The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase (LtrA protein), which binds the intron RNA to promote RNA splicing and intron mobility. Mobility occurs by intron RNA reverse splicing directly into a DNA strand and reverse transcription by LtrA. I used LtrA-GFP fusions and immunofluorescence microscopy to show that LtrA localizes to the cellular poles in both Escherichia coli and L. lactis. This polar localization occurs with or without co-expression of the intron RNA, is observed over a wide range of cellular growth rates and expression levels, and is independent of replication origin function. The same localization pattern was found for three non-overlapping LtrA subsegments, reflecting dependence on common redundant signals and/or protein physiochemical properties. When coexpressed in E. coli, LtrA interferes with the polar localization of the Shigella IcsA protein, which mediates polarized actin tail assembly, suggesting competition for a common localization determinant. In E. coli, the Ll.LtrB intron inserts preferentially into the chromosomal ori and ter regions, which are pole localized during much of the cell cycle. Thus, the polar localization of LtrA could account for the preferential insertion of the Ll.LtrB intron in these regions. I established a high throughput method using cellular array and automated fluorescence microscopy for screening transposon-induced mutants, and identified five E. coli genes (gppA, uhpT, wcaK, ynbC, and zntR) in which disruptions result in increased proportion of cells having diffuse LtrA distribution. This altered localization is correlated with a more uniform distribution of Ll.LtrB insertion sites throughout the E. coli genome. Finally, I find that altered LtrA localization in all five disruptants is correlated with accumulation and more diffuse intracellular distribution of polyphosphate, and that a ppx disruptant, which also results in polyphosphate accumulation, shows similar LtrA mislocalization. These findings may reflect interaction between LtrA and intracellular polyphosphate. My findings support the hypothesis that the intracellular localization of LtrA is a major determinant of Ll.LtrB insertion site preference in the E. coli genome. Further, they show that alterations in polyphosphate metabolism can lead to protein mislocalization, and suggest that polyphosphate is an important factor affecting intracellular protein localization.
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/3371 |
| Date | 28 August 2008 |
| Creators | Zhao, Junhua, 1976- |
| Contributors | Lambowitz, Alan |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | electronic |
| Rights | Copyright © is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works. |
Page generated in 0.0018 seconds