Isolation and preliminary characterization of amino acid substitution mutations that increase the activity of the osmoregulated ProP protein of Salmonella enterica serovar Typhimurium.

DNA and Cell Biology
Brittany J GasperStephanie M Gardner

Abstract

In Enterobacteriaceae, the ProP protein, which takes up proline and glycine betaine, is subject to a post-translational control mechanism that increases its activity at high osmolarity. In order to investigate the osmoregulatory mechanism of the Salmonella enterica ProP, we devised a positive selection for mutations that conferred increased activity on this protein at low osmolarity. The selection involved the isolation of mutations in a proline auxotroph that resulted in increased accumulation of proline via the ProP system in the presence of glycine betaine, which is a competitive inhibitor of proline uptake by this permease. This selection was performed by first-year undergraduates in two semesters of a research-based laboratory course. The students generated sixteen mutations resulting in six different single amino acids substitutions. They determined the effects of the mutations on the growth rates of the cells in media of high and low osmolarity in the presence of low concentrations of proline or glycine betaine. Furthermore, they identified the mutations by DNA sequencing and displayed the mutated amino acids on a putative three-dimensional structure of the protein. This analysis suggested that all six amino acid substit...Continue Reading

References

Apr 1, 1986·Journal of Bacteriology·S GrotheJ M Wood
Jul 1, 1985·Journal of Bacteriology·V J Dunlap, L N Csonka
Jun 1, 1969·Archives of Biochemistry and Biophysics·H R Kaback, F Deuel
Feb 1, 1996·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·M Sekiguchi
Oct 6, 1997·Microbiology·Min-Ken LiaoStanley Maloy
Feb 28, 2009·Nature Protocols·Lawrence A Kelley, Michael J E Sternberg

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Datasets Mentioned

BETA
AE006468.1

Methods Mentioned

BETA
light scattering

Software Mentioned

ProP
ClustalW2
PyMol
PyMol Molecular Graphics System
Phyre

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