Isolation and Preliminary Characterization of Amino Acid Substitution Mutations That Increase the Activity of the Osmoregulated ProP Protein ofSalmonella EntericaSerovar Typhimurium

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 substitutions are residues in trans-membrane helices that have been proposed to contribute to the formation of the transport pore, and, thus, may affect the substrate binding site of the protein.

Engaging Students in Authentic Microbiology Research in an Introductory Biology Laboratory Course is Correlated with Gains in Student Understanding of the Nature of Authentic Research and Critical Thinking

“…This course was recognized for its educational merit and was one of the 2012 AAAS/Science Prize winners for Inquiry-Based Instruction (15). Further, the data generated by the students in the 2010 and 2011 spring semesters led to a scientific publication with all of the students as authors (14)!…”