The Search for Novel Proteins Involved in the TonB-Dependent Energy Transduction System
Open Access
- Author:
- Lesnansky, Alana
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- March 06, 2020
- Committee Members:
- Kathleen Postle, Thesis Advisor/Co-Advisor
Timothy Charles Meredith, Committee Member
Timothy Iwao Miyashiro, Committee Member
Lorraine C Santy, Committee Member
Wendy Hanna-Rose, Program Head/Chair - Keywords:
- TonB
ExbB
Overproduction Lethality
Proline
Escherichia coli - Abstract:
- The semi-permeable outer membrane of Gram-negative bacteria affords some protection from harsh environmental agents, such as detergents and antibiotics. However, the size exclusion limit of the porins in the outer membrane (~600 Da) also precludes import of larger and essential nutrients. Iron is very scarce in most environments, and specifically in the human host serum. In order to remain competitive in the struggle to obtain iron, Gram-negative bacteria secrete molecules with extremely high affinities for iron, known as siderophores. The siderophores can effectively sequester scarce amounts of iron from the environment, including from host proteins. The iron-siderophore complex is too large to diffuse through the porins of the outer membrane. In addition, the outer membrane is unenergized, and iron is too scarce and too important to rely on diffusion alone. In order to actively transport the iron-siderophore complex across the semi permeable and unenergized outer membrane, Gram-negative bacteria must utilize the TonB dependent energy transduction system to harvest the energy of the proton motive force at the inner membrane and transduce it to the outer membrane to drive active import the iron siderophore complex. While it has been well established that the TonB-dependent energy transduction system of Gram-negative bacteria consists of at least 3 inner membrane proteins (TonB, ExbB, and ExbD) and an outer membrane transporter, we have several pieces of evidence to suggest that there are one or more cytoplasmically localized “mystery proteins” that play an important role in the function of the system. We know that overproduction of TonB from a strong promoter results in immediate cell death and growth arrest, a phenotype we call Overproduction Lethality. In this study, we attempt to not only understand Overproduction Lethality, but also to exploit the death phenotype in order to identify one or more “mystery proteins” interacting with the TonB-dependent energy transduction system.