Date of Award

1-1-1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology & Immunology

Abstract

Cosmid clones of E. coli chromosomal DNA were screened for the ability to complement a lysis deficient mutation that made the bacterium resistant to lysis by the E protein of bacteriophage $\phi\rm X174.$ From one of these clones a DNA fragment from the 74.8 minute region of the chromosome was cloned that contained two genes of interest, slyD and fkpA. Both genes encode proteins that are members of the family of proteins that bind the immunosuppressive drug FK506. The SlyD protein has very low sequence similarity to the FK506 binding proteins (FKBPs) and is the only E. coli protein that has been shown to be required for lysis by the E protein of $\phi\rm X174.$ The FkpA protein has very strong sequence similarity to the FKBPs and its carboxyl half matches the consensus of the FK506 binding motif in 40 of 48 positions, including those amino acids that form hydrogen bonds with the drug. FkpA has extensive similarity to a subclass of FKBPs, the macrophage infectivity potentiator (Mip) proteins of Legionella spp. and Chlamydia trachomatis. Overexpression of FkpA from a high copy number plasmid or inactivation of the chromosome copy of fkpA had no detrimental effect on bacterial growth, indicating that fkpA is not an essential gene. Hybridization of fkpA-specific DNA probes to genomic blots revealed that similar genes exist in other representatives of the Enterobacteriaceae. The ubiquity of the FKBPs in both prokaryotic and eukaryotic organisms indicates an important cellular function for these proteins. Most FKBPs have been shown to have peptidyl-prolyl cis-trans isomerase activity and their in vivo functions may involve protein folding and transport. The discovery of fkpA should make it easier to study the in vivo roles of FKBPs in a well-characterized bacterium, and may also mark the discovery of a new virulence gene shared by members of the Enterobacteriaceae.

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