Graduation date: 2004

Bibliography : leaves [1]-[5]. / [xiv], 66, [5] leaves, [20] 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 Biochemistry, 1991

Bibliography: leaves 120-138. / xi, 138, [41] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Characterises the 186 coliphage site-specific integration reaction in vitro with the expectation that it could be used in mammalian systems to insert genes or modify existing ones, to provide an alternative method for the production of transgenic livestock species. The analyzed system is the temperate bacteriophage 186. The in-vitro requirements for 186 integrative site-specific recombination were investigated. In vivo investigations were conducted whereby active 186-intasomes were microinjected into fertilised mouse eggs containing genomic copies of 186-attB. The 186 system has not been shown to work in vivo. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry 1999?

Bibliography: leaves 120-138. / xi, 138, [41] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Characterises the 186 coliphage site-specific integration reaction in vitro with the expectation that it could be used in mammalian systems to insert genes or modify existing ones, to provide an alternative method for the production of transgenic livestock species. The analyzed system is the temperate bacteriophage 186. The in-vitro requirements for 186 integrative site-specific recombination were investigated. In vivo investigations were conducted whereby active 186-intasomes were microinjected into fertilised mouse eggs containing genomic copies of 186-attB. The 186 system has not been shown to work in vivo. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry 1999?

Clostridium difficile is an important hospital-acquired pathogen causing C. difficile-associated diarrhoea (CDAD) in patients exposed to antibiotics. The lack of information on bacteriophages of C. difficile, and the potential of phages as therapeutic agents for the treatment of CDAD, prompted the isolation and characterisation of phages active against clinical isolates of C. difficile in order to determine the prevalence and significance of phages of this anaerobe. Three (5.4 %) of 56 clinical C. difficile isolates induced by mitomycin C yielded dsDNA phages C2, C5, C6 and C8. The four phages differed from previously described C. difficile phages in particle morphology, burst size and host range. C2, C5 and C8 particles were members of the family Myoviridae, while C6 belonged to Siphoviridae. The burst sizes were 5 for C2, 7 for C5, 19 for C6 and 33 for C8. C8 had the broadest host range, lysing 27 out of 56 (48 %) C. difficile isolates, followed by C6 (43 %), C5 (20 %) and C2 (20 %). Superinfection experiments, restriction enzyme analysis and Southern hybridisation showed C2 and C5 to be closely related with C8 somewhat related to them, however, C6 was distantly related to the other three phages. C2 was further characterised as a representative phage. Its genome did not possess cohesive ends, and was shown to integrate chromosomally via an attP site identified within a 1.9 kb HindIII fragment. However, an integrase gene, which is typically close to the attP region, was not located. Nine of 16 HindIII fragments of C2, including the 1.9 kb fragment, were cloned into pUC18. Approximately 9 kb of the estimated 43 kb genome of C2 was sequenced and analysed. Seven of the nine translated sequences were homologous to phage structural proteins, two sequences were not homologous to any relevant protein in the Genbank and EMBL databases, and one was homologous to proteins of Clostridium species. Nucleotide homology between the C2 sequences and the recently sequenced C. difficile strain CD630 was found in three regions within CD630 genome. Seven of the nine sequences, including the 1.9 kb fragment, were clustered in one region. These data suggest that the genes constitute a phage structural gene module. The presence of C2-like sequences in CD630, and Southern hybridisation of C. difficile strains using phage probes, suggested related prophage sequences may be commonly present in this bacterial species. An investigation was carried out to determine the presence of toxin genes tcdA and tcdB, and PaLoc-associated gene tcdE, in phage DNA. In addition, the effect of phage infection on toxin production of toxigenic C. difficile strains was studied. Of the three genes, tcdE only was detected in phages C2, C5 and C8, but not in C6. Strains that maintained phages in a stable manner (lysogens) were isolated and used in toxin studies. The amount of toxin B produced was measured by cytotoxic assays using Vero cells, and toxin A production was measured by ELISA. Although phages did not encode toxin A or B genes, there was a significant increase in toxin B production in some lysogens. There was no increase in toxin A production. Transcriptional analyses of tcdA and tcdB in lysogens and parental strains was performed by real-time RT-PCR and Northern hybridisation to determine whether phage was affecting regulation of toxin transcription. Phage did not appear to affect toxin gene transcription, although results from real-time RT-PCR and Northern hybridisation were conflicting. A phage induced from the highly toxigenic reference strain VPI 10463 was also briefly characterised and investigated for its effect on toxin production in VPI 10463. The phage, ΦCV, had similar particle morphology to C2, C5 and C8, and had some HindIII bands in common with C2 and C5. Two cured variant strains produced significantly less toxin B compared to VPI 10463. In conclusion, several important properties of C. difficile phages were characterised. It appears these temperate phages may play a role in toxin production making them unsuitable as therapeutic agents for the treatment of CDAD. However, C2 phage may have potential as the basis for an integrative vector that will add to the genetic tools available for clostridia.

