The halophilic bacterium Vibrio parahaemolyticus is widely distributed as a natural inhabitant of marine and estuarine environments. Some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Studies were undertaken to differentiate clinical (virulent) and environmental (mainly avirulent) forms of Vibrio parahaemolyticus. Initially, identification and confirmation of a total of 55 V. parahaemolyticus isolates (23 clinical and 32 environmental) was carried out by using selective media, biochemical and nutritional tests. Identity was confirmed by specific polymerase chain reaction (PCR) targeting the toxR gene. In an attempt to differentiate between virulent and avirulent forms V. parahaemolyticus, potential virulence factors (enzyme activities), presence of plasmids and analyses of whole cell protein profiles and extracellular products (ECPs) by SDS-PAGE were performed. The results suggested that the presence of plasmids in isolates was not linked to virulence and SDS-PAGE profiles did not differentiate between virulent and avirulent forms, but a combination of enzyme activities may contribute to virulence. The ECPs of all 55 V. parahaemolyticus isolates were tested for their cytotoxicity towards two types of cell lines, the clinical isolates showed that 21 out of 23 (91%) and 2 out of 23 (8.69%) showed high and medium cytotoxicity, respectively. Amongst the environmental isolates 2 out of 32 (6.25%), 2 out of 32 (6.25%) and 28 out of 32 (87.5%) showed high, medium and low cytotoxicity, respectively. Randomly amplified polymorphic DNA (RAPD)-PCR was used to analyse the two groups of isolates. Firstly a 600 bp band was recognised in mainly clinical isolates. This DNA fragment was cloned and sequenced and found to code for an outer membrane protein (OMP). Two PCR primers were designed to specifically amplify a 200bp unique sequence from presumptive virulent strains (PCR-OMP); however, not all clinical isolates were positive (21 out of 23, 91%). A second RAPD-PCR identified a further unique band of approximately 310 bp in mostly clinical isolates. After cloning this band’s DNA, the DNA sequence revealed a hypothetical gene, htp, whose function is not known. Specific primers, VPHTP1 and VPHTP2 were developed for the detection of the htp sequence, but again not all clinical isolates were positive (19 out of 23, 82.6%). This led to the development of a multiplex M-PCR which detects all isolates of V. parahaemolyticus and differentiates them into potentially virulent and avirulent forms. The M-PCR works by targeting the toxR gene, and sequences for omp and htp. The M-PCR was performed on all V. parahaemolyticus isolates used in this study, as well as other Vibrio species and a selection of non-Vibrio species. The amplification of toxR gene 367 bp fragment was found in all V. parahaemolyticus tested; all clinical isolates (100%) showed amplification of omp and/or htp. This multiplex PCR detected 3 (9.37%) environment isolates, which may potentially be able to cause disease. No amplification was seen for the other species tested. Thus, the M-PCR could be used for identifying V. parahaemolyticus and detecting / differentiating potentially virulent and avirulent forms. This method should be

The nuclease gene of Vibrio vulnificus, vvn, is 696 bp long encoding a protein¡]Vvn¡^of 232 amino acids. Vvn is a periplasmic protein and is active in the oxidized form. DNA shuffling is a powerful method for in vitro mutational mechanism by homologous recombination with a low level of point mutation . DNA shuffling consists of four steps¡G¡]1¡^preparation of genes to be shuffled, ¡]2¡^random fragmentation with DNase I, ¡]3¡^fragment reassembly by primerless polymerase chain reaction¡]PCR¡^, and¡]4¡^amplification of reassembled products by a conventional PCR. The advantage of this process is that it can be used to rapidly evolve any protein, without any knowledge of its structure. The goal of this work was using DNA shuffling to generate a diversity of mutation in vvn within a short time. Followed by analyzing the DNase activity of periplasmic protein or in vivo, the mutants were divided into three groups for increase, decrease or no change in DNase activity. Randomly DNA sequencing vvn gene of fourteen transformed clones from the three groups showed only one clone has one base change with comparison to wild-type sequence. The mutation is at amino acid 22 of the N-terminus of Vvn, the change is from serine to isoleucine. The relative activity of mutant Vvn was 82 % in DNase and 59 % in RNase. The effect of a single amino acid change on the DNase and RNase activity of Vvn is different. It supports the postulation that there are two distinct but overlapping active sites exist in Vvn.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 118-142).

