Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.

Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.

Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.

Date of Award


Access Type

Open Access Dissertation

Document type


Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

First Advisor

Alejandro P. Heuck

Second Advisor

Lila M. Gierasch

Third Advisor

Daniel N. Hebert

Subject Categories

Biochemistry | Biophysics


Transport of proteins across membranes is essential during many stages of pathogen infection and colonization of human cells. Many Gram-negative pathogens use a Type 3 Secretion (T3S) system to inject proteins into the target cell during infection. Substantial genetic and biochemical evidence suggest that proteins are translocated across the host plasma membrane through a proteinaceous pore or translocon formed by two bacterial secreted proteins: the T3S translocators. Despite its key role in pathogenesis, virtually nothing is known about the assembly mechanism, structure, and composition of this critical transmembrane complex.

To this end, a cell-free system for the structural and functional characterization of Pseudomonas aeruginosa T3S translocators PopB and PopD was established. PopB and PopD assemble discrete sized pores in liposomal membranes. These pores are stable and heteromeric in nature. Combining this reconstitution methodology with single-molecule fluorescence microscopy methods, the stoichiometry of the membrane-assembled hetero-complex was determined: PopB and PopD assemble an hexadecameric complex at the membrane, with a calculated molecular weight of 601 kDa. The obtained stoichiometry is consistent with ex vivo estimations of the translocon size, and represents the first report on the stoichiometric arrangement of a Type 3 Secretion System translocon.