Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 137-153).

This study presents the spatial and temporal variability of Salmonella enterica in urban and rural streams in a model watershed (Grand River watershed, Ontario, Canada), and examines the antimicrobial resistance (AMR) and genetic diversity of various serotypes. Using a swab collection method and various media types, Salmonella were detected in 78.4% of samples between November 2003 and July 2005. A diverse range of Salmonella serotypes (n=38) were isolated from water. Predominant serotypes and phagetypes (PT), including S. Typhimurium PT 104 and S. Heidelberg PT 19, and the proportion of isolates demonstrating AMR (33%), was similar to those for humans and farm animals locally and across Canada, a trend not commonly reported. There was a greater diversity of serotypes and AMR profiles in isolates from the urban stream compared to the rural/agricultural streams. Plasmid-borne resistance was observed in 28.6% of AMR isolates, with two different plasmids responsible for resistance; the TEM-1 plasmid (8.1Kb plasmids carrying blaTEM-1, responsible for ampicillin resistance) and CMY-2 plasmid (95.5Kb plasmids carrying blaCMY-2, responsible for 3rd generation cephalosporin resistance). CMY-2 plasmids were only found in the urban stream and did not create a biological burden under non-selective conditions, indicating the long-term permanence of these plasmids. Seasonal differences in the overall diversity of serotypes and predominance of serotypes of human health significance (S. Typhimurium and S. Heidelberg) were observed. The lower occurrence of S. Typhimurium and S. Heidelberg in February and March was not the result of lower survival of these serotypes at low temperatures. Peaks in occurrence of S. Typhimurium and S. Heidelberg in the summer and spring, respectively, were pronounced in the rural/agricultural streams, as opposed to the urban stream. Pulsed-field gel electrophoresis and plasmid-typing revealed diversity within multiple drug resistant S. Typhimurium PT 104 isolates, indicating genetic differences among tributaries. The ubiquitous nature of Salmonella in water and the predominance of serotypes/phagetypes of human or veterinary health significance suggest that environmental exposure through consumption or contact with contaminated water is plausible. These streams may act as a vehicle for the dissemination of these organisms and their resistance genes between different hosts or environments.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 179-191).

Viruses are the most abundant biological entities in the ocean and are believed to contribute to nutrient cycling, bacterial diversity, and horizontal gene exchange. However, little is known about the relationship between temperate phages and their hosts in marine environments. In this thesis, phage-host systems from the Gulf of Mexico were used to study the influence of temperate phages in bacteria. PhiHAP-1 is a temperate myovirus induced with mitomycin C from Halomonas aquamarina isolate. The genome of this phage was 39,245 nucleotides long and contained 46 predicted genes. Besides genes involved in lysogeny, PhiHAP-1 contained a protelomerase, which is responsible for resolution of telomeric ends in linear plasmid-like phages. Hybridization studies and PCR analysis indicated not only a lack of integration of the prophage in the host chromosome, but differences in genome arrangement between the prophage and virion forms of PhiHAP-1. These results suggest that PhiHAP-1 exists as a non-integrating linear phage with telomeric ends. Eleven pigmented Bacillus spp. isolates were examined for the occurrence of lysogeny and sporulation through induction with mitomycin C and decoyinine, respectively. The results from these experiments suggested a variety of interactions can occur between phages and their hosts, some of which may influence sporulation. The lysogenic strain B14905 had high frequency of sporulation and was selected for further analysis. The genome of B14905 contained 4 prophage-like regions, one of which was independently sequenced from an induced lysate. PCR and TEM analysis of a mitomycin C induced lysate indicated that two of these regions were inducible prophage, one was a defective phage, and one was a non-inducible phage remnant. One of the inducible prophages contained a transcriptional regulator that is hypothesized to be involved in regulation of host sporulation. The diversity of prophage and prophage-like elements found in B14905 suggest that the genetic diversity of phages in the oceans is vast. The studies of the temperate phages from H. aquamarina and Bacillus spp. isolates illustrates that integration of molecular, genomic, and function studies can be used to provide insight into the influence of prophage on host bacteria.

Lactic acid bacteria (LAB), a group of generally recognized as safe (GRAS) organisms that metabolize sugars into primarily lactic acid, have traditionally been used for the fermentation and preservation of various foods and beverages. There is increasing interest in the genetic manipulation of LAB to improve existing characteristics or introduce novel, industrially pertinent phenotypes. However, because these bacteria have food-related applications, their genetic modification requires the use of food-grade genetic engineering tools. LAB plasmids, self-replicating extrachromosomal DNA molecules, can be used to derive food-grade cloning vectors. The rationale of this research was to develop a food-grade cloning vector using a lactobacilli cryptic plasmid and to investigate its cloning and expression properties. The main objectives were to (i) screen Lactobacillus spp. for plasmids, (ii) isolate and characterize a plasmid, and (iii) use the plasmid replicon to construct a cloning vector and express heterologous genes in various hosts. This is the first step in the development of a new family of food-grade cloning vectors for the genetic modification of lactobacilli.

