title: Isolation and preliminary characterization of amino acid substitution mutations that increase the activity of the osmoregulated ProP protein of Salmonella enterica serovar 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.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, USA.
authors.forename: Brittany J
authors.initials: BJ
authors.lastname: Gasper
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: McCreight
authors.forename: Katelyn
authors.initials: K
authors.lastname: Banschbach
authors.forename: Annamarie
authors.initials: A
authors.lastname: Bustion
authors.forename: Chelsea
authors.initials: C
authors.lastname: Davis
authors.forename: Rohan
authors.initials: R
authors.lastname: Divecha
authors.forename: Madison
authors.initials: M
authors.lastname: Donoho
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Elmore
authors.forename: Curtis M
authors.initials: CM
authors.lastname: Garrison
authors.forename: Steve
authors.initials: S
authors.lastname: Glenn
authors.forename: Danielle C
authors.initials: DC
authors.lastname: Goeman
authors.forename: Michelle
authors.initials: M
authors.lastname: Haby
authors.forename: Terrice
authors.initials: T
authors.lastname: Hooks
authors.forename: Abraham M
authors.initials: AM
authors.lastname: Korman
authors.forename: Joseph
authors.initials: J
authors.lastname: Kowal
authors.forename: Samantha
authors.initials: S
authors.lastname: Kuschke
authors.forename: Jane E
authors.initials: JE
authors.lastname: Mellencamp
authors.forename: Melanie
authors.initials: M
authors.lastname: Meyer
authors.forename: Alake N
authors.initials: AN
authors.lastname: Myers
authors.forename: Monique F
authors.initials: MF
authors.lastname: Nichols
authors.forename: Allison
authors.initials: A
authors.lastname: Pfeifer
authors.forename: Alexander
authors.initials: A
authors.lastname: Porucznik
authors.forename: Xiao
authors.initials: X
authors.lastname: Qu
authors.forename: Margaret
authors.initials: M
authors.lastname: Ramos-Miller
authors.forename: Russell R
authors.initials: RR
authors.lastname: Reed
authors.forename: Adlet
authors.initials: A
authors.lastname: Sagintayev
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Singel
authors.forename: Anna
authors.initials: A
authors.lastname: Smith
authors.forename: Madeline E
authors.initials: ME
authors.lastname: Valle
authors.forename: Anne
authors.initials: A
authors.lastname: Venderley
authors.forename: Chelsea A
authors.initials: CA
authors.lastname: Weber
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Zaffino
authors.forename: Laszlo N
authors.initials: LN
authors.lastname: Csonka
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Gardner
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010641:Phenotype; D011392:Proline; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D012486:Salmonella typhimurium / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014882:Water-Electrolyte Balance / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D011392:Proline