Characterization of a novel tetracycline resistance gene from the Bacteroides transposons Tn4351 and Tn4400
Speer, Brenda Susan
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https://hdl.handle.net/2142/22461
Description
Title
Characterization of a novel tetracycline resistance gene from the Bacteroides transposons Tn4351 and Tn4400
Author(s)
Speer, Brenda Susan
Issue Date
1989
Doctoral Committee Chair(s)
Salyers, Abigail A.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Language
eng
Abstract
A tetracycline resistance ($\rm \sp{*}Tc\sp{r}$) gene that was originally found on two Bactetoides transposons, Tn4351 and Tn4400, confers resistance on Escherichia coli, but only when the cells are grown aerobically. I first showed that the product of the $\rm \sp{*}Tc\sp{r}$ gene is a 44 kDa cytoplasmic protein. This indicated that the mechanism might be different from efflux, which is mediated by a membrane protein. To determine if failure of the resistance to work under anaerobic conditions was due to lack of gene expression, I constructed a fusion of the $\rm \sp{*}Tc\sp{r}$ gene to 'lacZ and used it to show that the $\rm \sp{*}Tc\sp{r}$ gene was expressed under both aerobic and anaerobic conditions. Further experiments showed that the $\rm \sp{*}Tc\sp{r}$ gene product was chemically altering tetracycline and did not work anaerobically because the alteration reaction required oxygen. Altered tetracycline could be separated from tetracycline by thin layer chromotography or by reverse phase liquid chromotography. Altered tetracycline has a UV-vis absorption peak at 258nm, rather than a peak at 364nm which is typical of tetracycline. Attempts to solve the structure of altered tetracycline were unsuccessful. Previously, the only known mechanisms of $\rm Tc\sp{r}$ were active efflux of tetracycline and ribosomal modification to prevent binding of the antibiotic. My work revealed a third type of $\rm Tc\sp{r}$ mechanism. However, these findings produced a controversy. Park et al (27) observed that cells carrying Tn4400 exhibited tetracycline efflux activity and suggested that the resistance conferred by Tn4400 was due to efflux. I disproved this hypothesis by showing that Tn4351, which confers a high level of $\rm \sp{*}Tc\sp{r}$ than Tn4400, does not mediate tetracycline efflux. Moreover, I showed that the gene on Tn4400, which mediates efflux, is separate from the gene for tetracycline modification and that the efflux gene does not confer resistance to sensitive cells. The finding of a tetracycline efflux gene which does not confer resistance raises questions about the classical model of efflux mediated tetracycline resistance in which it was assumed that efflux alone is responsible for resistance.
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