Living organisms must monitor and respond to a diverse array of environmental stimuli. These responses, many of which are genetically-programmed, allow the organisms to obtain nutrients as well as cope with toxins. To identify such responses in Escherichia coli, a chromosomal luxAB gene fusion library was constructed. By screening this library with metals and monitoring for increases in luminescence, two metal-inducible clones were identified. Luminescence of clone LF20110 increases with increasing concentrations of added aluminum, iron, indium, gallium and vanadium, but not added calcium, magnesium or nickel. Luminescence of clone TBT1 increases with increasing concentrations of added tributyltin, triethyltin, tri-n-propyltin and carbonyl cyanide m-chlorophenyl hydrazone, but not added dibutyltin or tetrabutyltin. / Analysis of clone TBT1 revealed a luxAB fusion to the uhpT gene, which encodes a hexose-6-phosphate transport protein. Transcriptional regulation of uhpT by glucose-6-phosphate has been well characterized. However, it had not been previously demonstrated that addition of tributyltin could induce transcription of uhpT. This effect on uhpT is associated with the uncoupling of oxidative phosphorylation. / Isolation and sequencing of the luxAB gene fusion from clone LF20110 uncovered a previously uncharacterized gene, which we call ais (a&barbelow;luminum and i&barbelow;ron s&barbelow;timulated). The ais gene has been sequenced, the transcriptional start site has been identified, and the 22 kDa Ais protein has been overexpressed in vivo. Mutation of the basS gene abrogates metal-induced transcription of ais. However, expression of the BasR-BasS two-component regulatory system, in trans, restores the metal-inducible response. / The E. coli BasR-BasS regulon is poorly characterized. To identify genes regulated by this two-component system, a MudI (lac Ap) gene fusion library was constructed and screened Gene fusions to blc, glmUS, iap, and ygjlJK exhibited increased beta-galactosidase activity upon overexpression of BasR-BasS, while expression of a dsdXA fusion decreased. By searching the E. coli genome database for putative BasR binding sequences, four additional genes, seiC, yibD, yhhT and yaf were identified. Of these nine genes, four have not yet been characterized. The other five are associated with cell membrane functions or sugar metabolism.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37860 |
| Date | January 2001 |
| Creators | Alexander, David Charles. |
| Contributors | DuBow, Michael S. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001838390, proquestno: NQ75600, Theses scanned by UMI/ProQuest. |
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