Vibrio cholerae, the causative agent of cholera, has been a lethal enteric pathogen to humans for most of recorded history. Even though it is well studied, it still kills many people every year due to rapid and severe dehydrations from diarrhea. Part of what makes V. cholerae such an effective pathogen is its ability to control virulence factors depending on its environment. ToxR is a major virulence protein that has upstream control of most of the virulence genes that are turned on when in a human host. Two of the most critical virulence factors, toxin coregulated pilus and cholera toxin are controlled by ToxR. CsrA is a protein that regulates many cellular functions in V. cholerae, including glycogen synthesis, motility, and biofilm production. Preliminary data suggests a link between CsrA and the regulation of ToxR. In order to study CsrA as it relates to ToxR regulation, a csrA mutant must be generated in V. cholerae. CsrA plays such an important role in glycogen metabolism that a csrA mutant is not viable due to excessive glycogen levels. In order to make a viable csrA mutant, glycogen synthesis has to be turned off. In this research, I attempt to make a viable V. cholerae csrA mutant by deleting csrA in a strain that is deficient for glycogen synthesis (glg). Normally without CsrA, glycogen in the cell would increase to a detrimental level. Since a glg⁻ csrA⁻ mutant lacks the ability to make glycogen, the levels never reach a lethal level, allowing the mutant to survive without functional CsrA. Such a glg- csrA- double mutant's ToxR regulation can be studied by growth in various media by measuring OmpU and OmpT expression. Using PCR, restriction enzymes, and DNA ligase, a suicide plasmid was created containing sequences that flank the csrA gene but instead of the csrA gene, a chloramphenicol resistance cassette was inserted. Through bacterial conjugation this plasmid was introduced into three V. cholerae glg- strains. Allelic exchange was carried out utilizing the homology between the DNA flanking wild type csrA and the csrA deletion with chloramphenicol cassette. This first crossover event was initiated with the requirement of the [pi] protein for the plasmid to replicate. Without the pir gene to create [pi] protein, selection for antibiotic resistance required that the plasmid integrate into the genome. This was selected based on the plasmid encoded ampicillin resistance. After the second crossover event, there were two possible outcomes of excision: reverting to wild type csrA or retention of the csrA mutation. The csrA mutant was selected based on its sucrose and chloramphenicol resistance and ampicillin sensitivity. / text

Vibrio cholerae, the causative agent of the diarrheal disease cholera, must acquire iron to survive. Although iron is relatively abundant, it forms insoluble ferric complexes in the presence of oxygen. The more soluble ferrous iron is limited to anaerobic or reducing environments. To meet the nutritional needs of the cell, V. cholerae encodes many different ferric iron transport systems but only one characterized ferrous iron transporter, Feo. Feo is widely distributed in bacteria and archaea, but the mechanism for transport is not known. In this study, basic characterization of the V. cholerae feoABC operon was performed to gain further understanding about a critical iron transport system. Each gene in the operon, feoA, feoB, and feoC, was found to be required for ferrous iron uptake. FeoB, an inner membrane protein, is considered to be the ferrous permease but functions for FeoA and FeoC are not known. These studies show that neither FeoA nor FeoC is required for expression of feoB, suggesting that these proteins are required for Feo function. Analysis of the composition of the Feo transporter using a bacterial adenylate cyclase two-hybrid system indicated interactions between Feo proteins, specifically, between FeoC and the cytoplasmic portion of FeoB. This result indicates that feoC encodes a protein that interacts with FeoB and is necessary for ferrous iron transport. / text

Thesis (Ph. D.)--University of Hawaii at Manoa, 1991. / Includes bibliographical references (leaves 159-175). / Microfiche. / xiii, 175 leaves, bound ill. (some col.) 29 cm

Author's name on cover and spine: Helena M. Ward / Bibliography: leaves 136-157 / xi, 158 leaves, [31] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1989

xiii, 234 p. xxxiii, leaves : photos, tables, graphs ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1978) from the Dept. of Microbiology, University of Adelaide

Includes bibliography / xi, 123, [112] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1985

Thesis (Ph. D.)--University of New South Wales, 2000. / Also available online.