The Positional Requirement for the Aspartate Residue in the ExbD Transmembrane Domain

Open Access
Author:
Dubowy, Christine Marie
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Kathleen Postle, Thesis Supervisor
  • Ming Tien, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • Escherichia coli
  • iron transport
  • TonB
  • ExbD
  • transmembrane domain
Abstract:
The TonB system, found in E. coli and other Gram-negative bacteria, is responsible for harvesting the energy of proton motive force (pmf) at the inner membrane and transducing it to outer membrane transporters for the passage of iron-siderophore complexes and vitamin B12 into the cell. Known essential components of the TonB system are the inner membrane proteins TonB, ExbD, and ExbB. TonB is known to directly contact outer membrane transporters, and may enter an energized conformation in response to pmf from which the energy transduction event can take place. ExbB and ExbD are thought to harness pmf but their exact roles in this process are still unknown. ExbD has been proposed to have a signal transduction function, and it may also act as a chaperone to assist TonB in achieving the energized conformational state. ExbD contains a single, essential charged amino acid in its transmembrane domain, aspartate 25. This residue is highly conserved across species and in homologues of ExbD. Several possible functions for this residue have been suggested, including a function as a proton donor or acceptor that assists in the response of the system to pmf. In the work presented here, the spatial requirement for the aspartate residue in the transmembrane domain is tested. Plasmids encoding ExbD double substitution mutants, in which aspartate 25 is replaced with alanine and another transmembrane residue is replaced with aspartate, were constructed, and transformed into a strain lacking the exbD gene (as well as the tolQR operon of the homologous Tol system, because there is crosstalk between the systems). The strains were assayed for TonB system activity using spot titers of B group colicins and bacteriophage φ80. No TonB system activity was detected for any of the stable mutants constructed. Both ExbD(D25A, I33D) and ExbD(D25A, M35D) appeared to be highly unstable, and the mutant strain containing the pExbD(D25A, V43D) plasmid exhibited a severely growth-inhibited phenotype in the presence of ampicillin despite carrying an ampicillin resistance cassette on the plasmid.