The induction, decay and diffusion of a bacterial tetracycline efflux pump.

Format:

Book

Thesis/Dissertation

Author/Creator:

Chow, David G.

Contributor:

Yodh, Arjun, committee member.

Binns, Andrew, committee member.

Zhu, Jun, committee member.

Kamien, Randall, committee member.

Goulian, Mark, advisor.

University of Pennsylvania. Physics and Astronomy.

Language:

English

Subjects (All):

Biophysics.

Biophysics, General.

0786.

Penn dissertations--Physics and astronomy.

Physics and astronomy--Penn dissertations.

Local Subjects:

Biophysics, General.

Penn dissertations--Physics and astronomy.

Physics and astronomy--Penn dissertations.

0786.

Physical Description:

138 pages

Contained In:

Dissertation Abstracts International 74-06B(E).

System Details:

Mode of access: World Wide Web.

text file

Summary:

Drug efflux is the most common means of resistance against the antibiotic tetracycline. In the bacterium Escherichia coli, the tet system produces the tetracycline efflux pump TetA and a repressor that negatively autoregulates both the pump and itself. Though it is one of the best-studied gene regulatory circuits, little work has been done on its dynamics, especially in response to high levels of tetracycline.

The first part of this thesis explores the induction and decay dynamics of TetA. We constructed fluorescent protein fusions and used fluorescence microscopy with single-cell resolution, and found that surviving the sudden onset of a high concentration of tetracycline requires winning a race to produce sufficient TetA in a short time, which many cells lose. We explore what predisposing factors may help individual cells win this race, and find that the endogenous drug efflux pump AcrAB-TolC plays a vital role during early induction. We also find that TetA is not actively degraded after extracellular tetracycline is removed, and is produced for a surprisingly long time because of slow permeation of tetracycline out of the cytoplasm. We constructed a mathematical model that helps explain this.

The second part of this thesis explores the diffusion of TetA within the cell membrane. Structural inhomogeneities in biomembranes can lead to complex diffusive behavior of membrane proteins that depend on the length- or time- scales that are probed. This effect is well studied in eukaryotic cells, but has been explored only recently in bacteria. Here we used fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) to study diffusion of the membrane protein TetA-YFP. We find that the diffusion constant determined from FRAP is comparable to other reports of inner membrane protein diffusion constants in E. coli. However, FCS, which probes diffusion on shorter length scales, gives a value that is almost two orders of magnitude higher and is comparable to lipid diffusion constants. These results suggest there is a population of TetA-YFP molecules in the membrane that move rapidly over short length scales (∼400 nm) but move significantly more slowly over the longer length scales probed by FRAP.

Notes:

Thesis (Ph.D. in Physics and Astronomy) -- University of Pennsylvania, 2012.

Source: Dissertation Abstracts International, Volume: 74-06(E), Section: B.

Adviser: Mark Goulian.

Local Notes:

School code: 0175.

ISBN:

9781267882592

Access Restriction:

Restricted for use by site license.