosomal probes have generally been shared among different serotypes, whereas IS200 profiles have tended to be more serotype-specific. AP PCR and rep-PCR primers have been shown to discriminate Salmonella isolates, but fingerprints have been more difficult to reproduce and interpret than RFLP fingerprints. Several authors have reported bands of varying intensities, and some faint bands have not been reproducible. Improved methods of resolving and detecting PCR products are necessary. In a laboratory study, 85 environmental Salmonella isolates belonging to 22 serotypes were fingerprinted by 16S RFLP ribotyping, by rep-PCR, using ERIC (enterobacterial repetitive intergenic consensus) primers, and by AP PCR. Ribotypes were shared by isolates from different serotypes. ERIC PCR and one AP PCR primer produced fingerprints that discriminated among the different isolates, but did not identify serotypes. Another AP PCR primer produced simple patterns that neither discriminated isolates, nor identified serotypes. In a second related laboratory study, computer-assisted matching of AP PCR fingerprints of several known isolates was evaluated. Aliquots of the PCR reaction were run in the same and different gels, and the fingerprints bands were scored by two technicians on a presence-absence basis, and matched by creating dendrograms. Although replicate fingerprints of an isolate appeared reproducible, they were not always scored identically. Thus, the computer was not always able to correctly match fingerprints.

Organic matter amendments have long been known to improve native organic matter content, aggregation and structure of soils. In the laboratory, however, organic matter amendments to autoclaved soils have no such effect. This may explain the failure of many reclamation attempts on mine tailing wastes, which often proceed without regard for the microbiological processes necessary for soil formation and cycling of plant nutrients. In this study, incubation of tailing waste with soil microbes and a simple carbon source proved sufficient to increase the formation of water stable aggregates from tailing particles. Autoclaved control samples showed no change in aggregation. The incorporation of microbial cell mass into the mineral matrix of the tailing was observed using scanning electron microscopy. These results suggest that microbial activity is necessary in order to incorporate organic matter into the abiotic matrix of tailing, promoting aggregation and ultimately soil formation from this material.