Title

ynfA, a novel Escherichia coli gene of the small multidrug resistance superfamily

Date of Award

2005

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Rudolf Werner, Committee Chair

Abstract

ynfA, of Escherichia coli ( P76169 swissprot, g1787865 GenBank) is then newest Multidrug Resistance (SMR) gene family. It was identified with the aid of bioinformatics tools that allowed us to follow evolutionary principles of sequence conservation, in which functional similarity is inferred from sequence similarity, but surprisingly biochemical data yielded an unexpected mode of action for YnfA. The SMR gene family represents a prokaryotic transport system that is restricted to the eubacterial kingdom and has been so far identified in 63 bacterial species in both gram-negative and -positive bacterial species. Members of this family are composed of 100--120 amino acid residues in length and span the membrane four times as alpha-helices. A ynfA inframe deletion mutant was created from an isogenic wild type E. coli MG1655 that displayed no growth defect compared to its wild type isogenic pair. The ynfA deletion mutant appeared to have altered the partitioning of carbenicillin in such a way that it accumulated in the cytoplasm, thereby resulting in a carbenicillin resistant E. coli. Moreover the mutant resulted in an apparent alteration of the rate of uptake of carbenicillin into E. coli spheroplasts. A constructed multiple alignment revealed a highly conserved glutamic acid residue at position 14 among all of the members within the SMR superfamily. Predicted secondary structure and membrane topology localized the conserved glutamic acid residue to the first transmembrane alpha-helix. Upon mutation to alanine the substrate transport capability was abolished. The conserved glutamic acid residue at position 14 within the SMR family in E. coli corresponded to glutamic acid at position 15 in E. coli 's ynfA. ynfA's deletion or active site mutation of glutamic acid at position 15 to alanine-15 and expression in trans (by a plasmid) confers a gain of survival advantage, appearing as a recessive trait. Thereby, E. coli gains antibiotic resistance to penicillines and certain cephalosporins. Such a mechanism might play an important but currently unknown role in the rapid acquired resistance of human bacterial pathogens to many over-prescribed antibiotics. This is logically compelling since in this case, the gain in survivorship is not dependent on a horizontal transmission of genetic material, but on the loss of some internal genetic material.

Keywords

Biology, Molecular; Biology, Microbiology

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3168678