Thesis (M.S.)--University of Wisconsin--Madison, 1968. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

From Summary: A survey of collagenolytic aerobic bacteria from cured hides yielded three strains of Bacillus and eight of Achromobacter which degraded collagen at 0.4 M NaCl. Achromobacter sp. 2 was chosen for genetic studies due to its high collagenolytic activity and the lack of genetic information on Achromobacter. Four temperate bacteriophages specific for Achromobacter sp. 2 were isolated and their relationships studied. The phages caused lysogenic conversion resulting in the inability of lysogens to adsorb phage. Achromobacter sp. 2 was shown to be a cryptic lysogen as it was not immune to superinfection but had a very low rate of spontaneous induction which could be increased with mutagens. It is proposed that the cryptic lysogeny of this strain is maintained by a defective excision mechanism and the mode of prophage integration in the host chromosome. DNA extracted from phage α3a was used to transfect spheroplasts. The optimal conditions for the development of competence for transfection were determined. The presence of nuclease-attack on phage DNA under conditions of prolonged incubation of DNA and spheroplasts was proposed. A method for extracting Achromobacter DNA was devised which yielded purified, undegraded DNA, but it was not possible to transform Achromobacter sp. 2 with this DNA. The a phages were used to transduce a number of genetic markers into Achromobacter auxotrophs. The transduct ants had the ability to release the cryptic α3 prophage at a high rate while maintaining their sensitivity to homologous phage infection. It is proposed that this is due to complementation between the cryptic prophage and the residual phage functions in the transducing particles. The transductants segregated auxotrophs with a probability of 10⁻³ per cell per generation. It appears that an unusual system of generalised transduction is operating whereby the transducing particles contain both phage and bacterial DNA which is incorporated into the recipient genome by a single recombination event yielding unstable transductants. In a study on induction of Escherichia coli (λ), carcinogenic nitrosamines were shown to be inducers of phage development. This provides a screening system for potentially harmful nitrosamines.

The sequence of the rightmost 4,626 bp of the Bacillus phage φ29 genome is presented and analyzed. Nine large open reading frames (ORF's) have been found. Three of these ORF's are correlated with the late genes 14, 15 and 16. The remaining six ORF's are in the right early region. One of these early ORF's has been identified as gene 17 (g17), the only early gene to have been genetically mapped in this region. The remaining ORF's (16.5, 16.6, 16.7, 16.8 and 16.9) were previously unknown. The biological efficacies of some of these putative early ORF's were demonstrated using an in vitro E. coli transcription-translation system. The primary amino acid sequences, molecular weights, translational initiation sequences and genetic organization of these nine genes are presented and discussed. Gene product 15 (gp15) was found to have strong homology with Salmonella phage P22 gp19, a lysozyme. gp15 also has a lesser but possibly significant homology with T4 gene product e (gpe), also a lysozyme. Using a clone containing φ29 g15 it was shown that gp15 can complement T4 gene e (ge) mutant infections, leading to the conclusion that φ29 g15 encodes a lysozyme. Three transcriptional initiation sites (P(E)3, P(EC)3 and B2) were previously mapped in this region. The sequences of the putative P(EC)3 and B2 promoter sites are presented and shown to have homology with the Bacillus σ⁵⁵ concensus sequence. Sequences having homology to a minor Bacillus sigma factor recognition site, σ³², are also presented and discussed. The region between the last late gene (g16) and the last early gene (ORF-16.5) consists of only 30 bp. Analysis of potential secondary structures of transcripts across this region suggests that the same sequences may be involved in the termination of both late and early transcription.

Files on accompanying CD-ROM: Appendix III Phages ST64T and ST64B sequences, are in rtf format. Bibliography: leaves 279-324. System requirements for accompanying CD-ROM: IBM or compatible ; Microsoft Word or compatible to read rtf files.

Files on accompanying CD-ROM: Appendix III Phages ST64T and ST64B sequences, are in rtf format. / Bibliography: leaves 279-324. / System requirements for accompanying CD-ROM: IBM or compatible ; Microsoft Word or compatible to read rtf files. / xii, 325, [8] leaves, [116] leaves of plates : ill. (some col.) ; 30 cm. + 1 CD-ROM (4 3/4 in.) / 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 Molecular Biosciences, 2002?