An investigation of the mechanism responsible for genetic hypervariability in Halobacterium halobium gas vesicle synthesis was conducted. Four partially vacuolated mutants (Vac$\sp{\delta-})$ H. halobium mutants were analyzed by Southern hybridization, cloning, and DNA sequence analysis. In each mutant a different halobacterial insertion element was responsible for the observed phenotype. The insertions mapped upstream of the H. halobium gvpA gene. DNA sequence analysis of the 5$\sp\prime$ and 3$\sp\prime$ regions of gvpA revealed 10 open reading frames; gvpD, E, F, G, H, I, J, K, L, and M; 5$\sp\prime$ to the gvpA gene in the opposite strand and two open reading frames, gvpC and N, in the region 3$\sp\prime$ to gvpA with the same transcriptional orientation as gvpA. A study was conducted to determine if the products of the gvpA, gvpC, gvpD, gvpE, gvpF, gvpJ and gvpM genes could be detected in purified H. halobium gas vesicles or whole cell lysates using immunological techniques. To do so, LacZ-Gvp fusion proteins were produced in E. coli and used to immunize rabbits. The antisera and protein-A column purified antibodies were used in immunoblot analysis of purified gas vesicles and cell lysates. The antiserum directed against the LacZ-GvpC fusion protein was successful in identifying a protein present in both purified gas vesicles and whole cell lysates, and this indicates that the gvpC gene product is a structural gas vesicle protein. Techniques were developed to allow for genetic analysis of gas vesicle synthesis in H. halobium. An H. halobium/E. coli shuttle vector, pJHGV3, which contains the gvpA gene cluster was constructed. Transformation of Vac$\sp-$ H. halobium strains, in which the gvpA gene cluster is deleted, with pJHGV3 resulted in complementation of gas vesicle synthesis. Methods were developed to allow non-polar mutations to be introduced into gvp genes present on pJHGV3. A plasmid containing a disruption of the region 3$\sp\prime$ to the gvpN gene, pJHGV33$\sp\prime$::$\kappa,$ was constructed and used to transform Vac$\sp-$ deletion mutants. Resulting transformants were Vac$\sp+$ indicating that there are not additional contiguous gvp genes downstream from gvpN. Together these techniques will provide useful tools in further analysis of the gas vesicle structure and its assembly.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8521 |
| Date | 01 January 1992 |
| Creators | Halladay, John Thornton |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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