A hybrid vector, pANIC1, was to be constructed which was capable of regulating expression of cloned genes in both Escherichia coli and Anacystis nidulans R2 by controlling the iron content of the growth medium. Plasmid pANIC1 would have origins of replication for E. coli and A. nidulans R2, and a marker gene conferring ampicillin resistance. It would also contain the promoter for the irpA gene which is active only in low iron growth conditions.The first two stages of the construction were successfully completed, but unfortunately the final construction proved to be unstable. Recent information has shown that operator sequences upstream from the irpA gene's promoter result in an unstable message. This may be interfering with the normal functioning of the host cell, resulting in an unstable construction. In future experiments it may be neccessary to alter the growth conditions or remove the upstream sequences in order to stablize the construction. / Department of Biology

This study presents the spatial and temporal variability of Salmonella enterica in urban and rural streams in a model watershed (Grand River watershed, Ontario, Canada), and examines the antimicrobial resistance (AMR) and genetic diversity of various serotypes. Using a swab collection method and various media types, Salmonella were detected in 78.4% of samples between November 2003 and July 2005. A diverse range of Salmonella serotypes (n=38) were isolated from water. Predominant serotypes and phagetypes (PT), including S. Typhimurium PT 104 and S. Heidelberg PT 19, and the proportion of isolates demonstrating AMR (33%), was similar to those for humans and farm animals locally and across Canada, a trend not commonly reported. There was a greater diversity of serotypes and AMR profiles in isolates from the urban stream compared to the rural/agricultural streams. Plasmid-borne resistance was observed in 28.6% of AMR isolates, with two different plasmids responsible for resistance; the TEM-1 plasmid (8.1Kb plasmids carrying blaTEM-1, responsible for ampicillin resistance) and CMY-2 plasmid (95.5Kb plasmids carrying blaCMY-2, responsible for 3rd generation cephalosporin resistance). CMY-2 plasmids were only found in the urban stream and did not create a biological burden under non-selective conditions, indicating the long-term permanence of these plasmids. Seasonal differences in the overall diversity of serotypes and predominance of serotypes of human health significance (S. Typhimurium and S. Heidelberg) were observed. The lower occurrence of S. Typhimurium and S. Heidelberg in February and March was not the result of lower survival of these serotypes at low temperatures. Peaks in occurrence of S. Typhimurium and S. Heidelberg in the summer and spring, respectively, were pronounced in the rural/agricultural streams, as opposed to the urban stream. Pulsed-field gel electrophoresis and plasmid-typing revealed diversity within multiple drug resistant S. Typhimurium PT 104 isolates, indicating genetic differences among tributaries. The ubiquitous nature of Salmonella in water and the predominance of serotypes/phagetypes of human or veterinary health significance suggest that environmental exposure through consumption or contact with contaminated water is plausible. These streams may act as a vehicle for the dissemination of these organisms and their resistance genes between different hosts or environments.

DDT, an extremely widely used organochlorine pesticide, was banned in most developed countries more than 30 years ago. However, DDT residues, including 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), still persist in the environment and have been identified as priority pollutants due to their toxicity and their ability to bioaccumulate and biomagnify in the food chain. In particular, DDE was long believed to be &quotenon-biodegradable&quote, however some microorganisms have now been isolated that are able to metabolise DDE in pure culture. Terrabacter sp. strain DDE-1 was enriched from a DDT-contaminated agricultural soil from the Canterbury plains and is able to metabolise DDE to 4-chlorobenzoic acid when induced with biphenyl. The primary objective of this study was to identify the gene(s) responsible for Terrabacter sp. strain DDE-1�s ability to metabolise DDE and, in particular, to investigate the hypothesis that DDE-1 degrades DDE cometabolically via a biphenyl degradation pathway. Catabolism of biphenyl by strain DDE-1 was demonstrated, and a biphenyl degradation (bph) gene cluster containing bphDA1A2A3A4BCST genes was identified. The bphDA1A2A3A4BC genes are predicted to encode a biphenyl degradation upper pathway for the degradation of biphenyl to benzoate and cis-2-hydroxypenta-2,4-dienoate and the bphST genes are predicted to encode a two-component signal transduction system involved in regulation of biphenyl catabolism. The bph gene cluster was found to be located on a linear plasmid, designated pBPH1. A plasmid-cured strain (MJ-2) was unable to catabolise both biphenyl and DDE, supporting the hypothesis that strain DDE-1 degrades DDE cometabolically via the biphenyl degradation pathway. Furthermore, preliminary evidence from DDE overlayer agar plate assays suggested that Pseudomonas aeruginosa carrying the strain DDE-1 bphA1A2A3A4BC genes is able to catabolise DDE when grown in the presence of biphenyl. A second objective of this study was to characterise pBPH1. The complete 84,054-bp sequence of the plasmid was determined. Annotation of the DNA sequence data revealed seventy-six ORFs predicted to encode proteins, four pseudogenes, and ten gene fragments. Putative functions were assigned to forty-two of the ORF and pseudogenes. Besides biphenyl catabolism, the major functional classes of the predicted proteins were transposition, regulation, heavy metal transport/resistance, and plasmid maintenance and replication. It was shown that pBPH1 has the terminal structural features of an actinomycete invertron, including terminal proteins and terminal inverted repeats (TIRs). This is the first report detailing the nucleotide sequence and characterisation of a (linear) plasmid from the genus Terrabacter.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [139]-158).