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Updated: May 19, 2026
High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages
Published on: August 11, 2014
Reduced Na+ affinity increases turnover of Salmonella enterica serovar Typhimurium MelB
1Department of Cell Physiology & Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
Abstract:
The melibiose permease of Salmonella enterica serovar Typhimurium (MelB(St)) catalyzes symport of melibiose with Na(+), Li(+), or H(+). Bioinformatics and mutational analyses indicate that a conserved Gly117 (helix IV) is a component of the Na(+)-binding site. In this study, Gly117 was mutated to Ser, Asn, or Cys. All three mutations increase the maximum rate (V(max)) for melibiose transport in Escherichia coli DW2 and greatly decrease Na(+) affinity, indicating that intracellular release of Na(+) is facilitated. Rapid melibiose transport, particularly by the G117N mutant, triggers osmotic lysis in the lag phase of growth. The findings support the previous conclusion that Gly117 plays an important role in cation binding and translocation. Furthermore, a spontaneous second-site mutation (P148L between loop(4-5) and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases V(max) with little effect on cosubstrate binding in G117C, G117S, and G117N mutants. Thus, the P148L mutation specifically inhibits transport velocity and thereby blocks the lethal effect of elevated melibiose transport in the Gly117 mutants.
Insights
Mutations in the melibiose permease (MelB(St)) at Gly117 enhance transport but reduce sodium affinity. A secondary mutation, P148L, prevents lethal osmotic lysis by slowing transport velocity.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The melibiose permease (MelB(St)) from Salmonella Typhimurium facilitates the transport of melibiose coupled with cations.
- Conserved residue Gly117 in helix IV is implicated as a key component of the sodium-binding site.
Purpose of the Study:
- To investigate the role of Gly117 in cation binding and melibiose transport.
- To characterize the functional consequences of mutating Gly117 to Ser, Asn, or Cys.
- To identify mechanisms mitigating potential detrimental effects of altered transport.
Main Methods:
- Site-directed mutagenesis of Gly117 and P148.
- Transport assays in Escherichia coli to measure melibiose uptake kinetics (Vmax, affinity).
- Phenotypic analysis of cell viability and growth under stress conditions.
Main Results:
- Mutations at Gly117 (G117S, G117N, G117C) increased melibiose transport rates (Vmax) but decreased sodium affinity.
- Rapid transport by Gly117 mutants, especially G117N, induced osmotic lysis during the lag growth phase.
- A spontaneous P148L mutation in the G117C background prevented cell lysis by reducing Vmax without significantly affecting cosubstrate binding.
Conclusions:
- Glycine 117 is crucial for cation binding and translocation in MelB(St).
- Facilitated Na+ release at Gly117 enhances transport velocity but can lead to lethal osmotic stress.
- The P148L mutation acts as a suppressor, inhibiting transport velocity to prevent cell lysis, highlighting a balance between transport efficiency and cellular homeostasis.
