Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Gregory J. Phillips
The Ffh protein of Escherichia coli is a 48kd protein that is homologous to the 54kd subunit of the eukaryotic signal recognition particle (SRP). Ffh, similar to its eukaryotic counterpart, is important for localization of proteins to the bacterial inner membrane. Studies to understand Ffh function have largely depended on the use of E. coli strains that allow depletion of the wild type gene product. As an alternative approach to studying Ffh function, we have isolated a temperature-sensitive ffh mutant using a plasmid shuffling system developed to improve the efficiency of isolation of conditional mutations in essential genes. The mutant is the result of two amino acid changes in conserved regions of the Ffh protein. Characterization of the temperature-sensitive mutant revealed that the thermolabile protein is defective at the permissive temperature of 30°C, but cells rapidly lose viability upon growth at the non-permissive temperature of 42°C. While the ffhTS mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. Furthermore, the mutant shows elevated expression of heat shock proteins, and accumulates increased levels of insoluble proteins, especially at 42°C. Collectively these results are consistent with a model that ffh is required only for the insertion and translocation of inner membrane proteins.;The temperature sensitivity of the ffhTS mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial signal recognition particle. The suppression was due to stabilization of the thermolabile Ffh protein in the presence of elevated levels of 4.5S RNA. This observation can be exploited as a means to isolate and characterize 4.5S RNA mutants and should prove useful to better understand how the structure of the SRP contributes to its in vivo function.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Park, Sei-Kyoung, "Genetic analysis of ffh from Escherichia coli " (2001). Retrospective Theses and Dissertations. 1073.